Contents

Sponsors......................................................................................................................................4

Chairman message.......................................................................................................................5

Local Organizing Committee.......................................................................................................6

Scientific Advisory Committee....................................................................................................7

Student Poster Evaluation Committee.......................................................................................7

General Information.....................................................................................................................8

Program Day 1, Wednesday, August 15th.................................................................................10

AbstractsforVICWorkshopsandOpeningConference..................................................14

Program for Day 2, Thursday, August 16th...............................................................................18

AbstractsforPresentationsDay2....................................................................................21

Program for Day 3, Friday, August 17th..................................................................................... 26

AbstractsforPresentationsDay3....................................................................................31

Program for Day 4, Saturday, August 18th................................................................................35

AbstractsforSpeakersDay4...........................................................................................40

Program for Day 5, Sunday, August 19th..................................................................................44

AbstractsforSpeakersDay5...........................................................................................49

Abstracts for Posters Presentations........................................................................................52

Author index ............................................................................................................................146

Cross references for key words..............................................................................................153

Cross reference for species.....................................................................................................158

List of participants..................................................................................................................159

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Sponsors

DearVeterinaryImmunologists, TheBrazilianSocietyforImmunologywelcomesyoutothe8thInternationalVeterinaryImmunologySymposium.Participantsfrommorethan35countriescongregateinOuroPretotoshareknowledgeandexperience.

Thescientificprogram,theheartofthe8thIVIS,istheresultoftheeffortsoftheScientificAdvisoryCommittee,oftheChairsforthescientificsessionsandoftheLocalOrganizingCommittee.Wearetrulythankfulforourcolleagues’insightsandtime,whichproducedacomprehensiveandexcitingprogram.

Wewishallparticipantsaveryfruitfulmeeting.

IsabelK.F.deMirandaSantosChair

BeatrizRossettiFerreiraVice-Chair

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Local Organizing Committee

Isabel K.F. de Miranda Santos FaculdadedeMedicinadeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto

Beatriz Rossetti Ferreira EscoladeEnfermagemdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto

Maristela Martins Camargo InstitutodeCiênciasBiomédicas,UniversidadedeSãoPaulo,SãoPaulo

Rosangela Zacarias MachadoFaculdadedeCiênciasAgráriaseVeterinárias,UniversidadeEstadualPaulista,Jaboticabal

Alexandre Barbosa ReisFaculdadedeFarmácia,UniversidadeFederaldeOuroPreto,OuroPreto

Alexandre Rodrigues CaetanoEmbrapaRecursosGenéticoseBiotecnologia,Brasília

Aline Aparecida Rezende RodriguesValléeSA,SãoPaulo

Ana Paula Junqueira-KipnisFaculdadedeMedicinaVeterinária,UniversidadeFederaldeGoiás,Goiânia

Itabajara da Silva Vaz Jr. CentrodeBiotecnologia,UniversidadeFederaldoRioGrandedoSul,PortoAlegre

José Roberto Kfoury Jr.FaculdadedeMedicinaVeterinária,UniversidadedeSãoPaulo,SãoPaulo

Lygia Maria Friche PassosFaculdadedeMedicinaVeterinária,UniversidadeFederaldeMinasGerais

Maria Julia B. Flaminio CornellUniversity,Ithaca

Ricardo T. Fujiwara FundaçãoOswaldoCruz,BeloHorizonte

Ricardo Tostes Gazzinelli FundaçãoOswaldoCruz,BeloHorizonte

Valeria M.F. LimaUniversidadeEstadualPaulista,Araçatuba

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Scientific Advisory Committee

Bruce N. Wilkie UniversityofGuelph,Guelph,Canada Caroline Fossum SUAS,Uppsala,Sweden Chieko Kai UniversityofTokyo,TokyoJapan Jayne C. Hope InstituteforAnimalHealth,Compton,UK Falko Steinbach VLA,EVIG,Addlestone,UK Isabelle Schwartz-Cornil INRA,Jouy-en-Josas,France Jan Naessens VIC,ILRI,Nairobi,Kenya Javier Dominguez INIA,Madrid,Spain Joan K. Lunney ARS-USDA,Beltsville,USA Mark Jutila MSU,Bozeman,USA Paul Wood Pfizer,WestRyde,Australia Ricardo T. Gazzinelli FioCruz,BeloHorizonte,Brazil Wendy C. Brown WSU,Pullman,USA Wilmar Dias da Silva UENF,CamposdeGoitacazes,Brazil

Student Poster Evaluation Committee

Beatriz Rossetti Ferreira Chair,UniversityofSãoPaulo,Brazil Ana Paula Junqueira-Kipnis FederalUniversityofGoiás,Brazil Joan K. Lunney USDA-ARS,USA David Artis UniversityofPennsylvania,USA Ildiko van Rhijn UtrechtUniversity,TheNetherlands Isabelle Oswald NationalAgriculturalResearchInstitute,France Juan Anguita UniversityofMassachusetts,USA Maristela Martins Camargo UniversityofSãoPaulo,Brazil Preben Boysen NationalVeterinaryInstitute,Norway Ricardo Fujiwara OswaldoCruzFoundation,Brazil Waithaka Mwangi TexasA&M,USA Rosangela Zacarias Machado UniversidadeEstadualPaulista

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General Information

nIdentification Badgesarerequiredforaccesstoallsymposiumactivities.

nThe Registration DeskislocatedattheentrancetothePosterExhibitHall(SalãoDiamante).oStaffarereadytohelpyouandareidentifiedbytheirbadgesanduniforms(whiteshirtwiththe

symposiumlogo).

nParticipant Materials includeidentificationbadge,abookandCDwiththecompleteprogramandabstractsandacertificateofparticipation.

nTransportationfromOuroPretototheConventionCenterlocatedattheEstalagemdasMinasGeraisHotel.oTheshuttlesfromOuroPretototheConventionCenterareofferedbytheeventorganizersand

operatedbyBlumar/MasterTurismo.oAllShuttlesareidentifiedbysignswiththe8thIVISLOGO.oThedistancebetweenOuroPretoandtheConventioncenterisabout7Kilometers(5Miles).oTheshuttlerouteswilldepartfromthefollowinglocationsandbythefollowingschedule.

Route1–GrandeHoteldeOuroPreto.Route2–ColonialHotel.Route3–PousadadoMondego.Route4–CasadosContosHotelandPousadadosSinos.Route5–TiradentesSquare(PraçaTiradentes,intheHistoricalCenter)andPousadaSinháOlímpia;

oSchedulesfortheshuttles:August15th(Wednesday)oDeparture:08:00h/08:20h/08:40h/09:10h/09:20h/10:00hoReturn:19:30h/19:50h/20:10h/20:25h/20:40h/20:50h/21:15h/21:30h

August16th(Thursday)oDeparture:07:30h/07:50h/08:10h/08:40h/08:50h/09:30hoReturn:19:00h/19:20h/19:40h/19:55h/20:10h/20:20h/20:40h/21h00h

August17th(Friday)oDeparture:07:30h/07:50h/08:10h/08:40h/08:50h/09:30hoReturn:18:30h/18:50h/19:10h/19:25h/19:40h/19:50h/20:00h/20:20h/20:40h/21:00h/

21:20h/21:40h/22:00hAugust18th(Saturday)oDeparture:07:30h/07:50h/08:10h/08:40h/08:50h/09:30hoReturn:18h00h/18:20h/18:40h/18:55h/19:10h/19:20h/19h40h/20:00h

August19th(Sunday)oDeparture:07:40h/08:00h/08:20h/08:50h/09:00h/09:30hoReturn:19:00h/19:20h/19:40h/19:55h/20:10h/20:20h/20:40h/21:00h

oA taxi ride(oneway)betweentheConventionCenterandthemainhotelsintheOuroPretoHistoricalCentercostsapproximatelyR$25,00(USD13.00).

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nThe Media deskislocatedintheSalãoÁgata(lowerfloor)andisopendailyfrom07:50to18:00h.Speakersarekindlyrequestedtodelivertheirfilesatleast2hoursinadvanceoftheirpresentationor,inthecaseofsessionsbeginningat8:30am,thedaybefore.

nLunchoBoxluncheswithfruitjuicewillbesoldattheconventioncenterforR$11,20(approximatelyUSD

5.60).Softdrinks,beer,coffee,teaandmineralwaterwillbesoldseparately.oAbuffetwithsaladsandhotdishesisavailableattherestaurantlocatedattheHotelEstalagemdasMinasGerais(600mfromtheConventionCenter).EachmealcostsR$22,00(approximatelyUSD11.00)withjuice,softdrinksormineralwaterorR$28,00(approximatelyUSD14.00)withbeer.Therestauranthaslimitedseatingcapacity.

nLost and FounditemswillbehandledbytheRegistrationDesk.

nBulletin BoardsforMessagesandAnnouncementsarelocatedinthePosterExhibitHall.

nInternetiscomplimentaryandwirelessaccessisavailableattheConventionCenter.ComputerslinkedtointernetarelocatedintheSalãoÁgata(lowerfloor).

nBlumar AgencydeskislocatedintheSalãoÁgata(lowerfloor).TransfersfromOuroPretotoBeloHorizontemaybescheduledwithBlumarAgency.

nCertificatesoParticipationcertificateswillbeincludedintheSymposiummaterialshandedoutatregistration.oCertificatesforposterpresentationswillbedeliveredbytheSymposiumstaffduringthePoster

Sessions.oCertificatesforspeakersatplenaryandconcurrentsessionswillbedeliveredbytheSessionChairs.

Day 1 - August 15th - Wednesday

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8:30-20:30h RegistrationandPostersetup(allposters)

11:00-18:30h VeterinaryImmunologyCommittee(VIC)Workshops SALÃO OURO Allregisteredparticipantsarewelcome.

19:30-20:30h Opening of the 8th IVIS and SALÃO OURO Opening Conference

20:30h Welcomereception SALÃO DIAMANTE

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SALÃO OURO

11:00-13:00h VIC Workshop: NK cells Chairs:AnneK.Storset-NorwayandArminSaalmüeller-Austria

11:00-11:50h Basic Functions and Definitions AnneK.Storset NorwegianSchoolofVeterinaryScience,Norway NK cells - a brief overview. The role of NK cells in the immune system Definitions of NK cells.

PrebenBoysen NationalVeterinaryInstitute,Norway Characteristics and functions of natural killer cells in the cow. AbstractandPosterPR170,VA211

ReginaldoBastos WashingtonStateUniversity,USA Interaction of natural killer cells, monocytes and dendritic cell populations in cattle AbstractandPosterVA219

TimConnelley UniversityofEdinburgh,UK In vitro maintenance of bovine NK cells by culture with Theileria-infected lymphocytes AbstractandPosterAP127

MašaPintarič UniversityofVeterinaryMedicineVienna,Austria Synergistic effects of IL-2, IL-12 and IL-18 on cytolitic activity and IFN-γ production of porcine natural killer cells AbstractandPosterAP132

11:50-12:10h NK- cells: Role in infections BryceBuddle AgResearch,NewZealand Interactions between NK cells and Mycobacterium bovis.

DanielaDantasMoré UniversityofSãoPaulo,Brazil Tick infestations affect subpopulations of peripheral blood lymphocytes AbstractandPosterPR157

12:15-12:50h NK cell receptors ShirleyEllis InstituteforAnimalHealthatCompton,UK KIR receptors in cattle

LilianaJaso-Friedmann Georgia,USA A novel PRR on teleost NK cells and its function in antibacterial immunity

HarrisLewin UniversityofIllinoisatUrbana-Champaign,USA Organization and Evolution of Cattle ULBP Genes

12:50-13:00h Conclusions ArminSaalmüeller UniversityofVeterinaryMedicineVienna,Austria Summing up and future perspectives

13:00-14:00h Lunch

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SALÃO OURO14:00-16:00h VIC Workshop: Comparative MHC: levels of diversity and mechanisms involved in its generation Chairs:ShirleyEllis-UKandDeclanMcKeever-UK

ShirleyEllis InstituteforAnimalHealth,UK;chairofISAG/VICComparativeMHC nomenclaturecommittee Introduction and background

WendyBrown WashingtonStateUniversity,USA Direct detection of antigen-specific CD4+ T cells in peripheral blood with bovine DRB3 tetramers AbstractHC001

RebeccaBaxter RoslinInstitute,UK Quantifying the contribution of BoLA-DRB3 to immune responses in a cattle cross-population AbstractandPosterAP126

ChrisDavies WashingtonStateUniversity,USA A million years of evolution in the bovine MHC class I region

KeithBallingall MoredunResearchInstitute,UK Characterisation of diversity within the class I and II regions of four ovine MHC haplotypes

HirohideUenishi NIAS,Japan Differences of genomic structure among haplotypes of the SLA region AbstractandPosterIG018

16:00-16:30h Coffee Break

16:30-18:30h VIC Toolkit Workshop: The generation, maintenance and availability of reagents and technologies to study immune function in veterinary species Chairs:GaryEntrican-UKandVictorRutten-TheNetherlands

16:30-16:40h GaryEntrican MoredunInstitute,UK VictorRutten UtrechtUniversity,Netherlands Introduction and Workshop Objectives 16:40-17:00h JimKaufman UniversityofCambridge,UK The BBSRC/SEERAD Immunological Toolbox AbstractTK001

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17:00-17:20h CynthiaBaldwin UniversityofMassachusetts,USA The US Veterinary Immune Reagent Network AbstractandPosterTK004

17:20-17:40h EdwinTijhaar UtrechtUniversity,Netherlands The Cytokine Centre: Developing Tools for Research in Veterinary Immunology AbstractandPosterTK007

17:40-18:00h HarryDawson USDA-ARS,Maryland,USA The Porcine Immunology and Nutrition Resource Database AbstractandPosterTK005

18:00-18:30h Open Discussion

Tool Kit Workshop poster viewing: Friday 17 August, 8:30 – 10:30h

SALÃO OURO 19:30-19:45h Opening of the 8th IVIS IsabeldeMirandaSantos,Chair8thIVIS

19:45-20:30h Opening Conference Speaker:JulioScharfstein FederalUniversityofRiodeJaneiro,Brazil Bradykinin B2 receptors of dendritic cells: an innate pathway that promotes development of CD8 effector T cells.

SALÃO DIAMANTE 20:30h Welcome Reception

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HC001. IMPROVED SENSITIVITy ENABLES DETECTION OF ANTIGEN-SPECIFIC CD4+ T CELLS STAINED WITH

BOVINE DRB3 CLASS II TETRAMERS DIRECTLy IN PERIPHERAL BLOOD MONONUCLEAR CELLS

WEnDyCBRoWn1,WAITHAKAMWAnGI2,yAnZHUAnG1,GUyHPALMER1,JUnZonoRIMInE1

1VeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA99164;2VeterinaryPathobiology,

TexasA&MUniversity,CollegeStation,TX77843Wehavepreviously reported theuseofBoLADRB3 *1101

and *1201 tetramers to enumerate T cells specific for peptideepitopes of the cattle pathogen Anaplasma marginale in popu-lations of immune lymphocytes stimulated with antigen ex vivoforatleastoneweek(Norimineetal.,Immunogenetics,58:726).However,becausethefrequencyofMHCclassIIrestrictedTcellsis very low, the tetramers were unable to directly detect CD4+

Tcells inPBMC.To improve thesensitivityofdetectingantigenspecificTcellsinperipheralblood,weadoptedaprocedurethatenriches tetramerpositivecellsprior toFACSanalysis.The tet-ramerswereformedbybindingbiotinylatedDRB3/DRAheterodi-mers tophycoerythin (PE)-labeledstreptavidin.PBMCobtainedfromcalvesimmunizedwiththe30-merF2-5epitopeofthemajorsurfaceprotein(MSP)1aofA.marginalewerelabeledwithDRB3*1101tetramerlinkedtotheminimalpeptideepitopeF2-5B.CellswerewashedandincubatedwithFITC-MabspecificforCD4,withAlexa-fluor-647-conjugatedMabsspecificforCD14,CD8,CD21,andTcRdelta chain, andwith 7-AAD (which labels dead cells)and tetramer-positive cellswere selected by binding to anti-PEmagnetic beads. Cells were then analyzed by four-color flowcytometry.Thismethodpermitteddetectionof0.004 to0.1%ofCD4+Tcellsthatweretetramer-positiveandobtainedatdifferenttimesafterimmunizationandboosting.

AbstrACts for PresentAtions And tool Kit Posters

ViC WorKsHoP ComPArAtiVe mHC

tK001. THE BBSRC/SEERAD IMMUNOLOGICAL TOOLBOx

JIMKAUFMAn1,PETEKAISER1,JAynEHoPE1,PAULSoPP1,SHIRLEyELLIS1,DUnCAnHAnnAnT2,JULIAKyDD2,CoLIn

MCInnES3,DECLAnMCKEEVER3,GARyEnTRICAn31InstituteforAnimalHealth,Compton,Berkshire,RG207NN;2AnimalHealthTrust,LanwadesPark,Kentford,Newmarket,

SuffolkCB87UU;3MoredunResearchInstitute,PentlandsSciencePark,BushLoan,Midlothian,EH260PZ

Progressinunderstandingandutilisingtheimmuneresponseoflivestockanimalstopathogensandvaccineshaslongbeenlimitedby lack of appropriate reagents. There is a particular gap in suit-ablereagentstoidentify,isolateandmanipulateimmunemolecules(suchascytokines)andimmunecellpopulations(includingantigen-presenting cells and antigen-specific T cells). The ImmunologicalToolbox consortium, funded by BBSRC and SEERAD, addressestheproblembyaco-ordinatedefforttodevelopreagents(otherthanthose for functional genomics) necessary to study protective andpathologicalresponsesinhorses,cattle,sheep,pigsandchickens.Theconsortiumisorganisedandledbythreeinstituteswithahistoryofworkinreagentdevelopment:theInstituteforAnimalHealth,theAnimalHealthTrustandtheMoredunResearchInstitute.Currently,a large number of people contribute to the consortium at variouslevels,workingonseveralspeciesandinareasoftechnology.Theseincludesheep,horses,cattle,chickens,developmentoftetramerandco-stimulatory reagents (including for pigs), and website/databasedevelopment.Awebsite backed by a growing database has beenconstructed(http://www.immunologicaltoolbox.co.uk/).Key words: leukocyte antigens, monoclonal antibodies, cross-speciesreactivitySpecies:all

tK002. SCREENING OF COMMERCIALIZED MONOCLONAL ANTIBODIES FOR CROSS-SPECIES

REACTIVITy ON EQUINE WHOLE BLOOD AND PBMCCATHERInEMéRAnT,DAVIDWHoRoHoV

DepartmentofVeterinaryScience,UniversityofKentucky,108GluckEquineResearchCenter,Lexington,KY40546,USA.

catherine.merant@uky.eduInorder tofindnew reagentsagainstequine leukocytemark-

ers, lymphocyte sub-populations and costimulatory or adhesion

molecules,wescreenedcommerciallyavailablemonoclonalantibod-ies (mAbs) for reactivity toconservedepitopes,byflowcytometry.Equinewholeblood leukocytesandperipheralbloodmononuclearcells (PBMC)werestainedwithmAbs todefinedequine leukocytesurfaceantigens,withthemAbsofinterestorwithisotypecontrols.ThewholebloodwaslabeledinPBSatroomtemperature,whilethePBMCwerestainedinPBS-BSA-NaN3at4°C.Thecytometrygatesweredefinedon theexpressionofequineCD172(monocytesandgranulocytes),CD14(monocytes)andCD5(lymphocytes).Thefol-lowingmAbslabeledlessthan1%cellsfromeithergate:anti-mouseCD8 (clone KT14), human CD11a (HI111) and ruminant γδ-TCR(CACTB6A)andactivationmolecule2(CACT63A).MAbsspecifictomouseCD4(YTS19-1.1),F4/80(Cl:A3-1,macrophages)andLy-6G(1A8,neutrophils)labeledhorseleukocytes,butverydifferentlyfrommousecells.These reagentsareunlikely to recognizehomologuemolecules.However,theanti-humanCD49dmAb9F10usuallyrec-ognizedmorethan50%lymphocytesandmonocytes.Interestingly,the anti-human CD38 mAb HIT2 labeled 15 to 45% whole bloodlymphocytes,butuptotwiceasmanyPBMC.Similarly,lymphocyteswerestainedbymAbs3G8(5-19%)andCD28.2(0.5-7%),tohumanCD16andCD28,onlyinthepresenceofsodiumazide.Theweak-nessortheabsenceofstainingofwholebloodleukocyteswasprob-ablyduetoantigeninternalization.Theanti-humanCD16,CD38andCD49dmAbscould recognizehomologuemolecules in thehorse.Biochemicalcharacterization(byimmunoprecipitation)andstainingofleukocytesub-populationsarecurrentlyunderwaytoconfirmtheiridentity.Key words: leukocyte antigens, monoclonal antibodies, cross-speciesreactivitySpecies:domestichorse(Equuscaballus)Session:VICToolBoxworkshop

tK003. THE IMMUNOLOGICAL TOOLBOx: OVINE REAGENTS

SEAnWATTEGEDERA,DonnAWATSon,CATHERInEJEPSon,DAVIDDEAnE,KEITHBALLInGALL,DECLAnMCKEEVERCoLInMCInnES,GARyEnTRICAn

MoredunResearchInstitute,PentlandsSciencePark,BushLoan,Midlothian,EH260PZ.

sean.wattegedera@moredun.ac.ukInvestigationofimmuneresponsesinsheephasbeenrestricted

by a lack of appropriate reagents compared to those available to

ViC tool Kit:AbstrACts for PresentAtions And Posters

Poster PresentAtions: fridAy, August 17tH, 8:30-10:30H

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studyimmuneresponsesinlaboratoryrodentsandinhumans.Theobjective of this project is to develop tools and reagents to allowmoredetailedstudiesofimmuneresponsesinsheep,withparticularreference to responses that control viral infections.TheSEERADcomponent of the Immunological Toolbox funds a post at MRI toassist inthedevelopmentoftoolsandreagentstostudyimmunol-ogyinsheep.TheMRIprojecthasfocussedonthedevelopmentofreagentsandtechniquesthatfacilitatestudiesonimmuneactivationandimmuneregulation.Thisincludes:

1.Expressionofovinerecombinantcytokinesandotherimmu-nomodulatorymoleculessuchasIndolamine2,3-dioxygenase.

2. Development of quantitative assays to measure cytokineproductionatthemolecularandproteinlevel.

3.GenerationandcharacterisationofaflockofMHCclass I-typedsheep.

Progress todatehasbeentheexpressionofovine IL-1βandovineIL-8asmarkersofinnateimmunity.Micehavebeenimmun-ised with these cytokines. Five monoclonal antibodies (mabs) toIL-8andtwomabstoIL-1βarecurrentlybeingcharacterised.Theestablishedcytokineexpression technologyatMRIhasalsobeenusedtoexpressbovineIL-4andIL-10,generatingaddedvaluefromtheToolboxconsortiumwithourpartnersatIAH.Reciprocally,mabsproduced to bovine cytokinesat IAHhavebeen incorporated intoassaystomeasureovinecytokines.Variousexpressionsystemsforrecombinantcytokineshavealsobeeninvestigatedwithourcollabo-ratorsatAHT.Quantitativereal-timeRT-PCRhasbeendevelopedtodeterminethekineticsofmRNAexpressingovineIFN-γ(inflam-matory)andIL-10(regulatory)inantigen-specificovineTcells.ThishasbeencomparedtoproteinexpressionasmeasuredbyELISA.Sheep homozygous for four MHC class I haplotypes have beenestablished.MicehavebeenimmunisedwithleukocytesfromtheseMHC-defined sheep and fusions are being conducted to producehaplotype-specificmabs.TheToolbox initiativehasbothaddedto,andbenefitedfrom,theexistinginfrastructureinovineimmunologyatMRI.

tK004. US VETERINARy IMMUNE REAGENT NETWORK: PRIORITIZATION & PROGRESS

CBALDWIn*1,SJBLACK1,JLUnnEy2,HLILLEHoJ2,JLABRESH3,

DHoRoHoV4,JHAnSEn5,nMILLER6,EBEnGTEn6,GCHInCHAR6,MWILSon6,BWAGnER7

1UniversityofMassachusetts,Amherst,MA,2USDA-ARS,Beltsville,MD,3KingfisherBiotech,Minneapolis,MN,4UniversityofKentucky,Lexington,KY,5WesternFisheriersResCtrUSGS,Seattle,WA,6UniversityofMississippiMedicalCenter,Jackson,

MS,7CornellUniversity,Ithaca,NYImmunological reagents including recombinant cytokines and

chemokines and monoclonal and polyclonal antibodies (Ab) thatidentify the major leukocyte subsets (T and B lymphocytes, NKcells, macrophages, dendritic cells, neutrophils), that react withcytokines/chemokines and their receptors, and react with otherimportantreceptorsthatmodulateimmunefunctionsuchastoll-likereceptorsareusedtoevaluatechangesduringdiseaseincludingthecausesofimmune-pathology.Theyalsoallowscientiststoevaluatehost responses to vaccination. Finally, they provide themeans tomanipulateormodulateimmuneresponseseithertoenhancepro-tectiveimmuneresponsestovaccinesortoreduceimmune-system-mediatedpathology.AbroadcommunityeffortbeganintheUS18monthsagowith the targetspecies ruminants includingcattleandsheep, swine, poultry including chickens and turkeys, horses andcatfishandtrout.Theprojectdirectorsarecoordinatingtheireffortswithotherinternationalgroupsandarecontinuallyrevisingthepri-oritization listandseeking input fromscientistsworkingwith thesespecies.Alistofcurrentlytargetedreagentsandprogressregardingthesewillbepresented.Key words: reagents, monoclonal antibodies, cytokines, CDmoleculeSpecies:other

tK005. THE PORCINE IMMUNOLOGy AND NUTRITION RESOURCE DATABASE

HARRyDDAWSon,CATHERInEAGUIDRy,VAnDAnAVAnGIMALLA,JoSEPHFURBAnJR

UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,BeltsvilleHumanNutritionResearchCenter,NutrientRequirementsandFunctionsLaboratory,Beltsville,Maryland,

USA20705.harry.dawson@ars.usda.gov

Diverse genomics-baseddatabaseshavedeveloped to facili-tateresearchwithhumanandrodentmodels.Currentporcinegenedatabases, however, lack thenutritional and immunological orien-tationand robustannotation todesigneffectivemolecular tools tostudyrelevantpigmodels.Toaddressthisneed,acomprehensiveliterature-based survey was conducted that first identified genesrelated to nutrition and which were associated with macro- andmicronutrient metabolism, atherosclerosis, diabetes, and obesity.Italsoselectedgenes related to immunologyandassociatedwithallergy and asthma; chemokines, cytokines, and their receptors;dendritic,mastandNKcells; type1 IFN-inducedproteins; inflam-mation;tollreceptorsignalingpathways;andBandTcellactivationand development. The process identified 3,035 candidate genesusedtoselectpotentialporcinehomologuesbysearchingmultipleonlinesourcesofporcinegeneinformation.Wethenclonedfullorpartial lengthsequences for85missinghighpriority targetgenes,anddevelopedrealtimePCRassaysfor1,350highprioritygenesofparticularinterest.Asaresult,thedatabasealsocontainscompre-hensive informationonantibodyavailabilityandpublishedporcinegeneandproteinexpressiondata.Thisuniquedatabaselinksgeneexpressiontogenefunction,identifiesrelatedgenepathways,andconnectstootherporcinegenedatabases.Key words:cytokines,reagentsSpecies:swine

tK006. SWINE TOOLKIT PLANS AND PROGRESS FOR THE US VETERINARy IMMUNE REAGENT NETWORK

JoAnKLUnnEy1,PATRICIABoyD1,DAnTEZARLEnGA2,FEDERICoZUCKERMAnn3,WILLIAMSCHnITZLEIn3,JoAnnA

LABRESH4,BETTInAWAGnER5,CynTHIABALDWIn61APDL,BARC,USDA,Beltsville,MD;2BFGL,BARC,USDA,Beltsville,MD;3Univ.Illinois-Urbana,IL;4KingfisherBiotech,St.Paul,MN;5CornellUniversity,IthacaNY;6Universityof

Massachusetts,AmherstMA.jlunney@anri.barc.usda.gov

The US Veterinary Immune Reagent Network (http://www.umass.edu/vetimm/)wasestablishedtoaddressthelackof immu-nologicalreagentsspecificforruminant,porcine,poultry,equineandaquaculturespeciesandaccordinglyhassetaminimumgoalof20reagentsper speciesgroup.Currentplansare toproducesetsofimmune-relatedreagents:recombinantcytokinesandchemokines;monoclonalantibodies(mAb)tothemandtheirreceptors;andmAbthatidentifythemajorleukocytesurfaceantigens,theCDantigens,theTcellreceptors(TCR)andtheToll-likereceptors(TLR).Theseentitiesareneeded toevaluatechanges in the immunesystemofadiseasedorvaccinatedanimaland to testaspotentialbiothera-peutics.For theUSSwineToolkitportionof this initiative,wefirstcollateda listofexistingswinereagentssothatourpriority list fornew reagents could be developed. This priority list and planwasbasedon:1)importanceforswineimmunestudies;2)significancetoothertoolkitefforts;3)availabilityofswinesequenceinformation;and 4) likelihood of developing the respective protein/mAb.Sincemany swine cytokine and CD reagents are available, our priorityfocused on anti-TCRαβ, and on chemokines and their receptors.EffortsarealsounderwaytoproducebioactiveIFN-α,IL-7,IL-13andIL-15andrelevantmAb.Inaddition,sinceananti-CD45ROmAbhasnotbeenproduced fromtraditionalefforts,apeptide immunizationprotocolisnowbeingtested.Beforemakinganti-TLRmAb,thecrossreactivityofknownanti-humancounterpartswillbetestedtoreduceduplicationofeffort.OuroverallgoalistoproducereagentsthatwillfunctioninELISA,ELISpot,flowcytometricandimmunohistochemi-calapplications.Productsdevelopedinthisproposalwillbeopenly

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availabletocollaboratorsandwillbemadecommerciallyavailableusing non-exclusive licenses. These reagents are expected tobenefitalargegroupofresearchersincludingveterinaryimmunolo-gists,pathologists,microbiologistsandscientistsusingswineasabiomedical model for humans. This project was funded by USDANRICGPandARS.Key words:toolkitSpecies:swine

tK007. THE CyTOKINE CENTER: DEVELOPING TOOLS FOR CyTOKINE RESEARCH IN VETERINARy

IMMUNOLOGyEDWInJTIJHAAR1,2,DAPHnEVAnHAARLEM1,JUDITH

HEnDRIKS1,DARSHAnAMoRAR1,AURELnEGREA1,VICToRPMGRUTTEn1

DivisionofImmunology,DepartmentofInfectiousDiseasesandImmunology,FacultyofVeterinaryMedicineUtrecht1andCellBiologyandImmunologyGroup,WageningenUniversity,

Wageningen2,TheNetherlands.edwin.tijhaar@wur.nl

Researchinveterinaryimmunologyisfrequentlyhamperedbythelackofcytokinereagents.Toimprovethissituation,theCytokineCenter was established with the primary aim to produce tools forcytokineresearch in the followingspecies:cat,chicken,cow,dog,horse,man,mouse,pig, rat,andsheep.Due to the largenumberofcytokines thatareof interest,a technologyplatformwasdevel-oped to optimize the process of cloning, subcloning, and expres-sionofcytokine-genesandthegenerationofmono-andpolyclonalantibodies against the different cytokines. The IL4-, IL5-, IL6-,IL10-, IFNgamma-, GMCSF-, TNFalpha- and TGFbeta1-genes ofmostofthespeciesmentionedabovehavebeencloned.TheIL4-,IFNgamma-,GMCSF-andTNFalpha-geneshavebeenexpressedin bacterial and mammalian expression systems and the corre-spondingproteinshavebeenpurifiedandrefoldedwhennecessary.These recombinantcytokinesweresubsequentlyused toproducepolyclonalantibodiesinchickensandrabbitsandtogeneratemono-clonal(mouse)antibodies.Themono-andpolyclonalantibodiesareusedtodevelophighlysensitivecytokinedetectionassays.Thecur-rentstate-of-the-artwillbepresented.Key words:cytokine,antibodies,TNF,IFNSpecies:allspecies

tK008. GENERATION OF NOVEL REAGENTS: THE CHICKEN IMMUNOLOGICAL TOOLBOx

MUHAMMADIqBAL,LISARoTHWELL,ZHIGUAnGWU,UDAyPATHAnIA,SUCHARITHABALU,LAISHAnKWonG,PAULSoPP,JAynEHoPE,JoHnyoUnG,JIMKAUFMAn,PETE

KAISERInstituteforAnimalHealth,Compton,BerkshireRG207NN,

England,UK

tK009. THE IMMUNOLOGICAL TOOLBOx: EQUINE REAGENTS

JULIAHKyDD*,CARLRoBInSon,RUTHCASE,KELLySAUnDERS,nICoLAWRIGHT,SALLyDEBEnHAM,SHIRLEy

ELLIS#,DUnCAnHAnnAnT*AnimalHealthTrust,LanwadesPark,Kentford,Newmarket,SuffolkCB87UU.*Currentaddress:SchoolofVeterinary

Medicine&Science,UniversityofNottingham,SuttonBonington,Loughborough,LeicestershireLE125RD,UnitedKingdom.

#InstituteforAnimalHealth,Compton,Newbury,BerkshireRG207NN,UnitedKingdom.

julia.kydd@nottingham.ac.ukProgressinthecharacterisationofimmuneresponsestopatho-

gensoflivestockspeciesandthehorsehasbeenlimitedbythelackofappropriatereagentsandthisinturnhashinderedtheidentifica-tionofprotective immune responsesand their stimulationbyvac-cination.The ImmunologicalToolboxaims to redress thisproblemthrough a collaborative initiative involving the Moredun Research

Institute,InstituteforAnimalHealth,ComptonandtheAnimalHealthTrust(AHT;seeaccompanyingabstractsandposters).

Theaimsoftheequinecomponentofthisprojectareto:1.generateantibodiesagainstequinemoleculesofkeyimmu-

nologicalinterest;2.developarapidtechniquetoidentifyMHCclassIB2positive

horses;3.constructtetramersoftheequineMHCclassIB2geneand

CTLtargetpeptideinEquineherpesvirus-1gene64.Progress has been made in all three areas. Firstly, DNA

sequences encoding a selection of equine cytokines have beencloned into prokaryotic (pET21A Novagen and pGEX-3X GEHealthcare) and eukaryotic (pcDNA3IgHG1; Wagner et al 2003)expressionvectors.Polyclonalantiserum toTNFαand IL-15havebeenproducedandadditionalimmunisationswithCD14areunder-way.StableCHOcell lineswhichexpress IL6andRANTEShavebeenproducedincollaborationwithDrWagner(CornellUniversity)andrecombinantproteinwillbeusedformonoclonalantibodypro-duction.AntibodiesagainstbovinecytokinesfromourIAHcolleagueshavebeenscreenedagainstequineleucocytesanddifferenteukary-oticexpressionsystemsinvestigatedwithourMRIcolleagues.Foroursecondaim,aPCRwhichamplifiestheB2genefromgenomicandcomplementaryDNAofA3homozygousponieshasbeendevel-oped and its specificity is currently being tested in heterozygousanimals.Thirdly,theB2genehasbeenclonedandfragmentcloningofgene64isunderwaytoidentifyimmunogenicregions.

The reagents which are emerging from the BBSRC-fundedImmunological Toolbox (http://www.immunologicaltoolbox.co.uk/),together with the USDA-funded project (http://www.umass.edu/vetimm/) and the initial assembly from the Equine GenomeSequencingProject(BroadInstitute,Boston,USA(www.broad.mit.edu/ftp/pub/assemblies/mammals/horse/)willprovideequineimmu-nologistswithasubstantiallywiderpanelofreagentstoenhancethecharacterisationofinnateandadaptiveimmuneresponsesinducedbyinfectionorvaccinationagainstendemicandemergingviraldis-easesatthemolecularandcellularlevels.

Wagner,B.RobesonJ.McCrackenM.,Wattrang,E.,Anctzak,D.F.2005.Horsecytokine/IgGfusionproteins–mammalianexpres-sionofbiologicallyactivecytokinesandasystemtoverifyantibodyspecificity to equine cytokines. Vet. Immunol. Immunopathol.105:1-14.Key words:tetramers;equineMHCclassIB2;PCR;TNFα;IL-15;CD14;IL6;RANTESSpecies:equine

tK010. U.S. VETERINARy IMMUNE REAGENT NETWORK: POULTRy REAGENTS UPDATE

LILLEHoJ,HyUnAnDHonG,yEonGAnimalParasiticDiseasesLaboratory,USDA-ARS,Beltsville,MD

20705,USAAmajorobstacletoadvancepoultryimmunologyanddisease

research is the lack of adequate immunological reagents specificfor poultry. Although many immunological reagents which detectcell-surface antigens of immune system are commercially avail-able,thereisveryfewmonoclonalantibodies(mAb)andpolyclonalantibodieswhichcanidentifythemajorchickencytokinesandche-mokines,aswellas their receptors. Thenew initiative toaddressgenerallackofimmunologicalreagentsforveterinaryanimalspeciesincludingfishwasfundedbytheUSDA-CSREESNationalResearchInitiativegrantin2006.Thisprojectrepresentsabroadcommunityplantobegintosystematicallyaddressthe immunologicalreagentgapfortheU.S.veterinaryimmunologyresearchcommunityforthefollowing groups: ruminants, swine, poultry, horses, and aquacul-turespecies.Thegoalofthisprojectistodevelop20reagentspereachspeciesgroupincludingantibodiesthatfunctioninELISAandELISpotassays, for intracellularstaining, forblockingfunctionandsignaling,forflowcytometricanalysis,aswellasforimmunochem-istryusingtissuesections.Chickenfull-lengthgenesencodingIL-2,IL-15, IL-16, IL-17, IFN-γ, TNFSF15,NK-lysin, LPS-inducedTNF-like factor (LITAF), IL-4, IL-10, IL-18, lymphotactin,CCL4,CCL20,

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oPening ConferenCe

brAdyKinin b2 reCePtors of dendritiC Cells: An innAte PAtHWAy tHAt Promotes

deVeloPment of Cd8 effeCtor t CellsJULIoSCHARFSTEIn

FederalUniversityofRiodeJaneiro,BrazilStrategicallypositioned inperipheral tissues, immunesentinel

cellssuchasmacrophagesandmastcells,sensemicrobesand/ortheir products throughdifferent typesof pattern-recognition recep-tors.Uponsecretionofcytokinesandchemokines, inflammation israpidlyamplified throughcooperative involvementof themicrovas-culature,ofcirculatingleukocytes,peripheralneuronsanddendriticcells(DCs).Owingtodisturbancesofendotheliumbarrierfunction,theplasmaproteinsdiffuse intoextravascular tissues,allowing forproteolyticgenerationofshort-livedproinflammatorypeptides,suchas complement activation peptides and the kininogens (i.e., kinin-precursor proteins). While much emphasis has been placed onanalysis of the pro-inflammatory activity of complement peptides,the innate roleofkinins (eg.bradykinin,BK)was longoverlooked.A fewyearsago, however,we reported that thenonapeptidebra-dykinin (BK) is a potent inducer of dendritic cell (DC) maturation,driving IL-12-dependent Th1 responses through the activation of

G-proteincoupledbradykininB2 receptors (B2R).Thepremise thatthis endogenous signalingpathwaymaystimulate type-1adaptiveresponses was recently confirmed in the subcutaneous model ofTrypanosomacruzi infection.Analysisof thedynamicsofparasite-evoked edema formation revealed that activation of TLR2/neutro-philsdrives the influxofplasmaproteins,suchaskininogens, intoperipheraltissues.Afterdockingtosulfatedproteoglycans,thesur-face-associatedboundkininogensare turned into facilesubstratesforT.cruzicysteineproteases.Onceliberated,theshort-livedkininpeptidespotentlyactivateDCsviaB2R,convertingtheseAPCsintoTh1 inducers. Intensityof the innatesignalsconveyedbykinins istightlycontrolledbytheactivityofkinin-degradingmetallopeptidases,e.g.Angiotensin Converting Enzyme (ACE/CD143). Exploring theknowledgeaboutmechanismsunderlyingkiningenerationanddeg-radation,werecentlydevelopedavaccinationschemethatprotectsmice from lethal T. cruzi infection.An important lead coming fromthese vaccination studieswas theevidence that bradykinin steersdevelopmentoftype-1effector/memoryCD8+Tcellsthroughmodu-lationoftheDC/Tcellinterface.Insummary,lessonstakenfromstudiesofthemechanismsunderlyingbradykinin-adjuvanticitymaystimulatedevelopmentofnewvaccine formulationsagainst veteri-narydiseasescausedbyvirusand/orintracellularparasites.

and IL-12havebeenclonedandproteinsarebeingexpressed forantibodyproduction.Key words:cell-surfaceantigens;commercialantibodies;cytokines;chemokines;receptors.Species:Avian

tK011. ExPRESSION OF RECOMBINANT IMMUNOGLOBULINS AND CELL SURFACE MOLECULES

FOR SIx SPECIES: CATTLE, PIG, HORSE, CHICKEN, CATFISH AND TROUT.

BETTInAWAGnER1,JULIAM.HILLEGAS1,RICHARDICoM1,JoAnK.LUnnEy2,EVABEnGTEn3,noRMAnW.MILLER3,HyUnLILLEHoJ2,JoHnDHAnSEn4,CynTHIABALDWIn51CornellUniversity,IthacaNY;2APDL,BARC,USDA,Beltsville,MD;3UniversityofMississippi,Jackson,MS;4WesternFisheriesResearchCenter,Seattle,WA;5UniversityofMassachusetts,

AmherstMATheUSVeterinaryImmuneReagentNetworkseekstodevelop

new tools formolecules of the immune system in veterinary spe-cies (cattle, pig, horse, chicken and fish). Recombinant proteinsfor immunoglobulins and various cell surfacemolecules, includingT-cell receptorchains,CDmoleculesandcytokine receptorshavebeenexpressedatCornellUniversity.Twoexpressionmammaliansystemsareused;(1)anIgGfusionproteinand(2)anIL-4fusionprotein system. All proteins are expressed in Chinese HamsterOvary (CHO)cells.Purified recombinantproteinsare thensubmit-ted to theUniversity ofMassachusetts atAmherst formonoclonalantibodyproduction.Thefirstrecombinantproteinsthathavebeenexpressed with this system were T-cell receptor constant regiondomains.These include the bovineTCRγ andTCRd proteins andthe catfish TCRa and TCRγ constant regions. TCR genes of thehorse,pigandtroutarebeingprocessed.OthermoleculesthathavebeenexpressedwiththefusionproteinsystemincludeequineCD40and theFceRI a-chain.Expression cloningandproteinproductionisongoing forcattle IL-23Rand IL-10R,pig IL-4Raand IL-13Ra1,equineCD23,CD25,CD28and the IgDheavychain,andchickenIL-2RaandCXCR4.

ThisworkissupportedbyUSDAGrant#2005-01812(TheUS

VeterinaryImmuneReagentNetwork).Key words: Recombinant proteins; cell surface molecules; T-cellreceptorchains;CDmolecules;cytokinereceptorsSpecies:Ruminants;Equine;Swine;Avian;Fish

tK012. BOVINE: PROGRESS AND PLANS WITHIN THE U.S. VETERINARy IMMUNE REAGENT NETWORK

CynTHIABALDWIn1,EDWARDHUDGEnS1,DAnnIELLEToMPKInS1,JoAnnALABRESH2,BETTInAWAGnER3.

1UniversityofMassachusetts,Amherst,MA;2KingfisherBiotech,Minneapolis,MN;3CornellUniversity,Ithaca,NY

In an effort to overcome the lack of immunological reagentstargetedforruminants,therebyimprovingresearchintobovineandovineimmunologyanddisease,thebovinecomponentofthe“U.S.VeterinaryImmuneReagentNetwork”hasisolatedandsequencedthecompletecodingsequenceforalargenumberofgenes.Theseinclude the chemokines and cytokines IL-1β, IL-2, IL-4, IL-5, IL-6,IL-7, IL-8, IL-12p35, IL-12p40, IL-13, IL-17, IL-18, IL-23, IFN-γ,IFN-a A, IFN-β, TNF-a, CXCL9 (MIG), CXCL10 (IP-20), CXCL11(I-TAC), CCL2 (MCP2), CCL5 (RANTES), and CCL11 (eotaxin).Polymorphismsforsomeofthesegenesamongcattlebreedshavebeeninvestigated.Genesclonedforcellsurfacemoleculesinclude:thechemokinercceptorsCCR7,CCR5,CXCR3andCXCR5andthecytokinereceptorsIL-23RandIL-10Rβ,theTCRγ (6constantregiongenes and 8 variable genes), TCRd (1 constant and 4 variable),TCRa andβ(1constantgeneeach),andfinallyCD40andCD40L.Thegeneshavebeencloned intoexpressionvectorsandproteinswill be expression inmammalian cells (cell-surfacemolecules) oryeast (chemokinesandcytokines)and, subsequently, theproteinsused for monoclonal antibody (mAb) production and/or evaluatedfor bioactivity. TheC-domain of both TCRγ and TCRd have beenexpressed (Cornell) and mAb production toTRDC UMass begun.This isapilot study todeterminewhetherexpressionof individualC-domainswill yield a conformationally-preservedmolecule and ifsuccessfulwillbefollowedbyTRBCandTRAC.Key words: antibodies; chemokines; cytokines; chemokinercceptors;cytokinereceptors;TCRSpecies:Ruminants

Day 2 - August 16th - Thursday

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7:45-18:00h RegistrationandPosterSetup

8:30-10:30h PlenarySession:Immunogenetics SALÃO OURO

10:45-13:15h PlenarySession:BacterialDiseases SALÃO OURO

14:00-16:00h PlenarySession:Reproduction,Stress,Nutrition SALÃO OURO

16:30-17:30h PfizerAwardforDistinguished SALÃO OURO VeterinaryImmunologist

17:30-18:00h Introductiontothe9thIVIS; SALÃO OURO IntroductiontoIncomingPresidentofVIC

18:00-19:30h PlenarySession:Skin,Mucosae,MammaryGland SALÃO OURO

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SALÃO OURO8:30-10:30h Plenary Session: Immunogenetics and genomics of hosts; genomics of pathogens Chairs:FuadIraqi-IsraelandElizabethJ.Glass-UK

Speakers 8:30-9:00h AlbertoDavila OswaldoCruzFoundation,Brazil Exploring the genome of Trypanosoma vivax: towards markers for diagnosis and typing and relevance to immunity and disease resistance in livestock

9:00-9:30h JamesWomack TexasA&M,USA Bovine Genome Sequencing Update: Discovering Variation in the TLR Gene Family

9:30-10:00h AdrianSmith InstituteforAnimalHealth,UK Parasite genetics and the search for protective antigens

10:00-10:30h AllanCrawford AgResearch,NewZealand Transcriptomics: Its use in understanding resistance to nematode parasite infection in sheep

10:30-10-45 Coffee Break

10:45-13:15h Plenary Session: Immune Responses in Bacterial and Viral Diseases; Prions and BSE Chairs:WendyBrown-USAandPaulWigley-UK

Speakers 10:45-11:15h DonM.Estes UniversityofTexasMedicalBranch,USA Tuberculosis vaccine development and immunity in the neonatal calf model

11:15-11:45h ArthurSummerfield InstituteofVirologyandImmunoprophylaxisSwitzerland Immune Responses during infections with Foot and Mouth Disease Virus

12:00-12:30h PaulWigley UniversityofLiverpool,UK Salmonella and the macrophage: The Immunobiology of systemic avian salmonellosis

12:15-12:45h WendyBrown WashingtonStateUniversity,USA Immunoproteomic analysis of the protective outer membrane fraction of Anaplasma marginale

12:45-13:15h yasminBelkaid NIH,USA Role and origins of regulatory T cells during parasitic infections

13:15-14:00h Lunch

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SALÃO OURO14:00-16:00h Plenary Session: Immunoendocrinology, Stress and Immunology of Re production and Neonates; Microbial Flora, Nutrients and the Immune Response Chairs:GaryEntrican-UKandJoséRobertoKfouryJr.-Brazil

Speakers 14:00-14:30h AntonioLaCava UCLA,USA Cross-talk between neuroendocrine and immune system: the case of leptin

14:30-15:00h HarryDawson ARS-USDA,USA Regulation of porcine hepatic and pulmonary-associated immune responses by vitamin A

15:00-15:30h HarrisLewin UniversityofIllinois,USA Good Embryos Gone Bad: A Transcription Profile of What Goes Wrong

15:30-16:00h JosephUrban ARS-USDA,USA Use of parasitic infection to explore dietary components that regulate appropriate mucosal immune responses

16:00-16:30h Coffee break

16:30-17:30h Pfizer Award for Distinguished Veterinary Immunologist IntroductionbyWayneHein,ChairofAwardSelectionCommittee,NewZealand AwardpresentedbyPaulWood,Pfizer,Australia

Speaker: JohnE.Butler UniversityofIowa,USA Diversity, Frontiers and Careers in Immunology

17:30-18:00h Introduction to the 9th IVIS Dr.Takashionodera,Japan IntroductiontoIncomingPresidentofVIC IntroductionbyJannaessens,Kenya

18:00-19:30h Plenary Session: Immunology of the Mucosae, Skin and of the Mammary Gland; Mastitis Chair:MichaelP.Murtaugh-USA

Speakers 18:00-18:30h LorraineSordillo MichiganStateUniversity,USA Mammary Immunity and Susceptibility to Mastitis: role of oxidant stress

18:30-19:00h ElsMeeusen MonashUniversity,Australia Structure and protection of the ruminant lung

19:00-19:30h AdrianSmith InstituteforAnimalHealth,UK Structural and cellular aspects of immunity to pathogens in the gut

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ALBERToDAVILA(oSWALDoCRUZFoUnDATIon,BRAZIL)exPloring tHe genome of tryPAnosomA

ViVAx: toWArds mArKers for diAgnosis And tyPing And releVAnCe to immunity And

diseAse resistAnCe in liVestoCK

JAMESWoMACK(TExASA&M,USA)BoVInEgenome sequenCing uPdAte: disCoVering

VAriAtion in tHe tlr gene fAmilyApproximatelyoneyearago,theBovineGenomeSequencing

Project(http://genome.gov/12512284)releasedthethirdversionofthe bovine genome assembly, Btau_3.1, which is a 7.15Xmixedassemblythatcombineswholegenomeshotgun(WGS)sequencewithBACsequence.Thesequence,predominantlyfromaHerefordfemale, is available in GenBank, EMBL, and DDBJ. Sequencingskims for single nucleotide polymorphism (SNP) discovery weregeneratedfromrandomshotgunlibrariesfromindividualanimalsofHolstein,Angus,Brahman,Limousin,andJerseybreeds.

Toll-likereceptors(TLRs)playacrucialroleininnateimmunityinvertebratesaswellasininsectswheretheywerediscovered.Therecognitionofmicrobialelementsbydifferentmembersofthegenefamilyinitiatessignaltransductionpathwaysleadingtotheexpres-sionofspecificgenesimportanttotheinnateimmuneresponseandtodirectpathstoantigen-specificacquiredimmunity.Alargebodyofdata isemergingsuggestingrolesofsinglenucleotidepolymor-phisms (SNPs) inhumanTLRgenes insusceptibility to infectiousandinflammatorydiseases.

We,alongwithothers,havemappedthebovineTLRfamilyof10genestotheirrespectivechromosomepositionswhichcorrespondtoexpectedlocationsrelativetoconservedsyntenyinhumans.Wehave begun a search for bovine SNPs in coding regions of TLRgenes,utililzingdataavailablefromthebovinegenomesequencingprojectandalsobyresequencingthecodingregionsofallgenesin10animalsrepresenting10differentbreedsofcattle.

We sequenced approximately 35 kb of coding sequence ineightofthetenbovineTLRgenesfrom10animals.Morethan250SNPswererevealed,49ofwhichaccountfornon-synonomouscod-ing.SNPdistributionisnotuniformwithTLR-4and10revealing13SNPsperkbofcodingsequence. Asexpected,mostSNPswerediscoveredinBosTaurus/BosIndicuscomparisons.

We compared SNP discovery from genome sequence data-basesandresequencinganimalsfromdiversebreeds.Asexpectedat thisearlystageof thebovineSNPproject,mostSNPs forTLRgenesarenotyetavailablefromthedatabases.

ADRIAnSMITH(InSTITUTEFoRAnIMALHEALTH,UK)PArAsite genetiCs And tHe seArCH

for ProteCtiVe Antigens

EntericImmunology,InstituteforAnimalHealth,Compton,BerkshireRG207NN,UK

Development of effective sub-unit vaccines is dependenton selection of antigens capable of inducing protective immunity.Unfortunately, responsesagainstmostantigensarenotprotectiveand new strategies are required for antigen selection, especiallywith antigenically complex pathogens. For example, the genomesofparasiticpathogensencodemanythousandsofpotentialantigensandlargesubsetsoftheseinduceresponsesintheinfectedhost.Toavoidtheproblemof interpretingresponse-basedassayswehavedevelopedanapproachbaseduponparasitegenetics,selectivebar-riersandAFLP-basedfingerprinting.Theessential featuresof thisapproachwillbeillustratedwiththeintestinalapicomplexanparasiteEimeriamaxima.Thisparasite inducesextremelystrong immunityagainst rechallenge infection but immunity is strain-specific, i.e.fieldorlaboratorystrainscanbeidentifiedthatdonotcross-protect

againsteachother.Theprocessfirst involved identificationof twoantigenicallydistinctstrainsofE.maxima,oneofwhichwasselectedforresistancetothedrugrobenidine.Next,theseparentstrainswereusedtoestablishaco-infectionwhichresultsinthegenerationofageneticmixtureofoffspringasaresultofsexualrecombination.Theprogenyof thecrosswereexposedto thedouble-selectivebarrierofdrugexposureandimmunity(tothedrug-resistantstrain)whichkillsbothparentalgenotypesandanyirrelevantrecombinants.Thesurvivingparasiteshaverecombinedallthelocirequiredtoescapethedoublebarrierandwetermthese“relevantrecombinants”(RR).AnalysisoftheinheritancepatternsofAFLPfragmentsfrommultipleindependentpopulationsofRR,unselectedprogenyofthecrossandsingle-barrier selected populations revealed 36 polymorphic DNAmarkers that were consistently selected according to the immunebarrier. These markers group into five regions of the parasitegenomewhichareunderscrutinyto identifygenesresponsibleforhighlyeffectivestrain-specificimmunity.

These results have several implications: Firstly, that just fivegenomic regions(<1Mbp)areunderstrong immuneselectionoutofa60Mbpgenomeimpliesthatonlyasmallnumberofgenesareresponsible for protection. The simultaneous selection of all fiveregionsinallparasitepopulationssuggeststhathighlevelprotectionrequiresstimulationwithatleastoneantigenineachregion(i.e.5+antigens).Nonetheless,theabilitytoidentifyeachregionhassub-stantiallyreducedthecomplexityoftheantigendiscoveryprocess.Finally,wheretheabilitytoselectappropriatephenotypesexists,thisapproachmaybeapplicabletothesearchforprotectiveantigensinotherparasiticpathogens.

ALLAnCRAWFoRD(AGRESEARCH,nEWZEALAnD)

 trAnsCriPtomiCs: its use in understAnding resistAnCe to nemAtode PArAsite infeCtion

in sHeePALLAnMCRAWFoRD,oRLAMKEAnE,CRISTInADIEZ-

TASCon,JoHnCMCEWAnAgResearchInvermayResearchCentre,PuddleAlley,PrivateBag

50034,Mosgiel,NewZealand.Nematodeparasiteinfectionsofthealimentarytractarethelarg-

est,mostpervasivehealthproblemforsheepandgoatsproducedinagrazingenvironment.Over the last40yearschemicaldrencheshavebeenthemajortherapyforcontrolofnematodeparasiteinfec-tions,however,drenchesarenowfailing.Oneofthemostpromis-ingalternativemeansofcontrolhasbeentobreedsheepresistanttoparasite infection.Parasite resistance isamoderatelyheritabletrait and the benefits ofmeasuring parasite burden in lambs andincludingthisinformationinaproductionindexforbreedingisnowwell documented. Measuring parasite burden involves measuringparasiteeggs in faeces fromeachanimal.This isunderstandablyan unpopular chore with sheep breeders, made doubly so by thelow repeatability of each measurement necessitating duplicate ortriplicatesampleshavingtobetakenover3days.

TheappealofaDNAtest to identify thosesheepresistant toparasitesisobvious,especiallyasinadditiontosavingfaecalsam-plingitwouldmeananimalscouldbetestedearlyinlifeandsavedfromapotentiallydebilitatingparasitechallenge.QTLsearcheshavenotbeenencouraginginthatonlyQTLwithquitesmalleffectshavebeenidentified.

Wehave therefore triedanothergenediscoveryoptionwhichcomplements the QTL approach, and examines gene expressionusingarraysofcDNAsequencesonglassslides.Thesameresistantandsusceptibleselectionlinesorbreedsofsheephavebeenusedtocomparegeneexpression.Themajorityofthispresentationcon-cernswhatwehavelearntfromgeneexpressionstudiesinparasiteresistantandsusceptiblePerendalesheepandhowthiscompareswithstudiesinotheranimalsystems.

PlenAry session: immunogenetiCs And genomiCs of Hosts; genomiCs of PAtHogens.

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DonMESTES(UnIVERSITyoFTExASMEDICALBRAnCH,USA)

tuberCulosis VACCine deVeloPment And immunity in tHe neonAtAl CAlf model

ARTHURSUMMERFIELD(InSTITUTEoFVIRoLoGyAnDIMMUnoPRoPHyLAxISSWITZERLAnD)

immune resPonses during infeCtions WitH foot And moutH diseAse Virus

Foot-and-mouth disease (FMD) represents one of the mosteconomically important diseases of farm animals. Although suc-cessful eradication programs based on vaccination and stampingoutpolicieshasbeenappliedindevelopedcountries,thevirusisstillendemicinlargepartsofAfrica,SouthAsiaandSouthAmerica.Thebasisfortheconstantthreatcausedbythisvirusistheextremerateofreplication,shortincubationtime,contagiosity,virustenacityandahighmutationrateresultinginconstantantigenicchanges.Thus,althoughprotective immuneresponsesagainstFMDvirus(FMDV)arerapidandefficacious,thevirushasthecapacitytooverruntheimmunesystem.Thebasisforthisisnotonlyaparticularrapidrateof replicationbutalso theabilityofFMDV toshutdown thecellu-lar protein synthesis, including IFN type I, through the viral Lpro insusceptibleepithelialcellcultures.Thisappearsimportantforvirusevolution,asFMDVisquitesensitivetotheactionofIFN.Despitethis, innate immune responses can be detected in vivo indicatingthattheeffectofLproisnotabsolute.Invitroforexample,theporcineepithelial cell line PK-15 has some degree of resistance againstFMDVinvolvingthetriggeringoftheRNAhelicasemda-5andIFNtype I receptor signalling. Furthermore, the virus can induce IL-6responsesinthesecells.OfinterestistheinteractionofFMDVwithDC,inparticularlyplasmacytoidDC,whichresultsinthereleaseofrelatively large quantities of IFN.The cells do not allow completereplicationofFMDV,butcurrentdataindicatethatRNAreplicationinthecytoplasmandautophagosomaldeliverytoendosomescontain-ingTLR7wouldrepresentthemechanismbywhichthesecellsareactivated.Suchresponsesareamplifiedwhenthevirusispresentedinformofimmunecomplexes,whichneutralizethevirusforepithelialcellinfection,indicatingaparticipationofinnateimmuneresponsesalsoduringsecondaryimmuneresponses.RecentresearchhasalsofocussedonnovelFMDvaccines,whichprovideprotectionthroughinnate immunedefences,particularlybeneficial for thesituationofemergencyvaccination.

With respect toadaptive immune responsesagainstFMDValarge amount of data has accumulated.As a cytolytic virus infec-tion mainly neutralizing antibodies principally control FMDV.TheirdevelopmentisThelpercell-dependentandalsodependentonDCactivity. Even secondary in vitro antibody responses require bothIL-2andBAFF.TheroleofcytotoxicTcellsarelessclearandtheirtriggeringcouldbeofusefortheclearanceof“carrier”cattleinwhichtheviruspersistsinepithelialcellsofthedorsalpalate.

Amajorchallengeforthedevelopmentofnovelvaccines,whicharenotbasedontherequirementofgrowinglivevirus,istheefficientandrapidinductionofneutralizingantibodies.Asageneralrule,thisisachievedwithvaccinescontainingintactcapsids,enablingefficientcross-linkingBcellreceptorsandpresentationofconformationalepi-topes.ConsideringthatFMDVentersthroughthemucosalsurfacesof theupperrespiratory tract,and thatconventionalvaccinescan-notpreventlocalvirusreplicationwithestablishmentofpersistence,anothernewfocusisthedevelopmentofvaccineswiththecapac-ity to inducemucosal immuneresponses.Despitethese importantimpulsescomingfromimmunologicalresearch,successfulvaccineswillnotonlyneed toconfer rapidprotectionagainstseveralof thesevenknownserotypesbutwillalsoneedtobeconstantlyadaptedtocirculatingantigenicvariants.

PAULWIGLEy(UnIVERSITyoFLIVERPooL,UK)sAlmonellA And tHe mACroPHAge: tHe

immunobiology of systemiC AViAn sAlmonellosis

PAULWIGLEy1,CLAIREJoHnSTon1,LUCyCHAPPELL2,PETEKAISER2,RICHARDBEAL2,ADRIAn2SMITH,

PAULBARRoW31DepartmentofVeterinaryPathology,UniversityofLiverpool,

Leahurst,Neston,CH647TE,UK.2DivisionofImmunology,InstituteforAnimalHealth,Compton,UK

3SchoolofVeterinaryMedicineandScience,UniversityofNottingham,SuttonBonnington,UK.

SystemicsalmonellosisinthechickeniscausedprimarilybythetwoavianspecificserovarsSalmonellaentericaserovarGallinarumandserovarPullorum. InfectionwithS.Gallinarumresults inFowlTyphoid,asevereandacuteformofdiseasewithhighmortalityratesin birds of all ages. S. Pullorum causes Pullorum Disease, whichcauseshighmortality inyoungchicks,accompaniedbydistinctivewhitediarrhoea,butfrequentlyleadstopersistentinfectionwithoutclinicaldiseaseinolderbirdsresultinginreproductivetractinfectionandtransmissionofinfectiontoeggsandchicks.

Interactionwiththeimmunesystemduringinfectionwiththeseserovarshasthreedistinctphases.Phaseoneis invasionthroughthegastrointestinal tractandinitialexposuretotheinnate immunesystem.Thesecondphaseistheestablishmentofsystemicinfectionleading topersistence, immuneclearanceordeathof the infectedhost.Thefinalphaseis infectionofthereproductivetractandegginfection.

Data from in vitromodels has previously suggested that theinvasionprocessofbothS.GallianrumandS.Pullorumdoesnotinducetheexpressionofproinflammatorycytokinesorchemokines,unlikegastrointestinalassociatedserovarssuchasS.TyphimuriumorS.Enteritidis.Followingoralinfection,neitherS.GallinarumnorS.Pulloruminducegut-associatedinflammationorpathologywhereasS. Typhimurium and S. Enteritidis induce an influx of heterophils(PMNs)andseveredamagetotheileum.Comparisonofthecyto-kineandchemokinesexpressionprofile following infectionwithS.EnteritidisandS.Pulloruminchicksrevealsthatalthoughthereisnodifferencebetweenserovars intheexpressionofIL-6andIL-1β intheileum,andindeedlittlechangeovercontrols,thereissignificantincreasesinexpressionofCXCchemokinesfollowingS.Enteritidisinfection whilst there is downregulation of expression following S.Pullorum infection. These differences are most pronounced withCXCLi1, themajor chemokinesassociatedwith heterophil recruit-ment,and indicate thatS.Pullorumdoesnot induce inflammationduringinvasionforthegut.TheabsenceofflagellainS.Pullorumislikelytobeasmajorfactorinthis.

The interaction with macrophages is crucial to the establish-mentofsystemicinfectioninthespleenorliver.Salmonelladeficientin their ability to survive inmacrophages, (e.g. followingmutationin the SPI2 type III secretion system) are completely attenuatedin their ability to cause disease. Host genetic background has asubstantialinfluenceonthecourseofinfection.MacrophagesfromSalmonella-resistantinbredchickenlinesaremoreefficientatclear-ing intracellular Salmonella and express key cytokines, includingTh1-associated,more rapidlyandatahighermagnitude followingchallenge. Resistant line birds survive S. Gallinarum challenge,whereassusceptiblelinebirdsshowhighmortality.ClearanceofS.Gallinarum,determinedusing theattenuated9Rvaccinestrain, isprimarilymediatedthroughaTh1responsewithpeakantigen-spe-cificsplenicTcellproliferationandinterferon-γexpressioncoincid-ingwithbacterialclearance.AdoptivetransferofTcellsalsogivespartialprotectiontochallenge.Incontrasttothe‘killorbecleared’natureofS.Gallinarum,S.Pullorumisabletopersistwithinsplenicmacrophageswithlimitedclinicaldisease.Infectioninducesastrongantibody response,butcellular responsesareslow todevelop.S.EnteritidisorS.Typhimuriuminfectionresultsinpeakexpressionofboth interferon-γ and IL-18 around 14 days post challengewhichcoincides with subsequent clearance. In contrast expression ofthese cytokines is limited following S. Pullorum infection, thoughsomeexpressionof IL-4canbedetected,somethingnot found in

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other serovars.This suggests thatS.Pullorummaymodulate theimmune response favouring a Th2 response rather than the Th1responseassociatedwithclearance.

S.Pullorumpersistsinlownumbersinthespleenandliverofhensuntilthestartoftheegglayingperiodwherearecrudescenceof systemic infection and spread to the reproductive tract occurs.ThiscoincideswithadropinTcellactivitybothtospecificantigenicor mitogenic stimulation. We hypothesise that during the carrierstate the intracellular bacteria and the immune system reach a‘stalemate’.ThelossofTcellfunctionandpresumablyinterferon-γstimulationofmacrophgesallowstheSalmonellato‘breakout’ofthestalematemultiplyandspread.Ourrecentstudiessuggestthatthelackof functionmaybeaconsequenceofa large fall inThelper

cells,asnumbersofCD4+cellsinboththespleenandreproductivetractfalltoaroundathirdofthepriorlevelpriortothestartofthelay-ingperiod.AslayingbecomesestablishedCD4+numbersandTcellfunctionbegintorecoverleadingtoafallinbacterialnumbers.

WEnDyBRoWn(WASHInGTonSTATEUnIVERSITy,USA)immunoProteomiC AnAlysis of tHe

ProteCtiVe outer membrAne frACtion of AnAPlAsmA mArginAl

Rickettsialpathogens in thegeneraAnaplasmaandEhrlichiacauseacuteinfectioninimmunologicallynaïvehostsandaremajorcausesoftick-bornediseaseinanimalsandhumans.Immunization

withAnaplasmamarginalepurifiedoutermembranesinducescom-pleteprotectionagainstanaplasmosis in75%ofanimals,whereasimmunization with the well-studied and immunodominant majorsurfaceproteinsMSP1,MSP2,MSP3,MSP4,andMSP5haspro-vided little or noprotection. ThecompletedgenomesequenceofA. marginale facilitated the identification of subdominant and lessabundant immunogenic proteins in the outer membrane fraction,using two approaches. First, two-dimensional electrophoresis andimmunoblottingoftheoutermembranefractionwithimmuneserumidentified numerous antigenic protein spots.Analysis of individualproteinsexcisedfromthegelsbyliquidchromatographyandtandemmassspectrometryidentified21novelantigens.Ofparticularinterestis thefindingthatthreeproteinsfromthetypeIVsecretionsystem(TFSS),conjugaltransferprotein,VirB9,andVirB10wereantigenic.TFSSproteins formchannelsandare responsible forsecretionorcell-to-celltransferofmoleculesandDNA-proteincomplexesinothergram-negativebacterialpathogensTheseproteinswereexpressedandshowntostimulateIgG2,andCD4+TcellproliferationandIFN-gproduction,responsesassociatedwithprotectiveimmunity inoutermembranevaccinates.Asecondapproach tomoredirectlyscreenforantigensrecognizedbyTlymphocytesinvolvedinvitrotranscrip-tionandtranslation(IVTT)ofORFsencodingproteinspredictedtobe localized on the outer or innermembrane or to have a signalpeptide. PCR products of selected ORFs engineered to expressantibody-binding sequence tags were amplified and expressedusingIVTT.Asproofofprincipal,VirB9andoutermembraneprotein(OMP)7,OMP8,andOMP9,knowntostimulateTcellresponsesinoutermembrane vaccinates,were expressed by IVTT and affinitypurifiedbybindingtoanti-tagantibodycoupledtoprotein-Gboundbeads, and the beads were added toAPC and used to stimulateimmuneCD4+Tcellproliferation.Thisnoveltechnologycanbeusedtorapidlyscreenalargenumberofproteinsfromagivenpathogenfor recognitionbybothantibodyandT lymphocytes if thegenomesequenceisavailable.

yASMInBELKAID(nIH,USA)role And origins of regulAtory t Cells

during PArAsitiC infeCtions

AnTonIoLACAVA(UCLA,USA)Cross-tAlK betWeen neuroendoCrine And

immune system: tHe CAse of lePtin

HARRyDAWSon(ARS-USDA,USA)regulAtion of PorCine HePAtiC And

PulmonAry-AssoCiAted immune resPonses by VitAmin A

UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,BeltsvilleHumanNutritionResearchCenter,Diet,

GenomicsandImmunologyLaboratory,Beltsville,Maryland,20705USA

PigsinfectedwithAscarissuumorcontrolsweregiven100µg(LD)or1,000µg(HD)all-transretinoicacid(ATRA)/kgbodyweightincornoilorcornoilaloneperoson–1,1,and3daysafterinocula-tion (DAI)with infectiveeggs.PlasmaaspartateanimotransferaseincreasedinpigsgivenLD-ATRA,whileIL-4andIL-12p70increasedininfectedpigsgivenATRAat7DAI.TreatmentwithATRAaugmentedtheincreaseinbronchial-alveolarlavageeosinophilsobservedat7and14DAI.AquantitativerealtimeRT-PCRarraywasdesignedtotest thehypothesis thatATRAwouldenhancerobustgeneexpres-sionfollowingparasiteinfection.Infectedpigshadincreasedlevelsof hepaticmRNA forT helper 2 (Th2)-associated cytokines,mastcellmarkers,andTregulatory(Treg)cells,whileinfectedpigsgivenATRA had higher levels of hepatic IL4, IL13, CCR3 and CCR4ligands, and TPSB1 compared to controls. Gene expression forTh1-associated markers (IFNG, IL12B, and TBX21), the CXCR3ligand(CXCL9),IL1B,andtheputativeTregmarkerTNFRSF18wasincreasedbyLD-ATRAininfectedanimals.ExpressionofIL4,IL13,IL1B,IL6,CCL11,andCCL26wasincreasedinthelungsofinfectedpigstreatedwithATRA.Thus,ATRAaugmentsadiverseTh1-,Th2-,andinflammation-associatedresponseintheliverandlungsofswineinfectedwithAscaris.

HARRISLEWIn(UnIVERSITyoFILLInoIS,USA)good embryos gone bAd: A trAnsCriPtion

Profile of WHAt goes WrongRoBInEEVERTS1,SADIELSMITH2,AnTHonyRAZZAK1,PASCALECHAVATTE-PALMER3,ISABELLEHUE3,CHERyLA

GREEn1,RoSAnEoLIVEIRA1,SAnDRALRoDRIGUEZ-ZAS1,xCInDyTIAn2,xIAnGZHonGyAnG2,JEAn-PAULREnARD3,

HARRISALEWIn1,41DepartmentofAnimalSciences,UniversityofIllinois,Urbana,IL,USA,2CenterforRegenerativeBiology,UniversityofConnecticut,Storrs,CT,USA,3BiologieduDéveloppementetReproduction,INRA,JouyenJosas,France4,InstituteforGenomicBiology,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,US

h-lewin@uiuc.eduSomatic cell nuclear transfer (SCNT) is a unique model sys-

tem to explore nuclear reprogramming in early embryos, and toidentify thecriticalgenesandpathways thatproduceanomalies inplacental and fetal development. Cloning cattle using SCNT hasseveraladvantagesasamodelsystem,includinghighsuccessrateofblastocystdevelopment (30%-50%), relativelyhigh frequencyofliveoffspring(upto20%)andwell-establishedembryology.Atleast66%ofcattleembryosarelostbyday-60ofdevelopment.Placentas

PlenAry session: immunoendoCrinology, stress And immunology of reProduCtion And neonAtes; miCrobiAl florA, nutrients And tHe immune resPonse

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LoRRAInESoRDILLo(MICHIGAnSTATEUnIVERSITy,USA)mAmmAry immunity And susCePtibility to

mAstitis: role of oxidAnt stressMastitiscausessignificanteconomiclossestotheworld’sdairy

producers. Dairy cattle are more susceptible to mastitis duringtheperiparturientperiod. The incidenceofmastitiswith respecttolactationstageisdirectlyrelatedtochangesinthecomposition,magnitude,andefficiencyof themammaryglanddefensesystem.There exist numerous genetic, physiological, and environmentalfactorsthatcancompromisehostdefensemechanismsduringthefunctional transitionsof themammarygland. Forexample,physi-ological stresses associated with rapid differentiation of secretoryparenchyma, intense mammary gland growth, and the onset ofmilk synthesis and secretion are accompanied by a high energydemandandan increasedoxygen requirement. Increasedoxygenmetabolismaugments theproductionofoxygen-derivedreactants,collectively termed reactive oxygen species (ROS). The exces-

sive accumulation of ROS can lead to a condition referred to asoxidativestress thatplaysacentral role inmediatinguncontrolledinflammatory responsesand causes tissue injury.Data generatedthus farestablishesastrongcorrelationbetweenoxidativestress,suchasthatassociatedwiththeperiparturientperiod,andexagger-atedinflammatoryresponsesofbovinemammaryendothelialcells.HosttissuesdohaveseveralenzymesandsmallmoleculesthatcanreduceROS to less reactivemetabolites and it is this antioxidantcapability that will help to protect cells from the damaging effectsofoxidativestress.Forexample,thedegreeofvascularoxidativestress can be controlled by several important antioxidant seleno-proteins, including glutathione peroxidase (GPX) and thioredoxinreductase (TrxR). These antioxidant enzymes can either directlyreduceharmfulROSorplayaroleinredoxregulationofintracellularsignalingprocessesthatcontrolpro-inflammatorygeneresponses.ThispaperwilloutlinesomeofthemolecularpathwaystargetedbyGPXandTrxRthatcaninfluenceexcessiveinflammatoryresponsesofbovinemammaryendothelialcells.Theprospectsforcontrolling

fromclonedcattle typically showextensiveabnormalities, suchasincreased size and fewer placentomes. Of those SCNT-derivedclonesthatdeveloptoterm,~40%haveLargeOffspringSyndrome,which is characterizedbyenlargedorgans, hydropsof fetus, leth-argy,andrespiratoryproblems.LargeOffspringSyndromeissimilartohumanBeckwith-WiedemannSyndrome,whichisassociatedwithimprintingdefects.ThesedataindicateproblemsinreprogrammingofthesomaticnucleusduringtheprocessofSCNT.Wehavecon-ductedaseriesofexperimentstoidentifythemetabolicandcellularpathwaysthatcausethedeathofclonedcattleembryosandfetuses.TranscriptomicanalysisofplacentaederivedfromSCNTclones,IVFandartificial insemination(AI)wasperformedatdifferentstagesofdevelopmentinordertounderstandhowthecloningprocessleadstoplacentaldefectsandLOSincattleproducedbySCNTandIVF.Forthesestudies,microarraysconsistingofeither7,872cDNAsor13,25770-meroligonucleotideswereusedtoanalyzegeneexpres-sioninindividuald-7blastocysts,d-25embryonicdiskandextraem-bryonictissues,d-75fetaltissues,andtermplacentomes.Wehavedemonstratedmassive reprogramming of nuclear genes in SCNTembryosandidentifiedapproximately50genesthataredifferentiallyexpressedinclonedblastocystsascomparedtoblastocystsderivedbyAI. These data reveal candidate genes and cellular pathwaysforembryonicmortalityinclones,andforplacentaldefectsinthoseclonesthatsurvivetoterm.Wehaveshownthatthedefectsinclonesthatleadtoabnormalitiesinplacentaldevelopmentarelikelytobeintheformationandfunctioningofextraembryonictissues,whicharenecessary fornormalplacentation. Wepropose that theplacentaldefectsarepathway-dependent,maybelethalorcompensatedfordependingonthereprogrammabilityandextentofsuccessfulrepro-grammingof thedonorsomaticnucleus. Ourstudiesmay lead tonewtechniquesforimprovingcloningefficiencyandagreaterunder-standing of nuclear reprogramming and stem cell differentiation.Additionally, our work provides insight into the processes of earlydevelopment,implantation,andplacentalpathologies.

JoSEPHURBAn(ARS-USDA,USA)use of PArAsitiC infeCtion to exPlore

dietAry ComPonents tHAt regulAte APProPriAte muCosAl immune resPonses

Pfizer AWArd for distinguisHed VeterinAry immunologist

JoHnEBUTLER(UnIVERSITyoFIoWA,USA)diVersity, frontiers And CAreers in

immunologyImmunologicaldiversityhaslongbeenofacademicinterestto

evolutionarybiologists.Forveterinariansunderstandingsuchdiver-sityisofmorethanacademicinterestandisimportanttoanimalhealthandessentialtotheprofession.ThegeneticlociencodingtheTcellreceptorsandkappaandlambdalightchainsarehighlyconservedin higher vertebrates but major differences occur in the immuno-globulinheavychainlocus.Mostmammalshaveagenomecapableofexpressingallof themajorantibodyisotypesalthoughtherearemajordifferences insubclassdiversificationof IgAand IgGand intheheavychainvariablelocus.Therearealsoimportantdifferencesinhowtheseareused to formtheantibodyrepertoire.Differencesinclude thesiteof repertoirediversification, themechanismsused,thetimeofclass-switchrecombinationandperhapseventhenumberofBcellsubsets.Studiesonimmunologicaldiversityteachthatcau-tionisneededinextrapolatingparadigmsfrommouse-basedstudiestootherspecies.

Diversity among mammalian immune systems alsoextendstothetransmissionofimmunityfrommothertoyoungandinthematurityoftheoffspring’simmunesystematthetimeofbirth.Diversityof this typeallowscertainspecies tobeusedasmodelsandtoolstoaddresshypothesesandsolveproblemsthatcannotbeaddressedinrodentmodels.Examplesinvolvethestudyofnaturallyoccurring premature marsupials and isolator piglets. Both can beusefultoaddressmajorissueswithinthe“criticalwindow”ofneona-talimmunedevelopment.Theseincludetheroleofmaternalregula-tory factors, the impactofgutcolonizationand theearlyexposureto pathogenand thedevelopment of tolerance todietaryantigensandcommensalgutflora.Manyofthesefactorsarebelievedtobeimportanttothehygienehypothesisinwhichearlyexposuretobac-teriaandparasitescanlatereffectthedevelopmentofallergiesandinflammatoryboweldisorders.Addressingtheseissuesopensnewfrontiersforimmunology.Regardlessofhowpervasivemodelbuild-inghasbecomeintrainingimmunologystudents,onemustremaincognizant that while models provide useful information many arewrongsothebestmodelisthespeciesitself.Thereforetheneedtocharacterizetheimmunesystemofeachspecieswillpersist.

Veterinary species also provide new frontiers in medi-calscience.Theseinvolveengineeringoffarmanimalstoproduceimmunoglobulins to fill the dwindling supply of IVIG aswell as toproduce targetedantibodies for immunotherapy in caseswhere invitroproductionistoolow.Veterinaryspeciescanalsobeusedasdiseasemodelsinwhichrodentmodelsfail.

Old biologists often become old philosophers, usuallyto the benefit of younger scientists.They are able to share yearsof experience, ponder mistakes and even successes and passthat information on the young investigators. “Six commandments”fundamental to bulding a successful career in immunology will bediscussed.Thethreetopicscomprisingthislectureareallintertwinedandarerelevanttocareerbuilding.

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thedurationand severity ofmastitis bymanipulating these criticalhostantioxidantdefensemechanismsarediscussed.

18:30–19:00H-ELSMEEUSEn(MonASHUnIVERSITy,AUSTRALIA)

struCture And ProteCtion of tHe ruminAnt lung

AnimalBiotechnologyResearchLaboratories,DepartmentofPhysiology,MonashUniversity,Australia

e.meeusen@med.monash.edu.auProtectionofthelungfrominfectionandinjuryisprimarilyregu-

latedbymechanicaland innatedefencemechanisms thatpreventpathogensandobnoxioussubstancesfromreachingthemorevul-nerablelowerrespiratorytract.Cellsthatlineandinhabittheupperandlowerrespiratorytracthaveacrucialroleinsensingpathogenicorganismsforthegenerationofaprotectiveimmuneresponse,butalso in suppressing unnecessary inflammation that may interferewith the lung’sprimary functionofgasexchange.Thestructureofthelungsoflargeanimalsisdistinctfromsmallerlaboratorymodelsinanumberofaspectsthatinfluencethetypeofinnateandadaptiveresponsethatwillbegeneratedfollowinginfection.Thispresentationwillgiveanoverviewofthedifferentimmunemechanismsactiveatdistinctlevelsoftheruminantlungandhowthismayhaveimplica-tionsforthedesignofrespiratoryvaccines.

19:00–19:30H-ADRIAnSMITH(InSTITUTEFoRAnIMALHEALTH,UK)

struCturAl And CellulAr AsPeCts of immunity to PAtHogens in tHe gut

EntericImmunology,InstituteforAnimalHealth,Compton,BerkshireRG207NN,UK

The intestine is the largest immunecompartmentof thebodyand is thesiteof residenceorportalofentry formanypathogenicmicroorganisms.However,constantexposureof thegut to foreignmaterialderivedfromfoodandresidentnon-harmfulmicroorganismscomplicatestherulesofengagementfortheimmunesystem,neces-sitatingdevelopmentoftightregulatorynetworks.Focusingonstud-ies with the highly immunogenic apicomplexan protozoan Eimeriavermiformis Iwill exploreaspectsof theenteric immune responseto infectious challenge, including the requirements for differentlymphoidstructures, thecellular interactions thatmediateeffectiveimmunityandtheregulationofpathology.

EffectiveimmunityagainstprimaryinfectionwithE.vermiformisismediatedbyinductionofarapidTh1-typeresponsethatisdepen-dentonco-ordinatedinductioninboththePeyerspatches(PP)andthemesenteric lymphnodes(MLN).ThetimingofTh1-induction inbothMLNandgutwasdependentonthepresenceofPPsuggestingalevelofcooperationbetweenimmuneresponsesinducedinthesedistinct lymphoidstructures.Thedelay inTh1-inductionwasattrib-utedtothelatearrivalofabroadrangeofdendriticcell(DC)subsetsintheMLNandasubstantialreductionofCD8a-CD11bhiB220-Th1-responsepromotingDCinPP-deficientmice.TheeffectiveTCRaβ+CD4+T cell expressed IFNγ required expression of the IFNγR onstromalcellsratherthananybone-marrowderivedimmunecellsub-setbutthisinteractionalsoinducedtheentericpathologyassociatedwith infection.Hence,although IFNγ is anessential componentofcontrolofintracellularpathogensinthegutthiscytokinealsodriveslife-threateningpathologies.Hence, proinflammatory infection con-trolistemperedbytherapidinductionofregulatoryresponsesandwithE.vermiformisthisis,atleastinpart,mediatedbytheactivitiesofTCRγd+Tcells.IntheabsenceofthesecellstheTCRaβ+Tcellmediatedpathologiesaremoresevereandresult inovertbleedingintotheintestineofinfectedTCRγ-/-mice.

Day 3 - August 17th - Friday

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8:30-11:00h PosterViewing:Themes1–5 SALÃO DIAMANTE

11:00-13:00h PlenarySession:AntigenPresentation, SALÃO OURO EffectorCells,Immunoregulation

14:00-16:00h ConcurrentSession2:Bacterialand SALÃO OURO ViralDiseasesPrions

14:00-16:00h ConcurrentSession7:ComparativeImmunology SALÃO AMETISTA

14:30-16:30h ConcurrentSession9:Innateimmunity, SALÃO TURMALINA Inflammation,Adjuvants;Memory, AcquiredImmunity,Vaccines

14:30-16:30h ConcurrentSession10:ClinicalImmunology SALÃO ESMERALDA Immunopathology

17:00-19:00h PlenarySession:ComparativeImmunology SALÃO OURO

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SALÃO DIAMANTE

08:30-11:00h Poster Viewing

Coffee Break at 10:00h, during poster session

1. Immunogenetics and Genomics: postersIG001-IG026

2. Immune Responses in Bacterial and Viral Diseases; Prions and BSE: postersBV027–BV077

3. Immunoendocrinology; and Stress; Immunology of Reproduction and Neonates; Microbial Flora, Nutrients and the Immune Response: postersER078-ER094

4. Immunology of the Mucosae and Skin and of the Mammary Gland; Mastitis: postersSM095-SM118

5. Antigen Presentation and Dendritic Cells; Effector Cells, B and T cells, NK and NK T cells; Immunoregulatory cells: postersAP119-AP140

SALÃO OURO11:00-13:00h Plenary Session: Antigen Presentation, Dendritic Cells; Effector Cells, B and T cells, NK and NK T cells; Immunoregulation Chairs:MarcJutila -USAandIsabelleSchwartz-Cornil-France

Speakers 11:00-11:30h KenMcCullough InstituteofVirologyandImmunoprophylaxis,Switzerland Porcine Dendritic Cells: at the Front Line of Pathogen Attack

11:30-12:00h GregoryBorhach UniversityofIdaho,USA Bovine T Regulatory Cells

12:00-12:30h AnneK.Storset NorwegianSchoolofVeterinaryScience,Norway NK cells; general characteristics and immunity to infection

12:30-13:00h JoAnnFlynn UniversityofPittsburghSchoolofMedicine,USA IL-17-producing gamma delta T cells and Tuberculosis

13:00-14:00h Lunch

14:00-16:00h Concurrent Session #2: Immune Responses in Bacterial and Viral Diseases; Prions and BSE Chairs:TraceyJ.Coffey-UKandJoanLunney-USA

Speakers 14:00-14:30h JuergenRicht USDA,USA Prion Knockout cattle as a model to study prion disease

14:30h SandraSommer MichiganStateUniversity,USA Mycobacterium Paratuberculosis Suppresses CD40 Signaling Induced IL-12p40 and iNos Gene Expression In Bovine Monocyte-Derived Macrophages AbstractandPosterBV030

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14:45h CharlesJ.Czuprynski UniversityOfWisconsin-Madison,USA Prothrombotic Effects of Haemophilus Somnus on Bovine Endothelial Cells and Platelets AbstractandPosterBV037

15:00-15:30h KristienVanReeth GhentUniversity,Belgium Can Immunity To H1N1 Influenza Protect Against an H5N1 Avian Influenza Virus? - Experiments in Pigs as a Model for Humans

15:30h DavidM.Haig MoredunResearchInstitute,UK IL-15, TNF-a and the Autoimmune Pathogenesis of Malignant Catarrhal Fever AbstractandPosterBV075

15:45h ElizabethJ.Glass RoslinInstitute,UK Transcriptomic Analysis of the Chicken Anaemia Virus (CAV)-Induced Immunosuppression AbstractandPosterBV067

SALÃO AMETISTA14:00-16:00h Concurrent Session #7: Comparative Immunology Chairs:ChristopherJ.Secombes-UKandRobertKammerer-Germany

Speakers 14:00-14:30h GregoryWarr HollingsMarineLaboratory,USA Innate and Adaptive Immunity in a Marine Shrimp

14:30-15:00h RobertMiller UniversityofNewMexico,USA The marsupial immune system: novel adaptations and convergent evolution

15:00h TakayukiKubota NationalInstituteofAnimalHealth,Japan Gene expression of chicken interleukine-4 by baculovirus AbstractandPosterCI206

15:15h oliviaJHolland UniversityofAuckland,NewZealand MHC population structure in the New Zealand brushtail possum AbstractandPosterCI203

15:30h RobertKammerer LIFECenter,LMU,Germany The carcinoembryonic antigen (CEA) family in placental mammals of the superordinal clade Laurasiatheria AbstractandPosterCI202

15:45h HarryD.Dawson ARS-USDA,USA A Comparative Analysis of the Porcine, Murine, and Human Immune Systems AbstractandPosterCI205

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SALÃO TURMALINA14:30-16:30h Concurrent Session #9: Innate immunity, Inflammation and Adjuvants; Memory, Acquired Immunity and Vaccines Chairs:ThomasJungi-SwitzerlandandFalkoSteinbach-UK

Speakers 14:30-15:00h JanRombout WageningenUniversity,TheNetherlands Phylogeny and Ontogeny of Innate Immunity.

15:00-15:30h ReubenHarris UniversityofMinnesota,USA APOBEC3 proteins in artiodactyls constitute an innate retrovirus defense mechanism.

15:30-16:00h FalkoSteinbach VeterinaryLaboratoriesAgency,UK Dendritic cells and their long road to clinical application.

16:00h ReginaldoG.Bastos WashingtonStateUniversity,USA Interaction of natural killer cells, monocytes and dendritic cell populations in cattle. PosterandAbstractVA219

16:10h oliverBruhn UniversityofKiel,Denmark An equine a-defensin: gene transcription, recombinant expression and characterization of the structure and function PosterandAbstract232

16:20h JavierDominguez NationalInst.ForAgriculturalandFoodTechnology,Spain Targeting to sialoadhesin receptor improves antigen presentation to T cells PosterandAbstractVA243

SALÃO ESMERALDA 14:30-16:30h Concurrent Session #10: Clinical Immunology and Immunopathology Chair:MariaJuliaFlaminio-USA

Speakers 14:30-15:00h Jean-PierreLavoie UniversityofMontreal,Canada Th2-type cytokines and neutrophils: are they playing a role in heaves?

15:00-15:30h DennisHickstein NationalInstitutesofHealth,USA Correction of the phenotype in canine leukocyte adhesion deficiency using stem cell transplant and gene therapy: Canine Leukocyte Adhesion Deficiency Model for New Approaches to LAD

15:30-16:00h CorneliaDeeg LugwigMaximiliansUniversity,Germany A proteomic approach to the pathogenesis of spontaneous equine recurrent uveitis

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16:00-16:30h RicardoTostesGazzinelli OswaldoCruzFoundation,Brazil Towards an anti-amastigote vaccine for canine leishmaniasis: experimental and pre-clinical trials

16:00-17:00h Coffee Break

SALÃO OURO17:00-19:00h Plenary Session: Comparative Immunology Chairs:MaristelaMartinsCamargo-BrazilandMartinBilej-CzechRepublic

Speakers 17:00-17:30h Jean-MarcReichhart InstituteofMolecularandCellularBiology,France Evolution of the innate immune system, lessons from the Drosophila model

17:30-18:00h ChristopherJ.Secombes UniversityofAberdeen,UK How much have we learnt about the cytokine network of fish?

18:00-18:30h JimKaufman BiotechnologyandBiologicalSciencesResearchCouncil,UK Co-evolution between MHC genes determines alternative immune strategies in vertebrates

18:30-19:00h JohnButler UniversityofIowa,USA Piglet models in studies on antibody repertoire development.

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KEnMCCULLoUGH(InSTITUTEoFVIRoLoGyAnDIMMUnoPRoPHyLAxIS,

SWITZERLAnD)PorCine dendritiC Cells: At tHe front line

of PAtHogen AttACKKEnnETHCMCCULLoUGH,CARoLEBALMELLI,oLIVER

BAUHoFER,LAUREnCEGUZyLACK-PIRIoU,nICoLASRUGGLI,ARTURSUMMERFIELD,ISABELLEEVInCEnT

ResearchDepartment,InstituteofVirologyandImmunoprophylaxis,Sensemattstrasse293,CH-3147Mittelhäusern

Efficient immune defence function is dependent on the roleplayedbydendriticcells (DC),particularly the interactionbetweenconventional (“myeloid”) DC (cDC) and plasmacytoid DC (pDC),together with other monocytic cells.This determines the outcomeofimmuneresponsedevelopment,butthehostdefencecapacityisalsoopentomanipulationbyviralpathogensinfectingDC.Theman-nerbywhichdifferentvirusesinterferewithDCfunctiondependsonboththevirusandthesubsetofDCinvolved.

Classical swine fever virus (CSFV) is amonocytotropicRNAvirus infecting cDC and pDC.The viral non-structural Npro proteinantagonizestheTypeIinterferon(IFN)inductionpathway,promotingproteasomaldegradationofinterferonregulatoryfactor(IRF)3.WithCSFVvariantslackingNprowillinduceIFNaproductionbymeansoftheirdsRNAreplicativeintermediates.InfectionofpDCbywildtypevirusalsoresultsinIFNainduction,probablybecausetheNprodoesnotinterferewiththeIRF7whichismoreactivepDC.ThisabilityofthevirustoinhibitcDCproductionofIFNa,whileaugmentingtheIFNaproduction by pDC would lead to an exaggerated pDC response,relatingtotheimmunopathologicalcharacteristicsofthedisease.

ThessDNAvirus–porcinecircovirustype2(PCV2)–isalsoimmunomodulatory. Incontrast toCSFV,PCV2doesnot replicateinDC,butaccumulatestohighlevelsbothinvitroandinvivo.Thisaccumulationisdependentonviruscapsidprotein,butindependentof virus replication. Nevertheless, the presence of PCV2 in cDCdoes not interfere with processing of other antigens. In contrast,elevatedIL-10productionisobserved.Thisalonewouldnotexplainthe immunoregulatory characteristics of PCV2-induced diseases.PCV2will impair andevenabrogate “danger” recognitionby cellsoftheinnatedefences,apropertydependentontheviralgenome,particularlythedsDNAreplicativeform.TheDCfamilyrepresentsacriticalcentralelementintheefficientfunctioningofimmunedefensegenerationandmaintenance.Whilecertainvirusescaninterferewiththeefficientfunctioningofthesecells,immunedefencescanalsobeenhanced–byvaccination.Thisalsorequires interactionbetweencDC or pDC, towards building the immune defensive barrier forprotecting the host.TargetingDC has high potential for improvedvaccinationstrategies,protectingthehostagainstmanipulationofitsimmunedefensesbytheviralpathogeninquestion.Thecentralele-mentforsuccessistheefficienttargetingofDC,offeringasitdoeshighpotentialforimprovedvaccinesbothnowandinthefuture.

GREGoRy BoRHACH (UNIVERSITy oF IDAHo, USA)

BOVINE T REGULATORy CELLSboVine regulAtory t Cells induCed by A stAPHyloCoCCAl suPerAntigen in VitroKEUnSEoKSEo1,yonGHoPARK2,WILLIAMCDAVIS3,

GREGoRyABoHACH11DeptMicrobiol,MolecularBiol&Biochem,Univ,ofIdaho,

Moscow,ID,USA;2DeptMicrobiol,SeoulNationalUniv,Seoul,Korea;3DeptVetMicrobiol,&Pathology,WashingtonStateUniv,

Pullman,WA,USAgbohach@uidahoedu

Staphylococcalsuperantigens(SAgs),includingstaphylococcalenterotoxins (SEs)areexpressedbyahighpercentageofbovinemastitis isolates, suggesting that these toxinscontribute topatho-

genesisofthisinfection.EvidencefrommurineandhumananimalmodelssuggeststhatregulatoryTcells(Tregs)areinducedbyexpo-suretoSAgs.Recently,westudiedtheeffectsofexposingbovineperipheral blood mononuclear cells (PBMCs) to a physiologicallyrelevantdose(5ng/ml)ofSEtypeC1(SEC1)forupto10days.Weobserved thatSEC1caused initialproliferationofCD4+andCD8+Tcellsatsimilarrates.However,inprolongedcultures,nearlyallTcellproliferationoccurredindependentlyofVβexpression,althoughCD8+Tcellsproliferatedmorevigorously.ExpressionoftheCD25,CD152 genes increased concurrently with a decreased expres-sionof IL-2. IL-10andTGF-βgeneexpression inducedbySEC1occurred within the CD4+CD25+T cell subpopulation. ExpressionofFoxp3alsoincreasedasdeterminedbymeasuringmRNAlevelsandbyuseofabovineFoxp3-specificmonoclonalantibody. Thiseffectcoincidedwithanup-regulationofCD152anddown-regulationofIL-2transcription,characteristicofTregs.SEC1-stimulatedCD4

+Tcellswereimmunosuppressiveinvitroandsuppressedtheprolifera-tionofnaïvePBMCsinresponsetoheat-killed-fixedS.aureusinanIL-10andTGF-βdependantmanner.ActivatedCD8+Tcellswerealsoimmunosuppressiveinthisassay,althoughtheeffectwasnotmediatedbyIL-10orTGF-β.Theseresultssuggest1)theactivationof a bovine cell population characteristic of Tregs, 2) SAgs induceTregsinthebovinemodel,and3)inductionofthesecellsinvivocouldcontributetomastitisandpersistentinfectionsindairyanimals.

ANNE K. SToRSET (NoRWEGIAN SCHooL oF VETERINARy SCIENCE, NoRWAy)

nK Cells; generAl CHArACteristiCs And immunity to infeCtion

Naturalkillercellswerefirstknownintheearly1970saslargegranular lymphocytes that could spontaneously kill tumour cells- often ignored as disturbing sources of background in assays ofcancerimmunology.Asthenatureofthesecellsbecamecleartheyattracted increasingattention,andtheirability tokillcells that lackMHCclassIwasregardedastheirmostimportantfeature.StudiesofNKcellsandtheirreceptorsexplodedinthe1990s,andtodaytheyarerecognisedascentralplayersoftherodentandhumanimmunesystems. As NK cells have now been assigned roles exceedingthatofcancerandtransplantation immunology, theyhavebecameincreasinglyinterestingforveterinaryimmunologists.

NKcellsareinvolvedindefencemechanismsagainstseveralmicrobial infections,and theyhavea role inshaping theadaptiveimmune response, being of potential interest in vaccine develop-ment.Inprimates,theyalsohavearoleinplacentaldevelopment.NK cells belong to the innate immune system and work throughgermlineencodedreceptors.Familiesofreceptorswithbothinhibi-tory and activating functions have been the hallmark ofNK cells,althoughmostofthesereceptorsmayalsobeexpressedonotherleukocytes.Thecytokineandchemokineproducing capabilitiesofNKcellsisimportantintheirimmunoregulatoryfunctions.

Atpresent, thereisnoconsensusphenotypicdefinitionofNKcellsacrossspecies.Butveryrecentlythesurfaceexpressionofthenaturalcytottoxicityreceptor,NKp46,hasbeensuggestedasadefi-nitioncriterion.ThepresenceofthegeneforthisreceptorinseveralveterinaryspeciesmakesthisproposalanimportantstimulanttothedevelopmentoftheNKcellfieldinveterinaryimmunology.

NK cells in cattle and their functions have been studied forsomeyears,andalthoughthisfieldisstillinitsinfancy,possiblerolesin several infections have been described. Bovine NK cells haveshowed reactions against cells infected with intracellular bacteriaandmayalsobeofimportanceinparasiticinfections.

JoANN FLyNN (UNIVERSITy oF PITTSBURGH SCHooL oF MEDICINE, USA)

il-17-ProduCing gAmmA deltA t Cells And tuberCulosis

PlenAry session. ANTIGEN PRESENTATION, DENDRITIC CELLS; EFFECTOR CELLS, B AND T CELLS, NK AND NK T CELLS; IMMUNOREGULATION.

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JUERGEN RICHT (USDA, USA)

Prion KnoCKout CAttle As A model to study Prion diseAse

JARICHT1,AnHAMIR1,CSoTo2,JRoBL3,yKURoIWA31NationalAnimalDiseaseCenter,Ames,IA.,USA;2UTMB,Galveston,TX.,USA;3HematechInc.,SiouxFalls,S.D.,USAPrion diseases, such as bovine spongiform encephalopathy

(BSE)incattleandCreutzfeldt–Jakobdisease(CJD)inhuman,arecausedbypropagationofamisfoldedformofnormalcellularprionprotein,PrPC.DisruptionofPrPCexpressioninthemouse,aspeciesthatdoesnotnaturallycontractpriondiseases,resultsinnoappar-entdevelopmentalabnormalities,andinresistancetomousepriondisease.However,theimpactoftheablationofPrPCfunctioninanaturalhostspeciesofpriondiseasesisunknown.Here,wereportthegenerationandcharacterizationofPrPC-deficientcattle.Atover24monthsofage,thecattleareclinically,physiologically,histopatho-logicallyand immunologicallynormal, indicating that “lossof func-tion”ofendogenousbovinePrPCdoesnotdirectlycauseBSEandthatPrPCfunction itself isgenerallydispensablefornormalanimaldevelopment.Furthermore,theknockout(KO)cattleareresistanttoprionpropagation invitrobyproteinmisfoldingcyclicamplification.TheKOcattlecouldbeausefulmodelforprionresearchasanaturalhostspeciesofpriondiseasesandprovideprionprotein-freebovineproductsforbio-industries.

KRISTIEN VAN REETH (GHENT UNIVERSITy, BELGIUM)

studies on tHe immune resPonse to influenzA in Pigs: lessons for PAndemiC

influenzA VACCinesVAnREETHK1,BRAECKMAnSD1,CoxE2,DE

VLEESCHAUWERA1LaboratoryofVirology(1)andImmunology(2),Facultyof

VeterinaryMedicine,GhentUniversity,Belgium

SincenohumanshaveimmunitytotheH5influenzavirushae-magglutinin(HA),theappearanceofahighlypathogenic(HP)H5N1avian influenzavirus(AIV) inhumans isofmajorconcern.Ontheotherhand,theH5N1virussharesitsneuraminidase(NA)subtypewiththeendemichumanH1N1virusesandcell-mediatedimmunitytotherelativelyconservedinternalviralproteinsmayalsocontributetoprotection.Weusethepigmodelofinfluenzatostudytheextentof cross-protection between influenza viruses with unrelated HAsubtypesandtheunderlyingimmunemechanisms.InthislectureIwilldiscussrecentdataabouttheeffectofpriorinfectionwithH1N1swineinfluenzavirusonchallengewithalowpathogenic(LP)H5N1AIV.InfluenzavirusnaïvepigswereinoculatedwithH1N1andH5N1ata4-weekinterval(H1N1-H5N1group)orwithH5N1only(H5N1challenge control group). We examined serum antibody titres tobothvirusesinhaemagglutinationinhibition(HI),virusneutralisation(VN)andneuraminidaseinhibition(NI)tests,influenzavirusspecificlymphoproliferative responses of peripheral blood mononuclearcells,andclinicalandvirologicalprotectionagainstH5N1challenge.Beforechallenge,theH5N1challengecontrolpigswerenegativeinallserologicalassays.PigsoftheH1N1-H5N1grouphadHIandVNantibodiestoH1N1only,andNIantibodiestobothH1N1(meantitre80)andH5N1(meantitre61).Thisgroupalsoshowed20-foldhigherlymphoproliferative indexes to H5N1 than the challenge controlgroup.TheH5N1challengeproducedmildtomoderateclinicalsignsinallchallengecontrolpigsandtheviruswasisolatedfromthenasalmucosa(4/9pigs), trachea(7/9pigs)and lungs(9/9pigs)ofmostpigs.PigsoftheH1N1-H5N1group, incontrast,showedcompleteclinical protection and strongly reduced virus isolation rates. OurdataindicatethatimmunitytoanH1N1influenzavirusmaypartiallyprotectpigsfromavianH5N1influenza.Cross-protectionwasclearlyindependentofantibody to theviralHA,andmaybemediatedbyantibodytotheN1NAand/orcross-reactivecell-mediatedimmunity.Cross-reactiveN1antibodieswererecentlyshowntoaffordpartialprotectionagainstchallengewithHPH5N1inmice.Thepigisavalu-ablemodelforfurtherstudiesonthemechanismsandmodalitiesofcross-protectionbetweenH1N1andH5N1.SuchstudieswillrevealwhetherincreasinglevelsofimmunitytoH1N1inhumanscouldbeapossiblepandemicstrategyagainstH5N1.

ConCurrent session #2. IMMUNE RESPONSES IN BACTERIAL AND VIRAL DISEASES; PRIONS AND BSE

GREGoRy WARR (HoLLINGS MARINE LABoRAToRy, USA)

innAte And AdAPtiVe immunity in A mArine sHrimP

RoBERT MILLER (UNIVERSITy oF NEW MExICo, USA)

tHe mArsuPiAl immune system: noVel AdAPtAtions And ConVergent eVolution

ConCurrent session #7. COMPARATIVE IMMUNOLOGy

ConCurrent session #9. INNATE IMMUNITy, INFLAMMATION AND ADJUVANTS; MEMORy, ACQUIRED IMMUNITy AND VACCINES

JAN RoMBoUT (WAGENINGEN UNIVERSITy, THE NETHERLANDSPHylogeny And ontogeny of innAte

immunity

REUBEN HARRIS (UNIVERSITy oF MINNESoTA, USA)

APobeC3 Proteins in ArtiodACtyls Constitute An innAte retroVirus defense

meCHAnism

DepartmentofBiochemistry,MolecularBiologyandBiophysics,UniversityofMinnesota,Minneapolis,MN55455

TheAPOBEC3proteinsareuniquetomammals.ManyinhibitretrovirusinfectionthroughacDNAcytosinedeaminationmechanism.

At least twoof thehumanproteins,APOBEC3FandAPOBEC3G,caneffectivelyinhibitthereplicationofHIV-1.However,HIV-1usuallyneutralizesthishostdefensethroughVif,whichtriggersAPOBEC3ubiquitinationanddegradation.WehavediscoveredanAPOBEC3F-like,doubledeaminasedomainproteinfromthreeartiodactyls(cattle,pigs and sheep). Like their human counterparts,APOBEC3F andAPOBEC3G,theartiodactylAPOBEC3FproteinsareDNAcytosinedeaminasesthatlocatepredominantlytothecytosolandcaninhibitthereplicationofHIV-1andMLV.Retrovirusrestrictionisattributabletodeaminase-dependentand-independentmechanisms,asdeami-nase-defectivemutantsstill retainsignificantanti-retroviralactivity.However,unlikehumanAPOBEC3FandAPOBEC3G,theartiodac-tylAPOBEC3FproteinshaveanactiveaminoterminalDNAcytosinedeaminasedomain,whichelicitsabroaderdinucleotidedeaminationpreference,andtheyareresistanttoHIV-1Vif.ThesedataindicatethatDNAcytosinedeamination,subcellularlocalizationandretrovi-

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rusrestrictionactivitiesareconservedinmammals,whereasactivesitelocation,localmutationalpreferencesandVifsusceptibilityarenot. Together, these studies indicate that some properties of themammal-specific, APOBEC3-dependent retroelement restrictionsystemarenecessaryandconserved,butothersaresimultaneouslymodularandhighlyadaptable. Interestingly,artiodactylsappear tohaveanAPOBEC3genomicorganizationmorelikerodentsthanthatofprimates.ThishascontributedtoamodelforanoveralldynamicexpansionoftheAPOBEC3locusinmammals.

Key words: innate immunity, virus restriction, DNA cytosinedeaminationSpecies:ruminants

FALKo STEINBACH (VETERINARy LABoRAToRIES AGENCy, UK)

dendritiC Cells And tHeir long roAd to CliniCAl APPliCAtion

ConCurrent session #10. CLINICAL IMMUNOLOGy AND IMMUNOPATHOLOGy

JEAN-PIERRE LAVoIE (UNIVERSITy oF MoNTREAL, CANADA)

tH2-tyPe CytoKines And neutroPHils: Are tHey PlAying A role in HeAVes?

DENNIS HICKSTEIN (NIH, USA)

CorreCtion of tHe PHenotyPe in CAnine leuKoCyte AdHesion defiCienCy using stem Cell trAnsPlAnt And gene tHerAPy: CAnine leuKoCyte AdHesion defiCienCy model for

neW APProACHes to lAdChildren with the genetic immunodeficiency leukocyte adhe-

sion deficiencyor LAD have heterogeneous molecular defects inthe leukocyte integrin CD18 molecule and suffer life-threateningbacterialinfectionsduetotheinabilityoftheirleukocytestoadhereandmigratetositesofinfection.Hematopoieticstemcelltransplantremains the only curativetherapy for LAD, however the toxicity ofthistreatmenthaslimiteditsuseingeneticdiseasessuchasLAD.Wetestednewstemcell transplantandgenetherapyapproachesto LAD in a canine model of LAD. Matched littermate transplantfollowing a non-myeloablative conditioning regimen with 200 cGytotalbodyirradiationresultedinreversalofthephenotypeofCLADwithminimal toxicity.However, theoptimal therapy forLADwouldinvolve gene therapy since no donor is required and graft versushostdiseaseisnotanissue.Wefirstevaluatedexvivogammaretro-viral-mediatedgenetherapyusingtwonon-myeloablativecondition-ingregimens-200cGyTBIor10mg/kgbusulfan-withorwithoutpost-transplant immunosuppression.Sixof11treatedCLADdogsachievedtherapeuticlevelsofCD18+leukocytes.ConditioningwitheitherTBIorbusulfanallowed long-termengraftmentand immunesuppressionwasnotrequiredforefficacy.ThepercentageofCD18+leukocytesincreasedover6-8monthstolevelsrangingfrom0.72%to8.37%atone-yearfollow-upinthe6dogs.Theselevelsresultedin reversal of the severe CLAD phenotype. Linear amplification-mediated-PCR assays indicated polyclonality of insertion sites.

Sincegammaretroviralvectorshave led to insertionalactivationofnearbyoncogenesandleukemiainpreviousgenetherapytrials,wecarriedoutgenetherapyintheCLADmodelusingavectorbasedonfoamyvirus(FV).FouroffiveCLADdogsreceivingnon-myeloabla-tiveconditioningwith200cGyTBIandinfusionofautologousCD34+hematopoietic stemcells transducedby theFVvector expressingcanineCD18hadcompletereversaloftheCLADphenotype,whichwassustainedtwoyearsfollowingtreatment.Invitroassaysdem-onstratedcorrectionof the lymphocyteproliferationandneutrophiladhesiondefectsthatcharacterizeCLAD.Therewerenogenotoxiccomplicationsandintegrationsiteanalysisdemonstratedpolyclonalmarkingbytransducedcells.TheseresultssuggestthatFVvectorswillbeeffectiveintreatinghumanhematopoieticdiseasessuchasLAD.ThesestudiesindicatethatnewtherapeuticapproachesarebecomingavailabletotreatLADnow20yearsaftertheinitialcloningoftheCD18cDNA.

Dennis D. Hickstein, M.D. SeniorInvestigatorExperimentalTransplantationandImmunologyCenterforCancerResearch,NationalCancerInstituteNationalInstitutesofHealthBldg10CRC,Room3-3142(3EastLabs)10CenterDrive,MSC1203

CoRNELIA DEEG (LUGWIG MAxIMILIANS UNIVERSITy, GERMANy)

A ProteomiC APProACH to tHe PAtHogensis of sPontAneous equine reCurrent uVeitis

RICARDo ToSTES GAzzINELLI (oSWALDo CRUz FoUNDATIoN, BRAzIL)

toWArds An Anti-AmAstigote VACCine for CAnine leisHmAniAsis: exPerimentAl And

Pre-CliniCAl triAls

JEAN-MARC REICHHART (INSTITUTE oF MoLECULAR AND CELLULAR BIoLoGy,

FRANCE)eVolution of tHe innAte immune system,

lessons from tHe drosoPHilA model

CHRISToPHER J SECoMBES (UNIVERSITy oF ABERDEEN, UK)

HoW muCH HAVe We leArnt About tHe CytoKine netWorK of fisH?

JIM KAUFMAN, (BIoTECHNoLoGy AND BIoLoGICAL SCIENCES RESEARCH

CoUNCIL, UK)Co-eVolution betWeen mHC genes

determines AlternAtiVe immune strAtegies in VertebrAtes

JoHN BUTLER (UNIVERSITy oF IoWA, USA)

Piglet models in studies on Antibody rePertoire deVeloPment

The piglet was selected for studies on antibody repertoiredevelopment because no maternal antibodies cross the placentato influence fetaldevelopmentandbecause theirprecosial natureallows them to be raised in isolator units in which environmental

PlenAry session. COMPARATIVE IMMUNOLOGy

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influencesarecontrolledbytheexperimenter.Theiruseasmodelsrequiredcharacterizationof theirBandTcell repertoiresandtheirdevelopment.Discoveriesmadeduringthischaracterizationprocesscontributedtotheaccumulatinginformationonthediversityofanti-bodysystemsinothervertebratesandonthemechanismsinvolvedinthedevelopmentofantibodiesrepertoires.

Swine possess the same five isotypes of immunoglobu-linsasmostallmammalsbuthavegreatlydiversifiedtheirIgG(Cγ)genesofwhichatleastelevenareexpressed.Inaddition,therearealternativelysplicedformssopigscanpotentiallyexpressmorethan20Cγvariants.EspeciallyinterestingisIgG3thatisexpressedinanantigen-independentmanner in the ilealPeyerspatchesandmes-entericlymphnode.ClassswitchrecombinationoccursearlyinfetallifeandpigletsarebornwithBcellsexpressingallmajor isotypes.Uniquetoswine is theuseof fourVHgenes,2DHsegmentsandoneJHsegment to form>80%of thepre-immune repertoire.Thisuser-friendlysystemallowsdiversificationoftheantibodyrepertoiretobe readilymonitoredbyquantitative clonal hybridization.Unliketheheavychainlocus,thekappa,lambda,VβandVdlociaregreatly

conservedandsimilartothoseinothermammals. Theisolatorpigletmodelhasallowedustostudyevents

in antibody repertoire development that occur during the “criticalwindow”.Studieshaveshownthatthedevelopmentoftheadaptiveimmunesysteminthisspeciesdependsongutcolonizationactingthroughligandstheyproducethatarerecognizedbyreceptorsoftheinnateimmunesystem.Duringthe“criticalwindow”pigletsareespe-ciallysusceptibletoimmunedysregulationofthetypeproducedbypathogenslikeporcinereproductiveandrespiratorysyndromevirus(PRRSV).PRRSVinterfereswithnormaldevelopmentofadaptiveimmunitybyselectiveexpansionofaminorsubsetofpre-immuneBcellsbearinghydrophobic,germline-encodedHCDR3s.Thesearenotbelievedtocontributeanti-viralantibodies.ThisdiversionisbelievedtobeduetoaputativeBcellsuperantigensuchthatone-thirdofallBcellsinisolatorpigletscanbeofthistype.Thisisnotdissimilartopatientswithmyelomaandwebelievethisdiversionaccountsforthedelayinthedevelopmentofeffectiveadaptiveimmunity.

Day 4 - August 18th - Saturday

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7:45-18:00h Registration

8:30-10:30h PlenarySession:Immunoparasitology SALÃO OURO

11:00-13:00h PlenarySession:Mediators;Immunoglobulins SALÃO OURO andFcReceptors;SignalTransduction

14:00-18:00h ConcurrentSession4:Mucosae,Skinand SALÃO AMETISTA MammaryGland

14:00-18:00h ConcurrentSession8:MediatorsofRecruitmentand SALÃO OURO FunctionofCellsoftheImmuneSystem; Immunoglobulins;SignalTransduction

14:30-18:00h ConcurrentSession6:Immunoparasitology SALÃO TURMALINA

14:30-18:00h ConcurrentSession5:AntigenPresentation; SALÃO ESMERALDA EffectorCells;ImmunoregulatoryCells

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SALÃO OURO 8:30-10:30h Plenary Session: Immunoparasitology: Immune Responses to Protozoa, Helminths and Ectoparasites Chairs:Jannaessens-KenyaandElisabethA.Innes-UK

Speakers 8:30-9:10h DavidArtis UniversityofPennsylvania,USA A critical role for intestinal epithelial cells in regulating CD4 T cell responses and immunity to parasitic infection in the gastrointestinal tract

9:10-9:50h AlexLoukas QueenslandInstituteofMedicalResearch Using the dog as a model for developing a human hookworm vaccine

9:50-10:30h DeclanMcKeever RoyalVeterinaryCollege CTL immunity as a driver for diversity in Theileria parva parasites

10:30-11:00h Coffee Break

11:00-13:00h Plenary Session: Mediators of Recruitment and Function of Cells of the Immune System; Signal Transduction and Gene Expression Chairs:JayneC.Hope-UKandCynthiaBaldwin-USA

Speakers 11:00-11:30h StephanieWiddison InstituteforAnimalHealth,UK Macrophages and Mycobacteria: what’s the attraction?

11:30-12:00h JimHarris UniversityofDublin,Ireland TH1-Th2 polarisation and the control of intracellular mycobacteria by Autophagy

12:00-12:30h CynthiaBaldwin UniversityofMassachusetts-Amherst,USA Control of gamma delta T cell IFN-γ responses: the role of the TCR and co-receptor WC1

12:30-13:00h JayneHope InstituteforAnimalHealth-Compton,UK Dendritic cells, cytokines and mycobacteria

13:00-14:00h Lunch

SALÃO AMETISTA 14:00-17:30h Concurrent Session #4: Immunology of the Mucosae and Skin and of the Mammary Gland; Mastitis Chairs:DouglasBannermann-USAandTheoA.niewold-TheNetherlands

Speakers 14:00-14:30h MichaelMurtaugh UniversityofMinnesota,USA Genomic Analysis of Mucosal Immunobiology in the Porcine Small Intestine

14:30-15:00h EricCox GhentUniversity,Belgium

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Mucosal responses against fimbrial antigens of enterotoxigenic E. coli in pigs

15:00h Theoniewold CatholicUniversityofLeuven,Belgium The in vivo early transcriptional intestinal response to rotavirus infection in germ-free piglets AbstractandPosterSM107

15:15h AnneGoubier Merial,France Colostrum from sows vaccinated with an inactivated PCV2 vaccine contains antigen specific leukocytes AbstractandPosterSM111

15:30-16:00h Coffee Break 16:00-16:30h IsabelK.FerreiradeMirandaSantos UniversityofSãoPaulo,Brazil Sequential morphology and gene expression profiles of cutaneous reactions to tick antigens in bovines AbstractandPosterSM096,SM097,SM098

16:30h ArthurSummerfield InstituteofVirologyandImmunoprophylaxisSwitzerland Modulation of peripheral dendritic cells towards mucosa-type dendritic cells by all-trans retinoic acid AbstractandPosterSM109

16:45h Masahiroyasuda UniversityofMiyazaki,Japan Dynamics of B-cell repertoire in sheep jejunal and ileal Peyer’s patch single follicles AbstractandPosterSM101

17:00-17:30h Finish

SALÃO OURO14:00-18:00h Concurrent Session #8: Mediators of Recruitment and Function of Cells of the Immune System; Fc Receptors and Immunoglobulins; Signal Transduction and Gene Expression in cells of the immune system Chairs:VictorRutten-TheNetherlandsandIldikovanRhijn-TheNetherlands

Speakers14:00-14:30h IldikoVanRhijn UtrechtUniversity,TheNetherlands Trafficking and diversity of bovine gamma delta T cells

14:30-15:00h MarkJutila MontanaStateUniversity,USA Gene expression in bovine gamma/delta T cell subsets with distinct migratory patterns

15:00-15:30h JeanPierreScheerlinck TheUniversityofMelbourne,Australia Regulation of cell trafficking in a single lymph node

15:30-16:00h Coffee Break 16:00-16:30h EfrainGuzman InstituteforAnimalHealth,Compton,UK Identification and characterization of cattle MHC class I chain related (MIC)

16:30-17:00h EricDenkers

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CornellUniversity,USA Host responses to Toxoplasma gondii infections

17:00-17:30h yvettevanKoocyk VrijeUniversiteitMedicalCenter,TheNetherlands Interaction between neutrophils and dendritic cells

17:30-18:00h DougBannermann USDA-ARS,USA The role of cytokines in dictating the outcome of mastitis

SALÃO TURMALINA 14:30-18:00h Concurrent Session #6: Immunoparasitology: Immune Responses to Protozoa, Helminths and Ectoparasites Chairs:DavidArtis-USAandMisaoonuma-Japan

Speakers14:30-14:55h AlbertMulenga TexasA&M,USA Understanding how the tick attachment phase is initiated; current status and future

14:55-15:20h JuanAnguita UniversityofMassachusetts,USA Host-vector-pathogen interactions mediated by the multifaceted tick saliva protein, Salp15

15:20-15:33h SatoruKonnai HokkaidoUniversity,Japan Suppression of proliferation and cytokine expression by HeLIS, a tick salivary gland-derived protein of Haemaphysalis longicornis. Abstract and Poster PR195

15:33-15:46h SandraMaruyama UniversityofSãoPaulo,Brazil Transcriptomes of Ticks Fed on Resistant and Susceptible Cattle: Genes Affected By Host Immune Responses. Abstract and Poster PR149

15:46-16:00h ElizabethJ.Glass RoslinInstitute,UK TLR Expression in Bovine Monocytes derived from cattle breeds with differing susceptibility to tropical Theileriosis AbstractandPosterPR158

16:00-16:30h Coffee Break 16:30-16:55h WillGoff WashingtonStateUniversity,USA The Bovine Spleen: Interactions Between Dendritic Cells and both NK Cells and Gamma-Delta T-Cells in the Immunologic Control of Hemoparasitic Infections

16:55-17:20h MarkWilson NationalInstitutesofHealth,USA Regulation of Th2 responses- Cooperative mechanisms controlling acute inflammation and chronic immunopathology

17:20-17:33h PrebenBoysen NorwegianSchoolofVeterinaryScience Bovine natural killer cells act as primary responders in the early stages of Neospora caninum-infected cattle. PosterandAbstractPR170

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17:33-17:46h ValériaM.F.Lima SãoPauloStateUniversity,Brazil Leishmania vaccine-induced immune response in dogs from an endemic area of canine visceral leishmaniasis AbstractandPosterPR189

17:46-18:00h HarryDawson USDA-ARS,Maryland,USA Feeding probiotic bacteria to swine enhances immunity to Ascaris suum AbstractandPosterPR172

SALÃO ESMERALDA14:30-18:00h Concurrent Session #5: Antigen Presentation and Dendritic Cells; Effector Cells, B and T cells, NK and NK T cells; Immunoregulatory cells Chairs:D.MarkEstes-USAandSergeMuyldermans-Belgium

Speakers 14:30-15:00h SergeMuyldermans FlandersInstituteforBiotechnologyBelgium Nanobody technology and camelid Immunoglobulins

15:00-15:30h WaithakaMwangi TexasA&MUniversity,USA Enhancing Vaccine Efficacy by Directed Priming of CD4+ and CD8+ T Lymphocytes

15:30-16:00h ImreKacskovics EötvösLorándUniversityHungary The role of the neonatal Fc receptor in IgG catabolism and homeostasis: IgG metabolism in bovine FcRn transgenic mice

16:00-16:30h Coffee Break16:30-17:00h ShirleyEllis BiotechnologyandBiologicalSciencesResearchCouncil,UK Complexity in the cattle CD94/NKG2 gene families

17:00h AmandaStalker RoyalVeterinaryCollege,UK Identification of circulating lineage-negative type-I IFN producing plasmacytoid dendritic cell-like cells in the bovine blood PosterandAbstractAP122

17:15h MašaPintarič UniversityofVeterinaryMedicineofVienna,Austria Synergistic effects of IL-2, IL-12 and IL-18 on cytolitic activity and IFNγ production of porcine natural killer cells PosterandAbstractAP132

17:30h GervásioBechara SãoPauloStateUniversity,Brazil Draining lymph node APCs in the resistance of goats to Amblyomma cajennense (Fabricius, 1787) nymphs PosterandAbstractAP125

17:45h AadHoek UtrechtUniversity,TheNetherlands Characterization of bovine regulatory cells. PosterandAbstractAP124

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DAVIDARTIS(UnIVERSITyoFPEnnSyLVAnIA,USA)A CritiCAl role for intestinAl ePitHeliAl Cells in regulAting Cd4 t Cell resPonses

And immunity to PArAsitiC infeCtion in tHe gAstrointestinAl trACt

Intestinalepithelialcells(IEC)thatlinethemucosalsurfacepro-videanessentialbarrierbetweenthehostandresidentorinvadingmicroorganisms.Invitrostudieshaveshownthatfollowingexposureto enteric pathogens including intestinal nematodes, IEC exhibitNF-κBactivationandexpressionofinnateimmuneresponsegenes.However,theroleofIECininfluencinginnateandadaptiveimmuneresponsestointestinalnematodesinvivoisunknown.Totestthis,micewithIEC-specificdeletionsinIKKβ(IEC-ikkβ-/-)weregeneratedusingCre-loxtechnology.IKKβisacriticalkinaserequiredforclas-sicalNF-κBactivation. Micewere infectedwithTrichurismuris,anaturalpathogenofmice that livespartiallyembeddedwithinhostIECofthelargeintestine.PersistentchronicinfectionsarepromotedbyThelper type1 (Th1) responses,whilehost resistance is criti-callydependentonCD4TcellsthatproducetheTh2cytokinesIL-4andIL-13.Therefore, infectionprovidesamodelsystemto inves-tigate the regulation of CD4T cell-dependent immune responsesin thegut. Whilewild typemicemountedprotectiveTh2cytokineresponsesanderadictatedTrichuris,IEC-ikkβ-/-animalsexhibitedapolarizedIFN-γandIL-17cytokineresponseandharboredchronic

infections.Susceptibilitytoinfectionandexpressionofproinflamma-torycytokinesalsoresultedinthedevelopmentofsevereintestinalinflammation in infected IEC-ikkβ-/- mice. Gene profiling of IECresponsesduringTrichuris infectionrevealedacriticalroleforepi-thelial-derivedTSLPinregulatingintestinaldendriticcellresponses.Takentogether,thesestudiesimplicatetheIEC-intrinsicNF-κBpath-wayingoverningdendriticcellandCD4Tcellresponsesfollowingexposuretointestinalnematodeparasites.Harnessingthefunctionsof IECswill be an important consideration in the design of noveltherapeuticsandmucosalvaccines.

ALExLoUKAS(qUEEnSLAnDInSTITUTEoFMEDICALRESEARCH)

using tHe dog As A model for deVeloPing A HumAn HooKWorm VACCine

DECLAnMCKEEVER(RoyAL[DICK]SCHooLoFVETERInARySTUDIES)

Ctl immunity As A driVer for diVersity in tHeileriA PArVA PArAsites

STEPHAnIEWIDDISon(InSTITUTEFoRAnIMALHEALTH,UK)

PlenAry session. IMMUNOPARASITOLOGy: IMMUNE RESPONSES TO PROTOZOA, HELMINTHS AND ECTOPARASITES CHAIRS: JAN NAESSENS - KENyA AND ELISABETH A. INNES - UK

CHemoKines, mACroPHAges And myCobACteriA

JIMHARRIS(UnIVERSITyoFDUBLIn,IRELAnD)tH1-tH2 PolArisAtion And tHe Control of

intrACellulAr myCobACteriA by AutoPHAgyJAMESHARRIS1,SERGIoSDEHARo2,SHARonSMASTER2,

MonICADELGADo,ESTEBAnRoBERTS,JoSEPHKEAnE1,VoJoDERETIC2

1DepartmentofClinicalMedicine,TrinityCentreforHealthSciences,St.James’sHospital,Dublin8;2DepartmentofMolecularGeneticsandMicrobiology,UniversityofNewMexicoSchoolof

Medicine,Albuquerque,NM87131,USA.Autophagy is a major intracellular pathway for the lysosomal

degradationoflong-livedcytoplasmicmacromoleculesanddamagedor surplus organelles. Under conditions of amino-acid or specificgrowthfactordeprivation,autophagydegradesstablelong-livedpro-teinsandenablescellsurvivalbysupplyinganabolicneeds.Recently,autophagyhasalsobeenlinkedwithinnateandadaptiveimmunityagainstintracellularpathogens,includingMycobacteriumtuberculo-sis,whichisabletosurvivewithinmacrophagesbyblockingfusionof the phagosome with lysosomes. Induction of autophagy withaminoacidstarvationor IFN-γenablesmacrophages toovercomethisphagosomematurationblockandinhibittheintracellularsurvival

ofmycobacteria.Conversely,theTh2cytokinesIL-4andIL-13inhibitautophagyinmurineandhumanmacrophages.Inhibitionofstarva-tion-inducedautophagybyIL-4andIL-13isdependentonsignalingvia the Akt-pathway, while inhibition of IFN-γ-induced autophagyisAkt-independent. Inductionofautophagy leads to increasedkill-ingof intracellularmycobacteriabymacrophagesandthiseffect isabrogatedby theadditionof IL-4or IL-13. Thus, the inductionofautophagyinmacrophagescaninfluencethefateofphagocytosedmycobacteria,buttheseeffectsareinhibitedbyIL-4andIL-13.ThisrepresentsanovelpathwaybywhichTh-1/Th2polarizationcaninflu-encetooutcomeofmycobacterialinfection.

CynTHIABALDWIn(UnIVERSITyoFMASSACHUSETTS-AMHERST,USA)

Control of gamma delta t Cell Ifn-γ resPonses: tHe role of tHe tCr And Co-

reCePtor WC1JAynEHoPE(InSTITUTEFoRAnIMALHEALTH-CoMPTon,

UK)

dendritiC Cells, CytoKines And myCobACteriA

PlenAry session. MEDIATORS OF RECRUITMENT AND FUNCTION OF CELLS OF THE IMMUNE SySTEM; SIGNAL TRANSDUCTION AND GENE ExPRESSION.

ConCurrent session #4. IMMUNOLOGy OF THE MUCOSAE AND SKIN AND OF THE MAMMARy GLAND; MASTITIS

ERICCox(GHEnTUnIVERSITy,BELGIUM)“muCosAl resPonses AgAinst fimbriAl Antigens of enterotoxigeniC e. COLI”

MICHAELMURTAUGH(UnIVERSITyoFMInnESoTA,USA)“genomiCs of immune resPonses in tHe

intestinAl muCosA”

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ILDIKoVAnRHIJn(UTRECHTUnIVERSITy,THEnETHERLAnDS)

trAffiCKing And diVersity of boVine gAmmA deltA t Cells

ILDIKoVAnRHIJn,VICToRRUTTEn,BRyAnCHARLESTon,WILLEMVAnEDEn,ADKoETS

Tissue-specificdistributionofγδcellswithlimitedTCRdiversityisacommonphenomenoninspecieswithalowpercentageofγδTcellslikehumansandmice.Usingpseudoafferentlymphductcan-nulationincattle(Bostaurus),weshowedthatlargenumbersofγδTcells,butnotαβTcells,areconstitutivelypresentinpseudoafferentlymphdrainingbovineskin.Thecellsdidnothaveanactivatedphe-notypeasexemplifiedbytheabsenceofsurfaceexpressionofMHCclassIIandcostimulatorymolecules.ThelevelofγδTcellegresswasenoughtodepleteallγδTcellsfromtheskinwithin46hours.AsthismassiveγδTcellmigrationwasobservedduring14days,constantreplenishmentofthesecellsmusthavetakenplace.Thereasonandmechanismofthisextensivetraffickingisunknown,butthedatasuggestthatγδTcellsintissuesfulfillmorethanexclusivelylocalfunctions.

Subsequent analysis of δ chain sequences of these skin-derived,traffickingγδTcells,aswellasδchainsequencesofγδTcellsisolatedfromlymphnode,spleen,smallintestine,largeintes-tine,andblood,showednopreferentialusageofcertainVδsegmentsinanyofthesetissues,whichisquitedifferentfromthesituationinmice and humans. Awide variety of δ chainCDR3 lengthswasobservedineachofthebovinetissuestestedandwasconfirmedbyspectratyping.ThehighlyvariableCDR3lengthappearedtobeduetotheuseofuptofourdiversity(D)segmentsperbovineδchain.AhighnumberofVδsegments,incombinationwiththeuseofuptofouroutoffiveDsegments,andthepossibilityofusingnon-templateencoded(N)nucleotidesoneithersideofthese,makesthepotentialbovineδchainrepertoiremuchbiggerthananyknownTCRchain.

The reason and mechanism of γδ T cell trafficking and theimplicationsofthehighδchaindiversityremainssubjecttofurtherstudies.Key words:γδTcells;δchainrepertoire;skin;lymphnode;spleen;small intestine; large intestine; blood; pseudoafferent lymph ductcannulationSpecies:ruminants

MARKJUTILA(MonTAnASTATEUnIVERSITy,USA)gene exPression in boVine gAmmA/deltA t Cell subsets WitH distinCt migrAtory

PAtternsAbstract.Analysisofglobalgeneexpressioninimmunecells

hasprovideduniqueinsightsintoimmunefunctionandresponsetoinfection.Recently,microarrayandserialanalysisofgeneexpres-sion(SAGE)techniqueshavebeenappliedtothestudyofγdTcellfunctioninhumans,rodentsandcattle.Weappliedtheseapproachestothestudyofbovinetissue-restrictedCD8+andCD8-(WC1+)γdTcellsubsetsisolatedfromtheblood,andtotalγdTcellsandaβTcells isolated from mucosal lymphatics prior to and following oralSalmonellaserovarTyphimuriuminfectionincalves.ThesestudiesprovidednewinsightsintothefunctionofbovineγdTcellsingeneralanddifferencesinmucosal(CD8+)andperipheral(CD8-)γdTcellsubsets.Commontobothsubsetswasexpressionofmanymyeloidcellassociatedgenes,suchas receptors forpathogenassociatedmolecularpatterns(PAMPs).WefoundthatPAMPsprimeγdTcellsto more robustly respond to downstream cytokine and/or antigensignals.Thenatureoftheprimingresponseandexamplesofpotentprimingagentswillbediscussed.

JEAn PIERRE SCHEERLInCK (THE UnIVERSITy oF MEL-BoURnE,AUSTRALIA)

regulAtion of Cell trAffiCKing in A single lymPH node

EFRAInGUZMAn(InSTITUTEFoRAnIMALHEALTH,CoMPTon,UK)

identifiCAtion And CHArACterizAtion of CAttle mHC ClAss i CHAin relAted (miC)

EFRAInGUZMAn,CRISTInADEJUAnSAnJUAn,SHIRLEyELLIS

InstituteforAnimalHealth.DivisionofImmunology.Compton,Newbury.RG207NN.UnitedKingdom.

efrain.guzman@bbsrc.ac.ukIn humans, major histocompatibility complex (MHC) class I

chain-related(MIC)moleculesshowhomologywithclassicalhumanleukocyteantigen (HLA)molecules, but theydonot combinewithβ2 microglobulin, do not bind peptide and are not expressed oncirculatinglymphocytes.MappingstudiesinhumanshaveidentifiedsevenMICloci(MICA-MICG),ofwhichonlyMICAandMICBencodeexpressedtranscripts.ThepredicteddomainstructureofMICprod-uctsissimilartothatofclassicalMHCclassImolecules,includingthreeexternaldomains(α1-3)atransmembraneandacytoplasmicdomain.MICproteinsareexpressed inresponsetostress,onthecell surfaceof freshly isolatedgastric epithelium,endothelial cellsandfibroblastsandengagewiththeactivatingnaturalkillerreceptorNKG2D,whichisfoundonmanycellswithintheimmunesystem.WehaveidentifiedtwotranscribedcattleMICgeneswhichdifferbya21nucleotideindelintheα2domain,andthereisevidenceforatleastfour more bovineMIC genes or pseudogenes. Transcribed cattleMICgeneswerefoundtohave65%identityatthenucleotideleveltotheirhumanhomologues,and45%identityattheaminoacidlevel.TranscriptionofcattleMICwasfoundinstomach,lungandintestinetissues,butnotintheliverorheart.WehaveconstructedMICtetra-mersandusedthemtostaincirculatinglymphocytes.StainingofγδTcellsandαβCD8+Tcellswasdetected,butαβCD4+Tcellsdidnotstain,asseeninhumans.WealsoshowactivationofγδTcellsbyMICusinganinterferon-γELIspotassay.TheseresultsindicatethatthereareatleasttwofunctionalMICgenesincattlewhichdifferbya21nucleotideindel,andshowthepresenceofseveraladditionalMICgenesorpseudogenes.Key words:MHC class I chain-relatedmolecules; γδT cells; αβCD8+Tcells;αβCD4+Tcells;interferon-γSpecies:ruminants

ERICDEnKERS(CoRnELLUnIVERSITy,USA)Host resPonses to toxoPlAsmA gondii

infeCtions

yVETTEVAnKooCyK(VRIJEUnIVERSITEITMEDICALCEnTER,THEnETHERLAnDS

interACtion betWeen neutroPHils And dendritiC Cells

MolecularCellBiology,Vumc,v.d.Boechorststraat7,1081BTAmsterdam,TheNetherlands.

y.vankooyk@vumc.nlDendriticcells(DC)arespecializedintherecognitionofpatho-

gensandplayapivotalroleinthecontrolofimmunity.YetDCarealsoimportantforhomeostaticcontrolrecognizingselfantigensandtolerizingitsenvironment,indicatingthatthenatureoftheantigenitrecognizesmaysteeraDCtowardsimmunityortolerance.C-typelectin receptors expressed by DC are involved in the recognitionandcaptureglycosylatedselfantigensorpathogens.TodatesevendifferentC-typelectinshavebeenidentifiedonDC.Itisnowbecom-ingclear that theseC-type lectin receptorsmaynotonlyserveasantigenreceptorrecognizingpathogenstoallowinternalisationandantigenpresentation,butmayalsofunctionintherecognitionofself

ConCurrent session #8. MEDIATORS OF RECRUITMENT AND FUNCTION OF CELLS OF THE IMMUNE SySTEM; FC RECEPTORS AND IMMUNOGLOBULINS; SIGNAL TRANSDUCTION AND GENE ExPRESSION IN CELLS OF THE

IMMUNE SySTEM.

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antigen,orasadhesionmoleculesandsignalingmolecules.WehavestudiedingreatdetailthefunctionandtheglycanspecificityoftheDC-specificC-typelectinDC-SIGN.DC-SIGNrecognizeshighman-nosestructureandnon-sialylatedLewisantigens(Lex,Ley,LebandLea)whichareexpressedonmanypathogens,suchastheenvelopeprotein gp120 of HIV-1, andmany other viral envelope glycopro-teins,butalsoonthecellwallcomponentManLamofMycobacteria.TargetingofthesepathogenstoDC-SIGNhoweverleadstoimmuneescape.ThesefindingshinttoafunctionofDC-SIGNinrecognizingglycosylatedselfantigentotolerizeitenvironment.

TodatelittleisknowonthespecificitybywhichC-typelectinsinteractwith self-glycoproteins. Lewis antigens are recognized onglycoproteinspresent onPMNsandmediatea cellular interaction

betweenPMNandDCallowingproperantigendelivery.BothMac-1andCEACM1havebeenidentifiedasglycoproteinsonneutrophilsthatinteractwithDC-SIGNandregulateDCneutrophilinteractions.

UnderstandingthediversityofC-typelectinsbeingexpressedonDCaswellastheircarbohydratespecificrecognitionprofilewillbe instrumental to understand DC pathogen recognition in manypathogenicdisorders, aswell as the regulationof cellular interac-tionsofDCthatareessentialinthecontrolofimmunity.

DoUGBAnnERMAnn(USDA-ARS,USA)tHe role of CytoKines in diCtAting tHe

outCome of mAstitis

ALBERTMULEnGA(TExASA&M,USA)understAnding HoW tHe tiCK AttACHment

PHAse is initiAted; Current stAtus And future

AMULEnGA,ABMARIA,RKHUMTHonGTexasA&MUniversity,DepartmentofEntomology,Collegeofagriculture&LifeSciences,2475TAMU,CollegeStation,Texas

77843Thetickfeedingstyleoflaceratinghosttissuetocreateitsfeed-

ingsiteand thensuckingupblood from thehematoma that formsinthewoundedareashouldundernormalcircumstancesstimulatetissuerepairresponse,whichwillultimatelystopbleeding.However,ticksensureafullbloodmealbysecretingacocktailofpotentphar-macologically active enzymes that collectively disarm the host’stissuerepairmechanism.Sincethemammalianhost’stissuerepairresponse to prevent further bleeding is expected to be swift, it islogicaltoimaginethatthetickwillbereadytoevadehostdefenseresponsesuponpenetrationofhostskinforittosuccessfullyfeed.Tounderstandhowthetickisabletoinitiateitsfeedingprocess,weareusingtheLoneStartick,Amblyommaamericanumandbovinemodel.Towardsdiscoveryofmolecularsignalingcascadesthattriggerand/or facilitate the tickattachmentand formationof its feeding lesion,suppressive subtractive hybridization, high throughput sequencingandvalidationofdifferentialexpressionbycDNAdotblothybridiza-tionwereperformedonA.americanumticksthathadattainedappe-tenceandwereexposedtofeedingstimuli.Thisapproachallowedforidentificationof40genesthatareupregulatedbeforeticksbegintopenetratethehostskin.Amongthe40genes,wehaveidentifiedantimicrobial peptides, insulin-like growth-factor binding proteins,lipocalin/histaminebindingproteinandanextracellularmatrixmet-taloproteaseinducer.Putativebiologicalfunctionsoftheseproteinssuggestthattheyareinvolvedinmediationofmolecularmechanismthatunderpinformationandmaintenanceofthetick-feedinglesion.QuantitativerealtimeRT-PCRanalysishasshownthesegenesareupregulatedintheticksalivaryglandswithinthefirst24hrsofthetick initiating its feeding lesion. Research to determine molecularinteractionsofthesegenesatthetick-hostinterfaceisongoing.

JUAnAnGUITA(UnIVERSITyoFMASSACHUSETS,USA)Host-VeCtor-PAtHogen interACtions

mediAted by tHe multifACeted tiCK sAliVA Protein, sAlP15

WILLGoFF(WASHInGTonSTATEUnIVERSITy,USA)

tHe boVine sPleen: interACtions betWeen dendritiC Cells And botH nK Cells And

gAmmA-deltA t-Cells in tHe immunologiC Control of HemoPArAsitiC infeCtions

MARKWILSon(nATIonALInSTITUTESoFHEALTH,USA)immunoPAtHology during sCHistosomiAsis:treg-deriVed il-10 Controls inflAmmAtion

whIle Il-13rα2 CrItICally Controls immunoPAtHology And fibrosis.

MARKSWILSon,ELDADELnEKAVE,MARGARETMMEnTInK-KAnE,JoHnTPESCE,THIRUMALAIRRAMALInGAM,RoBERT

WTHoMPSon,ALLEnCHEEVER,THoMASWynnLaboratoryofParasiticDiseases,NationalInstituteofAllergyand

InfectiousDiseases.NationalInstitutesofHealth.Thedevelopmentof Immunopathology; suchasairwayhyper

responsiveness (AHR) in asthmatics and fibrosis during schisto-somiasis often result from superfluous inflammation, yet the tran-sition from inflammation to resultant immunopathology is poorlyunderstood. This study aimed to dissect critical mediators duringTh2-driveninflammation,followinginfectionorairwayallergenprovo-cation,whichmediatedown-streampathologyandtosubsequentlyidentify the regulatory mechanisms that control them.To separateinflammationfromimmunopathology,weusedtwoin-vivomodels-amodelofasthmaandSchistosomiasis,bothofwhichpresentdefini-tive cellular inflammation and downstream pathological changes.We observed an accumulation of IL-10+ regulatory T cells (Treg),usingIL-10gfpreportermiceandFoxp3expressioninthelungsfol-lowing allergen challenge or in the liver following infection. In theabsenceofIL-10overwhelminginflammationwithexacerbatedTh2responses,aswellastheemergenceofTh1responses,develops.Paradoxically,despiteincreasedinflammationinIL-10-/-micepathol-ogywasreduced.Inboththelungandtheliver,IL-13Ralpha2,anendogenousneutralizer of IL-13,waselevated.To test the roleofIL-13R alpha 2 in regulatingTh2 associated pathologywe gener-atedmicedeficientinbothIL-13Ralpha2andIL-10(IL-13Ralpha2-/-IL-10-/-).

WediscoveredthattheabsenceofIL-13Ralpha2,onanIL-10-/-background,increasedlungandliverpathology,comparedtoWTorIL-10-/-mice,indicatingthatIL-13Rα2isakeyregulatorofpathology.Thisstudypresentsanovel two-prongedmodelcontrolling inflam-mationandimmunopathologybyIL-10-secretingTregcellsandIL-13Rα2,respectively,whichcouldbeexploitedtocontrolavarietyofimportantTh2-dominantdiseases.

ConCurrent session #6. IMMUNOPARASITOLOGy: IMMUNE RESPONSES TO PROTOZOA, HELMINTHS AND ECTOPARASITES CHAIRS: DAVID ARTIS - USA; MISAO ONUMA - JAPAN

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SERGEMUyLDERMAnS(FLAnDERSInSTITUTEFoRBIoTECHnoLoGyBELGIUM)

nAnobody teCHnology And CAmelid immunoglobulins

WAITHAKAMWAnGI(TExASA&MUnIVERSITy,USA)enHAnCing VACCine effiCACy by direCted Priming of Cd4+ And Cd8+ t lymPHoCytes

IMREKACSKoVICS(EöTVöSLoRánDUnIVERSITyHUnGARy)

tHe role of tHe neonAtAl fC reCePtor in igg CAtAbolism And HomeostAsis: igg

metAbolism in boVine fCrn trAnsgeniC miCeBALáZSBEnDER1,LILLABoDRoGI1,JUDITCERVEnAK5,ZITASCHnEIDER2,BALáZSMAyER2,yAoFEnGZHAo3,LEnnARTHaMMarströM3,andréEggEn4,ZsuZsannaBősZE1,

IMREKACSKoVICS51AgriculturalBiotechnologyCenter,Gödöllő,Hungary;2Faculty

ofVeterinaryScience,SzentIstvánUniversity,Budapest,Hungary;3DivisionofClinicalImmunology,Departmentof

LaboratoryMedicine,KarolinskaInstitute,Stockholm,Sweden;4INRA,UR339,LaboratoiredeGénétiquebiochimiqueetdeCytogénétique,Jouy-en-Josas,France;5DepartmentofImmunology,FacultyofScience,EötvösLorándUniversity,

Budapest,HungaryIgG has the longest survival time in the circulation of the Ig

classes and the lowest fractional catabolic rate.The MHC class IrelatedFcreceptorforIgG(FcRn),whichiscomposedoftheFcRnheavychainandthebeta2-microglobulin (b2m),protects IgGfromintracellularcatabolicdegradation,playsimportantrolesinIgGtran-

scytosis inseveralmucosal layersand is involved in thematernalimmunetransport.Mostrecently,ithasbeenshownthatFcRnbindsalbuminandprotects it fromdegradation justas itdoes IgG.BothIgGandalbuminbindFcRnatlowpHatdistinctsites.WeclonedandcharacterizedthebovineFcRn(bFcRn)alphachainanddetecteditsexpressioninvariousepithelialcellsthatisinvolvedinIgGsecretion.Wehavealsoshownthat this receptor isexpressed in thebovinecapillaryendothelialcellsandinvolvedinIgGhomeostasisincattle.

InordertostudytheregulationofthebovineFcRnheavychaingeneandanalyzeitsroleinIgGandalbuminmetabolism,wegener-ated and characterized transgenicmice carrying a 102 kb bovinegenomic fragment,encoding thebFcRn.Abacterial artificial chro-mosomecontainingthebFcRnalpha-chaingene(bFCGRT)withits44 kb5’ and50 kb long3’ flanking sequenceswasmicroinjectedinto fertilized mouse oocytes. Two of the transgenic lines gener-ated,showedcopynumberrelatedandintegrationsiteindependentbFcRn expression based on Northern and Western blot studies.Pharmacokinetic studiesshowed that thehalf-livesof the injectedmouseandhumanIgGweresignificantlylongerintransgenicmicecompared to wild-type animals. These data indicate that bovineFcRn heavy chain is indeed expressed in themouse endothelialcells,formedafunctionalreceptorandprotectedIgGfromdegrada-tion.OurresultsunderlinethefeasibilityofcreatingBACtransgenicmousemodelsofeconomicallyimportantbovinegenes.

SUPPoRTEDByTHEGRAnTSoTKAT049015,oMFB1605/1606/2002,GAK-CALVES05

SHIRLEyELLIS(BIoTECHnoLoGyAnDBIoLoGICALSCIEnCESRESEARCHCoUnCIL,UK)

ComPlexity in tHe CAttle Cd94/nKg2 gene fAmilies

ConCurrent session #5. ANTIGEN PRESENTATION AND DENDRITIC CELLS; EFFECTOR CELLS, B AND T CELLS, NK AND NK T CELLS; IMMUNOREGULATORy CELLS. CHAIRS: D. MARK ESTES-USA AND SERGE

MUyLDERMANS- BELGIUM

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8:30-11:00h PosterViewingThemes6-10 SALÃO DIAMANTE

11:00-13:30h Mini-symposium:CanineVisceralLeishmaniasis SALÃO OURO

14:00-16:00h ConcurrentSession1:Immunogenetics SALÃO OURO

14:00-16:00h ConcurrentSession3:Stress;Reproduction; SALÃO TURMALINA Immunoendocrinology;MicrobialFlora,Nutrients

16:30-17:00h StudentAwards SALÃO OURO

17:00-19:30h PlenarySession:InnateImmunity,Inflammation, SALÃO OURO Adjuvants;Memory,AcquiredImmunity,Vaccines

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SALÃO DIAMANTE 8:30-11:00h Poster Viewing CoffeeBreakat10:00h,duringpostersession 6. Immunoparasitology: Immune Responses to Protozoa, Helminths and Ectoparasites; Canine Visceral Leishmaniasis: Posters PR141-PR196

7. Comparative Immunology; Immunoecology: Posters CI197-CI206

8. Mediators of Recruitment and Function of Cells of the Immune System; Fc Receptors and Immunoglobulins; Signal Transduction and Gene Expression in cells of the immune system: Posters MI207-MI210

9. Innate immunity, Inflammation and Adjuvants; Memory, Acquired Immunity and Vaccines: Posters VA211-VA250

10. Clinical Immunology and Immunopathology: Posters IP251-IP281

SALÃO OURO11:00-13:30h Mini-symposium Canine Visceral Leishmaniasis: Immunology and Vaccines Chairs:olindoMartinsFilho-BrazilandJavierMoreno-Spain

Speakers AlexandreBarbosaReis FederalUniversityofOuroPreto,Brazil Systemic and compartmentalized Immune Responses in Canine Visceral Leishmaniasis

JavierMoreno CenterforBiologicalInvestigations,Spain Cytokine profiles in Canine Visceral Leishmaniasis

WashingtonLuisdosSantos OswaldoCruzFoundation,Brazil Cell Migration in Tissues of Dogs Infected with Leishmania chagasi

olindoAssisMartinsFilho OswaldoCruzFoundation,Brazil Advances in flow cytometric serology to distinguish Leishmania(Leishmania)chagasi infected from Leishmune ®-vaccinated dogs

Dr.GérardMariePapierok BioVetoTest-VIRBAC,France Vaccinal interests of purified excreted-secreted antigens of Leishmania infantum against canine visceral leishmaniasis: explanation of success of experimental and efficacy field trials

13:00-14:00h Lunch

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SALÃO OURO

14:00-16:00h Concurrent Session #1: Immunogenetics and Genomics Chairs:Williamollier-UKandAlexandreCaetano-Brazil

Speakers 14:00-14:30h JoanK.Lunney AgriculturalResearchSystem,USDA,USA Comparative immune responses of pigs to infection with Salmonella enterica serovars of food safety (Typhimurium) and animal health (Choleraesuis) importance.

14:30h JohnWilliams ParcoTecnologicoPadano,Italy Identification of polymorphisms in bovine genes with immune function. AbstractandPosterIG007

14:40h DirkWerling RoyalVeterinaryCollege,UK Novel approaches to enhance disease resistance in ruminants? -Breeding for geo graphically important TLR SNPs. TheRuminantTLRConsortium. AbstractandPosterIG003

14:50h ElizabethGlass RoslinInstitute,UK Monocytes from disease resistant and susceptible cattle display distinct transcriptome profiles during Theileria annulata infection. AbstractandPosterIG011

15:00-15:30h Williamollier UniversityofManchester,UK Immunogenetic risk factors contributing to Canine Diabetes.

15:30h HirohideUenishi Natl.Institute.ofAgrobiologicalSciences,Japan Difference of genomic structure among haplotypes of swine leukocyte antigen region. AbstractandPosterIG019

15:40h CarlosPrudêncio FederalUniversityofUberlândia,Brazil Identification of Boophilus microplus phagotopes from phage displayed peptide libraries. AbstractandPosterIG022

15:50h TomokoEguchi-Togawa Natl.Institute.ofAgrobiologicalSciences,Japan Genomic analysis revealed the duplication model of porcine CD1 genes during evolution. AbstractandPosterIG020

16:00-16:30h Coffee Break

SALÃO AMETISTA14:00-16:00h Concurrent Session #3: Immunoendocrinology; and Stress; Immunology of Reproduction and Neonates; Microbial Flora, Nutrients and the Immune Response Chairs:nicolaLacetera-ItalyandIsabelleP.oswald-France

Speakers 14:00h JoséRobertoKfoury

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UniversityofSãoPaulo,Brazil Influence of hormones in the expression of indoleamine-2,3 dioxigenase in cultured cells from bovine placenta AbstractandposterER081

14:15-14:45h GaryEntrican MoredunResearchInstitute,UK The effect of pregnancy on maternal immunity in sheep

14:45-15:15h Isabelleoswald INRA,France Effect of some food contaminants, the mycotoxins, on the immune response of the pig 15:15h KatsuroHagiwara RakunoGakuenUniversity,Japan Colostral CD8 positive cell is a potent producing cell for IFN-gamma AbstractandPosterER079

15:30h JesúsHernández CIAD,Mexico Vitamin E modulates the expression of Th2 cytokines in porcine PBMC AbstractandPosterER086

15:45h AmandaA.Adams GluckEquineResearchCenter,USA Contribution of body condition score and percent body fat to the inflammatory response in aged horses AbstractandPosterER090

16:00-16:30h Coffee Break

SALÃO OURO 16:30-17:00h Student Awards Chairs:BeatrizR.Ferreira–BrazilandJoanK.Lunney-USA

17:00-19:30h Plenary Session Innate immunity, Inflammation and Adjuvants; Memory, Acquired Immunity and Vaccines Chairs:PhillipGriebel-CanadaandDirkWerling-UK

Speakers 17:00-17:30h GordonMacPherson OxfordUniversity,UK The Role of Dendritic Cells at mucosal surfaces and regulation of inflammation

17:30-18:00h VolkerGerdts VaccineandInfectiousDiseaseOrganization,Canada Strategies to link innate and adaptive immunity when designing vaccine adjuvants

18:00-18:30h PatShewen UniversityofGuelph,Canada Challenges in Development of Mucosal Vaccines

18:30-19:00h SarahDoyle TrinityCollege,Dublin TLR Signaling in Infection and Inflammation

19:00-19:30h SteveReiner UniversityofPennsylvania,USA Specifying the T cell fates required for immunity: Asymmetric Division of a T Lymphocyte in the Initiation of Adaptive Immunity

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9th IVIS: JAPAN, 2010

ThE 9Th INTERNATIONAL VETERINARy IMMUNOLOgy SyMpOSIUM

DearFellowVeterinaryImmunologists;

Averywarmwelcometothe9thInternationalVeterinaryImmunologySymposium(orthe9thIVIS).

JapaneseAssociationofVeterinaryImmunologistswouldliketocordiallyinviteyoutothe9thIVISscheduledonAugust16-20,2010atTowerHallFunabori(Edogawa-ku)inTokyo.

Wewishtocallforpapersonvariousscientificthemesofinterestrelatedwithallaspectsofveterinaryimmunology.Wesincerelyhopethatthe9thIVISwillbecomeanexciting,usefulandmemorablemeetingforallparticipantstodiscussrecentdevelopmentofveterinaryimmunologyandfutureprospect.

Thevenueforthe9thIVISislocatedinthecenterofTokyowitheasyaccessfromtheTokyoCentralStation,NaritaInternationalAirportandHanedaDomesticAirportbyefficienttransportationsystem.Theregionoffersvarioustraditionalcultureevents,excellentcuisine,andleisurefacilities,aswell.Wearesurethatyouwillbesatisfiedfrombothscientificandsocialaspects.

SimilarlyasthepreviousIVISmeetings,the9thIVISisorganizedtwodaysaheadoftheInternationalCongressofImmunology,whichwillbeheldinKobefromAugust22-27,fortheconvenienceofparticipantscomingforbothmeetings.

WelookforwardtoseeingyouinTokyo.

TakashiOnodera,Chairpersonofthe9thIVIS

aonoder@mail.ecc.u-tokyo.ac.jphttp://WWW.frc.a.u-tokyo.ac.jp/

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ALExAnDREBARBoSAREIS(FEDERALUnIVERSITyoFoURoPRETo,BRAZIL)

systemiC And ComPArtimentAlized immune resPonses in CAnine VisCerAl leisHmAniAsis

JAVIERMoREno(CEnTERFoRBIoLoGICALInVESTIGATIonS,SPAIn)

CytoKine Profiles in CAnine VisCerAl leisHmAniAsis

WASHInGTonLUISDoSSAnToS(oSWALDoCRUZFoUnDATIon,BRAZIL)

Cell migrAtion in tissues of dogs infeCted WitH leisHmAniA CHAgAsi

oLInDoASSISMARTInSFILHo(oSWALDoCRUZFoUnDATIon,BRAZIL)

AdVAnCes in floW CytometriC serology to distinguisH leisHmAniA (leisHmAniA)

CHAgAsi infeCted from leisHmune ®-VACCinAted dogs

GéRARDMARIEPAPIERoK(InSTITUTEPASTEUR,FRAnCE)VACCinAl interests of Purified exCreted-seCreted Antigens of LEISHMANIA INFANTUM AgAinst CAnine VisCerAl leisHmAniAsis:

exPlAnAtion of suCCess of exPerimentAl And effiCACy field triAls

GéRARD-MARIEPAPIERoK,CHRISToPHEHUGnET;GILLESBoURDoISSEAU,JEAn-LoUPLESMERE

BioVetoTestLaboratory83500LaSeynesurMer;Cliniquevétérinaire,26160,LaBegudedeMazenc,France;ServicedeParasitologie,ENVdeLyon,69280Lyon,France;Institutde

RecherchepourleDéveloppement,UR008,911AvenueAgropolis,BP64501,34394Montpelliercedex5,France

gpapiero@bvt.frWehaverecentlydemonstratedthatthecombinationofnaturally

excreted-secretedantigensfromculturesupernatantofLeishmania infantum promastigotes (LiESAp)asvaccineantigen in formula-tionwithmuramyldipeptide(MDP)hasthecapacitytoprotectdogs

not only against experimental L’infantum infection (Study on 18DOGS,(1))butalsoagainstvisceralleishmanisisinthefield(studyon414dogs)(2).

The good results obtained with this vaccinal candidate areduetothedoubleimmuneresponsespecificallydirectedagainstLiESAp:ananti-LiESAIgG2productionandacellmediated

immuneresponse(Th1response).Bothresponseshaveaspe-cificroleagainsttheparasite.

About the humoral response, the specific Ig G2 antibodiesinducealossofpromastigote’sandamastigote’sviabilityandprolif-erationinvitro(aninhibitionofthese2parasiticalformsproliferation“invitro”).

Leishmania viability was reduced to 50% (after contact withserumofimmunizeddogs)andnoproliferationwasevidencedafter30minofcontactofLeishmaniawithpureimmunizeddog(s)serum.ImmuneandcontroldogsseraweretestedbyastandardELISApro-cedureforIgG2antibodieslevelstoLiESAp.Fortheexperimentalstudyandthefieldtrial,allvaccinateddogsshowedincreasedanti-LiESApIgG2reactivityafterimmunization.

Thecellular immune responsewascharacterizedby theacti-vationofmacrophages in response tohigher IFN-gproductionbyTh1 lymphocytes.This responsewasstudiedbyseveralmethodsandespecially by the significant enhancedantileishmanial activityof canine macrophages co-cultivated with autologus lymphocytes(inresponsetohigherIFN-gproductionbyTcells).Anti-leishmanialactivity increased significantly after vaccineadministration (51,1 ±20,6%,P<0,01)andwashigherafterthebooster(60,7±10,4%,P<0,01) in field trial.Furthermorewedemonstrated that the lympho-cytesofdogsimmunisedwithLiESApco-incubatedwithLeishmaniainfected macrophages produce IFN-g resulting in NO-mediatedamastigoteapoptosis(3).

Allresultsduringtheexperimentaltrial(1)agreewiththeresultsobtainedinfeldtrial(2).

TodayweunderstandtherolesofLiESApforagoodprotectionagainstvisceralleishmaniasisinfectionindogsandhaveaspecificmethod of distinguishing between naturally infected animals fromvaccinated dogs (detection of specific Ig G2 antibodies).Anotherway of research appears : the study of Li ESAp and especially,amongthem,the identificationof themainspecificantigenabletoinducetheLIESApimmunopropectiveresponse.

(1) “Protection against experimental visceral leishmaniasisinfectionindogsimmunizedwithpurifiedexcretedsecretedantigensofLeishmaniainfantumpromastigotes.

JL.LEMESREetal,Vaccine23(2005)2825-2840

mini-symPosium CAnine VisCerAl leisHmAniAsis. IMMUNOLOGy AND VACCINES

JoAnK.LUnnEy(AGRICULTURALRESEARCHSySTEM,USDA,USA)

ComPArAtiVe immune resPonses of Pigs to infeCtion WitH sAlmonellA enteriCA

seroVArs of food sAfety (tyPHimurium) And AnimAl HeAltH (CHolerAesuis) imPortAnCe.

JKLUnnEy1,SMDBEARSon2,yFWAnG3,JJUTHE2,3,LqU3,oPCoUTURE3,SHZHAo4,DKUHAR1,DnETTLETon5,

JCDEKKERS3,CKTUGGLE31AnimalParasiticDiseasesLaboratory,BARC,USDA-ARS,Beltsville,MD20705,USA;2NationalAnimalDiseaseCenter,USDA-ARS,Ames,IAUSA;3DepartmentofAnimalScience,

2255KildeeHall,IowaStateUniversity,Ames,IAUSA;4KeyLabofAgAnimalGenetics,Breeding,andReproduction,HuazhongAgriculturalUniv.,Wuhan,PRChina;5DepartmentofStatistics,

124SnedecorHall,IowaStateUniversity,Ames,IA

Salmonellainfectionscausefoodsafetyconcernsforhumansaswellasproductionproblemsforswine.Ourteamhasusedsuppres-sionhybridization(SSH),longoligonucleotideQiagenandAffymetrixporcineGeneChip®arrays,andrealtimegeneexpression(Q-PCR)to understand the host response to, and control of, S. entericaserotype Typhimurium (ST) as compared to S. enterica serotypeCholeraesuis(SC).Weidentifieddifferentiallyexpressed(DE)genesinmesentericlymphnodes(MLN)andlungsofpigswithacute[8,24,48hourspost-inoculation(hpi)],andchronicstages[7,21days(dpi)]ofinfection.TheSSHanalysesidentifiedseveralgenesinvolvedinheatshockresponseandcytoskeletalrearrangements.Hierarchicalgeneclusterandpathwayanalysesofthemicroarraydatarevealedthathostproteintranslationwasrepressedbybothpathogens,withanespeciallystrongtranscriptionalresponseat48hpiwithSC.Ahighproportionofsignificantlyup-regulatedDEgenesinbothinfectionsareinvolvedinpathwaysforimmuneThelper1(Th1)differentiation,innate immune/inflammatory responses and antigen processing.

ConCurrent session #1. IMMUNOGENETICS AND GENOMICS

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Geneexpressioninductionwasweakerandoccurredearlier inST(24hpi)ascompared toSC. InSC the responsewasmaximalat48hpibutcontinuedtobeelevatedat7dpi.Thisdifferential tran-scriptionalresponsewasmirroredby interferongammaandtumornecrosisfactorserumproteinlevelsaswellasbacterialloadinthelymphnodes.Apoptosisandantigenpresentation/dendriticcellfunc-tionpathwaysweredown-regulatedat8hpi forST.Clusteranaly-ses,confirmedbyQ-PCRanalyses,revealedthatmanyDEgenesgroupedintoaspecificinducedsub-clusterareknownNFkBtargets.SuppressionofNFkBsignalingfrom24to48hpimayallowSTtoeludeananti-bacterialinflammatoryreaction.StudiesareunderwaytostudySalmonella-cellcultureinvasionfurtherusingIPECJ2epi-thelial cellsderived fromporcinesmall intestine.Wepropose thatNFkBsuppressioninantigenpresentingcellsmaybeamechanismbywhichST eludes a strong inflammatory response, thus settingthestageforestablishingacarrierstatusinpigs.ForSCinfections,therewasastrongNFkB-dependenttranscriptionalresponseandamoreintenseandextendedup-regulationofporcineimmunegeneexpression,potentiallyresultinginclearanceoftheSCinfection.

WILLIAMoLLIER(UnIVERSITyoFMAnCHESTER,UK)immunogenetiC risK fACtors Contributing

to CAnine diAbetes

WERoLLIER1,LJKEnnEDy1,ADSHoRT1,LJDAVIDSon2,ABARnES3,nFRETWELL4,CJonES4,BCATCHPoLE5

1.CIGMR,TheUniversityofManchester,UK;2.Dept.ClinicalVeterinaryScience,Cambridge,UK;3.FacultyofVeterinary

Sciences,UniversityofLiverpool,UK;4.WalthamCentreforPetNutrition,Leicester,UK;5.RoyalVeterinaryCollege,Hatfield,UK

DiabetesMellitusoccursspontaneouslyindomesticdogsandvirtually all affected dogs become dependent on insulin therapy.Caninediabetescanbeclassifiedintoanumberoftypesincludingcongenital,hormonal(dioestrous)andadultonsetinsulindependent

diabetes. There is no strongevidence for a canineequivalent ofhumantype2diabetesdespitecanineobesity.AdultonsetIDDisthemostcommontypeofcaninediabetesandthereisevidenceforinsulitisandanimmunemediatedbeta-celldestructiveprocess.

Whilst IDD can occur in many breeds, some (e.g SamoyedandTibetanTerriers)areparticularlypredisposed,whilstothers(e.gBoxer and German Shepherds) appear protected. These breeddifferences in susceptibility suggest a genetic component to IDDaetiologyinthisspecies.CanineIDDisthereforelikelytorepresenta complex condition where both multiple susceptibility genes andenvironmentalfactorsinteracttotriggerdisease.

We have examined this hypothesis by determining whethergenes previously shown to be associated with immunemediatedhuman diabetes also represent good candidate genes in caninediabetes and confer risk/resistance. To pursue this investigationwehaveexaminedcandidategenepolymorphismsandhaplotypeassociationsforawiderangeofcandidategenesofknownimmuno-logicalfunctioninwellcharacterisedcanineIDDcases(>500)andcontrols(>1,000).

Studies examining MHC (DLA) Class II haplotypes revealedclear riskassociationswithDLA*-DRB1*009-DQA1*001andDLA-DRB1*015-DQA1*006 haplotypes and resistance with the DLA-DQA1*004-DQB1*013haplotype.Theseassociationsappearedtorelatecloselywithbreedpredisposition.

Other immunologically related candidate genes examined,included CTLA-4, IL-4 and IL-10. SNPs were identified in thesegenes using dHPLC screening andDNA sequencing. ConfirmedSNP’s were genotyped in cases and controls using Sequenommass-arraytechnology.SNPhaplotypeanalysisrevealedbothIDDsusceptibility and resistance haplotypes. These results and theirimplicationsforcanineIDwillbepresentedanddiscussed.Key words:diabetes;DLA;Class IIhaplotypes;CTLA-4; IL-4; IL-10Species:canine

GARyEnTRICAn(MoREDUnRESEARCHInSTITUTE,UK)tHe effeCt of PregnAnCy on mAternAl

immunity in sHeeP

ISABELLEoSWALD(InRA,FRAnCE)effeCt of some food ContAminAnts, tHe

myCotoxins, on tHe immune resPonse of tHe Pig

ConCurrent session #3. IMMUNOENDOCRINOLOGy; AND STRESS; IMMUNOLOGy OF REPRODUCTION AND NEONATES; MICROBIAL FLORA, NUTRIENTS AND THE IMMUNE RESPONSE

GoRDonMACPHERSon(oxFoRDUnIVERSITy,UK)tHe role of dendritiC Cells At muCosAl

surfACes And regulAtion of inflAmmAtionGoRDonMACPHERSon,SIMonMILLInG,ULFyRLIDDendriticcells(DC)migrateconstitutivelyfromtheintestineto

drainingmesentericlymphnodes.TheseDCcompriseatleastthreephenotypicallyandfunctionallydistinctsubsets.Incontrasttosomecurrentdogma,theseDCarenotconstitutivelysuppressedbutarethestrongestactivatorsofnaiveTcellswehavefound.SuchactivatedTcellssecreteamixtureofTh1andTh2cytokines.Immuno-modula-toryagentsthatarepotentialintestinaladjuvants–R848(aTLR7/8ligand,LPS,E.coliheatlabiletoxinandschistosomeeggantigen)havebeenusedtoinvestigatechangesinintestinalDCassociatedwiththeswitchfromtolerancetoactiveimmunity.OurresultssuggestthatthisswitchisnotassociatedwithanyconspicuouschangesinthenumbersorpropertiesofmigratingDCs.

VoLKERGERDTS(VACCInEAnDInFECTIoUSDISEASEoRGAnIZATIon,CAnADA)

strAtegies to linK innAte And AdAPtiVe immunity WHen designing VACCine

AdjuVAnts

PATSHEWEn(UnIVERSITyoFGUELPH,CAnADA)muCosAl VACCines

SARAHDoyLE(TRInITyCoLLEGE,DUBLIn)tlr signAling in infeCtion And

inflAmmAtion

STEVEREInER(UnIVERSITyoFPEnnSyLVAnIA,USA)

sPeCifying tHe t Cell fAtes required for immunity: AsymmetriC diVision of A t lymPHoCyte in tHe initiAtion of AdAPtiVe

immunity

PlenAry session innAte immunity, inflAmmAtion And AdjuVAnts; memory, ACquired immunity And VACCines

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AbramsonFamilyCancerResearchInstitute,andDepartmentofMedicine,UniversityofPennsylvania,Philadelphia,PA19104

Ahallmarkofmammalianimmunityistheheterogeneityofcellfate that exists among pathogen-experienced lymphocytes. I willpresentevidencethatadividingTlymphocyteinitiallyrespondingtoamicrobeexhibitsunequalpartitioningofproteinsthatmediatesignal-ing,cellfatespecification,andasymmetriccelldivision.Asymmetric

segregationofdeterminantsappearstobecoordinatedbyprolongedinteractionbetweentheTcellanditsantigen-presentingcellpriortodivision.Additionally,thefirsttwodaughterTcellsdisplayphenotypicandfunctionalindicatorsofbeingdifferentiallyfatedtowardeffectorandmemorylineages.Theseresultssuggestamechanisminwhichasingle lymphocytecanapportiondiversecell fatesnecessary foradaptiveimmunity.

Abstracts for Posters

1. Immunogenetics and GenomicsPostersIG001-IG026..........................................................................................................................53

2. Immune Responses in Bacterial and Viral Diseases; Prions and BSEPostersBV027–BV077.......................................................................................................................62

3. Immunoendocrinology; and Stress; Immunology of Reproduction and Neonates; Microbial Flora, Nutrients and the Immune ResponsePostersIE078-ER094.........................................................................................................................79

4. Immunology of the Mucosae and Skin and of the Mammary Gland; MastitisPostersSM095-SM118.......................................................................................................................85

5. Antigen Presentation and Dendritic Cells; Effector Cells, B and T cells, NK and NK T cells; Immunoregulatory cellsPostersAP119-AP140.........................................................................................................................93

6. Immunoparasitology: Immune Responses to Protozoa, Helminths and Ectoparasites; Canine Visceral LeishmaniasisPostersPR141-PR196.....................................................................................................................100

7. Comparative Immunology; ImmunoecologyPostersCI197-CI206........................................................................................................................ 118

8. Mediators of Recruitment and Function of Cells of the Immune System; Fc Receptors and Immunoglobulins; Signal Transduction and Gene Expression in cells of the immune systemPostersMI207-MI210.......................................................................................................................121

9. Innate immunity, Inflammation and Adjuvants; Memory, Acquired Immunity and VaccinesPostersVA211-VA250........................................................................................................................123

10. Clinical Immunology and ImmunopathologyPostersIP251-IP281.......................................................................................................................136

ig001. IDENTIFICATION OF NOVEL BOVINE DH GENESMADHURIKoTI1,GALInAKATAEVA2,AZADKKAUSHIK1

1DepartmentofMolecularandCellularBiology,UniversityofGuelph,Canada;2McMasterUniversity,Hamilton,Canada

mkoti@uoguelph.caOurlaboratoryearlierdemonstratedthatlimitedgermlinesequence

divergence both at the heavy and light chain restricts combinatorialdiversity in cattle, similar to other species such as chicken, pig andsheep.Oneof themost importantcharacteristicsofbovineantibodyrepertoire is thatanextensiveCDR3Hlengthheterogeneityexists inbovineantibodiesthatisevidentfromfetalB-cellstage.ThegenerationofanexceptionallylongCDR3H(upto61aminoacids)withmultiplecysteineresiduesandsomatichypermutationscontributetoantibodydiversificationincattle.PartialcharacterizationofbovineDH-genelocusin our laboratorydemonstrated thepresenceof both short and longgermline DH genes with the potential to directly encode 49 codons.WehavenowcharacterizedthebovineDH-genelocusfromcattlethatdemonstratespolymorphismapartfromthefactthatbovineDHgenesareorganizedindistinctsubclusters.

(SupportedbyNSERCresearchgrant).Key words:AntibodyDiversity,CDR3H,DHgenesSpecies:Ruminants

ig002. GENERATION, HAPLOTyPE CHARACTERISATION AND PRACTICAL APPLICATIONS OF AN MHC DEFINED

SHEEP RESOURCE FLOCK. KEITHBALLInGALL1,DESPoInAMILTIADoU1,MARARoCCHI1,

DECLAnMCKEEVER1,21MoredunResearchInstitute,PentlandsSciencePark,Midlothian,

UK;2Royal(Dick)SchoolofVeterinaryStudies,EasterBushVeterinaryCentre,Midlothian,UKkeith.ballingall@moredun.ac.uk

InjawedvertebratestheTcellreceptorbindstoforeignpathogenfragments in association with classical self major histocompatibilitycomplex (MHC) molecules. This recognition event is fundamentalto the inductionand immune-mediated control of infectiousdisease.However, extensive allelic diversity within the genes encoding theMHCmoleculesisasignificantconstrainttolongtermstudiesofTcellimmunologyinout-bredspeciessincetheyrelyontheundefinedMHChaplotype carried by each experimental animal. This “MHC restric-tion”ledtothedevelopmentofinbredanimalmodelswithidenticalandwellcharacterisedMHCregionsthatarenowused inthemajorityofinfectiousdiseaseandbasicimmunologicalresearch.However,dataderivedfromstudiesinmodelorganismssuchasthelaboratorymouseareoftennotdirectly transferable to theoriginal targetspeciessuchashuman.Infectiousdiseaseresearchinlivestockontheotherhandhastheadvantageofbeingabletostudythediseaseandthemecha-nismsofimmunitywithinthetargetspecies.HowevertheproblemofMHCrestrictionremains.ToaddressthiswehavegeneratedanMHCdefinedresourceflockwhichisbasedaroundfourcommonbutdiversesheephaplotypes.AswellasalargecohortofMHCheterozygousani-malswehavealsopurposebredMHChomozygousanimalscoveringeachofthefourhaplotypes.InadditiontoprovidingavaluablecellularresourceforanalysisofTcellresponses,thesehomozygousanimalsalsoallowtheunambiguousmoleculargeneticcharacterisationofthefourtargetMHChaplotypes.HerewedescribethecharacterizationofthetranscribedMHCclassIandclassIIgenescarriedbythesedistincthaplotypes. Thisprovidessubstantialnew informationonallelicandhaplotypediversity insheep.TheMHCdefinedflock isavailableforcollaborativestudiesofTcellresponsestoinfectiouslivestockpatho-

gensandwillbevaluabletoolsforsubunitvaccinedevelopmentandcomparativestudiesofMHCdiversityandevolution.Key words:Sheep,MHC,haplotypesSpecies:ruminants

ig003. NOVEL APPROACHES TO ENHANCE DISEASE RESISTANCE IN RUMINANTS? - BREEDING FOR

GEOGRAPHICALLy IMPORTANT TLR SNPSTHERUMInAnTTLRConSoRTIUM:DWBURT1,TJCoFFEy3,SCHAnG2,EJGLASS1,DHAIG2,JCHoPE3,oJAnn1,JSALT1,C

WARKUP4,DWERLInG61RoslinInstitute(Edinburgh);2MoredunResearchInstitute

(Edinburgh);3InstituteforAnimalHealth(Compton);4GenesisFaraday(Edinburgh);5PfizerLtd(Sandwich);6RoyalVeterinary

College(London)Opportunisticinfectionsresultingfromintensivehusbandryoflive-

stockhavebecomeoneofthemajorproblemsinmodernanimalpro-duction.Asbacterialresistancetoantibioticsisexpectedtoescalate,novelapproaches todiseasepreventionwillneed tobeestablished.Oneapproach includesbreeding fordisease resistancebyselectingthe‘fittest’innateimmunesystem.Theinnateimmunesystemrecog-nises pathogens by means of pattern recognition receptors, suchas Toll-like receptors (TLRs). These interact with various microbialcomponentsand induceaspecific innate immuneresponse.SeveralpolymorphismsinTLRgeneshavebeendescribedforthehumanandmurinesystem that influence theabilitiesofaffectedTLRs to recog-nisepathogen-derivedmolecules-renderingindividualsmoreorlesssusceptibletoinfection.Thefirstnucleotidepolymorphisms(SNPs)inruminantTLRswere characterised in bovineTLR4, the receptor forgram-negativebacteria.Analysisofcodon-basedmodelsofselectionidentifiesmanysitesinTLR4underpositiveselectionintheregion261to375 residues.This region is located in themiddleof theextracel-lular domain between clusters of leucine-rich-repeats (LRRs) and ispredictedtobetheligandbindingdomain.WewouldexpectahigherfrequencyofnonsynonymousSNPstomaptohisdomain.Indeed,thefirstnonsynonymousSNPsmaptothisregion.

WehaveclonedandmappedtenbovineTLRs,andarecurrentlyintheprogressoffunctionallycharacterisingtheseTLRs.Inaddition,TLRgenesfromsheeparealsobeingclonedandcharacterised.TLRsfromdifferentcattleandsheepbreedsarecurrentlybeenanalysedforthepresenceof synonymousandnon-synonymousSNPs.OurdatasuggestaheterogeneityinextracellularregionsofTLRgenes,whichmaybeadvantageoustopromoteaspecificdiseaseresistance,andrepresentanewapproachtoselectdiseaseresistance-basedonageographical distribution of TLR SNPs. Breeding of ruminants withTLRsthatconfera ‘fitter’ innate immunesystemresulting indiseaseresistancecouldplayamajorroleinthefutureofthefarmingindustrybothintheUKandworldwide.

Funding and support from the BBSRC, Genesis Faraday andPfizerResearch.Key words: Pattern recognition receptors, TLR, Innate immuneresponse,GeneticresistanceSpecies:ruminants

ig004. THE DISCOVERy OF QTL FOR MASTITIS RESISTANCE IN NORDIC DAIRy CATTLE.

nICoLAHASTInGS1,AnAFERnAnDEZ2,MoGEnSS.LUnD3,GoUTAMSAHAnA3,BoTHoMSEn3,nInASCHULMAn4,

JoHnLWILLIAMS5,LEnAAnDERSSon-EKLUnD6,HALDJAVIInALASS7,JoHAnnAVILKKI4

1. IMMUNOGENETICS: POSTERS IG001-IG026

54

1RoslinInstitute,Roslin,Midlothian,EH259PS,U.K.;2MejoraGenéticaAnimal,SGIT-INIA,Crta.CoruñaKm.7.5,28040Madrid,

Spain;3DanishInstituteofAnimalSciences,BlichersAlle,8830Tjele,Denmark;4MTT,BiotechnologyandFoodResearch,Genomics,

31600Jokioinen,Finland;5ParcoTecnologicoPadano,ViaEinstein,PoloUniversitario,Lodi26900,Italy;6DepartmentofAnimalBreedingandGenetics,SwedishUniversityofAgriculturalSciences,Box7023,S-75007Uppsala,Sweden;7EstonianUniversityofLifeSciences,

Kreutzwaldi64,51014Tartu,Estonia.MastitisremainsthemostcostlydiseaseaffectingtheEuropean

dairycattleindustry.Thefrequencyofclinicalmastitisisincreasingduetoanunfavourablegeneticcorrelationbetweenincreasedproductivityandmastitisresistance.Thisandbecausethetraithasalowheritabil-itymake selective breeding formastitis resistance difficult.Mappingof quantitative trait loci (QTL) in livestock using existing populationstructures is restricted to the traits thatare recorded in thebreedingschemes.TheNordiccountrieshavetraditionallypaidparticularatten-tion to recording health traits, such as mastitis, in dairy cattle. Theextensive recording in theNordic herds provides a unique resourceformappingoflociaffectingfunctionaltraitsespeciallyclinicalmastitis.UsingtheNordicherdrecordsandDNAsamplesfromtheherds,whichincludedthethreebreedsFinnishAyrshire,SwedishRedandWhite,andDanishRed,weanalysedfivechromosomesforQTLaffectingclin-icalmastitis(CM)andsomaticcellscore(SCS).Jointsingle-traitandtwo-traitanalyseswereperformedusingvariancecomponentsmodels.QTLaffectingCMand/orSCSsegregateonBTA9,11,14and18whileaQTLonBTA29couldnotbeconfirmed.However,ouranalysescon-firmedatleasttwolinkedQTLonBTA9,onethatmainlyaffectsCMandasecondthatprimarilyaffectsSCS.InordertoobtainaccurateQTLpositioningonBTA9weimprovedthelevelofinformationintheanaly-sisbyemployinganumberofmethodstoincreasethemarkerdensityandqualityofthemappinginformation.Indevelopingnewmarkersweused in silicoandmolecular biological techniques.Themethodsweemployed includedmining thebovinegenome fornewmicrosatellitemarkersandre-sequencingofgenesandESTsfornewSNPs.Usingthis newmarker information alongwith previously reportedmarkersfrom USDA MARC (http://www.marc.usda.gov/genome/cattle/cattle.html)wecreateddenseradiationhybrid(RH)andlinkagemaps.Thenewmapswereusedtofine-maptheQTLusingalinkage/linkagedis-equilibriummappingapproach.Key words:Mastitis,Resistance,QTLSpecies:ruminant

ig005. GENOMIC AND CDNA ANALySIS OF THE BOVINE TCRβ GENE REPERTOIRE

TConnELLEy,JAERTS,ALAW,WIMoRRISon1DivisionofClinicalVeterinarySciences,UniversityofEdinburgh,

Edinburgh,United-Kingdom.Dept.ofBioinformatics,RoslinInstitute,RoslinBiocentre,Midlothian,UK,EH259RS.

ThediversityoftheTCRrepertoirethatisintegraltoeffectiveαβT-cellfunctionisgeneratedbysomaticrecombinationofvariable(V),diversity(D-βchainsonly)andjoining(J)genesegmentsofthecom-ponentαandβchains.TofacilitatethedevelopmentoftechniquestoexaminebovineT-cellresponsesweundertookanextensiveanalysisofgenomicandcDNAdatatocharacterisethebovineTCRβgenerep-ertoire.Analysisof the thirdbovinegenomeassemblydemonstratesthat although the TCRB locus is still incomplete, the TCRβ generepertoire in cattle is significantly larger than that of either humansormice.Most notably, several of the Vβ subfamilies, such as Vβ1,10 and 13 have undergone extensive duplication, containing 35, 16and40membersrespectively.Muchofthisexpansionappearstobedue toduplicationof ‘cassettes’containingmultiplegenes leading totandemlyarrangedduplicatesincorporatingmembersoftwoormoresubfamilies(e.g.Vβ18-Vβ17-Vβ2-Vβ10).Inall,130Vβgenesdistrib-utedover24subfamilieshavebeen identified in thegenomeso far.Compared to other species examined, the bovine genomealso hasexpanded Dβ and Jβ gene repertoires due to triplicate rather thanduplicatecopiesof theDJC regionof theTCRB locus.Themajority(126/130)oftheVβgenesandalloftheDβ,JβandCβgenespresentin thegenomeassemblyare locatedof4 largescaffolds,3ofwhichhavebeenmappedtochromosome4.Extensiveanalysisofover900

TCRβchainsequencesgeneratedfromcDNAstudiescompletedinourlaboratoryhaveidentifiedmorethan30Vβgenesthatarenotpresentinthecurrentbovinegenomeassembly,indicatingthatthefullgenomicVβ repertoire remains undefined. In contrast, comparison to cDNAandESTdatasuggeststhatthefullJβrepertoireisrepresentedinthegenomeassembly.Combinationof thecDNAandgenomicanalysessuggestthatthefunctionalbovineTCRβgenerepertoireiscomposedofover100Vβgenes,3Dβgenesand17Jβgenes.The resultsofthisworkindicatethatthebovineTCRβgenerepertoireisthelargestyetcharacterised.ThedramaticexpansionofmembershipofcertainVβsubfamiliesandcomparisontothehumanandmurineTCRβgenerepertoiresraisesinterestingquestionsconcerningtheforcesinfluenc-ingtheevolutionofthisimmunologicallyimportantlocus.Key words: TCR,GenomeSpecies:ruminant

ig006. SNPS IN BOVINE CANDIDATE GENES FOR MEDIATING RESISTANCE TO INFESTATIONS WITH THE

CATTLE TICKAnTonIoRRABATEPAULo1,ISABELKFDE

MIRAnDASAnToS1,DAnIELADMoRé1,WAnESSAACARVALHo1,ALExAnDRERCAETAno2

1Dept.ofBiochemistryandImmunology,RibeirãoPretoMedicalSchool,UniversityofSãoPaulo,RibeirãoPreto,SP,Brazil;2Embrapa

RecursosGenéticoseBiotecnologia,Brasília,DF,Brazil.abatepaulo@usp.br

IntroductionTicksarehematophagousarthropods thatcauseserious losses

inanimalproductivityandhealth,especiallyintropicalenvironments.Alternatives toacaricides, thecurrentmethodofcontrol,areneededbecauseof the resultingcontaminationof theenvironmentand foodproducts,andlossofeffectivenessensuingfromdevelopmentofresis-tancebytheticks.Bovinehostsexpressbreed-specific,heritable,con-trastingphenotypes(susceptible:Sorresistant:R)whenexposedtolarvaeofRhipicephalusmicroplus.Bosindicusbreedsaresignificantlymoreresistant thanBos taurusbreeds,whileanimals insegregatingpopulationsderivedfromcrossesbetweenthesegroupsshowvaryinglevelsofresistance.Thissuggestsapolygenicbasisforthetraitandoffers an opportunity to identify specific genes/mutations associatedwithtickresistance.

Objectives:Examinegenomicsequencesofcandidategenes,pre-viouslydeterminedtobedifferentiallyexpressedintick-infestedskin,for thepresenceof breed-specificSingleNucleotidePolymorphisms(SNPs) by comparing tick-resistant and tick–susceptible bovinebreeds

MethodsGenomicDNAfrom16resistenthots(Nelore)and16susceptble

hots (HPB) was PCR-amplified with specific primers designed togenerateanampliconanchoredontwoexonsandspanningat leastone intron. ThePCR fragmentswere purifiedwith ExoSapitTM andsequencedinanABI3100DNAanalyzer.

ResultsandDiscussionWe looked for breed-specific SNPs by comparing genomic

sequencesfromNelore(N=16)andHolstein(N=16)forcandidategenes(TGF-α,IFN-γ,IP-10,TNF-α,MIP-1α,IGF-1andMCP-1)wehadpreviouslydeterminedtobedifferentiallyexpressedinthetick-infestedskin of resistant hosts.A total of 40SNPswere found, at least fiveSNPspergene,andseveralalleleswerefoundtobebreed-specific.Theresultsprovideinformationthatwillallowforassociationstudiesincomposite/segregatingpopulationsresultingfromcrossesofB.taurusandB.indicusbreedstoascertainifthesemarkersareassociatedwithcausalmutationswhichconfertickresistancetoB.indicuscattle.

SupportedbyFAPESP,CNPq,TheWorldBankandValléeSA.Key words:SNP,Bovinegene,Resistance,TickSpecies:ruminants

ig007. IDENTIFICATION AND CONFIRMATION OF POLyMORPHISMS IN BOVINE GENES WITH IMMUNE

FUNCTION

55

ACAPRERA2,CCAMBULI1,RCAPoFERRI1,CGoRnI2,SVIoLInIFPAnZITTA2,BLAZZERI2,JLWILLIAMS2

IDRALaboratoryISLS1-PTP2,PoloUniversitario,viaEinstein,Lodi26900Italy.

Theidentificationofthegeneticvariationscontrollingphenotypes,includingimmunefunction,canbeachievedbyacombinationoflink-agemapping,associationstudiesorcandidategeneapproaches.Theavailability of the draft bovine genome sequence, progress towardsits’annotationandinformationonputativesinglenucleotidepolymor-phisms provides considerable new information to identify positionalcandidategenes.However,fromthe2.2millionbovineSNPsidentifiedfromthegenomesequencingproject,uptonow,fewhavebeencon-firmed.Therefore, theidentificationandvalidationofpolymorphismsinthefunctionalorregulatoryregionsofthegenesthatareputativelyinvolvedinregulatingimmunefunctionremainsanimportanttask.

Weareinterestedintheroleofvariousimmunegenesinthecon-trolofmycobacterialinfectionsincattle.ThereforewehaveselectedapanelofgenesinvolvedinmacrophagefunctionandareexaminingthelevelofpolymorphisminthesegenesThestartingpointistoidentifyputativepolymorphismsintargetgenesusingbovinesequenceinfor-mation.AsoftwareapplicationhasbeendevelopedtolocateputativeSNPsavailablefromDBSNPwithintargetgenesorsequencesusingpositional,ePCRandBLASTapproaches.ThepresenceofSNPsisthenconfirmedbydirectsequencingpriortodeterminingallelefrequen-ciesinapanelofanimalsfromarangeofgeneticallydiversebreeds.Key words:Polymorphisms,SNPSpecies:ruminants

ig008. GENOMIC RESPONSES OF THE BOVINE MAMMARy GLAND AND EPITHELIAL CELLS TO ACUTE LPS

CHALLENGEDEKERR1,MLATSHAW1,RPAREEK1,JZHEnG1,JPBonD21DepartmentofAnimalScience;2DepartmentofMicrobiologyand

MolecularGeneticsUniversityofVermont,BurlingtonVTThe early innate immune response to bacterial entry into the

mammaryglandisthoughtcriticalindeterminingtheoutcomeofmas-titis.Thegoalofidentifyinganimalswithenhancedgeneticresistancetomastitiswilldependongreaterknowledgeofthegenesinducedinresponsetopathogens.WehaveusedAffymetrixGeneChipstoprofilethe acute genomic response to an LPS challenge in lactating cowsand in cultured mammary epithelial cells. Sources of RNA includedbiopsy samples from LPS-challenged (1 ug/gland) and contralateralcontrolmammaryglandsof3 lactatingcows4hpost-challenge;andfrom LPS-stimulated (50 ng/ml) or control cultures of primarymam-maryepithelialcells3hpost-LPS.Thecellswerepreviouslyobtainedand cryopreserved from3 different cows.We identified 75 immune-associatedgenesthatweredifferentiallyregulated(74up,1down)inresponsetoLPS in the tissuebiopsysamples(P≤0.01, fold-change(FC)≥2.0).Inducedgenesincludedaremarkable17membersoftheCCL,CXCL,andCX3CLfamilyofchemokines.Alsoinducedwere,3inflammatory cytokines (IL1b, IL6,TNF), 3 acute phase (HP, SAA3,LTF), 2 antimicrobial (BNBD-4, LAP), and6membersof theS100Acalciumbindingproteins.ThecentralroleoftheNF-kBcomplexwasillustratedbyinductionofNFKB1andRELB,andinterestingly,acon-cordantupregulationof3membersoftheI-kBfamilythatappearstoindicateanegativemodulation.Thecellcultureresponserevealed44immune-associatedgenesdifferentiallyregulated(P≤0.01,fold-change(FC)≥2.0;allup)inresponsetoLPSstimulation.Thegenelistfromtheculturedcellsindicatedaclosereflectionoftheinvivoresponseinthat34upregulatedgeneswerecommonbetweenthetissueandcellcultureexperiments.Commonlyupregulatedgenesinclude10membersoftheCCL,CXCL,andCX3CLfamilyofchemokines,threeS100genes(A8,A9,A12),and the inflammatorycytokines,andNF-kB relatedgenespreviouslymentioned.Immune-associatedgenesupregulatedonly intheLPStreatedcellsincludedthreeadditionalinflammatorycytokines(IL1A,CSF1,CSF2)and theenzymesurinaryplasminogenactivator(PLAU) and nitric oxide synthase 2A (NOS2A). These studies haverevealed a rapid, robust genomic response of the bovinemammaryglandtoasmallquantityofE.coliLPS.ThegenomicresponseappearsdominatedbyactivationoftheNF-kBcomplexleadingtoupregulation

of inflammatorycytokinesandinparticulararemarkable inductionofmultiplemembersofthechemokinegenefamily.Key words: innateimmunity,mastitis,chemokine,infection.Species: ruminants

ig009. STRAIN SPECIFIC AND COMMON PATTERNS OF GENE ExPRESSION IN MACROPHAGES INFECTED WITH M.

PARATUBERCULoSIS ISOLATES. EKABARA*1,CKLoSS2,MWILSon2,SSREEVATSAn3,H

JAnAGAMA3,PCoUSSEnS21DepartmentofBiochemistry;2CenterforAnimalFunctional

Genomics,DepartmentofAnimalScience,MichiganStateUniversity,EastLansing,MI.;3CenterforAnimalHealthandFoodSafety,

UniversityofMinnesota,Minneapolis,MN.Mycobacteriumavium subspeciesparatuberculosis (MAP) isan

intracellularpathogenthatcausesaneconomicburdentotheUSdairyindustryestimatedatoveronebilliondollarsannually. AhallmarkofMAP infection is survival in host macrophages, cells that normallydestroy ingested microbes. As with other mycobacteria, survivalin macrophages appears to be a key determinant of pathogenesisassociated with MAP infections. Previously, we, and others, havedemonstratedthatinfectionwithbothMAPandthecloselyrelated,butnon-pathogenicMycobacteriumaviumsubspeciesavium(MAA)haveprofoundeffectsonmacrophagegeneexpression. Basedon thesestudies,wehypothesizedthatdifferentstrainsofMAPwouldhavebothcommonandstrain-specificeffectsonmacrophagecellgeneexpres-sion. To test thishypothesis,wehavenowstudied theeffect of 10differentMAPstrainsonmacrophagegeneexpressionprofiles,withtheultimategoalofrelatinggeneexpressiondifferencestovirulenceandgeneticsoftheMAPstrains.Ourinitialdataanalysissuggeststhatthereareover120macrophagegeneswhoseexpressionisgenerallyalteredfollowinginfectionwithanystrainofMAP.Ourdatahasbeenfurther scrutinized via mixed-model analysis to investigate potentialstrain-specific and/or host origin-specific differences in MAP-macro-phageinteractionsandremovethespecificeffectsofeachanimalusedinthestudy.Examplesincludegenestypicallyassociatedwithimmuneresponse,suchasIL-1a,IL-8,andCCL-3.Expressionpatternsofothergenes, not typically associated with immune responses, such as anADP-ribosyltransferase,werealsoaffectedbyinfectionwithallstrainsofMAP.Thisstudyalsorevealedtranscriptionlevelchangesinseveralpreviouslyunknowngenesthatmaybeimportantfortheprotectionofthepathogenandthusmeritfurtherinvestigation.Uponhierarchicalclusteringusingfold-changedata,MAPstrains isolatedfromhuman,cattle and sheep showed little initial host species similarity, but twosupershedder strains clustered tightly together, perhaps suggestingthesetwostrainshaveverysimilareffectsonbovinemacrophagecellsandpotentiallysuggestingarelationbetweenbacterialphenotypeandhostreactiontothepathogen.Key words:Pathogen-host interaction,microarrayanalysis, Johne’sdisease,Crohn’sdiseaseSpecies:ruminants

ig010. KNOCK-DOWN OF BOVINE LEUKEMIA VIRUS TAx By RNAI AND ITS EFFECTS ON HOST GENE ExPRESSION RoSAnEoLIVEIRA1,ALLISonSoMMERS1,RoBInEEVERTS1,

HARRISALEWIn1,21DepartmentofAnimalSciencesand2InstituteforGenomicBiology,

UniversityofIllinoisatUrbana-Champaign,Urbana,IL,USABovineLeukemiaVirus(BLV)taxproteinisatranscriptiontrans-

activatorofhostcellgenesthatmodulatescellgrowthandproliferation.By interfering with the transcription of host cell genes, tax expres-sion is believed to be an essential first step in the dysregulation ofhomeostasisleadingtothetransformationofB-lymphocytes.TobetterunderstandtheroleofBLVtaxindrivinglymphoproliferationandcelltransformationinthehost,BLVtaxgeneexpressioninaBLV-infectedbovine lymphoblastoid cell line (BL3*) was knocked down by RNAinterference (RNAi). Geneexpression profilingwas then performedusinga13,257-elementcattleoligonucleotidemicroarray.Atax-spe-cificsmall-interferingRNA(siRNA)andcontrolscrambledsiRNAwereused for transfections. Transfectionefficiencywasanalyzedbyflow

56

cytometry of cells transfectedwith aGFP-construct, and taxmRNAlevelswereassayedbyquantitativePCR.Sixbiologicalreplicatesoftheknock-downwereperformedwithbothBL3*andBL3º(uninfectedparentalcellline).Eachtreatmentcomprised5or6technicalreplicatestoprovideenoughRNAforthemicroarrays(totalof132transfections).Theaveragetransfectionandknock-downefficiencieswere92.9%and72.25%,respectively.RNAisolatedfromeachtreatmentwaspooled,and four independent transcriptome comparisons were carried outusingmicroarrayanalysis(totalof46slides). Thedifferentcompari-sonsallowedtheidentificationofgenesspecificallyknockeddownbytax-siRNA,aswellascellline-specificandoff-targeteffects.

InBL3*cellstreatedwithtax-siRNA,186geneswerefoundtobedifferentiallyexpressedafterremovalofoff-targeteffects(t-test,falsediscoveryrate,P-value<0.2).Thegenesaffectedbytaxknock-downwereminedforaffectedpathwaysandfunctionsusingIngenuityPathway Analysis. Among the canonical pathways significantlyaffectedbytheBLVtaxknock-downwereERK/MAPKsignaling,oxi-dative phosphorylation and glutathionemetabolism. Analysis of thedistributionofgenesaccordingtoGeneOntologyprocessesrevealedthatcriticalpathwaysincellgrowth(e.g.cellsignaling,cellcycleandcelldeath/apoptosis)areaffecteddirectlyorindirectlybyBLVtaxand/orothervirally-encodedgenes.Onthebasisoftheseresults,amodelfor BLV-induced lymphoprolferation and transformation is proposed.We postulate that persistent lymphocytosis in BLV-infected cattle iscausedbytax-induceddysregulationofaself-renewingpopulationofpre-B lymphocytes. Transformation leading to lymphosarcoma is arareeventthatiscausedbysecondarymutationsintheinfectedBcellprecursor. Our resultsdemonstrate thepowerofRNAicoupledwithmicroarrayanalysis fordissecting thegeneticandcellularprocessesleadingtocelltransformationbyretroviruses.Key words:BovineLeukemiaVirus,Taxgene,RNAinterferenceSpecies:ruminants

ig011. MONOCyTES FROM DISEASE RESISTANT AND SUSCEPTIBLE CATTLE DISPLAy DISTINCT

TRANSCRIPTOME PROFILES DURING THEILERIA ANNULATA INFECTION.

KJEnSEn1,RTALBoT2,DWADDInGTon1,EJGLASS11RoslinInstitute,Roslin,Midlothian,EH259PS,UK;2ARK-Genomics,

RoslinInstitute,Roslin,Midlothian,EH259PS,UKThe tick-borne protozoan parasite Theileria annulata causes a

debilitatingandoftenfataldiseaseofcattlecalledtropicaltheileriosis.ThediseasehasaglobaleconomicimpactonlivestockproductionasitisendemicinmanyareasoftheworldfromtheMediterraneanbasintoChina.ControlstrategieshavesofarfailedtoeradicateT.annulataor itsvector. Anattractivealternativecontrolstrategy is tobreedforcombined resistance and productivity in cattle by using pre-existinggeneticresistance. Wehave identifiedaBos indicusbreedofcattlethatoriginatesfromPakistan,theSahiwal,whichisresistanttotropicaltheileriosis. Ourcurrentstudiesaimto identify thegenesunderlyingtheresistanceofSahiwalcattletoT.annulata.

T.annulataprincipallyinfectsbovinemacrophagesandthepathologyofthediseaseisassociatedwiththeintramacrophagestageof theparasite. Thereforewehave focussedourstudiesonmacro-phagesandmonocytesderivedfromresistant(Sahiwal)andsusceptible(Holstein-Friesian,B.taurus)breedsofcattle.WehaveundertakenaglobalanalysisofthetranscriptionalresponseofSahiwalandHolstein-Friesian derived monocytes to T. annulata infection using a bovinemacrophage specific microarray developed in our laboratory. Thisapproachhas identifiedover60expressedgenesthatexhibitbreed-specific differential expression either in restingmonocytes or duringT.annulatainfection,whichmayberelatedtoT.annulataresistance.Manyofthedifferentiallyexpressedgenesarecell-surfaceexpressedproteins,e.g.prionproteinandICAM1,whichareinvolvedintheinter-actionofmacrophageswithotherimmunecells.Furtheranalysisofthepathways leadingtovariation inexpressionof thesemolecules,mayrevealtheunderlyingcausalgenesforresistanceandsusceptibilitytoT.annulata,andprovidenewapproachesfordiseasecontrol.Key words: macrophages, disease resistance, protozoan,transcriptomeSpecies:ruminants

ig012. COMPARATIVE GENOME ANALySIS OF TRyPANOTOLERANCE QTL

JoSEPHnGAnGA1,2,MABELIMBUGA2,FUADAIRAqI31InternationalLivestockResearchInstitute,P.O.Box30709,Nairobi,Kenya;2JomoKenyattaUniversityofAgricultureandTechnology,P.O.

Box62000,Nairobi,Kenya;3DepartmentofHumanMicrobiology,SacklerFacultyofMedicine,TelAvivUniversity,RamatAviv,TelAviv

69978,Israel.Certainbreedsofdomesticruminantsshowremarkableresistance

totheeffectsofAfricantrypano-somosis.Unlikesusceptibleanimals,trypanotolerantanimalscontrolparasitemiaanddonotshowsevereanaemiaor production loss. Identificationof trypanotolerancegenesincattle ishamperedbycostandbreeding time.Markeddifferencesbetween inbred strains of mice in their response to T. congolenseinfectioncanbeexploited in theanalysisof thegeneticbasisof theinfection.MurinetrypanotoleranceQTLshavebeenidentifiedonchro-mosome17,5and1,anddesignatedasTir1,2and3, respectively.Tir1and2havebeenfinemapped toaconfidence intervalof1cM.Inordertofindthemousehomologousregiononthebovinegenome,nucleotidesequenceacross95%CIofTir2and3wereused in theselectionof candidategenes.Homologous sequenceswereused inthedefinitionofsyntenyrelationshipsandsubsequentidentificationoftheshareddiseaseresponsegenes.Thehomologousgeneswithinthehumangenomewerethenidentifiedandalignedtothebovineradiationhybridmapinordertoidentifythemouse/bovinehomologousregions.ThisrevealedhomologybetweenmurineandbovineQTLonTir3whiletheregiononTir2islinkedtoinnateimmuneresponse.Key words:Trypanosomosis,Quantitativetraitloci,Homology,singlenucleotidepolymorphismSpecies:ruminants

ig013. ExPRESSION VARIATION OF TLR AS CANDIDATE GENES UNDERLyING TRyPANOTOLERANCE QTL IN MICE

JoSEPHnGAnGA1,2,FUADA.IRAqI31InternationalLivestockResearchInstitute,P.O.Box30709,Nairobi,Kenya;2JomoKenyattaUniversityofAgricultureandTechnology,P.O.

Box62000,Nairobi,Kenya;3DepartmentofHumanMicrobiology,SacklerFacultyofMedicine,TelAvivUniversity,RamatAviv,TelAviv

69978,Israel.AfterQTLmappingandphysical representationof theparticular

chromosomal fragment spanning trypanosomosis resistance lociTir2 and 3 in mice, possible candidate genes were selected.Theseappeared tobe linked to the innate immuneresponse.Plausible try-panotolerancecandidategeneswithinthelociincludeTLR1,5and6.TLRsarecriticalintheregulationofpro-inflammatorycytokinesecre-tions.Inanefforttofindanassociationbetweenthegenesandthedis-ease,expressionpatternsofTLRgenesmappingtotrypanotoleranceQTLwere investigatedusingquantitativereal timePCR.SusceptibleandresistantmiceinfectedwithT.congolenseportrayeddiverseTLRexpressionpatterns.UpregulationofdifferentTLRseemedtocoincidewithupregulationofIL-10andTNFinsusceptibleandresistantstrainsrespectively. T.congolense infection therefore induces a responsecharacterizedbychanges inTLR1,5and6expression in liverandspleen tissues which appear to regulate cytokine profiles in mice.Thesephenomenamayberesponsibleforthediversediseasepathol-ogyevidentindifferentmousestrainsandmayaccountsimilartrendsobservedinlivestockbreeds.Key words: Qtl, Expression Variation, Toll Like Receptors,InterleukinsSpecie:other(mice)

ig014. IMMUNOGLOBULIN ALLOTyPE-DEFINED PEDIGREED RABBITS AND GENES OF IMMUNOLOGICAL

INTEREST IN 2x RABBIT GENOME ASSEMBLIES (WORKSHOP)RoSEGMAGE

MolecularImmunogeneticsSection,LaboratoryofImmunology,NIAID,NIH,Bethesda,MD20892USAThisresearchwassupported

bytheIntramuralResearchProgramoftheNIH,NIAID.rmage@niaid.nih.gov

57

Although genomic sequence of the rabbit is currently “unfin-ished”at2x,deeper7xcoverageisexpectedsoon.TheJanuary2006document “Increasing sequence coverage from2x tohigh coverage(6-7x)forselectedmammalianspecies,”thatrecommendedrabbitbesequencedmoredeeply,describestheNIAIDallotype-definedrabbitsatp.14.TheseanimalsrepresentavaluableresourceforfutureSNPdiscovery.Theyhavepolymorphismsofavarietyof immunesystemgenesincludingvariants(allelicallotypes)oftheVH,CH,andCLregionsofantibodymolecules.Thecolonyalsocontainsdescendantsofrab-bitsformerlyattheBaselInstituteforImmunology,includingthemutantVH1a2-deletedAlicia,CK1splicingdefectiveBasilea,andseveralVH-CHrecombinantheavychaintypes.Theserabbitsarenowavailabletointerestedindividuals,particularlytositeswherebreedingcoloniescanbeestablished.Arelationaldatabase(computerprogram4D)containsmorethan45yearsofbreedingrecordsandother informationaboutanimalsinthecolony.Thewholegenomeshotgun(WGS)“unfinishedoryctolaguscuniculusdatabase”of2,076,044,328 letters in719,158sequences (ACCESSION AAGW00000000).hasseriousgaps, yetthe informationhasalreadyprovenuseful for immunological aswellasinsilicostudies.AttheBroadInstitute,theDNAofthe“ThorbeckeInbred Rabbit” chosen for sequencing was found by preliminarysequencing tohave lessheterozygosity thanoutbredNZW from thesamecompany(Covance).Ancestorsofthisstrainacceptedskingraftsafter20generationsofinbreeding.WhenItypedserumsamplesfromsuchanimalsin1995,6/12wereheterozygousfortheVH1atypesa1anda2andforthelinkedCgammahingeregiond11/12types.Twoof12rabbitswerealsoheterozygousforCK1allotypes(b4/b5).Asearchfor VH genes in theTraceArchive oforyctolagus cuniculus (WGS)finds perfect matches at http://www.ncbi.nlm.nih.gov/blast/mmtrace.shtml,topreviouslypublishedsequencesofbothVH1a1andVH1a2.Selectivemaintenanceofheterozygosityreportedinwildrabbitpopula-tions,alsooccurredat theheavychain locusduring inbreeding.ThegenomicsequencesofthehingeregionandrabbitCKwerenotfoundalthoughtherearesomeVKsequencesinthetracearchive.Linksatthe Rabbit Genome Resources sitehttp://www.ncbi.nlm.nih.gov/proj-ects/genome/guide/rabbit/tosearchesforgenesintheassembliesofthe2xWGSsequenceatEnsemblandUCSC(BLAT)arevaluablefordiscoveringpredictedmRNAsequencesandexonboundariesusefulindesigningprimersforquantitativereversetranscriptasePCR.Afullrabbitgenomesequencewillaiddiscoveryofgeneticcontributionstoanimalandhumandiseasesusceptibilities.Key words:PedigreedNIAIDRabbits,immunoglobulinallotypes,2xto7xcoverage,genomeassembliesSpecies: rabbit

ig015. GENOMIC ORGANIZATION AND ExPRESSION OF T CELL RECEPTORS (TCR) IN THE SOUTH AMERICAN

OPOSSUMZULyEPARRA,MICHELLELBAKER,JonATHAnTRUJILLo,

APRILLoPEZ,ALAnASHARP,JEnnIFERHATHAWAy,RoBERTDMILLER

DepartmentofBiology,UniversityofNewMexico,AlbuquerqueNM87131USA.

Tcellsplayanimportantroleinregulationandeffectorfunctionsintheadaptiveimmuneresponseofall jawedvertebrates.TherearetwosubsetsofTcellsaccordingtotheirantigenreceptors:aβTcellsandγdTcells.HerewepresentananalysisofthecompletegenomiccontentandorganizationoftheTCRlociinamodelmarsupialspecies.The SouthAmerican opossum (Monodelphis domestica) is used asmodelforbothUVinducedmelanomaandisreservoirfortheparasitethatcausesChagasdiseaseinhumans.Betterunderstandingofopos-sumTcelldiversitywould improvetheirutilityforsuchresearch.WefoundthattheorganizationandcomplexityoftheopossumTCRaandd loci are similar to that of human, withTCRd nested withinTCRa.Similarly,theopossumTCRβlocusresemblesthatofhumans,butwithtwoadditionalclustersofgenesegments.TheopossumTCRγ locushasatransloconorganization,differentfromthatofhumanwhereitisclustered.Aswereportedrecently,opossumshaveafifthTCR(TCRµ)whichdoesnothaveahomologueinplacentalmammals.TCRµoccu-piesadistinctlocusfromthoseencodingtheconventionalTCRsandithasaclusterratherthantheusualtransloconorganization.Opossums

likeallmarsupialsgivebirthtorelativelyimmatureyoungsoweinves-tigatedthetimingofTCRexpressioninontogeny.WefoundthatsomeTCRs are expressed as early as postnatal day 2, which appears toprecede thymusdevelopment.WithineachTCR isotype there isdif-ferentialexpressionofVsubgroupsatdifferenttimesofdevelopment,consistent with changes in repertoire diversity during development.Overall, thecontentandgenomicorganizationofconventionalTCRsintheopossumissimilartowhatisknownforplacentalmammalswithagreatpotentialtoexpressdiversifiedantigenreceptors.Inaddition,afifthTCRcouldrepresentanovelspecializedsubsetofTcellspresentinmarsupials.Key words:TCell,TCR,Opossum,marsupialSpecies:others

ig016. GENERATION OF PAIRED IMMUNE RECEPTORS By GENE CONVERSION WITHIN THE CANINE CEA GENE

FAMILy. RoBERTKAMMERER1,2,TAnJAPoPP1,STEFAnHäRTLE3,

BERnHARDBSInGER4,WoLFGAnGZIMMERMAnn11TumorImmunologyLaboratory,LIFECenter,KlinikumGrosshadern,LMU,Munich,Germany;2InstituteforMolecularImmunology,GSFNationalResearchCenterfortheEnvironmentandHealth,Munich,Germany;3InstituteforAnimalPhysiology,LMU,Munich,Germany;4InstituteforAnatomy,UniversityHospitalEssen,Essen,Germany

Immunecellsurfacereceptorssharinghighlysimilarextracellulardomainsandhavingcounteracting(activatingandinhibitory)signalingpropertiesarecalled“pairedimmunereceptors”.ThereisevidenceforpairedimmunereceptorswithintheKIR(Killercell Ig-likeRezeptors)andtheLy49receptorfamilies,thattheinhibitoryreceptorsareances-tralandtheactivatingreceptorsevolvedfromtheinhibitoryreceptorsbymutationtocounteractpathogenattack.Thisviewissupportedbythefinding, that themurinecytomegalovirus(MCMV)-encodedm157glycoprotein binds to the NK cell inhibitory receptor Ly49 and sup-pressanti-viralimmuneresponse,whilemiceexpressingtheactivatingreceptorLy49HareresistanttoMCMVinfection.Morerecently,pairedimmunereceptorswerealsofoundwithin theSiglecfamily.Thispairofreceptorshasundergoneconcertedevolutionviageneconversioninmultipleprimatespecies,however thedifferentdirectionsofgeneconversionbetweeninhibitoryandactivationreceptorsarguesagainsta pathogen-driven evolution. We have analyzed the evolution ofpaired immunereceptorswithin thecarcinoembryonicantigen(CEA)genefamily.OneoftheprimordialmembersoftheCEAfamilyistheITIM-bearingCEACAM1,whichbothregulatesimmuneresponsesandservesasacellularreceptorforpathogens,inhuman,miceandcattle.In the dog, theCEACAM1genegave rise to a recent expansion oftheCEAfamily,similartothatpreviouslyfoundinhumansandmice.However,whilethemurineandhumanCEACAM1-relatedCEACAMsare predominantly secreted and GPI-anchored, respectively, in thedog, most CEACAMs represent ITAM-bearing transmembrane pro-teins.TheN-domain of one of these proteins,CEACAM28, exhibitsnearly complete sequence identitywith the ligand-bindingN-domainof CEACAM1 but with antagonizing signaling motifs. PhylogeneticanalysesofthecanineCEACAMssuggestedthatthehighsequencesimilarityoftheN-domainsisduetoapartialgeneconversion,inwhichthe inhibitory receptor CEACAM1 converted the activating receptorCEACAM28.Comparisonofnonsynonymousandsynonymoussubsti-tutionsindicatesthattheCEACAM28N-domainisunderthestrongestpurifyingselectionofallcanineCEACAM1-relatedCEACAMs.Inaddi-tion,CEACAM28showsasimilarexpressionpatterninrestingimmunecells asCEACAM1.However, upon activationCEACAM28 is down-regulatedinTcellswhileCEACAM1isupregulated.ThusCEACAM1andCEACAM28arethefirstpairedimmunereceptorsidentifiedwithintheCEAgene family,most likely involved in thefine tuningofTcellresponses.Thedirectionofgeneconversionaccompaniedbyapurify-ingselectionsuggeststhepossibilitythatthegenerationofCEACAM28waspathogen-driven.Key words: cell surface molecules, costimulation, comparativeimmunology,carcinoembryonicantigenSpecies:canine

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ig017. GENETIC ANALySIS OF PORCINE TLR GENESInGRID-MARIABERGMAn1,AMELIEJoHAnSSon1,CARoLInE

FoSSUM2,LEIFAnDERSSon3,4,InGEREDFoRS-LILJA11SchoolofPureandAppliedNaturalSciences,Universityof

Kalmar,Kalmar,Sweden,2DepartmentofBiomedicalSciencesandVeterinaryPublicHealth,and3DepartmentofAnimalBreedingandGenetics,SwedishUniversityofAgriculturalSciences,Uppsala,

Sweden,4DepartmentofMedicalBiochemistryandMicrobiology,UppsalaUniversity,Uppsala,Sweden

Ingrid-Maria.Bergman@hik.seQuantitative trait loci (QTLs) influencing leukocytenumbersand

functionsandother immunerelatedparametershavebeen identifiedonpigchromosomes8(SSC8)and1(SSC1).TLRs1,2,4,6,and10areamongthecandidategenesmappedtotheseregions.TLRpoly-morphismsassociatedwithsusceptibility to infectiousdiseaseshavebeenfoundinmanandpig1.Severalofthesepolymorphismsaresinglenucleotidesubstitutions,butapolymorphicmicrosatellitewithinfluenceon invitroresponsehasalsobeenfoundupstreamthehumanTLR2gene2.Theaimof thisproject is toexploregeneticpolymorphism inporcineTLR genes and to relate these to functional studies in vitroandinvivo.PrimersweredesignedfortheupstreamregionofporcineTLR2andamicrosatellite(HIK10)wasidentifiedapproximately100bpupstreamtranslationstart.TwosetsofpigswereusedtoestablishageneticlinkagemapincludingTLRs1,2,6and10:47backcross(BC2)offspringofaBC1boar,originating fromaWildBoar (W)xSwedishYorkshire (Y) pedigree, and 191Swedish Landrace (L) xY crossesafter six L boars.Also, to compareTLRpolymorphismamongst dif-ferent breeds, additional tissue samples have been collected. Thefamilies were genotyped for the KIT gene and 14 microsatellites onSSC8.A linkagemapwasconstructedusing theCRI-MAPsoftware.Basedonrecombinationbetweenmarkers,therelativeorderSw1101–Sw1037–SJA7,SJA10,SJA11(TLR1,6,10)–Sw444–HIK2,HIK3,HIK4–KIT–S0086–Sw1679–SJ108(IL8)–S0069–S0225–HIK10wasdetermined.Severalnon-synonymousSNPsinTLR1,2,4,5and6havebeenfoundinastudycomprising11pigbreeds3.Theseaswellas thepolymorphicmicrosatellites inpig IL8,within theTLR1-TLR6-TLR10cluster,andupstreamTLR2willenable furtherstudiesof theinfluenceofthesecandidategenesonimmunerelatedtraits.

References1. Muneta et al. 2003. J Interferon Cytokine Research 23:

583-5902.Yimetal.2004.FEMSImmunologyandMedicalMicrobiology

40:163-1693.Shinkaietal.2006.Immunogenetics58:324-330

Key words:TLR,Gene,Polymorphism,InnateimmunitySpecies:swine

ig018. DIFFERENCE OF GENOMIC STRUCTURE AMONG HAPLOTyPES OF SWINE LEUKOCyTE ANTIGEN REGION

HIRoHIDEUEnISHI1,3,MAIKoTAnAKA2,3,TAKASHIAWATA1,3,ASAKoAnDo4,PATRICKCHARDon5

1NationalInstituteofAgrobiologicalSciences(NIAS),Tsukuba,Ibaraki,Japan;2InstituteofSocietyforTechno-InnovationofAgriculture,ForestryandFisheries(STAFF-Institute),Tsukuba,

Ibaraki,Japan;3AnimalGenomeResearchProgram,NIAS/STAFF,Tsukuba,IbarakiJapan;4TokaiUniversitySchoolofMedicine,Isehara,Kanagawa,Japan;5InstitutNationaldeRecherche

Agronomique(INRA),Commisariatal’EnergieAtomique(CEA),JouyenJosas,France

huenishi@affrc.go.jpSwinemajorhistocompatibilitycomplex (MHC),alsodesignated

as swine leukocyte antigen (SLA) region, is thought to have strongassociation with traits related to disease resistance. Elucidation ofassociationbetweenallelesofSLAgenesandthetraitsrequirespre-cise knowledge of the genomic structure of this highly polymorphicregion, and an efficient genotypingmethod for the SLA genes. Theentiregenomic regionofSLAwas recentlyelucidated inaparticularhaplotypenamedH01.However,ithasbeensuggestedthatthenum-berofSLAlocivariesaccordingtotheSLAhaplotypes.Toclarifysuchvariance,wesequencedthegenomicsegmentencodingSLAclassical

classIgenesfromtwodifferenthaplotypes.ThetwoSLAhaplotypesbelongedtoasingleLandraceindividualinwhichmicrosatellite-basedgenotyping suggested alteration of the SLA region in regard to thereferenceH01haplotype.Foreachof thetworelevantsegmentsweconstructedacontigofbacterialartificialchromosome(BAC)usingalibrarymadewithgenomicDNAfromtheselectedindividual.Southernblot analysis of the genomic DNA and the BAC containing contigsindicatedtherelevanceofcontigassembly.BACsequencesrevealedtheincrementofclassicalclassIgenesofsixandtwointherespec-tiveSLAhaplotypes.RT-PCRwithprimerstoamplifyspecificallyeachclassicalclassI locusindicatedthatthenumberofexpressedSLA-1orSLA-3geneswereseven,at least, in the individual,whereas twoSLA-1orSLA-3geneswereexpressed ineachchromosomeof theH01haplotype.TheprocessoftheduplicationofSLAclassicalclassI geneswere estimated usingDNA transposon-like sequences, andcharacteristicmicrosatelliterepeatsadjacenttotheduplicatedclassIgenes.Furthermore,sequencingof thegenomicregion fromDRAtoDOB inSLAclass II region revealed thecopynumbervariancealsoinDRBgenesbetweenhaplotypes.ThisstudyrevealedthedifferenceofstructureamongSLAhaplotypesbothintheclassIandIIregions,andimplieddifficultyofestimationofSLAhaplotypesincommercialpigbreedsbygenotypingofSLAgenesperse.Weproposeanalternativemethodofestimationofhaplotypesusingmicrosatellitemarkersdistrib-utedthroughout theentireSLAregion,anddemonstrateanexampleof thesignificantassociationbetweenantibody responsesafter vac-cinationagainstopportunisticinfectionsandparticularSLAhaplotypesreconstructedbymicrosatellitemarkersinpigs.Key words:MHC,SLA,genomics,copynumbervariationSpecies:swine

ig019. COMPARATIVE TRANSCRIPTOMIC ANALySIS OF PRV-HOST CELL INTERACTIONS IN PIG

FLoRILAUREnCE1,MARIAnIVALEnTInA2,CHARDonPATRICK1,LEMonnIERGAéTAn1,LEFEVRE

FRAnçoIS3,RoGEL-GAILLARDCLAIRE11INRACEA,LaboratoiredeRadiobiologieetEtudeduGénome,Jouy-en-Josas,France;2ParcoTecnologicoPadano,Lodi,Italy;

3INRA,LaboratoiredeVirologieetImmunologieMoléculaires,Jouy-en-Josas,France

Thepseudorabies(PrV)istheetiologicagentoftheAujezskydis-ease.ThediseasewaseradicatedthankstovaccinationbutthePrVisstillanexcellentmodeltosetupinvitrosystemstostudyinteractionsbetweenHerpesviruses and host cells due to efficient infection andpropagation in cell cultures. In vivo the first target cells aremucousepithelialcellsandimmaturedendriticcells(iDCs)thatfurtheractivateadaptive immune response.Thedialog betweenPrVand these twocelltypesareexpectedtodiffer.Theaimofourworkwastoanalyzethese differences during time course of infection and we performeda transcriptomeapproach thatallowedus tosimultaneouslyanalyzecellular and viral gene expression. Epithelial PK-15 cells and swineiDCsthatwereinvitrodifferentiatedfrombloodmonocytesusingIL-4andGM-CSFwereinfectedbythePrV.Infectedormock-infectedcellswerecollected0,1,2,4,8and12post infection(pi) forPK-15or0,12,18and24hourspi for iDCs.TotalRNA transcriptswere labeledandhybridizedontoDNAchipscomprising80PrVampliconscoveringthewhole viral genome and a set of 1663 cellular genes, including420 genes mapping to the extended major histocompatibility (MHC)locus, 73 immune genes outside the MHC and 1170 randomly cho-sengenes. InPK-15 cells, a high increase in viral geneexpressionwasfound4hourspiandmostviralgenesweredetecteddifferentiallyexpressed12hpiinbothcelltypes.Noearlyglobalcellulargeneshutoffoccurredandthehighestnumberofdifferentiallyexpressedcellulargenewasobserved8hpiinPK-15cells.TheresultsshowedthatMHCclassIgenesweredown-regulatedinbothinfectedcelltypesandthatMHCclassIIgeneswerealsodown-regulatediniDCs.Genesthatareinvolvedinotherpathwayssuchasapoptosis,proteinmetabolismandmodificationwerealsoidentifiedasdifferentiallyexpressed.RealtimequantitativePCRexperimentsconfirmedthedown-regulationofMHCclassIagenes,aswellasTAP1,TAP2,LMP2andLMP7allinvolvedinclassIantigenpresentationpathway,thedown-regulationofcyclophilinAandtheup-regulationofTNFAininfectedPK-15cells.Validationofthedown-regulationofclassIIantigenpresentationpathwayininfected

59

iDCs is inprogress.Thepresentcomparativestudywillprovidenewdataontime-dependantdifferencesaccordingto thehostcellduringPrVinfection.Key words:PrV,transcriptome,MHC,dendriticcellsSpecies:swine

ig020. GENOMIC ANALySIS REVEALED THE DUPLICATION MODEL OF PORCINE CD1 GENES DURING EVOLUTION

ToMoKoEGUCHI-oGAWA1,2,TAKEyAMoRoZUMI1,3,MAIKoTAnAKA1,3,HIRoKISHInKAI,1,3nAoHIKooKUMURA1,3,

KoHEISUZUKI1,3,TAKASHIAWATA1,2,HIRoHIDEUEnISHI1,21AnimalGenomeResearchProgram;2DivisionofAnimalSciences,

NationalInstituteofAgrobiologicalSciences,2-1-2Kannondai,Tsukuba,Ibaraki305-8602,Japan,3SecondResearchDivision,

STAFF-Institute,446-1Ippaizuka,Kamiyokoba,Tsukuba,Ibaraki305-0854,Japan

egutomo@affrc.go.jp(T.Eguchi-Ogawa)CD1 is anMHC class I-like protein that presents lipid antigens

to T cell receptors. To clarify the variety and genomic structure ofporcineCD1gene,weconstructedabacterial artificial chromosome(BAC)contig,anddetermined470,187bpoftheregionencodingtheCD1genes.Weidentified16genesinthisregionandnewlyidentifiedCD1A2,CD1B,CD1C,CD1D,andCD1Einadditiontoformerlyidenti-fied porcineCD1 gene,CD1.1 (CD1A1, homolog of humanCD1A).RT-PCR analysis showed CD1A1, CD1B, CD1D and CD1E wereexpressed,andsuggestedbothgroup1(CD1A1,CD1B,CD1E)andgroup2(CD1D)CD1genesarefunctionedinpig.Tyrosine-containingmotif involved inCD1intracellular traffickingwasconservedintheCterminiofporcineCD1BandCD1D.TheCterminusofporcineCD1bsharedasimilarmotifwithhumanCD1d,andporcineCD1dhadsimilarmotifwiththatofhumanCD1b;therefore,porcineCD1dmaycomple-mentthefunctionofhumanCD1binpigs.Southernblothybridizationwithseveralbreedsofpiggenomewasperformedusing theexon4sequence encoding the C-like-domain [D3] of CD1.1 as a probe.Althoughslightdifferenceinrestrictionsiteswasobservedamongthebreeds,we confirmed that nomore than sixCD1 genes existed ontheporcinegenome.GenomicsequenceanalysisshowedthatporcineCD1geneswere located in clusters betweenKIRREL and olfactoryreceptor (OR) genes, as observed in humans, although they weredividedintotworegionsbyaregionencodingORgenes.ComparisonofthegenomicstructureencodingCD1genesinpigswithothermam-mals showed that separation of the CD1 gene cluster by ORs wasobservedonly inpigs.To investigate theprocessofevolutionof theregionwheretheporcineCD1genesarelocated,weidentifiedcharac-teristicrepetitivesequencescommonlyfoundclosetothehumanandporcineCD1Agenes.Byestimationof the timeofCD1Aduplicationbyusing the repetitivesequences,weconcludeCD1Aduplication inthe porcine genomemight occur after the divergence of the humanandporcine.WeconstructedtheschematicmodelofporcineCD1andORgeneduplication,whichindicatedthattheuniquesplitstructureoftheCD1clusterinthepighadbeenestablishedbeforetheshufflingoftheORgenesintheartiodactyllineage.ThisanalysisofthegenomicsequenceoftheporcineCD1familywillcontributetoourunderstand-ingoftheevolutionofmammalianCD1genes.Key words:CD1Species:swine

ig021. SWINE IMMUNITy AND GENETIC RESISTANCE TO PORCINE REPRODUCTIVE AND RESPIRATORy SyNDROME

VIRUS (PRRSV) INFECTION JoAnKLUnnEy1,DEREKPETRy2,3,RoDGERJoHnSon2,DAnIELKUHAR1,RAMonMoLInA4,JAnECHRISToPHER-

HEnnInGS5,JEFFREyZIMMERMAn4,,RRRRoWLAnD61APDL,BARC,USDA,Beltsville,MD;2Univ.ofNebraska,Lincoln,NE;3TriumphFoods;4IowaStateUniversity,AmesIA;5South

DakotaStateUniversity;6KansasStateUniversity,Manhattan,KS.jlunney@anri.barc.usda.gov

Current vaccines are only partially effective against PorcineReproductive and Respiratory Syndrome (PRRS) virus infectionbecausetheyelicitaweak immuneresponsethat isnot fullyprotec-tive. PRRS is the most economically significant disease facing the

swineindustrytoday,costingU.S.porkproducersatleast$560millionannually. Despite substantial research efforts the exact componentsofaprotectiveanti-PRRSVimmuneresponsearestillnotknown,thuswearetestingalternateapproachestoevaluateimmunityandgeneticresistance to PRRSV. We used host genomics to compare differentlines of pigs and look for factors that correlated with PRRSV resis-tance/susceptibility. Viremia, weight change, and rectal temperatureat 0, 4, 7, and 14 days post-PRRSV infection (dpi) were recordedand genetic differences detected (Petry et al., 2005).We evaluatedimmunegeneexpressioninRNAfromfrozenlungandbronchiallymphnode (BLN) tissue of the 7 highest and lowest responders per line,and fromeachof theircontrol littermates,aswellasserumcytokineprotein levels.Genetic analyses of this data indicated that levels ofinterleukin-8 (IL8) may be predictive of resistance.Additionally, low(nottheexpectedhigh)levelsofseruminterferon-gamma(IFNG)afterinfectionmaybeassociatedwithaPRRSVresistantphenotype.ThesedataarecriticalforgeneticassociationstudiestofinemapcandidategenesanddeterminecausativeallelesofPRRSVresistance/suscepti-bility.Furthergeneticstudiesarerequiredtoaffirmtheseassociations.Fordirectimmunitystudieswe’veassessedimmunegeneexpressioninlung,BLN,andtonsilsamples,andproteinexpressioninserum,col-lectedfrompigsinfectedforover200daysafterPRRSVinfection.Wecomparedpigsthatapparentlyclearedtheviralinfectioninthefirst28dpitopigsthatevenat150dpihaveevidenceoflongtermpersistentPRRSVinfection.ResultsshowthatthereisupregulationofexpressionofIFNGassociatedThelper1(Th1)markersfrom14to84dpi;regula-toryIL10andapoptosisassociatedmarkersarealsoincreasedearly.To date, however, no significant differences between persistent andnon-persistentPRRSVinfectedpigshavebeendiscoveredinimmunegeneexpression;serumproteinexpressionstudiesareunderway.WehopetorevealdifferentialproteinexpressionassociatedwithPRRSVclearance.Overall,bycombiningthesediverseapproaches,weexpecttodevelopnewhypothesesaboutprotectiveanti-PRRSV responsesandtoidentifynovelregulatorypathwaysthatwouldstimulatePRRSVimmunity.SupportedbyUSDAARSandNRIPRRSCAP1funds.Key words: PorcineReproductive andRespiratorySyn, resistance/susceptibility,immunegeneexpression,cytokineregulation.Species:swine

ig022. IDENTIFICATION OF BOOPHILUS MICROPLUS PHAGOTOPES FROM PHAGE DISPLAyED PEPTIDE

LIBRARIES.CARLoSRoBERToPRUDEnCIo1,ALInEAPARECIDAREZEnDE

RoDRIGUES,GUILHERMERoCHALInoSoUZA1,JULIAnAFRAnCoALMEIDA1,AnAPAULAPERESFRESCHI,RonE

CARDoSo1,FAUSToEMÍLLIoCAPPARELLI1,LUIZRICARDoGoULART1

1InstitutodeGenéticaeBioquímica-UniversidadeFederaldeUberlândia-MG;2-ValléeS/A.

crprudencio@gmail.comThe ticks cause serious economic losses to animal production

worldwide,intheorderofbillionsofdollars.Phagedisplaytechniqueshavebeenwidelyemployedtomaptheepitopestructureswhichhaveservedas the basis for developingmolecular vaccines. In the pres-entstudy,weappliedthistechniquetomaptheepitopesofBoophilusmicroplus and directly evaluated the immune responses in miceto verify immunogenicity of the selected phage-displayed epitopes(phagotopes).Sevenphage-displayedrandompeptide librarieswerebiopannedindifferentsituationsofstringencywiththepurifiedIgYofchikenanti-B.microplushyperimmuneserumandtheselectedphageclones were sequenced and analyzed. Some of the inserts of theselectedphagotopesshowedagoodmatchwith theknownproteinsofB.microplus.Others,whichdidnotmatchwithanyknownproteins,but sharedextensivehomologywitheachother,wereclusteredandclassifiedastheconformationalepitopesofB.microplus.Toevaluatethepotentialofusingthesephagotopesaseffectivevaccines,severalphagecloneswerechosentoimmunizemice.Theserumraisedbythephageclonesclearlyrecognizedtickproteinsindicatingthatthephago-tope-induced immune responseswere antigen-specific. The presentwork demonstrates that the whole epitope profile can be obtainedthroughscreeningthephagedisplayedpeptidelibrarieswiththehyper-

60

immuneserumand reveals thepotential of usingepitope-displayingphagesaspeptidevaccines.

Finacialsupport:Finep,ValléeS/A,Cappes.Key words:epitopeprofile,B.microplus,PhageDisplay,Vaccine.Species:other

ig023. CONSTRUCTION OF AN ANTIBODIES LIBRARy (SCFV) FOR SELECTION AND CHARACTERIZATION OF

BOOPHILUS MICROPLUS ANTIGENSGUILHERMERoCHALInoDESoUZA1,CARLoSRoBERTo

PRUDEnCIo1,RonECARDoSo1,JULIAnAFRAnCoALMEIDA1,FAUSToEMÍLLIoCAPPARELLI1,AnAPAULAPEREZFRESCHI1,

AnDRéAqUEIRoZMARAnHÃo2,MARCELoDEMACEDoBRÍGIDo2,LIZIAnEMARIADELIMA3,MARCELoBEMqUERER4,

LUIZRICARDoGoULART11InstitutodeGenéticaeBioquímica-UniversidadeFederal

deUberlândia-MG;2DepartamentodeImunologiamolecular-UniversidadedeBrasília(UnB)-DF;3EmpresaBrasileiradePesquisa

Agropecuária,CentroNacionaldePesquisadeAlgodão,CampinaGrande-PB;4InstitutodeCiênciasBiológicas,DepartamentodeBioquímicaeImunologiaUniversidadeFederaldeMinasGerais

(UFMG)-MGgrlino@gmail.com

TheBoophilusmicroplustickisoneofthemostimportantarthro-podsthatcanparasitizebovines,causinggreatdamagestotheworldlivestockthroughdirectandindirecteffects.Theapplicationofchemi-calproductsistheprincipalmethodofcontrollingthisparasite,butinfunctionofthedisadvantagesofthispractice,theuseofvaccinesisagoodalternative,becausetheyareresiduefree,specificandpresentlowerpossibilityofdevelopingresistance.Withtheobjectiveofselect-ingandcharacterizingnewproteictargetsasvaccineagainstticks,wedeveloped a combinatorial antibody library (scFv), expressed in thecapsid of bacteriophages, produced from chickens immunoglobulinsdiversitypreviouslysensitizedwith total larvalandadultsproteinsoftheBoophilusmicroplus.Theseantibodies(scFv)recognizeaproteinofthetotallarvalandadult’sextractoftheproximally80kDa,bywest-ern blotting tests, and the sequence of these reactive proteins wascheckedbyN-terminalsequencing.ThebandcorrespondedtoaGP80proteinwithhighsimilarity(91%)andshownhugequantitiesinparasiteeggsofadultsandlarvalstages.Withtheobjectiveofselectingmimo-topes of B. microplus by phage displayed epitope characterization,therecombinantantibodieswithmajorfrequencyweresubmittedtoaselectionagainstaconstrictedpeptidelibrarydisplayedonphages.Thecrossreactivitywasconfirmedbytherecognitionofarecombinantanti-body(scFv)toacloneexpressingthemotifsimilartoGP80sequenceobtainedfrompeptidelibraryselection.ThisworkconfirmstheGP80asavaccinecandidateandrepresentstheselectionefficiencyofnewvaccinetargetsbyphagedisplayedlibrariesmethodologiestocontrolB.microplus.

Financialsupport:CNPqKey words: Phage display, Antibody libraries, Peptide libraries,Boophilusmicroplus.Species:other

ig024. ExPRESSION OF CEMENT-LIKE PROTEINS IN TRANSCRIPTOMES FROM TICKS FED ON RESISTANT AND

SUSCEPTIBLE CATTLE.MARUyAMASRC1,GARCIAGR1,BRAnDÃoLG1,RIBEIRoJMC2,AnDERSonJM2,VALEnZUELAJG2,FERREIRABR1,

DEMIRAnDASAnToSIKF11DepartamentodeBioquímicaeImunologia,FMRP-USP,Ribeirão

Preto,Brasil;2NIAID-NIH,Rockville-MD,USA.IntroductionandObjectives:Rhipicephalus(Boophilus)micro-

plus,thecattletick,causesenormouslossesforanimalproductionandhealth.Ticksinduceimmuneresponsesintheirhosts,indicat-ing that their immunobiological control is possible. Bovines pres-entdifferentphenotypesrelatedtointensityoftickinfestationsandthose phenotypes are mediated by qualitatively distinct immuneresponses.Ticksfedonresistantbovinesdonotfeedwellanddis-

playlowreproductiveefficiency.Inorderfortickstoefficientlyinfesttheir hosts, they must produce cement-like proteins that ensureattachmentandblood-feeding.Ourhypothesisisthatdifferentlev-elsofhostanti-tickimmunityaffectgeneexpressionincattleticks.Theobjectiveistoevaluateifandhowtheexpressionofcement-likeproteins isaffectedby immuneresponsesof tick-susceptible andresistantbovinehosts.

Methods and Results: cDNA libraries were constructed withSMART (Clontech-BD) technology andmRNA fromsalivary glandofnymphs,maleand female ticks fedonsusceptibleor resistant.CloneswererandomlyselectedforPCRamplificationandtheDNAinsertsweresequencedandanalyzedwithbioinformatic toolsthattrim ESTs of primer and vector sequences, clusterize them intocontigs and confront them against the NCBI non-redundant (NR)proteindatabaseandaprivateonecontainingsequencesforAcari.Developmentalstagesobtainedfromsusceptiblehosts(RmS)gen-erated4418ESTs,whilethosefromresistanthosts(RmR)generated2875ESTs.TheESTscombined fromalldevelopmentalstagesofRmSpresented618ESTs(expected574)similartocementproteins,whileESTsfromRmRexhibitednumberedonly322(expected365;P=0.003,χ2test).MaleswerethedevelopmentalstagepresentingthehighestexpressionofESTssimilartocement-likeproteins(596),but therewere no significant differences betweenRmSandRmRmales.However,RmSfemalesandnymphscontainmoreESTssim-ilartocementproteinsthanRmN,therebeing54ESTsfromfemaleRmS(expected36)and14ESTsfromfemaleRmR(expected30;P<0.001),279ESTsfromnymphsRmS(expected162)and29ESTsfromnymphsRmR(expected114;P<0.001).

Conclusions:Ourdataindicatethatthehostimmuneresponseoftick-resistantcattlenegativelyaffectsexpressionofgenescoding forcement-likeproteinsinR.microplusandthatthiscanbepreventingtheattachmentandfeedingofticksonthesehosts.

FinancialSupport:CNPq,FAPESPandValléeSA.Key words:Rhipicephalusmicroplus,transcriptome,salivaryglands,cement-likeproteins,vaccine.Species:others

ig025. CONSERVATION OF PEPTIDES 4822 AND 4823 CONSTITUENTS OF SyNTHETIC VACCINE SBM7462

AGAINST RHIPICEPHALUS (BOOPHILUS) MICRoPLUSAnAPAULAPEConICK,FLáVIAARAúJoGIRÃo,SIDIMARSoSSAI,MARInAqUADRIoRAPoUSo

BRAnCoRoDRIGUES,BREnoSoUZASALGADo,CARLoSHEnRyqUESoUZAESILVA,CARLALEITEMEDEIRoS,

HUGoGUIEIRoRIBEIRoRoCHA,KARLoSHEnRIqUEMKALKS,JoAqUÍnHERnAnPATARRoyo

LaboratoryofBiologyandControlofHaematozoaandVectors,InstituteofBiotechnologyAppliedtoAgricultureandAnimalScience(BIOAGRO/VeterinaryDepartament),FederalUniversityofViçosa,

36571-000,Viçosa,MG,Brazil.jpatarro@ufv.br

Rhipicephalus(Boophilus)microplusisoneofthemostimportantparasiteofcattleinCentralandSouthAmericaandAustraliafromaneconomical point of view. Vaccines derived fromBm86 glycoproteinhaveagreatpotentialofnon-chemicalcontrolofticks.TheSBm7462isasyntheticvaccinederivedfromBm86andhasthreeimmunogenicepitopes: 4822 (a.a. 398-411), 4824 (a.a. 123-145) and 4823 (a.a.21-35).Theknowledgeabout the conservationof thebm86gene isvery importanttoevaluateefficiencyofSBm7462.TwentysixR.(B.)microplus strains from Argentina, Colombia, Uruguay and variousregions from Brazil were analyzed for the bm86. Two fragments ofcDNAwereamplified, fragmentA (among thenucleotides39–438)andfragmentC(amongthenucleotides839-1600).Theywereclonedinto thepGEM-T®vectorand four clonesweresequenced foreachpopulation.Thenucleotidesanddeducedaminoacidsequenceswerecomparedwith thebm86andbm95genes.TheanalysiswasmadethroughalignmentofmultiplesequencesbytheprogramBioEditver-sion7.0.5.3and thepolymorphismsverification forvisual inspection.The results demonstrated the genetic conservations of the peptides4823and4822 foranalysedsamples. Insideof thegenebm86, the

61

aminoacidsvariabilitywasof5,49%and3,89%comparedwithBm86andBm95,respectively.Key words:peptides,vaccine,Rhipicephalus(Boophilus)microplus.Species:other

ig026. COMPARATIVE ANALySIS OF TRANSCRIPTOMES OF SALIVARy GLANDS FROM TICKS, AMBLyoMMA CAJENNESE AND RHIPICEPHALUS SANGUINEUS.

AnATRIELLoE1,FERREIRABR1,BRAnDÃoLG1,VALEnZUELAJG2,RIBEIRoJM2,SILVAJS1,DEMIRAnDASAnToSIKF1

1Dept.BiochemistryandImmunology,USP–RibeirãoPreto–SP,Brazil;2VectorMolecularBiologyUnit,LaboratoryofMalariaand

VectorResearch,NIAID,NIH–Bethesda–MD,USAIntroductionandobjectives:Ticksarehematophagousarthropod

vectors of disease.Vaccines are an alternative for their control, thepremise being that infestations with these parasites stimulate hostimmuneresponses,whichare implicated in theirrejection.Thetick’ssalivaryglandsareimportantforacquiringbloodmealsandforcounter-ingthehost’sdefenses.Inordertoelucidatethebiologyofthetick-hostinterfaceanddiscoverprotectiveantigensforamulticomponentvac-cine,transcriptomesofsalivaryglandsofthehorsetick,A.cajennense,anddogtick,R.sanguineus,arebeinganalyzed.

Methods andResults: Salivary glands frompartially fed femaletickswereused toobtainmRNA inorder to constructaPCR-based

library in lambda phages with theSMARTcDNAkit (Clontech).Theresultingplaqueswere randomlyselected forPCRamplificationandtheDNAinsertsweremasssequencedandanalyzedbybioinformat-ics.AcDNAlibrarywasconstructedfromsalivaryglandsofbothticks.Randomlyselectedclonesweresequencedandatotalof1803wereanalyzedbybioinformaticsprograms.Thesequencesweregroupedinto 867 clusters, which were confronted against the following data-bases:NCBInon-redundantprotein,GOandKOG.About90%ofthemRNA sequences showed significant similarity to known proteins inthenon-redundantproteindatabasebytheNCBIblastxprogramandappeared to be coding for functional predited proteins, whereas theremaining 10% had no similar sequences andmay represent novelgenes.

Discussion:Among the predicted protein sequences, we foundsimilaritiestocements,proteaseinhibitors,anticoagulants,metallopro-teases,anti-inflammatorymolecules,andpotentimmunosuppressants.Acomparativeanalysisofthetwolibrariesledtoidentificationofsev-eraltranscriptsthatarecommontobothspeciesofticks,suggestingthatcross-reactiveprotectiveantigensmayobtained.

Financialsupport:FAPESP,CNPqandValléeSA.Key words: Dog Tick, Horse TickTranscriptomes, ComparativeAnalysis,SalivaryGlands,VaccineSpecies:other

bV027. LONG-TERM STAPHyLOCOCCAL ENTEROTOxIN C1 ExPOSURE INDUCES SOLUBLE FACTOR MEDIATED

IMMUNOSUPPRESSION.KEUnSEoKSEo1,SAnGUnLEE1,yonGHoPARK2,WILLIAM

CDAVIS3,LAWREnCEKFox4,GREGoRyABoHACH11DepartmentofMicrobiology,MolecularBiologyandBiochemistry,

UniversityofIdaho,Moscow,ID83844;2DepartmentofMicrobiology,CollegeofVeterinaryMedicineandSchoolofAgricultural

Biotechnology,SeoulNationalUniversity,Seoul,151-742,Korea;3DepartmentofVeterinaryMicrobiologyandPathology,Washington

StateUniversity,Pullman,WA99164,USA;4DepartmentofVeterinaryMedicine,WashingtonStateUniversity,Pullman,WA

99164,USARegulatoryTcells(Tregs)helpcontroldevelopmentandmaintenance

of protective immunity and can lead to aberrant immune responsesto somepathogens.Several lines of evidence suggest thatTregs areinducedbyexposuretosuperantigens(SAgs)invitroorinvivo.Inthisstudy,bovinePBMCswereexposed invitro, toarelatively lowdose(5ng/ml)ofstaphylococcalenterotoxinC1(SEC1)forupto10days.Uponstimulation,CD4+andCD8+Tcellsinitiallyproliferatedatsimilarrates.Subsequently,fromdays6through10,mostCD4+andCD8+TcellsproliferatedregardlessofVβspecificity,butproliferationofCD8+Tcellsoccurredmorevigorously.TranscriptionofCD25andCD152genes increased, while that of IL-2 decreased. γd T cells appearedunresponsive. An increase in the transcription of IL-10 and TGF-βgenesinSEC1stimulatedcultureswasattributedtotheCD4+CD25+Tcellsubpopulation.ExpressionofFoxp3mRNAalsoincreased,andwasaccompaniedbyup-regulationofCD152anddown-regulationofIL-2transcription,suggestingthatcellsinthissubpopulationareTregs.Functionally,SEC1-stimulatedCD4+Tcellssuppressedtheprolifera-tionofnaïvePBMCs in response toheat-killed-fixedS.aureus.Thesuppression was partially mediated by IL-10 and TGF-β, anothercharacteristicofcertaintypesofTregs.TheCD8+TcellpopulationalsosuppressednaïvePBMCsthroughanothermechanismnotmediatedbyIL-10orTGF-β.TheseresultsprovidefurtherinsightintopotentialmechanismsbywhichSAgscouldcontributetoevasionoftheimmuneresponse,affectingtheoutcomeofinfectionorcolonization.Key words:Superantigen,RegulatioryTcell,Foxp3Species:bovines

bV028. PHENOTyPIC AND FUNCTIONAL ANALySIS OF STAPHyLOCOCCAL SUPERANTIGEN-STIMULATED BOVINE

MONOCyTESJooyoUnPARK1,2,KEUnSEoKSEo2,WILLIAMCDAVIS3,

LAWREnCEKFox1,,GREGoRyABoHACH21DepartmentofVeterinaryMedicine,WashingtonStateUniversity,

Pullman,Washington99164;2DepartmentofMicrobiology,MolecularBiology,andBiochemistry,UniversityofIdaho,Moscow,Idaho83844;3DepartmentofVeterinaryMicrobiologyandPathology,Washington

StateUniversity,Pullman,Washington99164Staphylococcal enterotoxins are prototype microbial superanti-

gens(SAgs)whichelicitextensiveTcellproliferationinanMHCclassII(MHCII)dependentmanner.Althoughantigenpresentingcells(APCs)providecrucialsignalstoTcellsthroughMHCIIandotherco-stimulatorysurfacemolecules,littleisknownabouttheirphenotypesandfunctionsduringSAgs-inducedactivation.Inthisstudy,bovineperipheralbloodmononuclearcellswereexposedtoalowdose(5ng/ml)ofstaphylo-coccalenterotoxinC1(SEC1)forupto10days.ThecharacteristicsofSEC1-stimulatedmonocytes(SSM)wereanalyzedbyflowcytometryandquantitativereal-timePCR.SSMhighlyexpressedCD40,CD11b,

MHCII andCD69 butwere negative forCD1a,CD11a,CD11c, andCD172a.Importantly,25%ofcells lackedCD14.Furthermore,SSMsdeveloped longdendriteson their surface,suggesting theyareden-driticcells.PinocytosisbySSMswassignificantlyhigherthanthatbynaïve monocyte. Upon stimulation, transcription of proinflammatorycytokines(IL-1α,β, IL-6,TNF-α)andchemokines(CXCL1,2,3,and6,CCL2and5)rapidlyincreased(within24h)butgraduallydecreasedlater.However, transcriptionofβchemokines(CCL3,CCL8,SDF-1),responsibleformononuclearcellmigration,wassustained.Consistentwiththesedata,invitrocellmigrationassays,showingvigorousearlymigration of granulocytes, followed by subsequent mononuclear cellmigration,demonstratedthattemporallydifferentialcellmigrationwasmediatedbySSM-derivedchemokines.Theseresultssuggestthatthestimulation by SAgs differentiate peripheral monocyte into dendriticcells.Thesefindingsprovidefurther insight intothepotentialrolesofAPCsduringSAg-mediatedimmunomodulation.Key words:Dendriticcells;chemokines;macrophagesSpecies:ruminants

bV029. EVALUATION OF THE AG85 SPECIFICITy IN THE HUMORAL AND CELLULAR IMMUNE RESPONSE IN BOVINE

TUBERCULOSIS.EDIAnEBSILVA,MARIAIMoURA,MARCoAMSILVA,ARIoLDo

CARVALHoJR,AnDRéKIPnIS,AnAPJUnqUEIRA-KIPnISDepartamentodeImunologia,LaboratóriodeImunopatologiadas

DoençasInfecciosas,FederalUniversityofGoiás–UFG.edianeveterinária@hotmail.com

BovinetuberculosiscausedbyMycobacteriumbovisaffectbovine,equineandseveralotherdomesticanimalsaswellashumanbeings.Theworldpresentsaround50millionsofinfectedanimalsthatdirectlyaffects milk and meat productivity. Although the cellular immuneresponseisthemainprotectiveresponseelicited,someproteinsfromM.bovisinduceantibodiesformationandthereforecouldbeusedasadiseasemarkers.Thedetectionofcellularimmuneresponsebynitricoxideproduction(NO)helpstounderstandthemacrophageactivationandthepresenceofspecificantibodiescoulddeterminetheevolutionoftheinfection.InordertoidentifythespecificityofNOproductionbymononuclearcellsagainstAg85,tuberculinandBCGamongaBrazilianherd,itwasused13cattle,adultsHolsteinwhichwerediagnosedastuberculosispositiveusing intradermal tuberculin test (ITT)andsixnegativecontrolanimals.Twelvemlofbloodwerecollectedinheparintubes.Theperipheralbloodmononuclearcells(PBMC)wereobtainedandincubatedwithrecombinantAg85a(20µg/ml),tuberculin(5µg/ml)orBCG(10µg/ml).TwentyeightserumsampleswereanalyzedinanELISA (20 fromTB positive cattle and 8 samples fromTB negativeones). In theevaluationofNOproduction therewerenodifferencesbetween the PBMC cultures from infected animals stimulated withAg85,(5,72nM±3,19),tuberculin(5,77nM± 3,11),andBCG,(7,73nM±5,99)whencompared to thecontrolgroup [Ag85 (10,13nM±10,49),tuberculin(7,32nM±3,02),BCG(8,65nM±4,90)].Evaluatingthehumoral immuneresponse, itwasobservedhigher levelsof IgGamong ITT-positive cattle (1,18 ± 0,13) when compared to the ITTnegativecattle (0,89± 0,10) (p<0,05). Thereforeevaluationof NOproductioncannotbeused for the discriminationbetweennaturallyinfected bovines from healthy ones. In contrast, humoral immuneresponsewithrecombinantAg85isagoodcandidatetobeusedinthediagnosisofM.bovisinfection.Key words:Micobacteriumbovis,nitricoxide,recombinantantigenSpecies:ruminants

2. IMMUNE RESPONSES IN BACTERIAL AND VIRAL DISEASES; PRIONS AND BSE: POSTERS BV027-BV077

bV030. MyCoBACTERIUM PARATUBERCULoSIS SUPPRESSES CD40 SIGNALING INDUCED IL-12P40 AND

INOS GENE ExPRESSION IN BOVINE MONOCyTE-DERIVED MACROPHAGES.

SAnDRASoMMER,CHARLESBPUDRITH,CHRISCoLVIn,PAULMCoUSSEnS

Dept.ofAnimalScience,MolecularPathogenesis,MichiganStateUniversity,EastLansing,MI,USA.

Mycobacteriumaviumssp.paratuberculosis(MAP),thecausativeagentofJohne`sdisease,isafacultativeintracellularpathogen,resid-inginsubepithelialmacrophages.ClearanceofMAPcriticallydependsupon an appropriate pro-inflammatory and cytotoxic Th-1 immuneresponseleadingtoactivationand/orlysisofpersistentlyinfectedmac-rophagestopromotebacterialkilling.Workinvivohasshown,thattheappropriateTh-1immuneresponseoccurringearlyinMAPinfectionislost,followedbyanineffective,antibody-mediatedTh-2response.OuroverallhypothesisisthatonceMAPpersistswithinnaïvemacrophages,itreducestheabilityofinfectedmacrophagestoreacttonormalTcellsignaling,failingtobeactivatedanddestroyMAP,andfailingtoproperlysignalTcellstorespond.Totestthishypothesis,weinvestigatedtheeffectofMAPinfectiononCD40signaling,amainpathwayusedbyTcellstoactivatemacrophages.Ourrecentstudiesdemonstratethatashort-livedresponseofbovinemonocyte-derivedmacrophages(MDM)toMAPinfectioninvitroisapparentlyfollowedbyablockintheabilityofinfectedcellstorespondnormallytosubsequentexternalactivation.WehavedemonstratedbyusingQ-RT-PCRthatnormalMDMrespondto CD40 ligand (CD40L) stimulation by up-regulation of immuneresponsegenes,includingthoseencodingIL-6,TNF-α,iNOS,andIL-12p40.Consistentwiththeseresults,westernblotanalysesindicatedthat CD40L stimulation causes a rapid, but short-lived activation ofJNK,ERK1/2andp38MAPK.Studieswithspecificinhibitorsrevealedthat theCD40L-mediated increase in IL-6and IL-8geneexpressionisdependentuponactivationofERK1/2andJNK,while increasesinIL-12p40andiNOSgeneexpressionaredependentuponactivationofp38.OnceinfectedwithMAP,however,MDMcellsfailtoup-regulatetheexpressionofiNOSandIL-12p40encodinggenesinresponsetoCD40L,whereas the expression of the other tested genes, suchasIL-8andTNF-α is not repressed.Using flowcytometric analysiswedetermined,thatfailureofinfectedmacrophagestorespondtoCD40Lwasnotdue todown-regulationofCD40on thecellsurfaceofMAPinfectedMDM.Westernblot analysisalso revealed that interferencewithCD40L-mediatedincreasesingeneexpressiondoesnotappeartobedue topreventionofp38,ERK1/2,orJNKactivation,suggest-ingtheblockisdownstreamofthesekinases.ContinuingstudiesareunderwaytouncoverthemechanismresponsibleforMAPinterferencewithCD40signalingininfectedmacrophages.Key words: Johne´s disease, macrophage, CD40 signaling,mycobacteriaSpecies:ruminants

bV031. IMBALANCE OF TUMOR NECROSIS FACTOR RECEPTORS DURING PROGRESSION IN BOVINE LEUKEMIA

VIRUS INFECTIONSAToRUKonnAI1,TATSUFUMIUSUI1,MAnABUIKEDA2,JUnKo

KoHARA3,KoSUKEoKADA2,KAZUHIKooHASHI1,MISAoonUMA1

1DepartmentofDiseaseControl,GraduateSchoolofVeterinaryMedicine,HokkaidoUniversity,Sapporo,Hokkaido060-0818,Japan;2FacultyofAgriculture,IwateUniversity,Morioka,Iwate020-8550,

Japan;3HokkaidoAnimalResearchCenter,Shintoku,Hokkaido081-0038,Japan.

konnai@vetmed.hokudai.ac.jpBovine leukemia virus (BLV), the genusDeltaretrovirus, family

Retroviridae,isthecausativeagentofenzooticbovineleukosis(EBL),whichisthemostcommonneoplasticdiseaseofcattle.Theprogres-sion of BLV infection is divided into three stages: aleukemic (AL),persistentlymphocytosis(PL)andlymphosarcoma(LS).Althoughthepathogenesisofinfectionclearlyinvolvesimmunoregulatoryhostfac-tors,includingexpressionofthecytokines,theexactmechanismofthediseaseprogressionfromALtoPLorPLtoLSinBLV-infectedcattleis

notyetknown.Inthepreviousstudies,wefoundthattheexpressionoftumornecrosisfactor(TNF)-αanditsreceptorscloselyassociatedwithdiseaseprogression in sheepexperimentally infectedwithBLV.Interestingly,wefoundaconflictingroleofTNF–ainsheepexperimen-tally infectedwithBLV. In theearlyphaseof infection,TNF-amRNAexpressionwassignificantlyup-regulatedinBLV-resistantsheep;how-ever, down-regulationofTNF-awas found in susceptible sheep. Incontrast,TNF-astronglyinducedtheproliferativeresponseofperiph-eral bloodmononuclear cells (PBMCs) in sheepwithhigher level ofBLVinthelatephaseofinfection.InordertoinvestigatethedifferentTNF-a-inducedresponses,weexaminedtheTNF-a-inducedprolifera-tiveresponsesandtheexpressionlevelsoftwodistinctTNFreceptorsofPBMCsderivedfromcattlewithdifferentstageofBLV-infection(ALorPL).TheproliferativeresponseofPBMCisolatedfromthosecattlewithPL in thepresenceof recombinantbovineTNF-a (rTNF-a)wassignificantlyhigherthanthosefromALanduninfectedcattle.ThecellsfromPLcattleexpressedsignificanthighermRNAlevelsofTNFrecep-tortypeII(TNF-RII) thanthosefromALandBLV-infectedcattle. NodifferencewasfoundinTNF-RImRNAlevelsamongtheanimals.MostcellsexpressingTNF-RII inPLcattlewereCD5+orsIgM+cellsandthesecellsshowedresistancetoTNF-a-inducedapoptosis.Moreover,thereweresignificantpositivecorrelationsbetweentheTNF-RIImRNAlevelswitheitherthechangesinprovirusloadandtheTNF-a-inducedproliferation.ThesedatasuggestthatimbalanceintheexpressionofTNF receptorscouldat least inpartcontribute to theprogressionoflymphocytosisinBLVinfection.Key words:BLV,TNF-a,TNF-receptors,cellproliferationSpecies:ruminants

bV032. EARLy LOCAL IMMUNE RESPONSES TO MyCOBACTERIAL 70 KD HEAT-SHOCK PROTEIN

VACCINATIONFEMKEBRoERE2,WILLEMVAnEDEn2,VICToRRUTTEn2,AD

KoETS1,21DepartmentofFarmAnimalHealth,P.O.Box80.165,FacultyofVeterinaryMedicine,UtrechtUniversity,3508TDUtrecht,TheNetherlands;2ImmunologyDivision,DepartmentofInfectious

DiseasesandImmunology,P.O.Box80.165,FacultyofVeterinaryMedicine,UtrechtUniversity,3508TDUtrecht,TheNetherlandsParatuberculosisisachronicgranulomatousinflammationofthe

smallintestineofcattleandotherruminants,causedbyinfectionwithMycobacteriumaviumssp.paratuberculosis(MAP).Thediseasecanbefound inruminantherdsworldwide,causingsubstantialeconomiclossesatfarmlevelduetoprematurecullingandproductionlosses.

Wehavedocumentedpreviously thatmycobacterial heat-shockproteins (Hsp) are dominant antigens in various stages of bovineparatuberculosis.Especiallythe70kDHsp(Hsp70)inducescellmedi-ated responses in natural infection. Furthermore, recombinant MAPHsp70has been shown to be a successful subunit vaccine againstbovine paratuberculosis. Surprisingly the main hallmark of vaccina-tion induced immunity was antibody production rather then T cellimmunity.

Toexplore the immunologicalmechanismsof inductionof earlycellularresponsesat thelocaldraining lymphnodeswithindaysaftervaccinationweadoptedamurinemodel.MicewerevaccinatedwithHsp70 orOVA in DDA, comparable to the cattle vaccine and BrdUincorporationwasmeasuredbyflowcytometry4and7daysaftervacci-nation.Inadditionlymphnodecellsandsplenocyteswererestimulatedinvitrotoaddressthefunctionaldifferentiationoftheimmuneresponseasmeasuredbycytokineprofileandantibodyproduction.

Enhanced BrdU incorporation was observed in draining lym-phnodes of mice that were immunized with Hsp70 compared toOVAtreatedmice7daysafter immunization.Nodifferences inBrdUincorporation were observed in non-draining lymphnodes or at day4after immunization. Inaddition, invitroBcell restimulationshowedanenhancedantigenspecificBcellsresponseinthedraining lymphnodesonlyafterHsp70vaccinationatday7,whereasBcellsisolatedfromOVAtreatedmicedidnotproduceantibodiesinanantigenspe-cificfashion.

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SimilartotheimmunizationoutcomeincattleinthemurinemodelthereisapreferentialactivationofBcellactivityfollowingsubcutane-ousHsp70/DDAvaccination.Therefore, themurinemodelpresentedinthisstudyoffersaconvenientmeanstostudythemechanismlead-ingtothisimmuneresponsebiaswhichisoppositetoHsp70immuneresponses innatural infectionandyetconfersprotective immunity toparatuberculosis.Key words:mycobacteriumavium ssp.Paratuberculosi,heat shockprotein,vaccinationSpecies:ruminants

bV033. IMMUNOGENICITy OF A CHIMERIC SUBCELLULAR VACCINE IN OVINE BRUCELLOSIS

SILVIAMESTEIn1*,MARÍAAFIoREnTIno2*,FERnAnDoAPAoLICCHI3,JULIAnACASSATARo3*GUILLERMo

GIAMBARToLoMEI3*,VAnESAZyLBERMAn4*,CARLoSAFoSSATI3*,FERnAnDoAGoLDBAUM4*

1LaboratoriodeInmunología,FacultaddeCienciasVeterinarias,UNCPBA,(7000)Tandil,BuenosAires,Argentina.Tel./fax:+542293439850;2LaboratoriodeBacteriología,ÁreadeProducciónAnimal,INTA-Balcarce;3LaboratoriodeInmunogenética,HospitaldeClínicas“JosédeSanMartín”,FacultaddeMedicina.UBA,Bs.As.,Argentina,InstitutodeEstudiosdelaInmunidadHumoral(IDEHU-CONICET),FacultaddeFarmaciayBioquímica,UBA,BuenosAires,Argentina,4FundaciónInstitutoLeloir,BuenosAires,Argentina.*CONICET,Bs.

As.Argentina.

silmares@vet.unicen.edu.arBrucellaoviscausesaninfectiousdiseaseinsheepcharacterized

byepididymitisandinfertilityinramsandabortioninewes.VaccinationprogramsaretheonlyviablemeansforthecontrolofB.ovisincoun-trieswithahighincidence.B.melitensisRev.1isconsideredthebestvaccinefortheprophylaxisofovinebrucellosisbuthasimportantdis-advantages.Accordingly, research isunderway todevelopeffectivesubcellularvaccines.Detergent-extractedrecombinantOmp31fromB.melitensiswaspreviouslyidentifiedasaprotectiveimmunogenagainstB. ovis in rams. Moreover, our previous results demonstrate that achimeraofBrucellalumazinesynthase(BLS)thatcontainsanimmu-nodominantepitopeofOmp31deliveredasaprotein(BLS-Omp31)orplasmidicDNAvaccine (pCIbls-omp31) conferredprotectionagainstB.ovisinfectioninmice.Inthiswork,weevaluatedtheimmunogenic-ityof thesevaccines in ramsusingdifferent strategiesof immunisa-tion. Seventy rams four-five- months-old were randomly distributedin following groups of immunisation: G1) BLS-Omp31 in Freund´sincompleteadjuvant(FIA),G2)BLS-Omp31inQUILAadjuvant,G3)pCIbls-omp31 with electroporation (INOVIO, Norway), G4) pCIbls-omp31withoutelectroporation,G5)Prime-boost,G6)HS(hotsaline)extract in FIA,G7) PBS.Animalswere vaccinated three times, fourweeksapart.G5 received four injections (DNAprime-proteinboost).Ramswerebledaftereachimmunization.Antibodyresponsesinserumwereevaluated in indirectELISAagainstBLS-Omp31. ImmunisationwiththeproteinchimeraformulatedwithadjuvantinducedIgGspecificantibodiessignificantlyhigherthanchimericalDNAvaccine.However,humoral immune response was enhanced in electroporated sheep.CombinationofaplasmidDNAprimingstepfollowedbyaboostwiththehomologousproteinresultedinimprovedhumoralantigen-specificresponse.These results indicate that the chimerical subunit vaccinewasimmunogenicinramsandwouldbeconsideredaspotentialvac-cineinovinebrucellosis.

ReferencesBlascoJ.M.1990.Brucellaovis,In:NielsenK.DuncanJ.R.,edi-

tors.AnimalBrucellosis. EsteinS.M.et al., 2003,Microbes Infect.Estein S.M. et al., 2004, Vet. Microbiol. Cassataro, J. et al., 2007,Vaccine.CassataroJ.etal.,2007,ClinicalandVaccineImmunology.Key words:ovinebrucellosis,subcellularvaccines,rams,chimericSpecies:ruminants

bV034. INTERACTION OF BOVINE TOLL-LIKE RECEPTORS WITH MOLECULAR PATTERNS AS ASSESSED By STABLy

TRANSDUCED CELLS

KAy-SSAUTER,MARIJABRCIC,,THoMASWJUnGIInstituteofVeterinaryVirology,UniversityofBern,Bern,Switzerland

thomas.jungi@ivv.unibe.chThe concept that cells of the innate immune system recognize

pathogen-associatedmicrobialpatterns (PAMPs)bypattern recogni-tionreceptors(PRRs)hasreceivedwideacceptanceinrecentyears.AnimportantfamilyofPRRisthatofToll-likereceptors(TLR).Someof these,e.g.TLR2,TLR4orTLR5mediatea response tobacterialPAMPsandmaybe implicated inmastitis.Rapidprogresshasbeenmadewith regard to tissuedistribution, agonist specificity, signallingpathwaysandeffectorfunctionsmediatedbyTLRs.However,knowl-edgeisrestrictedtospeciessuchashumanormouse,andwhetherknowledgeacquiredinthesespeciescanbeextrapolatedtothoseofveterinaryinterest is largelyunknown.Thisisduetothefactsthat inthese species the knockout technology and TLR-specific antibodiesaregenerallyunavailable,andthesiRNAknockdownmethodologycre-atesartefactssuchasinterferontypeIinduction.TogaininsightsintotheinteractionofbacterialpathogenswithPRRswestartedaprogramgenerating HEK293 cells being deficient for endogenous (human)TLRs but stably transduced with bovine TLR members. Here, IL-8productionasaresultofanengagementoftransducedwholebovineTLR2orwholebovineTLR4withorwithoutMD-2 is reported.TLR4ispartof theLPS receptorcomplex, togetherwithCD14andMD-2.Cells stably transduced with TLR4 failed to respond to LPS unlesscotransducedwithbovineorporcineMD-2.Doublytransduced,clonedcells reacted to LPS by IL-8 production, in decreasing order, in thepresenceoffoetalbovineserum,humanABserumandhumanserumalbumin(HSA).ThepartialresponseinthepresenceofHSAwascom-plementedbytheadditionofhighlypurifiedsolublehumanCD14,butnotbytheadditionofhighlypurifiedhumanlipopolysaccharidebindingprotein. Stimulation by partial LPS structures being recognized in aspecies-specificmanner suggested that cells resembled the reactiv-ityofbovinemonocyte-derivedmacrophagesbutreactedinamannerdistinctfromhumans,miceorhamsters.TLR2isengagedinrecogni-tion of variousPAMPsexpressed, e.g., by gram-positive bacteria.Aclonewasgenerated that responded, indecreasingorder, toStaph.aureus-derived peptidoglycan, toPalm3-Cys, lipoteichoic acid, heat-killed Listeriamonocytogenes and LPS fromE. coli (TLR4agonist).The response of this clone to causative agents ofmastitis is understudy.ThesestablytransducedclonesmightbeusefulwhensearchingforantibodiesspecificfororcrossreactivewithbovineTLRmembers,andwhenanalysingtheinteractionofbovinebacterialpathogenswithTLR-expressingcells.Key words:TLR4,TLR2,MD-2,stabletransductionSpecies:ruminants

bV035. PROTECTIVE ROLE FOR γδ T CELLS IN THE INNATE IMMUNE RESPONSE AGAINST BRUCELLA

ABoRTUS JERoDASKyBERG,MARKAJUTILA,DAVIDWPASCUALDepartmentofVeterinaryMolecularBiology;MontanaState

University;Bozeman,Montana,59718USAγdTcellshavebeenpostulated toactasafirst lineofdefense

against infectious disease, particularly intracellular pathogens, andappeartobeanimportantlinkbetweentheinnateandadaptiveimmuneresponses..WhileγdTcellsrepresentasmallpercentageofcirculatingTcellsinadulthumansandmice,γdTcellsconstituteamajorsubsetoflymphocytesinadultruminantsandmakeupamajority(upto70%ofcirculatinglymphocytes) inneonatalcalves,suggestinganenhancedimportanceofthissubsetintheimmuneresponseofruminants.IndeedpaststudieshaveintimatedthatbovineγdTcellsplayapivotalearlyroleintheresponsetotheintracellularpathogenMycobacteriumbovis.PreviousworkconductedwithhumanshasshownthatγdTcellsexpandintheperipheralbloodofpatientsinfectedwithBrucellaandthatγdTcellsareactiveagainstBrucellainvitro.However,theroleofγdTcellsinexperimentalbrucellosismodelshasyettobestudied.DuetotheabundanceofγdTcellsinruminants,alongwiththeestablishedroleofγdTcellsincontrollingintracellularinfections,wehypothesizethatγdTcellsmaybeanimportantresourcetoresolveBrucellainfectionsofcattle.HerewereportthatγdTcell-deficient(TCRγd-/-)micearemoresusceptible toB.abortus infection thanagematchedC57BL/6mice

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atoneweekpost-infection.B.abortus-infectedTCRγd-/-micehadsig-nificantlygreatersplenomegalyattributedtoincreasedbrucellaethandidC57BL/6wild-typemice.AnincreaseinγdTcellswasobservedin thespleensofB.abortus-infectedC57BL/6micewhichpeakedattwoweekspost-infection,andoccurredconcomitantlywithdiminishedbrucellae.IntracellularcytokineanalysisofspleniclymphocytesfrominfectedC57BL/6micerevealedthattheCD8-γdTcellsarethemajorsource of IL-17 andTNF-α amongst lymphocytes duringB. abortusinfection.TheseresultsindicateaprotectiveroleforγdTcells,possiblyviaIL-17and/orTNF-a,andthatroleofbovineγdTcellsintheimmuneresponsetoBrucellawarrantsfurtherinvestigation.Key words:Brucella,γδTcells,ImmunitySpecies: ruminants

bV036. CONSTRUCTION OF A BOVINE HERPESVIRUS 5 WITH A DELETION ON THE GENE VIRION HOST SHUTOFF

MARCoSSPRoFES1,FRAnSAMRIJSEWIJK1,PAULoMRoEHE1,AnACFRAnCo1

1LaboratóriodeVirologia,DepartamentodeMicrobiologia,InstitutodeCiênciasBásicasdaSaúde,UFRGS;RuaSarmentoLeite,500,

CEP90050-170,PortoAlegre,RS.marcos_s_p@yahoo.com.br

Bovineherpesvirustype5(BHV-5)isamemberofthesub-fam-ilyAlphaherpesvirinae,ofthefamilyHerpesviridae,andisoneofthemajorcausesofbovineviralencephalitis inBrazil.Allalphaherpesvi-rusescarryahomologofthevirionhostshutoff(vhs)proteinencodedbytheUL41geneofprototypealphaherpesvirus:herpessimplexvirus1.ThevhsproteinispresentintheviraltegumentanduponinfectionofthehostcellinitiatesdegradationofmRNAs.Consequently,vhsdown-regulatesproteinsynthesisand impairs theexpressionofmoleculessuch as MHC class I. vhs also interferes with the interferon type Iresponsesofinfectedcellsandblockstheactivationofdendriticcells.Theseeffectsofvhsinterferewiththeimmuneresponsesofthehostagainsttheinfectionvirus.Consideringthis,vhsnegativealphaherpes-virusesmaybe lesspathogenicandmay induceastronger immuneresponse,characteristicswhichcouldmakethemstrongcandidatesforthedevelopmentofattenuatedvaccines.AimingtheconstructionofanattenuatedBHV-5recombinantthatcouldalsobeusedasdifferentialvaccine,wedescribeherethedeletionoftheUL41genefromtheBHV-5genomeof tripledeletionmutantofBHV-5 (EVI88/95gI/gE/US9-).Forthis,theupstream(5´)anddownstream(3´)regionsoftheUL41geneofEVI88/95wereamplifiedandeachonewascloned into thepCR2.1-TOPO vector.After that, the 3´ region was sub-cloned intothevectorwiththe5´regionandtheorientationofthefragmentswasverifiedbyrestrictionenzymeanalyses.Oneclonewithbothregionsinthesameorientationasintheviralgenomewaschosentoinserttheenhanced green fluorescent protein gene (EGFP) under the controloftheimmediateearlyie1/2promoterofhumancytomegalovirus.TheEGFPgenewillbeclonedbetweenthe5´and3´fragments.Afterthis,theconstructwillbeco-transfectedwithEVI88/95gI/gE/US9-genomicDNA intobovinecells toallow the recombinationand,consequently,thedeletionoftheUL41gene.Theco-transfectionwillbeperformedusingembryonicbovinetracheacellsapplyingthecalciumphosphatemethod.MutantsexpressingEGFPintheUL41locuswillbeselectedundertheUVmicroscopeandplaquepurifiedandmultiplied.TheDNAofoneoftherecombinantswillbeanalyzedbyrestrictionendonucleaseanalysisandpartialsequencing,andfurthercharacterizedbystudyingit’sinvitrogrowthproperties.Thisrecombinantviruswilleventuallybeusedinfutureanimalexperimentsinordertoestablishitsimmunogenicproperties.Key words: Bovine herpesvirus 5, virion host shutoff, immuneevasionSpecies:ruminants

bV037. PROTHROMBOTIC EFFECTS OF HAEMOPHILUS SOMNUS ON BOVINE ENDOTHELIAL CELLS AND

PLATELETSERICABEHLInG-KELLy1,CHRISToPHERJKUCKLEBURG1,

SHAADIFELSWAIFI2,THoMASJInZAnA2,CHARLESJCZUPRynSKI1

1Dept.PathobiologicalSciences,UniversityofWisconsin-Madison,MadisonWI53706;2CenterforMolecularMedicineandInfectious

Diseases,Virginia-MarylandRegionalCollegeofVeterinaryMedicine,VirginiaPolytechnicInstituteandStateUniversity,Blacksburg,VA.

Haemophilus somnus (also known as Histophilus somnii) is animportantpathogenofcattleandsheepthatcancauserespiratoryandreproductive infections, and an acute thrombomeningoencephalitis(TME).AcommonalityofH.somnus infectionsisvasculitis inbothlargeandsmallbloodvessels.Wehavebeeninvolvedinaseriesofinvestigationsofmechanisms related to thedevelopmentof vasculi-tis.PreviousworkinourlaboratoryshowedthatH.somnusbindsto,but isnot internalized,bybovineendothelial cells.Thisprocesscanresultinendothelialcelldeathbyapoptosis.WealsofoundthatbovineplateletsareactivatedbyH.somnus,andcaninturnamplifytheapop-totic responseof endothelial cells.More recent studies have lookedat earlier events in the response of endothelial cells toH. somnus,andhowplateletsmight influence theseresponses.EndothelialcellsincubatedwithH.somnusproducethe inflammatorymediatorsIL-1βandTNF-a,andexpress increasedamountsof tissue factoron theirsurface.They also produce less thrombomodulin, and therefore arelessabletoactivateproteinC.Thesumofthesechangeswouldbeexpected toaugment thrombus formation. Incubation of endothelialcell monolayerswithH.somnuscausedcytoskeletalalterationsthatdecreased electrical resistance and increased monolayer perme-ability.Wehaveevidence thatphosphorylationofmyosin light chainkinase is an important step in this process.When bovine plateletsthathadbeenactivatedbyincubationwithH.somnuswereincubatedwithendothelialcells,thelatterincreasedtheirsurfaceexpressionofICAM-1,E-selectinandtissuefactor,andtheirproductionofseveralinflammatorymediators(IL-1β,MCP-1andMIP-1a).Tooursurpriseweobservedthatsomeactivatedplateletswereinternalizedbybovineendothelialcells.ActivationofbovineplateletsbyH.somnuscanbeprevented by inhibitors of the platelet activating factor receptor.Webelievethisdoesnotreflectaroleofplateletactivatingfactorperseintheprocess.Ratheritappearstoidentifyaroleforphosphorylcholineon the lipooligosaccharide of H. somnus. H. somnus cells that lackphosphorylcholinedonotcauseplateletaggregation.

ThesestudiesillustratedynamicinteractionsbetweenH.somnusandseveralofthecellularelementsofthevasculature(i.e.plateletsand endothelial cells). It is hoped that further investigation of theseinteractionswillhelpusbetterunderstandthepathogenicmechanismsofvasculitisinH.somnusinfectedanimals.Key words:endothelial,platelet,thrombus,vasculitisSpecies:ruminants

bV038. EVALUATION OF NOVEL COMBINATIONS OF ADJUVANTS AND IMMUNOMODULATORS FOR BOVINE

TUBERCULOSIS VACCINESBRyCEMBUDDLE1,MICHELDEnIS1,DnEILWEDLoCK1,H

MARTInVoRDERMEIER2,RGLynHEWInSon21AgResearch,HopkirkResearchInstitute,PalmerstonNorth,New

Zealand;2VeterinaryLaboratoryAgency,Weybridge,UKbryce.buddle@agresearch.co.nz

OurpreviousstudieshaveshownthatvaccinationofcattlewithacombinationofBCGandatuberculosisproteinvaccine(Mycobacteriumbovisculturefiltrateprotein,CFP)inducedbetterprotectionthanBCGvaccinealone.Furtherstudiesarenowrequiredtooptimisetheadju-vantandimmunostimulantusedinthevaccinetomaximiseprotectionagainst bovine tuberculosis andminimise vaccination site reactions.The adjuvant used in the current study was dimethyldioctyldecylammoniumchloride(DDA),whichhasbeenusedinaMycobacteriumparatuberculosisproteinvaccinetoprotectcattleagainstJohne’sdis-ease.Thethreeimmunostimulantstobetested,allpotentstimulantsofTh-1 type immune responses, included a syntheticmycobacterialPhosphatidylinositolMannoside(PIM),MonophosphorylLipidA(MPL)andasyntheticLipopeptide (LP).Sixtycalves,5-6monthsoldwererandomlydividedintosixgroupscomprising:

1. Non-vaccinated2. BCGalone3. BCG+M.bovisCFP/DDA/PIM

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4. BCG+M.bovisCFP/DDA/MPL5. BCG+M.bovisCFP/DDA/LP6. BCG+DDA/MPLBCGvaccine(106CFU/dose)wasadministeredsubcutaneously

ononeoccasion,whiletheM.bovisCFPortheadjuvant/immunostimu-lantvaccineswereadministeredsubcutaneouslyatWeeks0,3and6.AllcalveswerechallengedintratracheallywithalowdoseofM.bovisat14weeksaftertheinitialvaccinationandwereeuthanizedandexam-inedfortuberculouslesionsat15weeksafterchallenge.Thehighestlevel of protection against challengewithM. bovis was observed ingroupsvaccinatedwiththecombinationofBCGplusaM.bovisCFPvaccine, with significant reductions in four to five lesion parametersin the lungsandlymphnodes.Themostpromising immunostimulantwas the synthetic lipopeptide,withonlyoneof 10animals from thisgroupshowinglunglesionsandtwoanimalswithlymphnodelesions.DDAprovedtobeavaluableadjuvantforaM.bovisproteinvaccineby promoting protective immune responses, while inducing minimalvaccination site reaction. Combining the adjuvant/ immunostimulantvaccineplusBCGvaccineappeared tonegate theprotectiveeffectsofBCG.Thedifferent levelsofprotectionobservedamongst thefivevaccinatedgroupsdidnotrelatetopost-vaccinationinterferon-gamma(IFN-γ)levelsreleasedfrombovinePPD-stimulatedbloodcultures.Key words:Bovine,tuberculosis,vaccineSpecie:ruminants

bV039. EFFECTS OF CULTURE CONDITIONS AND TUBERCULIN SOURCE ON INTERFERON-γ PRODUCTION IN WHOLE BLOOD CULTURES FROM MyCOBACTERIUM BOVIS

INFECTED CATTLEIREnESCHILLER1,RAyWATERS2,MARTInVoRDERMEIER3,

MITCHELLPALMER2,TEKLUEGnUnI3,RoLAnDHARDEGGER1,AnnIKAKyBURZ1,ALExRAEBER1,BRUnooESCH1

1PrionicsAG,Schlieren,Switzerland;2NationalAnimalDiseaseCenter,Ames,UnitedStates;3VeterinaryLaboratoryAgency,

Addlestone,GreatBritainirene.schiller@prionics.com

The BOVIGAM® interferon (IFN) - γ assay constitutes an ante-mortem,invitrolaboratory-basedtuberculosistestandiswidelyusedcomplementarytothetuberculinskintest.Theassayisperformedintwo stages: firstly, whole blood is cultured with antigens stimulatingblood leucocytes toproduce IFN-γwhich isquantifiedbyELISA inasecondstep.Environmental conditionsbeforeandduring thecultur-ingoftheleucocytesinfluencetheefficacyofinvitroIFN-γproduction.Optimalconditionsarethereforeessential. Inthisstudyweanalyzedtheeffectofstimulationvesselgeometry,temperaturesduringstimula-tionandthestabilityofantigensstoredatdifferenttemperatures.Bloodfrom experimentally infected cattle and from tuberculosis-negativecattlewasstimulatedin24-welltissueculturetrays(standard),48-welland96-wellcultureplateswiththefollowingantigens:PurifiedproteinderivatefromMycobacteriumbovis(PPD-B)andfromMycobacteriumavium(PPD-A),afusionproteinfromearlysecretoryantigenictarget6(ESAT-6)andculturefiltrateprotein10(CFP-10),andpokeweedmito-gen.Stimulationwasequally efficient in all threeplate formats.Theresultswithspecificantigenscorrelatewithmitogeninducedstimula-tion.CO2isnotrequiredduringincubation,asculturesfromanincuba-torwith5%CO2producedsimilaramountsof IFN-γaswithoutCO2.However,thetemperatureusedforstimulationwascritical:Stimulationat37°Cand33°Cwereequallyefficient,butaculture temperatureof29 °C reduced IFN-γ productionsignificantly.At25 °Cand22 °Cnostimulationwasdetectable.Antigensareusuallystoredat2-8°C(tuberculins)orat-80°C(recombinantproteins)untilusage.Wetestedin parallel antigen storage of recombinant proteins (ESAT-6:CFP-10fusionprotein,TB10.4,TB27.4,MPB83)at4 °C for24horat20°Cfor8hpriortouseincellculture.Ourresultsshowthatantigensmaybestoredateitheroftheseconditionswithoutaffectingtheefficacyofstimulation.Finally,wecomparedtheactivitiesoftuberculinsfromfivedifferent sources innaturally infectedcattle (n=10).MatchedPPD-BandPPD-Atuberculinswereusedateightdilutionseach.Relativepotency 30 (RP30) was defined as the tuberculin concentrationrequired to induce30%of thepeak responsevalues.RP30differedbyafactorofmorethan10betweenthePPD-Bwiththehighestand

lowestpotency.Thereforetuberculinsofdifferentsourcesmaygivedif-ferentresultsandtheoverallassayperformancemaybeimprovedbyoptimizingtuberculinconcentrations.Key words: tuberculosis, daignosis, Bovigam®, tuberculin earlysecretoryantigenictarget6,culturefiltrateprotein10Species:ruminants

bV040. DETECTION OF ELEVATED LEVELS OF INTERLEUKIN-10 IN CATTLE FOLLOWING A TUBERCULIN

SKIN-TESTMCoAD,AoWHELAn,SGRHoDES,DJCLIFFoRD,RG

HEWInSon,HMVoRDERMEIERTBResearchGroup,VeterinaryLaboratoriesAgency;Woodham

Lane,Addlestone,SurreyKT153RW,UKBovinetuberculosis(BTB)isadiseaseofeconomicandzoonotic

importance caused by the bacterial pathogen Mycobacterium bovis.Control of BTB in the UK centres on the application of the SingleIntradermalComparativeTuberculinTest(SICTT)andthesubsequentslaughterofanimalstestingpositive(reactors).

In this studywe selected naturally infected field cattle of differ-entagesandbreedsandhousedtheminsecureaccommodationandinvestigatedtheeffectsofrepeatskintesting.Duringthestudyperiod,animalsweresubjectedtorepeatskintestingataminimumintervalof60daysinlinewithEUfieldpractice(meannumberoftestsperanimal=2.9).Regularbloodsamplesweretakenforcytokinetestingandani-malswerethoroughlyexaminedatpost-mortemwithtissuesexaminedbycultureandhistopathologyforthepresenceofBTB.

Ofthe30cattletested,21(70%)showedadropofatleast3mmbetween the disclosing and final SICTT. Eight (27%) of these reac-tors became inconclusive under the standard test interpretation. Offourteenanimals thathaveso farbeenculled,allhavebeenshowntobepositiveforBTB.ThesedatasuggestthatanimalsmaybecomedesensitisedtobovinePPDwithrepeattesting(althoughitisacceptedthatthestudyanimalswouldhavebeenremovedfromtheherdfollow-ingtheirdisclosingtestresult).

Investigations into a possible mechanism for this effect haveshown that production of the anti-inflammatory cytokine Interleukin-10 increases following the SICTT.We did not observe a consistentdecrease in Interferon-γproduction followingpeaks in IL-10.Work iscontinuing to lookat thepossibility that numbersof IL-10producingcells do not fall sufficiently before the next SICTT thus suppressingthedelayed-typehypersensitivity responsewhichwouldnormallybeobservedinBTBinfectedcattle.Key words:Tuberculosis;Cattle;Interleukin-10;Skin-testSpecies:ruminants

bV041. TRUNCATED E2 GLICOPROTEIN ExPRESSION IN CHO-K1 CELLS TO PRODUCE A SUBUNIT VACCINE

AGAINST BVDVSEBASTIánMCHIAVEnnA1,AGUSTÍnoSTACHUK1,AnDREA

PECoRA1,MSUSAnALEVy2,MARInAJDUSSAnToS1,AnDRéSWIGDoRoVITZ1

1InstitutodeVirología,CICVyA,INTA-Castelar,Argentina;2Biogénesis-Bago,Argentinaschiavenna@cnia.inta.gov.ar

BovineViralDiarrheaVirus(BVDV)isresponsibleforworldwideeconomic losses incattlepopulationmainlydue to reproductive fail-ure.Control of fetalBVDV infection throughvaccination is critical toavoidtheappearanceofpersistently infectedcalvesatbirth.Currentvaccinesuseinactivatedvirusasimmunogen.However,viralantigenproductionincellculturesisdifficultandmayaffecttheeffectivenessof the resulting vaccines.To overcome this problem,we decided todevelopatruncatedversionofBVDVE2glicoproteinexpressedinamammaliancellsystemtobetestedasimmunogenforproductionofanexperimentalsubunitvaccine.HerewereportthedevelopmentofaChineseHamsterOvary(CHO-K1-E2T)celllinewhichisstablytrans-fected. It expresses a truncated version of E2 (E2T). RecombinantE2T lacks the transmembranedomainof thenativeproteinand it isfusedtoasecretionpeptidetoallowrecoveryoftherecombinantpro-

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teinincellculturesupernatants.TranscriptionalfusiontoaC-terminalhistine tag also permits its purification by immobilizedmetal affinitychromatography (IMAC). CHO-K1-E2T supernatants were analyzedandarecombinantproteinoftheexpectedelectrophoreticmobilitywasdetectedusingamonoclonalantibodyagainstE2(CA3)asprobe.TheE2TwaspurifiedfromculturesupernatantbyIMACanditwasquanti-fiedusingacolorimetricassay.ThisproteinwasusedasstandardforcuantificationoftheE2TobtainedineachcellcultureusinganELISAassay. Preliminary results obtained using a guinea-pig experimentalmodelindicatetheabilityoftherecombinantE2TtoinduceBVDV-spe-cificantibodies.FurthertestingiscurrentlybeingconductedtoobtainamoredetailedcharacterizationoftheimmunogenicpropertiesofthisBVDVsubunitantigen.Key words:BVDV,Vaccine,MammaliansystemSpecies:ruminants

bV042. BLS PROTEIN AS BRV ANTIGEN DELIVERy SySTEM IN A SUCKLING MICE MODEL.

DEMIAnBELLIDo1,PATRICIooCRAIG2,MARInAVMoZGoVoJ1,DIEGoDGonZALEZ1,AnDRESWIGDoRoWITZ1,

FERnAnDoGoLDBAUM2,MARIAJDUSSAnToS11LaboratoriodeBiologíaMolecular,InstitutodeVirologia,INTACastelar,Argentina;2LaboratoriodeInmunologíaEstructuraly

Molecular.FundaciónInstitutoLeloir,Argentina1CorrespondingAuthor:demianbellido@gmail.com

Brucellaspp lumazinesynthase(BLS)presentsaveryresistentandhighorderdecamerycstructure.Itallowsthefusionofpeptidesinits10N-terminus,givingchimericproteinswithhighorderarrangementthatareabletoinduceefficientimmuneresponses.TheobjectiveofthisworkistoproducestablefusionproteinswhenbiggerproteindomainsareinsertedintheBLSstructureandtoevaluatetheimmuneresponseinduced by the chimeric protein.We believe that BovineRotavirus(BRV)VP8protein isanaccurateprotein tobe fused toBLSbasedonitsphysical,chemicalandimmunologicalproperties.TheinnerpartofVP8,VP8d, isahighstructuredsolubledomain thatpreserve thesialicacidbindingsiteandtherelevantepitopeswichinduceneutral-izingantibodies.ThepresentworkshowsthattheBLS-VP8dchimeraisabletoefficientlystimulatetheimmunesystem.Theimmunologicalpropertiesofthechimericproteinanditscapabilityforinducingpassiveprotectionwere tested inasucklingmicemodel.Theseresultswerecompared with those obtained with the single VP8d protein. Damsimmunizedwiththechimericproteingavebirthtopupsthatafterchal-lengewithBRVC486 [P1]G6showed100%protection level;on theotherhand,pupsborntodamsimmunizedwithVP8dpresentedalevelofprotectionof30%.Theantibodypatternwasalsosignificatlydifferentinbothexperimentalgroups,as itwasevaluatedbyELISA inserumandmilk.Key words: BLS (Brucella Lumazine Synthase),Bovine Rotavirus,VP8,sukclingmicemodelSpecies:ruminants

bV043. CHARACTERIZATION OF MyCoBACTERIUM AVIUM SUBSP. PARATUBERCULoSIS ANTIGEN-SPECIFIC

REGULATORy T CELLS.DEnISEEDEALMEIDA,CHRISToPHERJCoLVIn,PAULM

CoUSSEnSMolecularPathogenesisLaboratory,Dept.ofAnimalScience,

MichiganStateUniversity,EastLansing,MI,USA.Johne’sdiseaseiscausedbyMycobacteriumaviumsubspecies

paratuberculosis (MAP)and isoneof themostcostly infectiousdis-easesindairycattle.MAPisanintracellularpathogenthatsurvivesinhostintestinalmacrophagesleadingtochronicenteritisinruminants.Following initialexposure toMAP,aThelper (TH)1 responsedevel-ops.However,duringthelatesubclinicalphaseofMAPinfection,thispro-inflammatoryTH1responseislost,andanantibody-mediatedTH2responsebecomespredominate,butisineffective.TheimmunologicalmechanismsresponsibleforsuppressionofMAP-specificTH1immuneresponsesareunknown.However,understandingthesemechanismsiscriticaltoaccuratelydiagnoseJohne’sdiseaseandfordevelopmentofeffectivetherapiesandvaccines.Thehypothesisofthisstudyisthat

dairy cattle infectedwithMAPdevelop regulatoryT cells capableofproducing IL-10 (Tr1)orTGFβ (Th3), thereby limitingperipheralandtissue-specificTH1 immuneresponses. Insupportof thishypothesis,our recent studiesdemonstrated that stimulationof peripheral bloodmononuclear cells (PBMCs) from subclinical MAP-infected cattleresults in enhanced expression of IL-10mRNA. Furthermore, IL-10suppressesIFN-γexpressioninMAPantigen-reactiveeffectorTcells,suggesting that IL-10up regulation favorsMAPproliferation inmac-rophages. Depletion studies in MAP-infected cattle also revealedthat the MAP-responsive and IL-10 producing T cell population islikely CD4+ and CD25+. In order to test our hypothesis, we havefirstbeguntoprofilebovineregulatoryTcellsinhealthycows(n=4).Using primary and secondary monoclonal antibodies including anti(aCD4, aCD25, aCD45RO, aCD8, and aIL-10, dual flowcytometricanalysesofPBMCswereperformed.PreliminaryresultsindicatethatrestingbovinePBMCsarecomposedof2.7%ofCD4+CD25+Tcells,28.9%ofCD4+CD45RO+Tcells,3.3%ofCD4+IL-10+Tcells,3.0%of CD8+CD25+T cells, 26.8% of CD8+CD45RO+T cells, 13.3% ofCD25+CD45RO+Tcells,and3.3%ofCD25+IL-10+Tcells.FurtheranalysisofregulatoryTcellsinMAPinfectedandhealthycontrolcattleisinprogressusingadditionalantibodies,suchasaFoxp3(atranscrip-tionfactorspecificforregulatoryTcellscapableofdifferentiatingthemfrom Th2 cells), and three-color flow cytometry. Characterization ofMAP-responsiveregulatoryTcellsisessentialtodeterminetheirroleinlimitingappropriateTH1immuneresponsesinMAP-infectedcattleanddeterminingtheirroleinprogressiontoclinicaldisease.Key words: Paratuberculosis, Regulatory T cells, Mycobacteriumaviumparatuberculosis,CattleSpecies: ruminants

bV044. CLONING AND CHARACTERIZATION OF BIGHORN SHEEP INFLAMMATORy CyTOKINES INTERLEUKIN-1β,

TUMOR NECROSIS FACTOR-α, AND INTERLEUKIN-8. CARoLInEnHERnDon,RoHAnAPDASSAnAyAKE,WEIGUo

LIU,WILLIAMJFoREyT,SUBRAMAnIUMSRIKUMARAnDepartmentofVeterinaryMicrobiologyandPathology,Washington

StateUniversity,Pullman,WA99164-7040,USA.cherndon@vetmed.wsu.edu

PneumoniacausedbytheGramnegativebacteriumMannheimiahaemolyticaisadevastatingdiseasetothebighornsheeppopulationinNorthAmerica.AlthoughM.haemolyticacancausepneumoniainbothdomesticandbighornsheep, the latteraremoresusceptible tothis disease than are domestic sheep. Pro-inflammatory cytokineshavebeenimplicatedinthepathogenesisofanumberoflungdiseasesofbothhumansandanimals.Studiesincattlehaveshownthatalveolarmacrophages,uponstimulationwith the lipopolysaccharide (LPS)ofM. haemolytica, secrete pro-inflammatory cytokines IL-8, IL-1β, andTNF-a. IL-8attractspolymorphonuclear leukocytes (PMNs) into thelungs, and IL-1β andTNF-a enhance the expression of β2-integrinsby PMNs. Enhanced expression of β2-integrins onPMNs results inenhancedcytolysisbyleukotoxin.WehypothesizethatbighornsheeparemoresusceptibletoM.haemolyticapneumoniathanaredomesticsheepbecauseofanenhancedpro-inflammatorycytokineresponse.The long termgoalof thisstudy is tocompare thepro-inflammatorycytokinesecretionbyalveolarmacrophagesofbighornanddomesticsheep in response to M. haemolytica infection. Characterization ofthe cytokine response of bighorn and domestic sheep necessitatesthecloningandsequencingofcDNAencodingIL-8, IL-1β,andTNF-α, which forms the objective of this study. Bighorn sheep alveolarmacrophagesandperipheralbloodmononuclearcellswerestimulatedwithLPSfromEscherichiacoli. TotalRNAwasextractedandcDNAwasmadebyRT-PCR.ThePCR-amplifiedcDNAwascloned intoamammalianexpressionvectorandsequenced.ThecDNAofbighornsheepIL-1βencodes266aminoacidsandexhibits99%,92%,60%,and59%identitytoovine,bovine,human,andmurineIL-1β,respec-tively.ThecDNAofbighornsheepTNF-αencodes235aminoacidsandexhibits96%,88%,and76%identitytoovine,bovine,andhumanTNF-α, respectively. ThecDNAofbighornsheep IL-8encodes102aminoacidsandexhibits98%,94%,and80%identitytoovine,bovine,andhumanIL-8,respectively.Theclonedcytokinesarecurrentlybeingtestedinbioassays.AvailabilityofIL-1β,TNF-a,andIL-8willhelpin

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thecharacterizationofthepro-inflammatorycytokineresponseofbig-hornanddomesticsheepinM.haemolytica-inducedpneumonia.Key words:bighorn,inflammatory,cytokine,cloningSpecies:ruminants

bV045. ExPRESSION OF IL-8 AND MIP-1α CHEMOKINES IN HELA CELLS INFECTED WITH CAMPyLoBACTER FETUS

SUBSP. VENEREALISAPCCoTToRELLo1,TMALVES2,MMMACHADo3,BSARAUJo4,

EFBSTAnCIoLI5,RLSAnToS6,APLAGE61PostDoc–EscoladeVeterinária–UFMG;2PhDstudent–EscoladeVeterinária–UFMG;3Masterstudent–EscoladeVeterinária–

UFMG;4Gradstudent–EscoladeEnfermagem–UFMG;5Professor–ICB–UFMG;6Professor–EscoladeVeterinária–UFMGBovine genital campylobacteriosis, caused by Campylobacter

fetus subsp. venerealis (Cfv), is a venereal disease responsible foreconomicallossesincountrieslikeBrazil,whereartificialinseminationisstillrestrictedtoasmallpercentageofherds.Althoughtherearesev-eralpreviouslypublishedstudiesonthehosthumoralresponseagainstthisorganism,noinformationisavailableontheinteractionofCfvwithhost epithelial cells in vitro or in vivo as well as about chemokinesinvolved in the innate immune response.Theaimof this studywastoevaluate thekineticsexpressionof two inflammatorychemokines,IL-8andMIP-1α,inHeLacellsinfectedwithCfv.ThestrainsusedinthisstudywereCfvNCTC10354andstrainPN,whichwas isolatedsevendaysaftervaginalinoculationofaheiferwithCfvNCTC10354.Thesestrainswere incubatedwithcells for0.5,1,2,4,6,8,and12hours.AfterRNAextractionandcDNAsynthesis,PCRforamplificationofIL-8(IL-8F-5’CTTGGCAGCCTTCCTGATTT–3’,IL-8R-5’TCAAAAACTTCTCCACAACC3’)andMIP-1a(MIP-1aF-5’ACCATGGCTCTCTGCAACCA-3’,MIP-1aR-5’TGTGGAGGTCACACGCATGTT-3’)wasperformed.IL-8mRNAtranscriptionwasevidentinHeLacells within half an hour of exposure to Cfv. Induction was maximalafter 2 and 8 hours of incubation for PN and NCTC 10354 strains,respectively.ThePNstrain inducedhigher levelsof IL-8mRNA(P<0.05)whencomparedtoNCTC10354fromhalfanhourupto4hoursafterinfection.MIP-1aexpressionoccurredlaterincellsinfectedbyPNstrain,withmaximalinduction12hoursafterinfection.InfectionofHeLacellswiththeNCTC10354straindidnotresultedininductionofMIP-1αintheperiodstudied.ThislaterexpressionofMIP-1αcomparedtoIL-8wasexpectedsinceitactsaschemoattractantformonocytesandmacrophages,whichisingoodagreementwiththehistologicalfindingsininfectedheifers.IL-8expressionwashigher(P<0.05)thanMIP-1αexpressioninthemajorityofincubationtimesstudiedforbothstrains.Inconclusion,thesedatasuggestthatthesechemokinesareimportantintheinitialimmuneresponseagainstC.fetussubsp.venerealis.Key words:C.fetussubsp.Venerealis,chemokinesexpression,HeLacellsSpecies:ruminants

bV046. MONOMERIC ExPRESSION OF BOVINE β2-INTEGRIN SUBUNITS CLARIFIES THEIR ROLE IN

MANNHEIMIA HAEMOLyTICA LEUKOTOxIN-INDUCED CyTOLySIS.

RoHAnAPDASSAnAyAKE,SUBRAMAnIAMSRIKUMARAnDepartmentofVeterinaryMicrobiologyandPathology,Washington

StateUniversity,Pullman,WA99164-7040,USA.ssrikumaran@vetmed.wsu.edu

β2-integrinsareleukocyte-specificintegrinsthatfacilitatehominginto areas of inflammation, phagocytosis, antigen presentation, andcytotoxicity.Theyareexpressedonthecell-surfaceasaheterodimercomposedofαandβsubunits.Thecommonβsubunit,CD18,associ-ateswith3distinctαchains,CD11a,CD11b,andCD11ctogiveriseto 3different β2-integrins:CD11a/CD18 (lymphocyte function-associ-ated antigen 1, LFA-1), CD11b/CD18 (complement receptor 3, CR3or Mac-1); CD11c/CD18 (complement receptor 4, CR4). Previousstudiesbyusandothersidentifiedβ2-integrinsasthereceptorsforleu-kotoxin(Lkt)whichisthemostimportantvirulencefactorproducedbyMannheimia(Pasteurella)haemolytica,theprimarybacterialpathogenofbovinerespiratorydiseasecomplex.ByrenderinganLkt-non-sus-

ceptiblemurinecell-linesusceptibletoLkt-inducedcytolysis,wehavepreviously demonstrated that CD18 mediates Lkt-induced cytolysis.However, in that study, bovineCD18was expressed as a heterodi-merwithmurineCD11awhichprecludedtheelucidationoftheroleofbovineCD11a.Therefore,theobjectiveofthisstudywastopreciselyidentifytheroleofbovineCD11aandCD18inLkt-bindingandcytolysisoftargetcells.cDNAforbovineCD11aandCD18,eitherindividuallyortogether,wastransfectedintothehumanembryonickidneycell-line(HEK-293) which does not express any β2-integrins. TransfectantsstablyexpressingmonomericCD11a,CD18,orheterodimericLFA-1(CD11a/CD18)ontheircellsurfacewereselectedbyflowcytometricanalysis with monoclonal antibodies specific for bovine CD11a orCD18. InLkt-bindingassays,all three transfectants,butnot thepar-entcells,effectivelyboundLkt. However,Lkt-inducedcytolysiswasobservedonlywithtransfectantsexpressingmonomericCD18orLFA-1.Furthermore,intracellular[Ca2+]ielevationfollowingexposuretoLkt,whichisacknowledgedasanindicationofLkt-receptorinteraction,wasseenonlywith transfectants expressingmonomericCD18or LFA-1.Takentogether,theseresultsclearlyindicatethatitistheCD18subunitthat is involved in theLkt-inducedcytolysisof targetcells. AlthoughCD11abindstoLkt,itisnotinvolvedintheeventsleadingtointracel-lular[Ca2+]ielevationandcytolysis.TheLFA-1expressioninthetrans-fectantswasstableforlongerperiodsthaneitheroneofthesubunitssuggestingthatassociationofthetwosubunitswasnecessaryfortheirstableexpression.Key words:Mannheimiahaemolytica,Leukotoxin,Receptor,C18Species: ruminants

bV047. DETECTION OF ANTIBODIES TO oVINE HERPESVIRUS-2 INTERLEUKIN-10HOMOLOGUE IN SHEEP-

ASSOCIATED MALIGNANT CATARRHAL FEVERRoBInLCISSELL1,SHAHIRAABDELWAHAB2,RoBERTL

DonnELL3,STEPHEnAKAnIA2UniversityofTennesseeVeterinaryTeachingHospital,Knoxville;

2DepartmentofComparativeMedicine;3DepartmentofPathobiology;1ComparativeandExperimentalMedicineProgram

Interleukin-10 (IL-10) interfereswithmonocyteandmacrophageactivationofTh1helperlymphocyteproductionofnitricoxideandsyn-thesisofvariousinflammatorymediators.AnIL-10homologue(vIL-10)isproducedbyseveralherpesvirusesandishypothesizedtohelpthevirusdown regulate,and thusevade,host immune responses. Thegammaherpesrhadinovirusovineherpesvirus-2(OvHV-2)encodesanIL-10likemoleculehighlyhomologoustomammalianIL-10.Thisviruscausessheep-associatedmalignant catarrhal fever (MCF), themostcommonformofMCFintheUnitedStates.MCFisalymphoprolifera-tiveandinflammatorysyndromewhichhasdelayedclinicalpresenta-tionhypothesized tobe influencedby theproductionof vIL-10. Forthis study, the gene encoding vIL-10,Ov2.5ORF,was amplified byPCR,cloned,sequenced,andapredicted30aminoacidsegmentfromtheaminoterminussynthesized.Thissyntheticpeptidewasusedtodevelopanoveldirectenzyme-linkedimmunosorbantassay(ELISA)todetectisotype-specificantibodiestoOvHV-2vIL-10.Worktodateindi-catesthat lambsdonothavedetectable levelsofmaternallyderivedantibodytovIL-10duringthefirstfewweeksofage.Ewes,whicharerefractory toclinical infection,generallyhavehigh levelsofantibodytovIL-10.AweakcorrelationexistsbetweenthevIL-10ELISAandacommercially available competitive-inhibitionELISA (CI-ELISA). Webelieve the vIL-10 ELISA will refine the ability to identify ruminantsexposedtosheep-associatedMCF,provideanimportanttoolfordeter-miningtheroleofvIL-10indiseasepathogenesis,andmaycontributetoward thedevelopmentofanewvaccinestrategy for thecontrolofmalignantcatarrhalfever.Key words:viralIL-10,MalignantCatarrhalFever,Ovineherpesvirus-2,IL-10homologueSpecies:ruminants

bV048. IMMUNOPROTEOMIC ANALySIS OF THE PROTECTIVE OUTER MEMBRANE FRACTION OF

ANAPLASMA MARGINALEWEnDyCBRoWn

69

DepartmentofVeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA99164

wbrown@vetmed.wsu.eduRickettsial pathogens in the genera Anaplasma and Ehrlichia

cause acute infection in immunologically naïve hosts and aremajorcausesof tick-bornedisease inanimalsandhumans. ImmunizationwithAnaplasmamarginalepurifiedoutermembranesinducescompleteprotectionagainstanaplasmosisin75%ofcattle,whereasimmunizationwiththewell-studiedandimmunodominantmajorsurfaceproteinssuchasMSP2hasprovidedlittleornoprotection.ThecompletedgenomesequenceofA.marginalefacilitatedtheidentificationofsubdominantand less abundant immunogenic proteins in the outer membranefraction, using two approaches. First, two-dimensional electrophore-sisand immunoblottingof theoutermembrane fractionwith immunebovine sera identified numerous antigenic protein spots.Analysis ofindividualproteinsexcisedfromthegelsbyliquidchromatographyandtandemmassspectrometryidentified21novelantigens.Ofparticularinterest is the finding that three proteins from the type IV secretionsystem, conjugal transfer protein,VirB9, andVirB10wereantigenic.TFSS proteins form channels and are responsible for secretion orcell-to-celltransferofmoleculesandDNA-proteincomplexesinothergram-negativebacterialpathogens,buthavenotbeenstudiedasvac-cineantigens.TheseproteinswereexpressedandshowntostimulateIgG2,andCD4+TcellproliferationandIFN-γproduction,responsesassociatedwithprotectiveimmunityinoutermembranevaccinates.Asecondapproach tomoredirectlyscreen forantigensrecognizedbyTlymphocytesinvolvedaninvitrotranscriptionandtranslation(IVTT)ofORFsencodingproteinspredicted tobe localizedon theouterorinnermembraneortohaveasignalpeptide.PCRproductsofselectedORFs engineered to express antibody-binding sequence tags wereamplifiedandexpressedusingIVTT.Asproofofprincipal,VirB9andoutermembraneprotein(OMP)7,OMP8,andOMP9,knowntostimu-lateTcellresponsesinoutermembranevaccinates,wereexpressedbyIVTTandaffinitypurifiedbybindingtoanti-tagantibodycoupledtoprotein-Gboundbeads,andthebeadswereaddedtoAPCandusedtostimulate immuneCD4+Tcellproliferation.Thisnovel technologycanbeusedtorapidlyscreenalargenumberofproteinsfromagivenpathogen for recognition by both antibody andT lymphocytes if thegenomesequenceisavailable.Key words: mass spectrometry, Anaplasma marginale, outermembranes,TlymphocytesSpecies:ruminants

bV049. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST CAMPyLoBACTER

FETUS SUBSP. VENEREALISTELMAMARIAALVES1,LUIZGUILHERMEDIASHEnEInE2,

BáRBARASILVEIRAARAúJo1,LUCIAnAMARIASILVA3,PATRÍCIACoTACAMPoS2,MáRCIASILVAHERMoGEnES3,

AnDREyPEREIRALAGE1*1LaboratóriodeBacteriologiaAplicada-NúcleodePesquisaemSaúdeAnimal-DepartamentodeMedicinaVeterináriaPreventiva-EscoladeVeterinária-UniversidadeFederaldeMinasGerais-

BeloHorizonte-MinasGerais–Brazil;2LaboratóriodeImunologiaeBioprodutos-FundaçãoEzequielDias,BeloHorizonteMinasGerais-Brazil;3LaboratóriodeBiologiaCelulareMolecular-Fundação

EzequielDias,BeloHorizonteMinasGerais-Brazil.MyelomacellsSp2/0-Ag14andspleencellsfromBALB/cmouse

immunized with sonicated Campylobacter fetus subsp. venerealisNCTC10354werefusedwithpolyethyleneglycol (PEG)for thepro-duction of monoclonal antibodies (MAb’s). Clones were obtained bylimiting dilution and screened for specific MAb’s toC. fetus subsp.venerealisNCTC10354 by indirectELISAandwestern blot againstapanelofbacteria:C.fetussubsp.venerealisNCTC10354,C.fetussubspfetusADRI1812,C.sputorumbiovarsputorumLMG6647,C.lariNCTC11352,andArcobacterskirrowiiLMG6621fortheELISAandC.fetussubsp.venerealisNCTC10354andC.sputorumbiovarsputorumLMG6647forthewesternblotting.FifteenclonesproducingMAb’santi–C.fetussubsp.venerealisoftheIgM(1)andIgG(14)classeswerefurtherscreenedforspecies-specificity.Fourclonesofthe15obtainedwereproducersofspeciesspecificMAb’s:2werespecificforC.fetus

subspvenerealisand2specificforC.fetussubspfetus.NoneofthecloneswerereactiveagainstC.sputorumbiovarsputorumLMG6647.Allclonesrecognizedaproteinwithmolecularmassofapproximately148kDafromlisedC.fetussubsp.venerealisNCTC10354.Key words:Monoclonalantibodies,C.fetussubsp.venerealis,cattle,bovinegenitalcampylobacteriosisSpecies:ruminants

bV050. CHARACTERIZATION OF THE IMMUNE RESPONSE INDUCED By LIPOARABINOMANNAN (LAM) FROM MyCoBACTERIUM SP. IN VACCINATED CALVES

JoLLyA,STEMPLERA,CoLAVECCHIAS,FERnAnDEZE,MUnDoS

FacultaddeCienciasVeterinarias,UniversidaddeBuenosAiresParatuberculosis,causedbyMycobacteriumaviumsubsp.para-

tuberculosis(Map),isachronicgranulomatousenteritisincattle.LAMisthemostabundantpolysaccharideinmycobacteriacellwallandalsothemajorimmunodominantsurfaceantigen.Currentlyavailablevac-cinesdonotprovidefullyprotection(1).LAMvaccinesweretestedinlaboratoryanimalswithpromisingresults(2).InordertoevaluatetheuseofLAMasavaccineincattle,thisworkcharacterizestheimmuneresponseofcalvesimmunizedwithLAM.

Mycobacterium avium subsp. avium were sonicated (S) and aglycolipid fraction containing LAM (L)was obtained through phenol-chloroform extraction. Both extracts were emulsified with Freund’sIncompleteAdjuvant(FIA)andusedtoimmunize3months-calves.Twodosesof immunogenwereinoculatedatday0and35(2mgoftotalcarbohydrateseach)inSgroup(n=4)andLgroup(n=6).Anothergroup(n=3)waskeptasnegativecontrolandreceivedPBS-FIA(ACgroup).Serumsampleswereevaluatedatday0and60.ELISAwasperformedtodetecttotalandisotypesanti-LAMantibodiesinducedbyvaccination.Cellularimmuneresponsewasevaluatedinvivoatday60byIntradermalreactiontest(IDR),usingbovinePPD.

Anti-LAM antibodies were detected in all vaccinated calves. InLgroup,83.3%ofcalvesshowedtiters ≥2000,whereasallcalves ingroupShadtiters≥2000.Isotypeprofileanalysisisshownintable1.

Table1.Anti-LAMantibodiesProfiles

IgM IgG1 IgG2 IgALgroup ≤0,001 0,177±0,030 ≤0,04 ≤0,04Sgroup 0,247±0,310 0,448±0,110 0,477±0,260 ≤0,07

ResultsareexpressedasDOmeanvalueafterACgroupmeanvaluesubstraction±standarderror

Serumsamplesdilutionwasanalyzedat1/100NopositivereactionsweredetectedbyIDRinLvaccinatedgroup,

whileallSvaccinatedcalvesreacted.Our results demonstrate that immunizationwith LAM is able to

induce a specific humoral immune responsewithout interferingwithIDRtestusuallyusedforbovineTuberculosisdiagnosis. IthasbeendescribedthatIgG1isthepredominantisotypeintheimmuneresponsetoLAMofinfectedanimals(3).Ourresultsindicatethatthesameiso-typeprofileisobtainedwhenLAMisinoculatedasapurifiedantigen.

TheseresultscouldsupportfurtherinvestigationsevaluatingLAMasacandidateforaParatuberculosissubunitvaccine.

References1.UzonnaJE,Vaccine2003.2.HamasurB,ClinExpImmunol2004.3.KoetsAD,InfectandImmun2001.

Key words:(cattle),(Paratuberculosis),(LAM),(subunitvaccine)Species:ruminants

bV051. BOVINE ROTAVIRUS VACCINES: IMMUNOGENICITy OF TWO STRAINS OF BOVINE ROTAVIRUS IN CATTLE AND

GUINEA PIGSVIVIAnAPARREño1,DAnIELARoDRIGUEZ1,MERCEDES

IZUEL2,JoRGEFILIPPI2,LAURAMARAnGUnICH3,VIRGInIA

70

LóPEZ4,FERnAnDoFERnánDEZ1,RoDoLFoBELLInZonI2,MARÍAM.VEnA2

1InstitutodeVirologia,INTA,Castelar;2BiogenesisBagóS.A.;3UNTREF;4UBA,Argentina.

maria.vena@biogenesisbago.comBovine Rotavirus (BRV) is amain cause of severe diarrhea in

neonatalcalves,worldwide. InArgentinaBRVwasdiagnosedasthecauseofneonataldiarrheain71and58%oftheoutbreaksregisteredinbeefanddairyfarms,respectively.TheBRVstraintypedasP[5]G6was prevalent in beef herds, while P[11] was the prevalent P-type(71%),associatedinsimilarproportionswithvariantsofG6andG10,in dairy herds (Garaioechea et al, 2006). Prevention strategies arebasedon thevaccinationofpregnant cows inorder to transferhightitersofmaternalantibodies(Ab)tothecalfthroughcolostrumintake.Threeexperimentalvaccinescontaining10^7FFU/doseofUKP[5]G6,orB223P[11]G10orbothstrainsformulatedinoiladjuvantweretestedtogetherwithacommercialvaccine(RotatecJ5®) inbeefcattle(withlowAbtiterduetonaturalRVinfection)andseronegativeguineapigs.ImmuneresponsewasevaluatedbyELISAandvirusneutralizationtest(VN).Bovinetrialconsistedoftwovaccinationsandserumsamplingat0,30,60and90dpv.whereasguineapigsreceivedoneortwodosesofvaccineandweresampledweeklyuntil60dpv.ThekineticofRVAbresponsesbyELISA,mostlydirected toVP6,wassimilaramongallthevaccinestestedinbothspecies,withseroconvertionregisteredat60dpvincattleandat21dpvinguineapigs.AllvaccinatedbovinesdevelopedVNAbsagainstUKindependentlyofthevaccineformula-tion, indicating thepreviouscontactwith that strain. In contrastonlythegroupsvaccinatedwithvaccinescontainingB223RVdevelopedaVNAbresponsetothatstrain,suggestingminimumfieldexposureto P[11]G10. In guinea pigs optimal Ab response was obtained byexperimental and commercial vaccines.TheVN responses in naïveguineapigallowedtodemonstratedthat there isnocrossprotectionbetweenUKandB223strainsconfirmingthatinordertoobtainprotec-tion against the prevalentRV types circulating inArgentina all P[5],P[11],G6andG10typesshouldbeincludedinthevaccine.Key words: bovine rotavirus, Cattle immune response, Guinea pigimmuneresponse,vaccinesSpecies:ruminants;other

bV052. INVESTIGATION THE SEROPREVALENCE OF BOVINE BRUCELLOSIS USING BLOOD SERUM AND

SEMINAL PLASMADAnILoGJUnqUEIRAJúnIoR,GILSonPMoRAES,AnnA

MCLIMA,GERMAnoKFARIA,FERnAnDAKMARqUES,PAULoRoLIVEIRA,CéSARAGARCIA

FaculdadedeMedicinaVeterináriadaUniversidadeFederaldeUberlândia-FAMEV-UFU

dan_hp2002@yahoo.com.brBrucellosis is an important public health problem is a zoonotic

diseasethatcauseseconomicloss,andseenallovertheworld.Theinfection causedbymembersofgenusBrucella, asmaleas femalebovine,induceshumoralandcellularimmuneresponse.Thehumoralresponseagainstnaturalinfectioncharacterizedbyalmostsimultane-ousincreasingofantibodies(Ig)concentrationofkindsIgMandIgG.Intheseanimals,theIgofIgMkindreduces,tendingtowardsdisappear-inginafewweeksaftertheinfection.Ontheotherhand,theIgofIgGkindestablishedcontinuesinchronicallyinfectedanimals.Theofficialtests inBrazilarebased in theseantibodydetectionsonly inbovinebloodserum.Tamponatedacidifiedantigentest(TAAorRose-Bengal,RB)detectstheIgG1whilethetubeagglutinationwith2-mercaptoeta-nol(2-ME)detectsIgG.Although,thefavoredplaceofBrucellaintes-ticlesandaccessoryglansinmalebovinecanformlowserumIgtitles;whichmakesextremelyhardthediagnosisbyserologicaltechniquesoftriage.Thelawdoesn´testablishanytestforIgdetectioninsemenofreproductivebulls.Thepurposeofthisstudywastoinvestigatetheseroprevalenceofbrucellosisusingbloodserumandseminalplasmafrom71bullsprovidedof8propertiesinUberlandiaCounty–MGState,Brazil.Thisworkhascomparedtheresultsoftwotestsfordiagnosisofbovinebrucellosis,applyingthetestsofRB(oftriage)and2-ME(con-firmatory).Theresultsfoundinthesetwotestspresentanegativecor-relation,consideringthat:whenusedthebloodserum,ithaspresented

adecreaseinnumberofpositivebullsforthebrucellosisdiagnosis(2positiveanimals).Ontheotherhand,whenusedtheseminalplasma,thenumberofanimalswithpositivediagnosishasincreased(7positiveanimals).ThisstudyresultshighlighttheimmediatenecessitytoalsoinvestigateantibodiestoBrucellainseminalplasma.

Keywords:Seminal,Bovine,Brucellosis,BullsSpecies:ruminants

bV053. EFFICIENCy OF AN INDIRECT ELISA USING TWO ANTIGEN PREPARATIONS OF BRUCELLA CANIS FOR

IMMUNODIAGNOSISMARIAZDoLIVEIRA1,3*,SonGELIMFREIRE3,RoBERTo

MEyER3,LARAKEID4,STELLAMBARRoUIn-MELo1,2,PALIS,PAULo

1LaboratóriodeInfectologiaVeterinária,EscoladeMedicinaVeterinária,UniversidadeFederaldaBahia(UFBA),Av.Ademarde

Barros,500,Salvador,Bahia,Brasil,CEP:40170-000;2DepartamentodePatologiaeClínicas,EscoladeMedicinaVeterinária,UFBA;

3LaboratóriodeImunologiaeBiologiaMolecular,InstitutodeCiênciasdaSaúde,UFBA,AvReitorMiguelCalmonS/NValedoCanelaSalvador,Bahia,Brasil,CEP40.110-100;4FaculdadedeMedicinaVeterinária

eZootecniadaUniversidadedeSãoPaulo.zoraida.vet@gmail.com

Introduction: Canine brucellosis is a zoonotic bacterial infectionwhoseclinicaldiagnosisisdifficulttoperformduetofrequentasymp-tomatic cases. The disease is important for Public Health. Humaninfectionshavebeen reported,being laboratory techniciansanddogownersthemostexposedtoB.canis.Havingaworldwidedistribution,the infection representsagreat causeofeconomic losses inbreed-ing kennels, since it causes abortion and infertility in dogs. Despitemolecular techniques have been recently described, blood cultureis still considered theonly routinedefinitive test for thediagnosisofcanineinfection.Differentserologicaltestsareroutinelyused,butthereisaneedforafast,reliableandlowcostimmunologicaltesttoimprovethe immunodiagnosis and surveillance studies of caninebrucellosis.Theaimofthisstudywastocomparetwoantigenpreparationsmadefroma cultureofB. canis inELISA testsusing sera fromdogswithdistinctprofilesofinfection,definedaspositiveandnegative.Materialand Methods:A B. canis strain, isolated from aborted placenta andfetusesofabitch,wasused forproducing twoantigenpreparations:aheatsolublebacterialextractandasonicallyobtainedextract.Aftercentrifugation steps, the soluble fractions of both preparations wereappliedintwoindirectELISAtests,performedwithserafrom85dogs,fordetectionofB.canis-specificIgG.Fiftyonefromthese85animalswerediagnosedpositiveforcaninebrucellosis,bybloodculturesandserologyinagar-gelimmunodiffusiontest(AGID).Thirtyfourofthesedogswerehealthynegativeanimals,withnohistoryofbreeding.Theopticaldensity readingswereused tocalculatespecificity,sensitivityandaccuracyoftheELISAsbyROCanalysis.Results:Thesensitivity,specificityandaccuracywereof100,84.30and90.6%fortheELISAtestwithsonicallyobtainedextract,andof91.2,100%and94.4%forthe ELISA test with heat soluble extract, respectively. Conclusions:BothELISAtestsusingthedifferentantigenpreparationsobtainedfromawildB.caniswereconsideredapplicableforserodiagnosisofcaninebrucellosis,showinggoodspecificityandsensitivity.Key words:Brucellacanis,Immunodiagnostics,ELISA,DogSpecies:canine

bV054. INHIBITION OF EHRLICHIA CANIS INFECTION By INTERFERON GAMMA IN VITRo

ToMoKoTAJIMA1,MAKoToWADA1,MISAoonUMA21LaboratoryofVeterinaryMicrobiology,GraduateSchoolofLifeandEnvironmentalScience,OsakaPrefectureUniversity,Osaka,Japan;2LaboratoryofInfectiousDisease,GraduateSchoolofVeterinary

Medicine,HokkaidoUniversity,Sapporo,Japane-mailaddress:tajima@vet.osakafu-u.ac.jp

Ehrlichia canis infects to macrophages-monocytes of dog andcause canine monocytic ehrlichiosis, a persistent infection that con-tinues for a long period even after treatment with antibiotics. We

71

previously reported that expression ofmRNA for interferon-γ (IFN-γ)wasdetected inperipheralbloodmononuclearcells (PBMC) fromE.canis-infecteddogswithinthreedaysafterinfectionandcontinuedformorethan50days.TodeterminetheroleofIFN-γinE.canisinfection,E.caniswasculturedwithwhitebloodcells(WBC)orPBMCfromE.canis-infecteddogs. TwoSPF femalebeagledogswere inoculatedintravenously with E. canis Oklahoma strain and the infection wasconfirmedbyPCRusingperipheralbloodDNAofthesedogs14dayspost-inoculation (DPI). Bloodwasdrawn fromeachdogon16DPI,andisolatedWBCorPBMCwereaddedtoE.canis-inoculatedDH82cells.WBCandPBMCfromanuninfectedbeagledogwereusedasnegativecontrols.Theculturedcellswerecollecteddaily,stained,andincidenceof inclusion-positivecellswascalculatedbycountingcellsunder microscope. Incidence of inclusion-positive cells was signifi-cantlyreducedintheculturewithWBCorPBMCfromE.canis-infecteddogsbutnotwiththosefromuninfecteddog.Suchreductionofinclu-sion-positivecellswasinhibitedbyadditionofanti-dogIFN-γantibody.Ontheotherhand,culturedsupernatantwascollectedandassayedforIFN-γbyELISA.ThoughIFN-γwasdetected inallsupernatant fromcultureswithWBCorPBMCfrominfecteddogs,theconcentrationwasreducedwhenthecellswereculturedwithanti-dogIFN-γantibody.ToexaminethedirecteffectofIFN-γ,recombinantcanineIFN-γ(r-IFN-γ)wasaddedtoE.canis-inoculatedDH82cellsandcultured.Significantreduction in incidence of inclusions is observed in E. canis-infectedDH-82cellsculturedwithr-IFN-γ. Presentresultsshowedanimpor-tantroleofIFN-γintheimmunityofE.canisinfection.CounterbalancebetweenIFN-γactivityandE.canisgrowthmayleadtoestablishmentofpersistentinfection.EnhancementofIFN-γproductionmaybeeffec-tivetopreventpersistentinfectionindogs.Key words:Ehrlichiacanis,interferongammaSpecies:canine

bV055. INTESTINAL CD134+ T AND B LyMPHOCyTES: SITES FOR ACTIVE FIV REPLICATION IN CHRONICALLy

INFECTED CATSHELGARCH1,AGoUBIER1,HPoULET1,yMUELLE,S

RICHARD1,LFoREST1,LCHAPAT1,CAnDREonI1,VJUILLARD11MerialS.A.S.,DiscoveryResearch,254rueMarcelMérieux,69342

Cedex07Lyon,FranceFelineImmunodeficiencyVirus(FIV)isamajorpathogenofcats,

responsible for an acquired immuno-deficiency syndrome (AIDS),comparable to Human Immunodeficiency Virus (HIV)-associatedAIDSinhumans.Therefore,notonlyisFIVamajorissueinveterinarymedicine,butitalsoprovidesanaturalmodeltostudylentiviruspatho-genesis.EntryofHIVandFIVinhostcellsfollowsatwo-stepmodelin which binding to a primary receptor induces conformational rear-rangements exposing the co-receptor binding site.Both viruses useCXCR4asaco-receptorbutdiffer in theirprimaryreceptor(CD4forHIVandCD134forFIV)andintheirtropism,restrictedtoCD4+TcellsforHIVandbroaderforFIV.Gut-associatedlymphoidtissue(GALT)isareservoirforthevirusandasiteofCD4+Tcellsdepletioninchroni-callyHIV-infectedhumans,butitsroleinFIVpathogenesisneedstobeinvestigatedfurther.

Our objectiveswere to characterizeFIV-induced immunedisor-derandFIVtargetpopulationinGALTofchronicallyinfectedcats.WethereforedevelopedamethodtoisolateGALTimmunecellsandana-lyzed immunologicalchangesandvirusmultiplication throughdetec-tionofp24.WefirstobservedadepletionofmucosalCD4+TcellsandBcells.AsinHIVinfection,thisdepletionispreferentiallyobservedinGALTeffectorsites(intestinalepitheliumandlaminapropria),ascom-pared to the inductive compartments (mesenteric lymph nodes andPeyer’sPatches).ByfocusingonCXCR4andCD134expressingcells,weobservedapreferentialdepletionofmucosalCD134+BcellsandCD4+CD134+Tcells,especially in thoseco-expressingCD134andCXCR4.Whetherthesedepletionswereassociatedwiththeinductionofapoptosiswasnotinvestigated.

To further characterize FIV replication in the gut of chronicallyinfectedcats,theexpressionofp24wasanalyzedbyflowcytometryinmucosalimmunecells.Weobservedthatp24positivecellswerewithintheCD134+cellpopulationandwerepreferentiallyobservedinGALTeffectorcompartments,aT-cellrichsite.

AltogetherourdatashowedthatmucosalCD4+TcellsandBcellsconstituteasiteofactivereplicationofthevirusinchronicallyinfectedcats. Our results emphasize the role of GALT in FIV pathogenesis,further reinforcing the homologies observed between FIV and HIVinfection.Key words: FIV,chronicinfection,CD134,GALT,p24,reservoirSpecies: felines

bV056. MICROARRAy ANALySIS OF SySTEMIC PIG INNATE IMMUNE RESPONSES AFTER ExPERIMENTAL INFECTION

WITH ACTINOBACILLUS PLEUROPNEUMONIAEKERSTInSKoVGAARD,SHILAMoRTEnSEn,KARInTARP

PoULSEn,GREGERSJUnGERSEn,PETERMHHEEGAARDNationalveterinaryInstitute,TechnicalUniversityofDenmark,

Bülowsvej27,DK-1790CopenhagenV,Denmarkpmhh@vet.dtu.uk

Actinobacillus pleuropneumoniae (Ap) is the causative agentof porcine pleuropneumonia, a highly infectious respiratory disease.Theroleoftheporcineinnateimmuneresponseinthepathogenesisand clinical outcome of this infection remains poorly understood. Inthisstudyweexaminedtheinnatetranscriptionalresponseintheliver,14hoursafterinoculationwithApusingoligonucleotidemicroarrays.

Specificpathogenfreepigs(8-10-week-old)werechallengedwithApserotype5B(isolateL20).Microarrayswereusedtorevealgenesbeing differentially expressed in liver tissue from infected animals(N=10)versusliversamplesfromnon-infectedanimals(N=5).

Themicroarrayusedinthisstudy(POM-4)isafocusedlowdensityin-housespottedmicroarraycomprising immunologically relevantoli-gonucleotideprobescorrespondingtogenescodingforinterferonsandinterleukins(andreceptors),chemokines(andreceptors),acutephaseproteins, apoptosis-related factors and sequenceswith relevance toToll-like receptors and their intracellular signalling pathways.Probesfordifferenthousekeepinggenesarealsoincludedfornormalisationpurposes.Themicroarrayholds384differentoligonucleotidesrepre-sentingmorethan200differentimmune-relatedgenes.

Atotalof61geneswerefoundtobesignificantly(P<0.05; foldchange>2)differentiallyexpressedinlivertissuefrominfectedanimalscompared tocontrol.Of thesegenes,36wereexpressedata lowerleveland24geneswereexpressedatahigherlevel.

Amongthedifferentiallyexpressedgeneswereseveraltobelong-ingtotheacutephaseproteins(APP).C-reactiveprotein,fibrinogen,haptoglobinandserumamyloidAwereup-regulatedandalpha-1acidglycoprotein,albumin,apolipoproteinA-1,transferrin,andtransthyretinwere down-regulated in liver samples of infected animals comparedto control animals. In addition, the expression of genes encodingpro-inflammatory mediators of the innate immune response includ-ingTNF-alpha, IFN-gamma, IL-1, IL-10and IL-18wasalsochangedsignificantly.

Geneexpressiondifferencesof the four up-regulatedAPPs (C-reactiveprotein,fibrinogen,haptoglobinandserumamyloidA)aswellasthreeofthedown-regulatedAPPs(apolipoproteinA-1,transferrin,andtransthyretin)wereverifiedbyquantitativereal-timePCR.

Thus,theimmunefocusedporcinemicroarrayPOM-4allowedthestudyofhundredsof immunerelatedgenes inasingleanalysisanda number of geneswere significantly up- or down-regulated in liversamplesaccordingtoinfectionstatusandreflectingasystemicinnateimmuneresponsetotheinfection.Key words:pighostresponse,microarrays,innateimmunologySpecies:swine

bV057. PROTECTION AGAINST LAWSONIA INTRACELLULARIS RE-INFECTION DID NOT CORRELATE

WITH HUMORAL OR CELL-MEDIATED IMMUNE RESPONSE1ULLARIBER,1ToRSTEnSBoUTRUP,1LIEnTHInGUyEn,

1JEAnnETJAKoBSEn,1TIMKJEnSEn,1PETERMHHEEGAARD,1GREGERSJUnGERSEn

1NationalVeterinaryInstitute,TechnicalUniversityofDenmark,27,Bülowsvej,DK-1790CopenhagenV,Denmark

72

Background: Lawsonia intracellularis is an obligate intracellularbacteriumcausingproliferativeenteropathy(PE)inpigs.Clinicalsignsof affected pigs afterweaning include diarrhoea and unevenweightgain.

Experimentaldesign:Threegroupsofpigswereestablished:“Re-infectedpigs”(Re-i;N=8)receivedaprimaryL.intracellularisinfectionat5weeksofagebyoralinoculationwithatleast1010bacteriainaninfectedmucosahomogenate.After3weekstheinfectionwasclearedbyTiamulintreatmentforoneweek,andat12weeksofage,thepigswere re-infectedwith L. intracellularis. “Challenge control pigs” (Cc;N=10)werenotinfectedat5weeksofage,butreceivedtheTiamulintreatment and a (primary) infection at 12weeks of age. “Treatmentcontrolpigs” (Tc;N=4)didnot receive there-infectionafterantibiotictreatment.

Protectionagainstre-infection:WhilefaecalexcretionofL.intra-cellulariswasdetectedbyPCRfrom6to21daysafterprimaryinocula-tionsat5weeks(Re-iandTc)or12weeks(Cc)ofage,excretionofL.intracellulariswasnotdetected following re-infection. Inaddition,PEwasonlyobserved intheprimary infected,Cc,pigsatnecropsyandhistopathologicalevaluation.

Immune response: Acute phase protein measurements wereemployedtocharacterisethemagnitudeandextentofimmediatetissuedestructionbytheinfectionasameasureofthecapacityoftheanimaltohandle the infection.Lawsoniaspecific IgGinserum(ELISA)wasmeasuredinallpigs,butaboostwasnotobviousintheRe-igroup.

IFN-γwasmeasured inwholebloodand ileocaecal lymphnode(ILN)cellculturesafter invitrostimulationwithsonicatedL. intracel-lularis, followed by quantification of produced IFN-γ byELISA.OnlylowlevelsofantigenspecificIFN-γresponsesweredetectedinwholebloodcultures,whilethelevelswerehigherinILNcellcultures.FlowcytometricmeasurementofT-cellproliferationafterinvitrostimulationwithsonicatedL.intracellularisrevealedproliferativeCD4+responsesinall3groupsofpigs.Differencesincellsubsetphenotypesinbloodsamplesandincellpreparationsfromileumidentifiedsometransloca-tionofCD4+andCD8+cellsintotheaffectedtissues.

Conclusion:Protectionagainstre-infectionwasclearlyobservedby lack of faecal excretion of L. intracellularis and absence of PE.Differences in immuneresponsedueto thisprotectionagainst infec-tioncouldnotclearlybe identified from theparametersmeasured inthisexperimentKey words:Lawsonia,protection,immunity,pigsSpecies:swine

bV058. IGG ISOTyPES RATIO AND REPRODUCTIVE PERFORMANCE IN SOWS NATURALLy INFECTED WITH

BRUCELLA SUISARESTEGUIMB1,GUALTIERICAS1,ToRIonIDEECHAIDES2,AGUIRREn2,DELGADoG3,PERALTAL1,BESSoR1,GEnTILE

n1,SCHARoVSKyoG41CátedradeSuerosyVacunas,3CátedradeObstetricia,Fac.Cs.Veterinarias,Casilda;2INTA,EEARafaela;4Inst.Genética

Experimental,Fac.Cs.Médicas,ConsejodeInvestigaciones,U.N.R.,Rosario,Argentina.

maresteg@fveter.unr.edu.arDifferentialimmunoglobulin(Ig)isotypeexpressionallowsdiverse

isotype-relatedfunctions,whichcanberelatedtoinfectionortoprotec-tion,dependingupontheprevalentIg isotype.IthasbeenconfirmedthedifferencesinbiologicalfunctionsfordifferentIgGisotypesofthepig. Inpreviousstudieswehaveshown, ina farmnaturally infectedwithB.suis, thatB.suisantibodyconcentrationwasassociatedwithan increased individual animal risk of non-pregnancy and abortion.Porcinesfromherdswithdiverselevelsofreproductivesuccess,thatwereseropositive(S+)forbrucellosisbyBPA,2ME,FPA,C-ELISAandCF(complementfixation)analysis,hadahigherriskofabortionthanseronegative (S-)animals. In thisstudy,weaimed to investigate thehost–parasiterelationshipwithinthepregnanthost,inordertoincreasetheunderstandingofthefactorsthatinfluencetheinfectiondynamics;also,westudiediftheIgG1/IgG2serologicalrelationshipisassociatedtothetypeofresponsetoBrucellainfectionanditsroleininfectionsatpregnancyand reproductiveperformance.Westudied the IgG1/Gg2

isotypes ratio in sows from a herd naturally infected with B. suis inpregnant(groupI),non-pregnant:irregularoestrusreturn(groupII)andfemalesthathadaborted(groupIII).Alltheanimalssharedthesameenvironment.Serasamplesweretakenandreproductivevariableslikepregnancy, abortions and infertility were controlled, simultaneously,duringtheexperience.IgGisotypewasdeterminedbyIndirectELISAwithmouseantiporcineIgG1andIgG2in320femalepigs.Thediffer-encesbetweengroupswereanalyzedwiththeunpairedttestusingtheGraphPadInStatstatisticalsoftware.TheIgG1/IgG2ratiofromgroupI[Median(Range):0.28(0.05-0.72)]wassignificantlylowerfromthatofgroupII[1.21(0.41-4.10)]andgroupIII[0.90(0.53-1.35)](p<0.0005andp<0.0001, respectively).These resultsagreewith the functionalandphenotypicdifferencespreviouslyfoundintheseanimals.WecanconcludethatalowIgG1/IgG2ratioor,inotherwords,aisotypebalancefavoringIgG2,wouldbeassociatedtoaprotectiveimmuneresponseleading to the absence of reproductive alterations and/or to a goodreproductiveperformance.Theseresultscontributetoabetterunder-standingofdiseasecontrolmechanismsandvaccineresponses.Key words: IgG Isotypes, Brucella suis, Sows, ReproductiveperformanceSpecies:swine

bV059. PRRSV-INDUCED IMMUNE DySREGULATION SELECTS B CELLS WITH HyDROPHOBIC HCDR3S FOR

ExPANSIONCAITLInDLEMKE1,JoHnEBUTLER2,PATRICKWEBER2,

MAREKSInKoRA3,AMyVInCEnT4,KELLyDLAGER4 1RobartsInstitute,London,Ontario,CAN;2DeptofMicrobiol.,Univ.ofIowa,USA;3CzechAcademyofScience,NovyHradek,CZ;4NationalAnimalDiseaseCenter,Ames,Iowa,USAGermfree isolator piglets infected with PRRS virus (PRRSV)

develop lymphoid hyperplasia, extreme hypergammaglobulinemia,depositionof immunoglobulincomplexesofall isotypesinthekidneyandautoimmunity(Lemkeetal2004).Hypergammaglobulinemiaofallmajorisotypeswasseeninthebronchial-alveolarlavage(BAL)aswellasserum.AnalysisofVHusagerevealedthelackofrepertoirediversi-ficationwhilespectratypicanalysesshowedthatcloneshavingcertainHCDR3lengthswerepreferentiallyexpanded.Interestingly,thesameclones were expanded in most tissues examined in contrast to thepatternobserved inSIV-infected littermatesorshamcontrols.Thesesameexpandedclonesexpressed IgGand IgAandsometimes IgM.SequenceanalysesofHCDR3sinPRRSV-infectedanimalsshowedastrongbiasfavoringsequencesinthehydrophobicregionofthehydro-pathicityprofile.AhighproportionsharetheAMVLVandrelatedmotifsencodedbythegermlinesequenceofreadingframe3ofDHA(Butleretal2007).SincethespectratypicpatternofcellsfromtheBALandthetracheo-bronchial lymphnodeswereidentical,weconsideredBcellsfromtheBALtoberepresentativeoflymphnodesassociatedwiththelung.BcellsinBALweresignificantlyelevatedandmostallretainedCD2,amarkerofnaïveBcells.AdditionalphenotypicanalysesofBALlymphocytesindicatedthataβTcellsweregreatlyexpandedandthatbothaβandγdTcellsdisplayedactivationmarkers.Wesuggestthatinisolatorpiglets,PRRSVinfectionresultsinaproperTcellresponsebutactsasaBcellsuperantigenthatcausesdysregulationofthenormalanti-viralimmuneresponse.

Lemke,C.D.,J.S.Haynes,R.Spaete,D.Adolphson,A.Vorwald,K.D.LagerandJ.E. Butler. 2004. J. Immunol. 172:1916-1925

Butler,J.E. ,C.D.Lemke,P.Weber,A.Vincent,M.SinkoraandK.D.Lager.2007J.Immunol.178(inpress)Key words:Immunedysregulation;virus;HCDR3;superantigenSpecies:swine

bV060. TRANSFECTION OF PORCINE SIALOADHESIN INTO A MURINE MACROPHAGE CELL-LINE RENDERS IT

PERMISSIVE FOR PRRSV REPLICATIONSUDARVILISHAnTHALInGAM1,WEIGUoLIU1,KEVInSnEKVIK1,

ASITPATTnAIK2,FERnAnDoAoSoRIo2,SUBRAMAnIAMSRIKUMARAn1

73

1DepartmentofVeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA99164-7040,USAand2DepartmentofVeterinaryandBiomedicalSciences,UniversityofNebraska,Lincoln,

NE68583-0905,USA.Contactsudar@vetmed.wsu.edu

Sialoadhesin isamacrophage-specificadhesionmolecule. It istheprototypicmemberof thesialicacidbinding immunoglobulin-likelectin (siglec) family, andhence referred toas siglec-1, andalsoasCD169.Itbindssialylatedligandsonotherhematopoieticcells,pre-dominantlyneutrophils,butalsomonocytes,naturalkillercells,B-cellsandcytotoxicTlymphocytes.Itisanon-phagocyticreceptor,butmayaid in phagocytosis. Other functions of sialoadhesin are not clear.Earlierstudieshaveidentifiedsialoadhesinasthecell-surfaceproteinon porcine alveolar macrophages (PAMs) that mediates internaliza-tionofporcinereproductiveandrespiratorysyndromevirus(PRRSV).In these studies, transfection of porcine sialoadhesin into a porcinekidney cell-line (PK-15) rendered it susceptible to PRRSV infection.However,theinternalizedvirusdidnotreplicate.WehypothesizedthatreplicationofPRRSV requiresporcinesialoadhesinaswell asaddi-tionalmacrophage-specificfactors.TheobjectiveofthisstudywastoclonethecDNAencodingporcinesialoadhesin,transfectintoamurinemacrophagecell-line,anddeterminethepermissivenessofthetrans-fectantcell-lineforPRRSVreplication.

TotalRNAfromPAMswasextractedandcDNAwasmadebyRT-PCR.ThePCR-amplifiedcDNAforsialoadhesinwasclonedintoa mammalian expression vector and sequenced. Four independentclonesweresequenced.Comparisonofoursequencedata(Genbankaccession no. DQ176853) with that published previously (Genbankaccessionno.NM_214346)revealed15aminoaciddifference,whichlikelyrepresentspolymorphism.ThecDNAforsialoadhesinwastrans-fected into a mouse macrophage cell-line. The transfectants werelabeledwithamonoclonalantibodytosialoadhesin,andsubjectedtofluorescence-activatedcellsorting.Weobtained51singlecellcloneswhichexpressedporcinesialoadhesinonthecellsurface.Ofthese,17clonescontinuedtoexpressporcinesialoadhesinontheirsurface,tovaryingdegrees.Threeof theseclonesstablyexpressingsialoadhe-sinwerefoundtobesusceptibletoPRRSVinfection.ThecloneAA9was tested further todetermine itsability tosupportPRRSVreplica-tion.AA9cellssupportthereplicationofPRRSV.ThetiterofPRRSVobtainable inAA9cells isbetween2x105and2x106TCID50/ml. Thiscell-lineshouldbevaluableforlargescalepropagationofPRRSVforvaccineproduction.Key words:PRRSV,Sialoadhesin,Receptor,TrasfectantSpecies:swine

bV061. INHIBITION OF IFN-α PRODUCTION By PORCINE CIRCOVIRUS-DERIVED DNA MOTIFS

TAnJALöVGREn,FRIDAHASSLUnGWIKSTRöM,LISBETHFUxLER,SIRJETIMMUSK,EVAWATTRAnG,CARoLInEFoSSUM

Dept.ofBiomedicineandVeterinaryPublicHealth,SectofImmunology,SwedishUniversityofAgriculturalSciences,Uppsala,

Sweden.tanja.lovgren@bvf.slu.se

Porcinecircovirusesaresmall,non-envelopedviruseswithsingle-stranded, circular DNA genomes of around 1760 nucleotides.Thereare two typesofporcinecircoviruses; type1 (PCV1)and2 (PCV2).PCV1hasnotbeenassociated todiseasewhereasPCV2hasbeenproposed to be the causative agent of postweaning multisystemicwasting syndrome (PMWS) and porcine dermatitis and nephropathysyndrome(PDNS).ThereareseveralisolatesofPCV2andwhilethesequenceidentitybetweenPCV2andPCV1isaround80%,theiden-titybetweenPCV2 isolates ismore than96%.The immunedefenceagainstvirusesdependstronglyontype1interferon(IFN-a/β)produc-tionbyleukocytesrespondingto,forexample,viralnucleicacids.InthePCV2genome,severalmotifs inducingproductionof IFN-αbutalsoa20-nucleotidemotif(nucleotides1481-1500)thatinhibitsproductionof IFN-αhasbeen identified.Aphosphodiesteroligodeoxynucleotide(ODN) representing this motif inhibits IFN-a production induced bythestimulatoryODN2216,plasmidDNAorDNAvirus(Aujeszky’sdis-easevirus),butnotthatinducedbydouble-strandedRNA(polyI:C)orRNA-virus(Sendaivirus).Theinhibitorymotifwasfirstidentifiedinthe

PCV2 isolate Imp1010-Stoon (AF055392), from a Canadian PMWScase,and is identical inan isolate fromahealthySwedishSPFpigherd(EF184220).However,thecorrespondingPCV1motifdifferat8nucleotidepositionsandthemotifinthePCV2isolateN1(EF184226),fromthefirstSwedishPMWScase,differattwopositions.Inthepres-entstudy,wedesignedODNsrepresentingthismotiffromStoon,N1orPCV1.TheinhibitoryeffectwastestedinculturesofporcineperipheralbloodmononuclearcellsstimulatedwithODN2216,pcDNA3plasmidorpoly I:C.BothStoonandN1motifsdecreased IFN-aproduced inresponse toODN2216andpcDNA3butnot topoly I:C.ThePCV1motifalsodecreasedinductionofIFN-aproductiontopcDNA3butwaslesseffective indecreasingIFN-aproduction inducedbyODN2216.Using a secondary structure-predicting program, an ability to formhairpin-likestructureswasfoundinthemotifs fromall three isolates.However,whiletheN1motifformedthemoststablehairpin,thehair-pinformedbythePCV1motifwastheleaststable.ThePCV1motifsinabilitytoformastablehairpinstructuremaycontributetoitsreducedinhibitionofODN2216 induced IFN-αproduction,butstill it remainsto establish if this discrepancy is of importance for the difference invirulencebetweenPCV1andPCV2.Key words:IFN-a,circovirusesSpecies:swine

bV062. MICE VACCINATION WITH VAPA: CHALLENGE WITH RHoDoCoCCUS EqUI IS FOLLOWED By PRODUCTION OF

TH1 CyTOKINES. ALInEFoLIVEIRA1,SAnDRoGSoARES1,MARIA-CRISTInA

RoqUE-BARREIRA11DepartmentofCellularandMolecularBiology.SchoolofMedicineof

RibeirãoPreto,USP-SP.IntroductionandObjectives:Rhodococcusequiisthemostimpor-

tantcauseofbacterialinfectioninyoungfoalsallovertheworld,andhas emerged as an opportunistic pathogen in immunocompromisedhumans. The severe pneumonia caused byR. equi, constitutes animportanteconomicandpublichealthproblem,generatingtheinterestinthedevelopmentofefficientvaccinesagainstthebacteria.TheVapAproteincorrespondstoanimportantvirulencefactorofR.equi(Jainetal.,Mol.Microbiol.50:115-28,2003),and isconsideredaprotectiveimmunogen.WehavedevelopedanattenuatedSalmonellaentericastrainexpressingtheVapAprotein,whoseadministrationtomicehasinducedprotectionagainstR.equiinfection,asdemonstratedbyhighersurvivalratesandbacterialclearanceincomparisonwiththenon-vac-cinatedanimals.Thisprotectionwasassociatedwithdetectionofhighlevels of serum specific antibodies, predominantly of IgG2a isotype(Oliveiraetal.,MicobesInfect.9(3):382-90,2007).Moreover,invitroantigenstimulationofcellsobtainedfromvaccinatedmiceresultedinhighproductionofIL-12p40,TNF-a,IL-1β,andIFN-γ,whereasnoIL-4wasdetected.SinceTh1immuneresponseisextremelyimportanttoachieveprotectionagainstrhodococcosis,thepresentstudyaimedtoevaluatetheprofileofcytokinesproducedinvivobyimmunizedmicefollowingchallengewithvirulentR.equistrain.

MethodsandResults:GroupsofBALB/cmicewereorallyimmu-nizedwithtwodosesof1x109CFUofS.entericaTyphimuriumvapA+orvapA-.AthirdgroupofmicereceivedonlyPBS.Twoweeksafterthelastimmunization,allmicewereintravenouslychallengedwithasub-lethaldoseofvirulentR.equi.Ondays2,4,8,and10post-challenge,groupsof4miceweresacrificedandtheirspleens, livers,andlungswereremoved.Tissueshomogenateswereusedforcytokinesquan-tization byELISA.The vaccinatedmice (inoculatedwithS. entericaTyphimurium vapA+), in contrast with the control groups (inoculatedwithS.entericaTyphimuriumvapA- strainorPBS),producedhigherlevels of IL-12p40 and IFN-γ in all organs analyzed, whereas lowerlevelsofTNF-aandIL-4weredetected.

Conclusion:MiceprotectionagainstR.equiinfectionprovidedbyvaccinationwithS.entericaTyphimuriumvapA+isassociatedwithaTh1biasedspecificimmuneresponse.TheimmunizationusingattenuatedrecombinantSalmonellastrainsasvectorforthedeliveryofheterolo-gousantigenshadalreadybeenshowntoinduceaTh1-typeresponsepreferentially(Langeetal.,Infect.Immun.72:4924-28,2004).

FinancialSupport:FAPESPandCNPq.

74

Key words: vaccines, Rhodococcus equi, Salmonella enterica,VapAproteinSpecies:equine

bV063. RECOMBINANT PROTEIN M DETECTS ANTIBODIES INDUCED By STREPToCoCCUS EqUI STRAINS ISOLATED

FROM CASES OF STRANGLESCARInAMMoRAES1*,AnDRéASRRoCHA1,LUAnAA

DUMMER1,ALCEUGSJUnIoR1,CARLoSEWnoGUEIRA2,AGUEDAPCVARGAS3,FáBIoPLLEITE4,FABRÍCIoR

ConCEIçÃo1,CARLoSGIL-TURnES1-21CENBIOT/UFPel;2FV/UFPel;3DVP/UFSM;4DMP/UFPel

carinamoraes@terra.com.brStranglesisacontagiousdiseaseoftheanteriorrespiratorytract

ofEquidæcausedbyStreptococcusequi.Asymptomaticcarriersareresponsibleforthemaintenanceoftheinfectionintheherds,andarerecognizedonlybyserologicalormicrobiologicalmethods.Vaccinesusedinitscontrolinducelowprotectionlevels,generallybelow50%.S.equiproteinM(SeM)isconsideredthemdostpromisingprotectingantigenagainsttheinfection.Inthisresearchwestudiedtheantigenic-ityoftwocommercialvaccinesinuseinsouthernBrazilandofothertenmonovalentvaccinespreparedwithstrainsofS.equirecoveredfromclinicalcasesofStrangles.Allthevaccineshadthesameconcentra-tionofinactivatedantigenandusedAluminumHydroxideasadjuvant.Isogenic Balb-c mice were randomly grouped and vaccinated with1/20thofanequinedoseoftherespectivevaccineondays0and14.Serumantibodiescollectedondays0,14,28,56and70weretitratedbyELISAusingrecombinantSeMasantigen(0,12µg/well).RecombinantSeMwasproducedinourlaboratorybyatransformedE.coliharboringtheSeMgeneobtainedbyPCR(GenBankaccessionNºU73162).Theampliconwas cloned in the pAE vector, expressed in competentE.colipLyss,andtheprotein,purifiedbyaffinitychromatography(his-tag)usingAKTAPrimeTMPlus (GEHealthcare),wasdetectedbyWesternBlot.All the vaccines induced antibodies againstSeM, although thetiters differed among them. Mean seroconversions induced by theexperimentalvaccinesvariedbetween3.1and9.2,whilethoseofthecommercialvaccineswere1.7and3,3.Seronconversionsinducedbytheexperimentalvaccineswerehigherthanthoseof thecommercialvaccines.OurresultsshowedthattherearequantitativedifferencesintheimmunogenicityofdifferentstrainsofS.equi,andthatthispropertymaybeusefulintheselectionofvaccinalstrains.

Financialsupport:CNPq(474509/2004-4);FAPERGS(0523299)Key words: M protein, Strangles, Streptococcus equi, recombinantproteinSpecies:equine

bV064. MAPPING THE ANTIBODy RESPONSE OF THE NATURAL HOST TO EQUINE RHINITIS A VIRUS CAPSID

PROTEINS.FAnLI1,BREnDAnSCRABB2,MICHAELJSTUDDERT1,JAMES

RGILKERSon1,CARoLAHARTLEy1*1SchoolofVeterinaryScience,TheUniversityofMelbourne,Victoria

3010,Australia;2TheWalterandElizaHallInstituteofMedicalResearch,Parkville,Victoria3052,Australia.

carolah@unimelb.edu.auEquinerhinitisAvirus(ERAV)isasignificantpathogenofhorses

andisalsocloselyrelatedtoFoot-andmouthdiseasevirus (FMDV).Wehaveevidenceshowing50%ofhorsesbecomeinfectedwithERAVby5yearsofage,and itappearsmanyof theseseroconversions toERAVmayoccurcoincidentwith1-2yearoldhorsesenteringtrainingstables.Despitethis,knowledgeoftheprevalenceandimportanceofERAVinfectionsremainslimitedlargelyduetotheabsenceofasimple,robustdiagnosticassay.EffortstodevelopsuitableantigensforsuchanassayhaveconcentratedonmappingtheantigenicstructureoftheERAVcapsidandthekineticsofthehumoralimmuneresponsetothoseproteins. The antigenicity of recombinant full-length and fragmentedERAVcapsidproteinsexpressedinE.coli,havebeenusedtomaptheantibodyresponseofserafromexperimentallyinfectedandnaturally-exposedhorses.Wefoundthateachofthefiveexperimentallyinfectedhorsesexaminedproducedantibodies that reactedagainst recombi-nantproteinsencompassingtheC-terminalregionofVP1,full-length

VP2and theN-terminal regionofVP2.Antibody toepitopesonVP2(both native and recombinant forms) persisted longer post-infection(>105days)thanantibodiesspecificforepitopesonotherfragments.OurdataalsosuggestthatBcellepitopeswithinC-terminusofVP1andN-terminusofVP2contributetoalargeproportionofthetotalreactivityof recombinantVP1andVP2, respectively,The reactivity inenzymelinked immunosorbentassay (ELISA)of individual linearepitopesorarecombinantantigenengineeredtoexpressamosaicoftheselinearepitopes,showedvariablecorrelationwithotherassays. Importantlyhowever, the reactivity in ELISA of combinedVP1 andVP2 recom-binant proteins correlatedwellwith a range of native antigen-basedserologicalassaysusingserafrom12fieldhorses.ThisstudyprovidespromisingcandidatesfordevelopmentofanERAVELISAcapableofdetectingantibodieselicitedduringinfectionofthenaturalhost.Key words:equine,respiratoryvirus,antibody,ELISASpecies:equine

bV065. CELLULAR AND FUNCTIONAL CHANGES IN THE REPRODUCTIVE TRACT ASSOCIATED WITH ONSET-OF-LAy

IN CHICKENS. CLAIREEJoHnSTon,PAULWIGLEy

DepartmentofVeterinaryPathology,FacultyofVeterinaryScience,UniversityofLiverpool,UnitedKingdom.

c.e.johnston@liv.ac.ukInfection of the chicken reproductive tract and transmission to

developingeggscanresultindiseaseintheprogenyandinfectionofeggsforhumanconsumption.SalmonellaentericaserovarEnteritidis,for example, is an important food-bornezoonosis,withpoultrymeatand eggs being a major source of infection in humans. In the U.K.,vaccination of laying hens and improved biosecurity has resulted inadeclineinS.Enteritidisinfection,butthroughouttherestofEuropeinfectionremainsprevalent.SystemicinfectionwithS.Enteritidiscanresultincolonisationoftheovaryandoviductleadingtocontaminationofboththeyolkandalbumen.Asyethowever,theprecisemechanismof this transmission is unclear. It is now well-known that systemicimmunosuppression occurs at the point-of-lay, and in S. Pullorum-infectedhensthis immunosuppression isassociatedwith infectionofthereproductivetract.ChickensmaycarryS.Pullorumasymptomati-callyinsplenicmacrophagesformanymonths.However,uponsexualmaturity,T-cell responsiveness significantly decreases in the spleenandnumbersofS.Pullorumincrease.Furthermore,Salmonellaecanberecoveredfromthereproductivetractanddevelopingeggs.Asyet,verylittleisknownaboutthelocalcellularchangeswhichmayoccurinthereproductivetractduringthisimmunosuppressedstateandwhichmayfacilitateSalmonellatransmissiontotheegg.Studieshaveshownmacrophages,CD4+andCD8+Tlymphocytestobepresentthrough-outthereproductivetractofhens,withBcellsmainlyrestrictedtotheoviduct.

Inordertocharacterisethecellularandfunctionalchangesassoci-atedwithimmunosuppressionatthepoint-of-lay,wehavecarriedoutadetailedimmunohistochemicalandgeneexpressionstudyoftherepro-ductivetract-associatedimmunesystemofhens.Betweenweeks15and24post-hatch,wemeasuredthechangeinimmunecellnumbersintheovary,infundibulumandmagnumbyimmunohistochemistry,andlinkedthiswithsystemicchangesinthespleen.Furthermore,wehavebegun to functionallycharacterise thesecells,measuringexpressionofanarrayofcytokinesandchemokines.Thus,thesestudiesprovideimportantinformationonthelocalimmunologyofthereproductivetractinlayinghensandthenatureofthepoint-of-layimmunosuppression.Also,characterisingthesecellswillprovideaninsightintotheroleofdifferentcell-typesinfutureSalmonellainfectionstudiesandhowwecanenhanceimmunityinordertopreventtransmissiontothedevelop-ingegg.Key words: Reproductive tract, Immunosuppression, Cytokines,SalmonellaSpecies:avian

bV066. CLONING, ExPRESSION AND CHARACTERIZATION OF NUCLEOCAPSID PROTEIN FROM INFECTIOUS

BRONCHITIS VIRUS IN ESCHERICHIA CoLI

75

ALIAnDRAMGIBERTonI,CAMILACFERnAnDES,MARIADEFATIMASMonTASSIER,HéLIoJMonTASSIER

LaboratóriodeVirologiaeImunologia,DepartamentodePatologiaVeterinária;FaculdadedeCiênciasAgráriaseVeterinárias,

UniversidadeEstadualPaulista,14884-900,Jaboticabal,SP,Brazil.ProjetoFinanciadopelaFundaçãodeAuxílioàPesquisadoEstadodeSãoPaulo(FAPESP),processosnº05/54275-4enº01/14950-3.

aligiber@fcav.unesp.br;heliojm@fcav.unesp.brTheavian infectiousbronchitisvirus (IBV) is theetiologicagent

of avian infectious bronchitis, which causes an acute, highly conta-gious disease in chickens that affects the respiratory, reproductiveand renal systems. IBV is the prototype of the Coronaviridae familyand isdistributedworldwide, influencingnegatively theproductionofthepoultry industry,so that rapidandaccuratediagnosis is requiredfortheadoptionofthemostefficientcontrolmeasures.TheIBVRNAgenomeencodesthreemajorstructuralproteins:thespikeprotein(S),themembraneprotein(M)andthenucleocapsidprotein(N).Thelattercontaining409aminoacidsandamolecularmassof50kDa,ishighlyconserved among different IBV strains and is abundantly producedduringinfection,whichmakesitanidealantigenictargetfortheimmu-nodiagnosis of IBV infection. This study aims to clone and expressthe IBVN protein, using the vector pET28a(+) andEscherichia coliashost-cellsandtocharacterizeimmunochemicallythisrecombinantprotein.Firstly,theNproteingenefromM41strainofIBVwasamplifiedusingprimerscontainingspecificrestrictionsitesandflankingtheopenreadingframeofthisgenebyreversetranscription-polymerasechainreaction(RT-PCR).TheampliconwasclonedintothepGEM-TEasy(Promega)plasmidwasdigestedwithNdeIandxhoI,gel-purifiedforsubcloning into the Escherichia coli expression plasmid pET28a(+)(Novagen),usingT4DNAligase.TheexpressionoftheHis-Tag-Ngenefusedproteinwasinducedbytheadditionofisopropyl-1-thio-D-galac-toside(IPTG),andtherecombinantproteinwasdetectedandcharac-terizedbySDS-PAGEandWestern-Blotting,whichdemonstratedthepresenceoftheNproteinwithapproximately54kDaandcarryingthepoly-histidinetagandmostlyoftheviralNproteinepitopes,sinceboththeanti-His6MAbandthechickenpolyclonalantibodiesfromchickenshyperimmunizedagainstIBV,reactedwiththisrecombinantprotein.Inconclusion, our results indicated that the E. coli - expressedN IBVproteinhashighhomologytotheviralNproteinandcouldbeagoodsourceofantigentodevelopimmunodiagnosticassaysforthespecificdetectionofantibodiesinchickensinfectedwithIBV.Key words: avianinfectiousbronchitisvirus,recombinantnucleoprotein,Escherichiacoli,expressionSpecies: avian

bV067. TRANSCRIPTOMIC ANALySIS OF THE CHICKEN ANAEMIA VIRUS (CAV)-INDUCED IMMUNOSUPPRESSION.

EFSTATHIoSSGIoTIS1,2,5,DAVEWBURT2,ALISTAIRnJSCoTT1,5,ALISonDoWnInG3,RICHARDTTALBoT3,LISA

RoTHWELL4,PETEKAISER4,ELIZABETHJGLASS2,DAnIELToDD1,5

1DepartmentofVeterinarySciences,Queen’sUniversityofBelfast;2DepartmentofGeneticsandGenomics,RoslinInstitute,Roslin,Midlothian;3ArkGenomics.Roslin;4InstituteforAnimalHealth,

Compton,Berkshire;5AgrifoodandBiosciencesInstitute,Stormont,Belfast.

Chicken anaemia virus (CAV) is an unusually small virus thatcauses severe anaemia and immunosuppression in young chick-ens.Sucheffects reduce theefficiencyof routinevaccinationswhileaggravatingtheeffectsofotherpathogensinchickenpopulations,con-stitutingaseriouseconomicthreattopoultryindustry.Thereplication/pathogenicityofCAVreliesontheexpressionofonlyasinglestructuralproteinVP1and twonon-structuralproteinsVP2andVP3(apoptin).Thefunctionofeachindividualproteinisasyetunclear.Thisstudyhasusedtranscriptionalprofilingtoidentifypathwaysthataredramaticallymodulatedafter48hrof infection inan invitromodel(MDCC-MSB1cells)andafter4daysofexperimentalinfection(1dayoldchickens-invivomodel),usingAffymetrixchickenoligonucleotidearrays.Changesin transcript levels between infected and uninfected cells indicate adysfunction in theMitogenActivatedProtein kinases (MAPK) andTcellreceptorsignallingcascadesimplyingthatCAVmightsubverthost

signallingresponses to itsownbenefit.Twohighly-passagedattenu-ated cloned isolates and three VP3 CAVmutant viruses were alsousedinthisstudywiththeaimofelucidatingtheinteractionsofeachoftheseproteinswiththehostcell.VP3mutationsresultedinlowerlev-elsoftranscriptsencodingsignalling,transcriptionandmitosisrelatedproteins.Overall,thesedataextendourunderstandingofhowbroadlyCAValterstheregulationofhostgeneproductsandhighlightthevirus’‘need’toutilisethehostcellmachinerytoreplicateitself.Key words: transcriptome,chickenanaemiaimmunosuppressionSpecies: avian

bV068. CLONING AND ExPRESSION OF S1 GLyCOPROTEIN OF INFECTIOUS BRONCHITIS VíRUS (IBV)

IN SACCHAROMyCES CEREVISIAEAPoLIVEIRA,AMGIBERTonI,MFSMonTASSIER,AGCAETAno,

HJMonTASSIERLaboratóriodeVirologiaeImunologia.DepartamentodeMicrobiologia

-FaculdadedeCiênciasAgráriaseVeterinárias,UniversidadeEstadualPaulista,Rod.Prof.PauloD.Castellane,s/n.Jaboticabal,SP,Brasil,ProjetoFinanciadopelaFundaçãodeAuxílioàPesquisadoEstadodeSãoPaulo(FAPESP),processosnº02/08649-1enº

01/14950-3ebolsadeestudosconcedidapelaCAPES.heliojm@fcav.unesp.br

Theinfectiousbronchitisvirus(IBV)isoneofthemostimportantrespiratory pathogens, causing an infectious disease in chickens,which is characterized by severe losses on the productive perfor-manceofthepoultryflocks.HighgeneticandantigenicvariationsarecommonfeaturesofIBV,bringingseriousdifficultiesforthecontrolofthisdisease.Inadditiontothis,suchgeneticandantigenicmodifica-tionaffectsmainlytheS1glycoproteinandparticularlyitsneutralizingantigenicsites.ThediagnosisandimmunoprophylaxisofIBVrequiresthe search for more effective serological reagents and immunoges,respectively.Thus,wecarriedoutthisstudyaimingtoclonetheentireopenreadingframeofS1genefromM41strainofIBVintopYES2.1/V5-His-TOPOvectorand,followingcloninginEscherichiacoiandsub-cloninginSaccharomycescerevisiae,toinducetheexpressionofthisproteininhostyeastcells.TheS1recombinatproteinwassuccessfullyexpressedandcharacterizedasaproteinwithapproximatemolecularweightof87kDainPAGE-SDSandcarryingspecificreactivityagainstanti-His6MAbor polyclonal antibodies of laying hens that had beenimmunizedwithM41strainofIBVwasdetectedintheWestern-blottinganalysis.Therefore, such recombinant polypeptide, due to the pres-enceofcommonepitopeswith theM41strainof IBV,mayhave thepotentialtobeusedsuccessfullyintheimuno-diagnosisofIBVandinimmunizationagainstthisvirus.Key words:S1Glycoprotein,InfectiousBronchitisVírus(IBV),CloningandExpression,SaccharomycescerevisiaeSpecies:avian

bV069. CONSTRUCTION AND ExPRESSION OF SCFV ANTIBODy FRAGMENT SPECIFIC FOR INFECTIOUS

BRONCHITIS VIRUS IN ESCHERICHIA CoLIALInEGCAETAno,ALIAnDRAMGIBERTonI,MARIAnACM

GonçALVES,HéLIoJMonTASSIERLaboratóriodeVirologiaeImunologia.DepartamentodeMicrobiologia

-FaculdadedeCiênciasAgráriaseVeterinárias,UniversidadeEstadualPaulista,Rod.Prof.PauloD.Castellane,s/n.Jaboticabal,SP,Brasil,ProjetoFinanciadopelaFundaçãodeAuxílioàPesquisadoEstadodeSãoPaulo(FAPESP),processosnº02/08649-1enº

01/14950-3ebolsadeestudosconcedidapelaCAPES.alinegcaetano@yahoo.com.br/heliojm@fcav.unesp.br

ABronchitisVirus(IBV)wasproducedathighlevelinEscherichiacoli.Thecodifyinggenesfunctionalsingle-chainFvantibodyfragment(scFv) specific for theH120 vaccine strain of Infectious for variableregionsofimmunoglobulin(Ig)heavychain(VH)andlightchain(VL)wereamplifiedfromspleenRNAextractscollectedfromSPFchickensimmunizedagainstIBV,usingtwospecificpairofprimersbyRT-PCRandtheseampliconswereconnectedtoaflexibleoligo-peptidelinker,usinganotherspecificsetofprimersandPCR.Thefinalconstructofthisrecombinantgene(VH-linker-VL),whichisabletocodifytheScFv

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antibody fragments,was inserted intoaphagemidpCANTAB5Efol-lowedbypanning - selectionwith theRecombinantPhageAntibodySystem(RPAS)againstpurifiedIBVparticles.ThescFvgeneinsertedinto the recombinant phagemid pCANTAB 5E –H11 and harvestedfromapositiveclonecarryinganti-IBVspecificitywassubclonedintopET28afusedtoN-terminalHis-tagsequenceinframeand,followingIPTGinduction,itwasoverexpressedinE.coliBL21.ThepresenceofscFvanti-IBVantibodieswasdeterminedbysodiumdodecylsulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) followedbyWesternblottinganalyses.Aproteinfractionwiththeexpectedmolecularweightofapproximately32KDaandcarryingthepoly-histidinetagwasrecog-nizedbyanti-His6MAb.ThismonoclonalscFvantibodyproducedherecombinedspecificallywiththesubunit1ofspikeglycoprotein(S1)ofIBVanddidnotreactwithothervirusstructuralproteins.Thus,suchrecombinantantibody fragmentcouldbeuseful tocharacterizemorepreciselytheconformationalstructureofS1glycoproteinanditsimmu-nogenicity,aswellasitsvariability,sincethisproteincarriesthemajorvirus-neutralizing epitopes and suffers preferentially more variabilityduringvirusevolution.Key words: Infectious Bronchitis Virus, Monoclonal scFv antibody,Recombinantantibody,EscharichiacoliSpecies:avian

bV070. TLR7 AND INOS RNA ExPRESSION IN SPLEEN CELLS OF COCONUT MEAL-FED BROILERS AFTER A VIRAL

CHALLENGE WITH IBVD SUSAnDEICHER1,TSAnGLLIn2,CHInGCWU2ToDDJ

APPLEGATE3,JoHnAPATTERSon31USDA-ARS,2DepartmentofComparativePathobiology,3SchoolofVeterinaryMedicineandDepartmentofAnimalScience,Purdue

University,WestLafayette,IN.USAspruiett@purdue.edu

Coconut meal is immunomodulating in part because of its lau-ric acid content.Whether this dietary immunomoulator canenhanceinnateimmunityofchickenstocircumventorcombataviralchallengehasnotbeenstudied.Chickensarefrequentlyraised inconfinementbuildings,butagrowingtrendforpastureraisedbirdsisevident.Theobjectiveofthisworkwastodeterminetheeffectofcoconutmealoninnate immune functionof pastureand confinement raised chickenswithout and with Infectious Bursal Disease (IBDV). Sixty day-oldbroilerchickenswereassignedtoeitheracoconutmealdietorastan-dardcorn/soydietcontainingantibiotics ineachof twostudies. Thefirststudywasconductedwithpastureraisedbroilersandthesecondwithbroilersintypicalconfinementhousing.Afterreceivingthedietsfor4weeks,birdswerechallengedorallywith4.1x103EID50IBDVvirus.Sixchickensfromeachdietarygroupwereeuthanizedat0,24and96hafterchallengeandspleensharvested fordeterminationoftoll-like receptor (TLR) 7 and inducible nitric oxide synthase (iNOS)RNAexpression.Toll-likereceptor7increased(P<0.05)followingtheviralchallengeinbothpastureraisedandconfinementraisedchickensandwasgreater(P<0.05)forthecoconutdietat96h.Theexpres-sion of iNOS in the spleen cells did not change in the confinementraisedbirds(P>0.10),butwasinitiallygreater inthepastureraisedbirds,suggestingexposuretobacterialpathogensintheenvironment.iNOSdecreasedby1.67foldby96hcomparedto0hforbirdsfedthecorn/soydietanddecreasedbymorethan10,000foldforcoconutmealfedbirds.iNOSwasless(P<0.05)inthebirdsfedthecoconutdietthanforthecorn/soyfedbirdsat96hpostchallenge.ThesedatashowtheviralchallengeenhancedTLR7expressionby96hafterchallengeandwasaffectedbydietarytreatmentbythattime.ExpressionofiNOSwasvariablyaffectedbytheviralchallengeinbirdsraisedunderdiffer-entconditionsandfeddifferentdiets.Theseresultswillbeusefulforproducersindeterminingtherisksandbenefitswhenseekingpossibleimmune modulators to replace antibiotics.This study was funded inpartbyTropicalTraditions.Key words:TLR7,iNOS,Coconutmeal,IBVDSpecies:avian

bV071. FLUORESCENT ANTIBODy TEST IN SPLEEN OF RABIES VIRUS INFECTED MICE SUBMITTED TO

PRoPIoNIBACTERIUM ACNES

CMAPPoLInáRIo,AMMAZInI,JMEGIDUniversidadeEstadualPaulista“JúliodeMesquitaFilho”-Campusde

BotucatuFaculdadedeMedicinaVeterináriaeZootecniaDepartamentodeHigieneVeterináriaeSaúdePública

jane@fmvz.unesp.brTheuseof immunomodulators, antiviral drugsand interferon in

rabies was reported by some authors. Propionibacterium acnes (P.acnes), as immunomodulator, has been evaluated in rabies virusinfected mice and researchers observed greater survival rates inanimals treated with the immunomodulator and Fuenzalida Palaciosantirabiesvaccine.Thepercentageofsurvivalwascorrelatedtohigherpositivitytofluorescentantibodytest(FAT)inlymphoidtissuesintheinfectedvaccinatedmicesubmittedtoP.acnes,followedbythegroupsubmitted toP.acnes suggesting macrophageactivation inducedbyP.acnes and viral sequester inside the someones. Considering thepossibilityofviruspersistenceinspleenofinfectedmiceandthesub-stitutionofFuenzalidaPalaciosbyVEROantirabiesvaccineinBraziltheobjectiveofthisworkwastocomparethepercentageofpositivitytoFATinspleenofrabiesvirusinfectedmicesubmittedornottoantira-biesVEROorFuenzalidaPalaciosvaccineindifferentmomentspostinoculation.Thistrialwasconductedusing16experimentalgroupswith40mice in each, divided in 2 subgroups, being 8 using FuenzalidaPalaciosvaccineand8usingVEROvaccine.Animalswere infectedbyintramuscularyrouteandafter24hoursreceivedornotanti-rabiesvaccine.P.acneswasusedisolatedorinassociationwithvaccineinone, two or three doses.After immunomodulator treatment, 6 micesfromeachgroupwerekilledafter5d,10d,14dand21dpostinfectionand spleenandbrain collected.Thematerialwas submitted toFAT.Thepercentageoflethalitywasevaluatedinanumberof8micefromeach groupmaintained in a separate cage, observed for symptomsanddeathduring30days.Clinicalsignsanddeathwereobserved7-12daftervirusinoculation.Greaterpercentageofpositivitywasobservedinspleen,comparativelytobrain, inalmostallgroupsandmoments.ThepositivitywasnotrelatedtotreatmentwithP.acnesorantirabiesvaccine. Rabies virus was detected in spleen and brain before thepresenceofclinicalsignsandpositivitywasalsodetected21daftervirusinoculationsuggestingrabiesviruspersistenceinagreementtoauthorsthatconsidervírusreplicationandpersistenceinmacrophagescorrelatedwithalongerincubationperiodwithpossibilityofsubsequentvirusreplication.Key words: rabies, macrophages, Propionibacterium acnes,FluorescentBodyTestSpecies:other

bV072. PRODUCTION OF TNF-A AND IL-6, ANTIBODy RESPONSE, AND BACTERIAL RECOVERy, DURING

LEPTOSPIROSIS INFECTION*MMARInHo,CSILVA,VMFLIMA,GFMACHADo,JRPEIRo,

SHVPERRIDepartamentodeApoioProduçãoeSaudeAnimal,FaculdadedeOdontologiadeAraçatuba,UniversidadeEstadualPaulistaJúliode

MesquitaFilho*mmarinho@fmva.unesp.br

The aims of the present studywere to investigate the humoralandcellularresponsekineticsinleptospirosis.Itwasobservedthatthepresenceofthetumornecrosisfactor-alpha(TNF-a)andinterleukin-6(IL-6)wereassociatedtotheproductionofantibodiesandthebacterialrecovery,andthecompromisingofbothintheimmunopathogenesisofleptospirosisinanexperimentalinfectionofBalb/cmiceinoculatedwithLeptospira interrogans serovar canicola. The analysis of the resultsshowedhigherlevelsofTNFitandIL-6intheinitialphaseoftheinfec-tion, periodwhere itwas observed the greatest bacterial clearence.However, when comparing the bacterial recovery with the kineticsof theproductionof antibodies, the results revealedaproportionallyinvertedkinecticstotheproductionofantibodies.weconcludedthatinleptospirosisthereisagreatermobilizationoftheactivityofthecellularimmuneresponse,mainlyintheinitialphaseoftheinfectiousprocess,forposteriorinvolvementofthehumoralresponse.Thisfactcouldbeassociated to some inhibitory factor which could be responsible fortheselectivesuppressionofthecellularimmuneresponseandthatasmuchtheTNF-αastheIL-6couldbeassociatedtotheimmunopatho-

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genesisofthedisease.Weconcludedthat in leptospirosisthereisagreatermobilization of the activity of the cellular immune response,mainlyintheinitialphaseoftheinfectiousprocess,andthatasmuchtheTNF-αastheIL-6couldbeassociatedtotheimmunepathogenesisofthedisease.Key words: cytokines, cellular immune response, Balb/c mice,leptospirosisSpecies:other

bV073. EFFECT OF BACILLUS CEREUS AND SACCHARoMyCES BoULARDII ON THE HUMORAL

RESPONSE OF VACCINATED MICE TALITABAnDEIRARooS1,AGSAnToS2,GFISCHER2,T

VIDoR3,FPLLEITE4,CGIL-TURnES1FV/UFPel,2CENBIOT/UFPel,3LaboratóriodeVirologia,FV/UFPel,

4LaboratóriodeImunologia,IB/UFPelProbioticshavesomekindoeffectonboththeinnateandacquired

immuneresponses,probablyduetotheaccelerationoftheformationofmemoryclones,increasingthespecificimmuneresponse.However,studiesontheeffectofprobioticsonimmunity,arescarce.Theobjec-tiveofthisresearchwastoevaluatetheeffectofBacilluscereusvar.toyoi and of Saccharomyces boulardii on the humoral response ofmicetoaninactivated,oiladjuvantedBovineHerpesVirus-5vaccine.IsogenicBalb-cmicewererandomlygrouped.Theanimalswere fedwith a commercial feed free of antibacterials supplemented with S.boulardii(1x107UFCgr-1)forgroupA,B.cereusvar.toyoi(1x106viablesporesgr-1)forgroupBandwithoutsupplementationforgroupC.Theanimalswerevaccinatedondays0,28and114with1/20thofabovinedose.Bloodwascollectedfromtheorbitalsinusondays0,42,114and144.The humoral responsewas quantified byELISAusing thehomologousvirusasantigen.Resultsweretransformedinseroconver-sionsdividing theactualabsorbenciesby thatofday0of thesameanimal.Meanseroconversionsondays42,114e144ofgroupAwere8.9,8.5and9.9,thoseofgroupsBwere10.1,7.2and7.7,andthoseofgroupCwere8.2,6.7e7.0, respectively.Seroconversionsof theanimals that receivedprobioticsweresignificantly (P<0.01)higher.Thefactthattheresponseinsupplementedanimalsremainedhighereven after suspending probiotic administration suggests that theimmuomodulationwastriggeredatthebeginningofitsadministration.We concluded that both probiotics enhanced the humoral responseagainstthevaccine.

SupportedbyCNPq(474509/2004-4)andFAPERGS(0523299)Key words:Probiotics,Immunomodulation,BHV-5Species:other(mice)

bV074. A NEW ADJUVANT ENHANCES THE PR OF THE COMMERCIAL INFLUENZA VACCINE IN THE FERRET MODEL

CyRILJMARTEL1,TRInEHJEnSEn1,LARSPnIELSEn2,BIRGITTEVIUFF1,ELSE-MARIEAGGER3,MERETE

BLIxEnKRonE-MØLLER1,PETERAnDERSEn3,BEnTAASTED11InstituteofVeterinaryPathobiology,FacultyofLifeSciences,UniversityofCopenhagen,Denmark,2DepartmentofVirology,StatensSerumInstitute,Copenhagen,Denmark,3DepartmentofImmunologyofInfectiousDiseases,StatensSerumInstitute,

Copenhagen,DenmarkDDA-TDB is a cationic liposome-based adjuvant known to pro-

duceaverysubstantialCMIandat thesametimeastronghumoralresponse,desirableforahighnumberofdiseasetargets.Wetestedtheeffectofthisadjuvantwhencombinedtoacommerciallyavailableinactivated influenza vaccine. When challenged with H1N1 A/NewCaledonia/20/99, ferrets immunizedwith theadjuvantedvaccinedis-playedamuchstrongerhumoralresponseandlowerviral titersthanthe ones that received only the regular vaccine. Gamma-interferonproduction,assessedbybothRT-PCRandflowcytometry,andpathol-ogystudiesontheupperandlowerrespiratorytractconfirmedthosefindings.ThisstudyindicatesthatDDA/TDBhasastrongpotentialtobeusedasanadjuvantforinactivatedinfluenzavaccines.Key words:Vaccine,adjuvant,influenza,ferret,modelSpecies:other(humanandferret)

bV075. IL-15, TNF-α AND THE AUTOIMMUNE PATHOGENESIS OF MALIGNANT CATARRHAL FEVER

DAVIDMHAIG,IAnAnDERSon,DAVIDDEAnE,SAnDISWA1,x-qWEI2,GEoRGERUSSELL

MoredunResearchInstitute,Edinburgh,1CancerResearchUK,London,U.K.,2UniversityofCardiff,Wales.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferativediseaseof cattle, deer, pigsandbison. It is causedbyeither of theγ-herpesvirusesAlHV-1orOvHV-2thatdonotcausediseaseintheirreservoirhosts(wildebeestandsheeprespectively)butcausediseasein susceptible species characterised by lymphocyte accumulationandareasofnecrosis inmultiple tissues.Webelieve that the tissuedestruction in MCF is due to the indiscriminate cytotoxic activity oflymphocytesactivatedasaconsequenceof infection.ThepathologyofMCFissimilartothatseeninsomeautoimmunediseasesinvolvingdysregulatedexpressionofIL-15(andconsequentlyTNF-a)thatmain-tainsactivecytotoxiccells. Ina rabbitmodelofAlHV-1andOvHV-2infection,wediscoveredthatthereisabundantexpressionofIL-15andTNF-αintissuesaffectedbyMCF.TheIL-15-producingcellsappearedto be predominantly non-T non-B lymphocytes. Large granular lym-phocytes (LGL), which are virus-infected cells obtained from MCF-affected tissues in culture,didnotproduce IL-15butdid respond tothe cytokine in bioassays.Experiments to deplete IL-15 in vivowithanIL-15receptorproteingaveequivocalresults.Thiscouldbeduetothe recent discovery that such treatment can enhance IL-15 activityratherthanblockit.OtherexperimentsareunderwaytoblockTNF-αwithEtanercept®,aTNF-αreceptorprotein.WeconcludethatIL-15isproducedinabundanceinMCFandthatcytotoxiccellsmaintaintheiractivephenotype in thepresenceof IL-15, contributing to the tissuedamageseeninMCF.Key words: Herpesvirus, autoimmunity, interleukin-15, malignantcatarrhalfeverSpecies:rabbit

bV076. IMMUNE RESPONSES AGAINST MEASLES VIRUS IN CyNOMOLGUS MONKEyS

HIRoKISATo,FUMIoKoBUnE,yASUSHIAMI,MISAKoyonEDA,CHIEKoKAI

LaboratoryAnimalResearchCenter,InstituteofMedicalScience,TheUniversityofTokyo,Tokyo,Japan

Measlesvirus(MV)inducesprofoundsuppressionoftheimmuneresponseduringandforweeksafteracuteinfection.Ontheotherhand,virus-specificimmuneresponsesthatmediateviralclearanceandcon-fer long-lasting immunityareefficientlygenerated.Toinvestigatethisparadoxwestudied the immune responses incynomolgusmonkeysagainst MV infection. Cynomolgus monkeys experimentally infectedwithwild-typeMV(MV-HL)showedmarkedleukopeniaassociatedwithasteadyreductioninCD4+Tcellnumbersfor18dayspostinoculation.Transientexpressionofinterferonandinterleukin(IL)-6wereobservedintheserumbetweenfourandsixdayspostinoculation,indicatingthatMVreplicationinducedearlyantiviralresponse.IL-10levelsincreasedafter11dayspostinoculation,suggestingthattheprolongedimmuno-suppressionobservedduringmeaslesinfectionisduetotheinhibitoryeffectsofIL-10ontype1CD4+Tcells.Interestingly,serumlevelsofIL-8showedconsiderablevariationthatwascharacteristicofthreepeaksatday3,5-6,and11postinoculation.IL-8mRNAinperipheralbloodmononuclearcellspeakedatday2,4,and9post inoculation,whichcorrelatedwithIL-8serumproteinlevels.AlthoughthefirstIL-8serumproteinpeakwasthehighestofthethreeproteinpeaksobserved,theIL-8mRNApeakwas lower than estimated from the serumprotein.Invitrostudyusingarespiratoryepithelialcell linerevealed thatMVparticle induces IL-8 production via binding to and/or incorporationinto cells without virus replication. These suggested that rapid IL-8secretionatdaythreepostinoculationisprimarilyinducedbyasmallamountofcirculatingMVparticlesthatarosefromrespiratoryepithelialcells.The cynomolgusmonkey is valuable for the study ofmeaslespathogenesis, themechanismof long-termimmunememory,andforthedevelopmentofanewmeaslesvaccinethatwouldstillbeeffectiveinthepresenceofmaternalneutralizingantibodies.Key words:measlesvirus,cytokine,immunosuppression,cynomolgusmonkey

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Species:other(monkeys)

bV077. VETERINARy VACCINE ADJUVANTS LDUPUIS1,SDEVILLE1,JAUCoUTURIER1,SASCARATEIL1,A

LAVAL2,VGAnnE31TourKupkaC7boulevardFranckKupka92039ParisLaDefensecedex,France,2NationalveterinaryschoolofNantes,Atlanpole-LaChantrerie-BP40706Nantescedex03France,3SEPPICChinaRoom510JinTaiBuilding58SouthMoaMingRoad.Shanghai

200020China.Thedevelopmentofefficaciousandsafevaccineismoreandmore

linkedtotheselectionofanappropriateadjuvant.Specificadjuvantsforveterinaryvaccineshavetobeselectedaccordingtovariouscriterialikethetargetspecies,kindofantigen,thetypeanddurationofimmuneresponseneededforprotection.Thereisnoknownuniversaladjuvantformula.Mostcommonlyusedadjuvantsinveterinaryvaccinesareoiladjuvantsandaluminumhydroxide.Oiladjuvantsaregenerallywater inoil for-mulations inducing strong and long term immunity.Adjuvants basedonmineraloilsareknowntobeefficaciousbutcansometimesinducelocalreactionswithreactiveantigens.Multiphasicemulsionshavealsoprovedtheirefficacyinvaccineastheycaninduceshortandlongterm

immune response with various antigens. Specific range of adjuvantbasedonmetabolisableoilscanbeusedtoreducelocalandgeneralreactionslinkedtoreactiveantigensassociationtomineraloil.Asec-ondgenerationofadjuvant suitable for veterinaryvaccines isbasedon nanoparticles with a new immunostimulant and combined theirimmunostimulating properties to induce a positive synergistic effect.Differentmechanismsofactioncanexplain theefficacyofadjuvant:-The depot effect where the emulsion entraps the antigens andinduce a slow release of it at the injection site. Inflammatory reac-tion correlated with the induction of an immune response can bealso observed. -The recruitment of immuno-competent cells bymicro diffusion of the droplets via the lymphatic system, or bythe facilitation of the antigen uptake by antigen presenting cells.-Adjuvantscanalsoenhancethehumoralandcellmediatedimmuneresponse. Therefore, the choice of the adjuvant should be doneaccording to several criteriawith the goal to obtain a good balancebetween safety and immunogenicity. For example, a well toleratedadjuvantshouldberecommendedforusewithacrudebacterialextractandalsoforusewithLPS,bothofwhicharereactive.Conversely,arecombinantviralproteincanbeaweakimmunogenthusrequiringastrongadjuvant.Key words:vaccine,adjuvant,MontanideSpecies:allspecies

er078. EFFECTS OF BOVINE RECOMBINANT LEPTIN ON PROLIFERATION OF HEAT SHOCKED LyMPHOCyTES IN

DAIRy COWSnICoLALACETERA,GIoRGInAKUZMInSKy,UMBERTo

BERnABUCCI,PATRIZIAMoRERA,LoREDAnABASIRICò,ALESSAnDRonARDonE

DipartimentodiProduzioniAnimali,UniversitàdellaTuscia,Viterbo,Italy

nicgio@unitus.itWe have previously reported that heat shock alters mitogen

driven proliferation and gene expression of leptin and leptin recep-torsinperipheralbloodmononuclearcells(PBMC)ofdairycows,andthatunderconditionsofelevatedtemperaturesPBMCproliferationinresponsetomitogensispositivelycorrelatedwithleptinmRNA.Presentstudywascarriedouttoestablishwhetheradditionofbovinerecombi-nantleptintobovinePBMCculturedunderelevatedtemperaturesmaycounteracttheimpairmentoftheproliferativeresponseofthesecellsto concanavalinA (ConA). Six Holstein early pregnant heifers wereutilisedasblooddonors.PBMCwereculturedunder39or42°Cfor65 hours, in the presence of the following concentrations of bovinerecombinant leptin: 0, 9, 18, 27, or 150 ng/ml. ConA was added toPBMCculturesatconcentrationsof2.5or0.25µg/ml.AdditionalwellswerealsoarrangedtocontainthefiveconcentrationsofleptinwithoutConA.CultivationofPBMCat39°Cwasintendedtomimicconditionsofnormothermia,whereascultivationat42°Cwasrealisedtosimulateconditionsofhyperthermia.AdditionofleptininabsenceofConAdidnotaffect proliferationofPBMC.ProliferationofPBMCunder39 °Cwaspositivelyaffectedbythehighestconcentrationofleptin(150ng/ml)onlywhenasuboptimalconcentrationofConAwasutilized(0.25µg/ml).Theseresultsareinlinewiththosealreadyreportedforotherspecies.ExposureofPBMCto42°Cwasresponsiblefordecreasedproliferation only when the suboptimal concentration of ConA wasaddedtoculturemedia,andundertheseconditions,additionofleptindid not interfere with the negative effects of elevated temperatureonPBMCproliferation.Verificationof thehypothesis thatadditionofleptinmaycounteractthenegativeeffectsofelevatedtemperaturesonPBMCproliferationprovidednegativeresults.Reducedexpressionofleptinreceptorsduetoheatshockislikelytoexplainpresentfindings.Key words:leptin,bovine,heatshock,lymphocyteproliferationSpecies:ruminants

er079. COLOSTRAL CD8 POSITIVE CELL IS A POTENT PRODUCING CELL FOR IFN-γ

KATSURoHAGIWARA,MAyUMIDoMI,JUnICHIAnDoRakunoGakuenUniversity

k-hagi@rakuno.ac.jpIFN-γplaysanimportantroleincellularimmunityleadingtomicro-

organismelimination.WehavereportedthatbovinecolostrumcontainshighlevelsofIFN-γaswellasimmunoglobulin(1).LymphocytesarepotentcellsforIFN-γproduction,therefore,clarificationofthelympho-cyte population in the colostrum would help to clarify the source ofcolostralcytokines.Inthisstudy,weclarifiedthepopulationoflympho-cytesubsets incolostrum-namely,CD4 (Th)cells,CD8 (cytotoxicT)cellsandγd-Tcellsbyflowcytometricanalysis(EPICSXL,BeckmanCoulter),andquantifiedtheconcentrationofcolostralIFN-γbyELISA.IFN-γwasdetected in thecolostrumfromall the96healthyHolsteincows,thelevelstendedtodecreaseonthedaysubsequenttoparturi-tion.Flowcytometricanalysisshowedthatmanyγd-TandCD8positivecellswerecontainedinthecolostrum,andCD4/CD8ratiowasalow.TheratioofCD8andγd-Tpositivecellsdecreasedduringthe5days

afterparturition,buttheCD4positivecellsshowednochangeduringtheobservationperiod.Toclarify the IFN-γexpressingcolostral lym-phocytes,magneticseparationtechnology(Dynabeads,DYNAL)wasemployedtosortthelymphocytes(CD4,CD8andγd-T)fromthecolos-tralcells.Thesepositivelyselectedlymphocyteshavebeenexaminedfor IFN-γ mRNA expression by RT-real time PCR (RtPCR). RtPCRanalysis showedapotentexpressionof IFN-γgene inCD8positivecellsandthegeneexpressionwashigher thanCD4orγd-Tpositivecells.TheseresultssuggestthatCD8positiveTcellsincolostrumplayaroleinIFN-γproducingcells.References:

1.Hagiwara,K.,S.Kataoka.,H,Yamanaka.,K,R,KirisawaandH,Iwai.2000.DetectionofCytokinesinBovinecolostrum.Vet.Immunol.Immunopathol.76:183-190.Key words:IFN-γ,,γδ-Tcell,CD8+Tcell,colostrumSpecies:ruminants

er080. THE EFFECTS OF LOW LEVELS OF DIETARy COBALT ON SELECT PARAMETERS OF THE SPECIFIC AND

NON-SPECIFIC IMMUNE RESPONSES OF GOATSEUGEnEHJoHnSon*,KHALIDAL-HABSI,RASHILDMAL-

BUSAIDyDepartmentofAnimalandVeterinarySciences,CollegeofAgriculturalandMarineSciences,SultanQaboosUniversity,P.O.Box34,Al-Khod

123,Oman*ejohnson@squ.edu.om

Thetraceelementcobalt(Co)issynthesizedbyrumenbacteriatoproducevitaminB12whichassists theenzymesmethylmalonyl-coen-zymeAmutaseintheformationofglucoseandmethioninesynthaseneededformethane,acetateandmethioninesynthesis.Goats,incon-trasttosheephavebeenreportedtoberatherresistanttolowlevelsofdietaryCo.However,inOman,wehavedescribedingoats,fedlowlevelsofdietarycobal,acommonlyoccurringconditionreferredtoashepatic lipidosis, as well as anemia, poor weight gains, reductionsin their serum protein levels, a decrease in their apparent nutrientdigestibility coefficients and poorer meat quality. The present studyexpandsontheseobservationsinordertoascertainwhethergoatsfedlowlevelsofCoexhibitalterations inselectparametersof theirnon-specificandspecificimmunesystems.Inthefirstphaseofthisstudyweutilized twenty, ten-weekold,newlyweanedmaleBatinahgoats.Theywererandomlydividedintotwogroups,namelyacontrol(n=10)andatreatedgroup(n=10).Eachgroupwashousedinseparatepensandfedadietof150g/dayofaspeciallyformulatedconcentrateandRhodegrasshayadlibitumcontaining0.12mg/kgand0.1mg/kgDMofCo,respectively.Goatsinthetreatedgrouprecevedbi-monthlysubcu-taneousinjectionsof2000µgofhydroxycobalamin.Wecomparedthechemiluminescence(CL)responseofneutrophils(PMN)isolatedfromtheseanimalsduringinvitrophagocytosisofthetargetzymosanandutilizedaWST-8assaytocomparetheirT-cellblastogenicresponsestothemitogens,pokeweedmitoge(PWM)andphytohaemagultininA(PHA).ThecontrolgoatsexhibitedasearlyasthreeweeksaftertheonsetofthisstudyasignificantlylowerCLresponsetozymosanandthis lowered responsewas still evident after two and threemonths.Similarly,controlgoatsexhibitedlowerblastogenicresponsestobothPWMandPHA.Thesepreliminaryresultssuggestthatlowdietarylev-elsofcobaltleadtoareductioninbothphagocyticactivityaswellasinTcellresponsesandaresensitiveindicatorsofdevelopingvitaminB12deficiencyKey words: cobalt, goats, phagocytosis, neutrophils, lymphocyticproliferation,,hepaticlipidosisSpecies:ruminants

3. IMMUNOENDOCRINOLOGy, AND STRESS, IMMUNOLOGy OF REPRODUCTION AND NEONATES, MICROBIAL FLORA, NUTRIENTS AND THE IMMUNE RESPONSE: POSTERS ER078 - ER094

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er081. INFLUENCE OF HORMONES IN THE ExPRESSION OF INDOLEAMINE-2,3 DIOxIGENASE IN CULTURED CELLS

FROM BOVINE PLACENTAARLIMA,JMMonTEIRo,RVBoSCH,RSIUnES,ETFIoRETTo,

LJoLIVEIRA,JRKFoURyJR.Thematernal immunesystem ischallenged to tolerate thecon-

ceptus as a semi-allogeneic graft during pregnancy. The enzymeindoleamine2,3dioxygenase(IDO)hasbeenshowntoplayaroleinthe maternal immune-tolerance, mainly through catabolism of tryp-tophan, resulting in the inhibitionofT-cell proliferationbystarvation.In addition the maternal immune system hormonal regulation is stillnotcompletelyunderstood.Forexample,progesteroneincreasestheproductionofinterleukines(IL4andIL-10)byTcells,andthepredomi-nanceofestrogenisassociatedtotheTh2response.Thepurposeofthisstudywasto investigatethepotentialeffectofprogesteroneandestrogen in IDO expression in cultured bovine placental cells alongthepregnancy.Explantsofplacentomesfromfouranimalsfromeachtrimesterofpregnancywereculturedusingsupplementedmediawithprogesterone(medroxiprogesteroneacetate,PromoneE®)orestrogen(Stradiol Cipionate, E.C.P. ®).The IDO expression was assessed atdayzero(D0)anddayone(D1)ofculturebyflowcytometryusinganti-IDOmousemonoclonalantibody(Upstate®,USA).AnalysisoftheIDOlevels inthecontrolgroupshowedanincreasefromD0toD1inthesecondandthird(64to78%and67to82%,respectively)butnotinthefirst trimester(74 to75%).TheIDOexpression inplacentalexplantsinthepresenceofestrogenwassimilartothecontrolgroupinthefirsttrimester (73% to76%),nevertheless thesecondand third trimesterrevealedlowervalues,76%to67%and84%to73%,respectively.InapresenceofprogesteroneofIDOexpressionwaslowerwhencompar-ingtheD0toD1inthefirstandsecondtrimesters(72%to68%and79%to73%),andhighervalues inthethirdtrimester(69%to80%).The resultssuggest that invitroplacentalexpressionof IDOcanbeaffectedbythepresenceofprogesteroneandestrogen.Key words:indoleamine2,3dioxygenase•oestrogen•progesterone•immunetoleranceSpecies:ruminants

er082. ISOLATION AND PROLIFERATION OF T LyMPHOCyTES FROM BOVINE PLACENTA

JMMonTEIRo,RVBoSCH,ARLIMA,MSAKAI,KKIELInG,GMCoSTA,RSIUnES,CCARAúJo,JRKFoURyJR

J.R.UniversityofSãoPauloSchoolofVeterinaryMedicineLeucocytesintheplacentabehavedistinctivelyfromthoseofthe

peripheralbloodandthisfactisdirectlyrelatedtotheimmune-responseobservedinthematernal-fetaltoleranceprocess.ThisbehaviorincludespredominanceaTh2orientated immunoresponse,a temporary toler-anceto theembryoor fetalantigens, involvementof immunosupres-sionrelatedlymphocytepopulations,etc.Thedistributionandfunctionofplacental lymphocytes inbovinesare still unclear, therefore,asafirststeptoconductstudiesonthissubject,weattemptedtoestablishisolationandproliferation techniques for lymphocytes fromplacenta.Placentomesweresampledfromcowsindifferenttrimestersofpreg-nancyandplaced in culturebyexplant technique.After twodaysofculture,cellswereincubatedwithprimaryantibodyanti-bovine-CD4+(VRMD,Inc.Pullman,USA),washed,andlabeledwithsecondaryanti-bodyanti-mouseIgGconjugatedwithmagneticbeads(MiltenyiBiotec,Germany).CD4+positivecellswereobtainedaftermagneticsepara-tion(VarioMACS,MiltenyiBiotec,Germany)andincubatedfor2hourstoallowmonocytestoadheretotheculturemicroplate.Afterthat,cellsinsuspension (lymphocytesCD4+)werecollectedandaddedCFSE(Carboxyfluoescein Diacetate Succinimidyl Ester). After 4 days ofincubation,cellswereanalyzedbyflowcytometry.Resultsshowthatlymphoproliferationwere13,81±9,19,28,33± 6,07,3,40± 15,3 forlymphocytesfromfirst,secondandthirdtrimesterpregnancy,respec-tively. A basal placental T lymphocyte proliferation without specificstimulationwasobserveddifferentlyfromthatofmouselymphocytesfromspleenorperipheralblood.Ourresultsshowedthatitispossibletoisolatelymphocytesfrombovineplacentaandtoconductprolifera-tionassaybyusingmagneticcellseparationandCFSEassay.Key words:Placenta,Lymphocyte,CFSE,CD4+.

er083. THE EFFECT OF PREGNANCy ON MATERNAL IMMUNITy IN SHEEP

SEAnWATTEGEDERA,MARARoCCHI,JAynEHoPE,GARyEnTRICAn

gary.entrican@moredun.ac.uk

MoredunResearchInstitute,PentlandsSciencePark,BushLoan,Midlothian,EH260PZ,UK,1InstituteforAnimalHealth,Compton,

Newbury,BerkshireRG207NN,UK.The failure of eutherianmammals to reject the semi-allogeneic

fetus isan immunologicalparadox in thecontextof self-nonselfdis-crimination.InthefiftyorsoyearssincePeterMedawarcommentedonthisparadox,multiplemechanismsformaternalacceptanceofthefetus have been postulated and tested.One of the original theorieswas that the maternal immune system was suppressed. There hasbeenverylittleexperimentalevidencetosupportgeneralisedmaternalimmunosuppression,butthereisevidenceforamoresubtlemodulationofmaternalimmunity,notablythedown-regulationofIFN-γproduction.Thishasbeenobservedinhumansandrodents,andinsomecaseslinkedtopregnancyfailureduring infection.Weareparticularly inter-estedinhost-pathogeninteractionsduringchlamydialabortioninsheepsincethebacterialpathogenChlamydophilaabortusisthesinglemostcommondiagnosedcauseofinfectiousabortioninsheepintheUK.InvitrodataindicatethathostcontrolofC.abortusismediatedthroughIFN-γproduction,inductionofindoleamine2,3-dioxygenase(IDO)andsubsequentdeprivationoftryptophan.SinceC.abortusestablishesapersistentinfectionthatusuallyonlymanifestsitselfduringpregnancy,wewereinterestedtoknowifTcellsfrompregnantsheepexhibitedareducedcapacityofIFN-γthatcouldofferanexplanationforrecrudes-cenceof theorganismand invasionof theplacenta.We immunisedsheepwith thenominal antigenovalbumin (Ova) prior to pregnancyandmonitoredanti-OvaT cell responses throughoutpregnancyandcomparedthesetonon-pregnantcontrols.WealsoanalysedresponsestotheTcellmitogenconcanavalinA(ConA).WefoundthatpregnancydidnotsignificantlyalterIFN-γproductionbyperipheralmaternalovineTcellsinresponsetoinvitrorestimulationwithOva(P=0.565)orConA(P=0.110).TheeffectsofIFN-γcanbecounteractedbyIL-4andIL-10,twocytokinesthathavebeenreportedtobeincreasedinpregnantmammals.Analyses revealed low levels of both these cytokines inrestimulated cultures, and neither were elevated during pregnancy.Thesedata indicate thatT cell responses inpregnant sheepdonotfollowtheTh1/Th2paradigmandthatotherfactorsarelikelytobemoreimportantinthepathogenesisofovinechlamydialabortion.Key words: abortion, Chlamydophila abortus ,• indoleamine 2,3-dioxygenase(IDO),IFN-γSpecies:ruminants

er084. IN VITRO ExPRESSION OF BOVINE CLASSICAL AND NON-CLASSICAL MHC CLASS I PROTEINS

PPARASAR1,CSUAREZ2,DDnEW1,WCDAVIS1,CJDAVIES11WashingtonStateUniversity,Pullman,WAUSA,2Agricultural

ResearchService,USDA,Pullman,WAUSAIn humans, cattle and other viviparous species, placentalMHC

classI(MHC-I)expressionistightlyregulated.Bovineinterplacento-maltrophoblastcellsexpressbothclassicalandnon-classicalMHC-Igenes during the third trimester of gestation (Davies et al.Placenta2000, 21:194-202). Classical MHC-I antigens have the potentialto trigger immune-mediated abortion and appear to be involved inplacental release during parturition (Hill et al. Biol. Reprod. 2002,67:55-63,Daviesetal.Anim.Reprod.Sci.2004,82-83:267-280). Incontrast,non-classicalMHC-Iantigens,suchasHLA-G,seem tobeimmunosuppressive. Inapreviousstudy,weanalyzedexpressionofMHC-I genes inPBMCand interplacentomal trophoblast cells usingcDNAcloning,microarrayanalysisandsequencing(Daviesetal.Am.J.Reprod.Immunol.2006,55:188-200).Interplacentomaltrophoblastcellsfromlatepregnancyexpressedfournon-classicalMHC-Ilociwithdifferentialexpressionindifferenthaplotypes.ThelevelofMHC-Itran-scripts encoded at non-classicalMHC-I loci was significantly higherin trophoblast cells than inPBMC.Nevertheless, all of the classicalMHC-IgenesexpressedinPBMCwerealsoexpressedintrophoblastcells.Wearenowstudyingexpressionof bovine classical andnon-

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classicalMHC-IproteinsinmouseP815cells.Twoclassicalandfournon-classicalMHC-Ialleles from theAH11haplotypewereamplifiedwithPlatinumPfxDNAPolymeraseandsubclonedintothepcDNA3.1expressionvector.ThepcDNA3.1subclonesweresequencedusingthe T-7 forward and BGH reverse sequencing primers and perfectcloneswere selected for expression. By using two different reverseprimersbothcloneswiththenormalstopcodonandclonesexpressinga3’6xHis tagwereproduced.P815mousemastocytomacellsweretransfectedwitheitheraclassicalornon-classicalMHC-Igeneusinglipofectamine.BovineMHC-ItranscriptsweredetectedbyRT-PCRinalltransfectedcelllines.TransfectantswerescreenedforcellsurfaceexpressionofbovineMHC-Iproteinsbyflowcytometrywithanti-bovineMHC-Imonoclonalantibodies,48hoursafterbeingtransfectedand/orafterselectionwithgeneticin (Gentamycin).The twoclassicalMHC-Iproteins (N*01802 andN*01701) and one non-classicalMHC-I pro-tein (NC3*50201)were detected on the cellmembrane. Isolation ofHis tagged proteins and Western blotting are being used to identifyintracellularandsecretedclassicalandnon-classicalMHC-Iproteins.Thisstudyprovidesthefirstevidencefortheexpressionofcellsurfaceandsecretedbovinenon-classicalMHC-Iproteins.Key words: Classical MHC Class I, Non-classical MHC Class I,transfectedmouseP815cellsSpecies:ruminants

er085. T CELLS DISTRIBUTION AND FUNCTION IN PERIPHERAL BLOOD AND UTERUS DURING

PERIIMPLANTATIONAL PERIOD IN PIGMARIAFCUELLo,MARIACGRoSSo,RAMIRoAMARTInEZ,

ADRIAnABVIVAS,CECILIARGRECo1FacultaddeAgronomíayVeterinaria.1FacultaddeCienciasExactas.UniversidadNacionaldeRíoCuarto.5800RíoCuarto.Argentina.

cgreco@exa.unrc.edu.arIthasbeensuggested thatsuccessfulpregnancy inpigdepend

on adequate immuno-endocrine modulation at conceptus-maternalinterfaceandsystemiclevels.TheaimwastostudytheTcelldis-tributions inperipheralbloodanduterusand its functionalpropertiesduringperi-implantationalperiod.Bloodsampleswereobtained frompregnant sows in10days (n=15)and30days (n=15)of pregnancyfromabreedingfarm.Pregnantstatewasdeterminedbyultrasound.Non-pregnant sows (n=15) in the luteal period of the oestrus cyclewereusedascontrol.Uterinetractswereobtainedfromnonpregnantandpregnantsowsin10days(n=10)and30days(n=10)ofpregnancyfromaslaughterhouse.Peripheralbloodmononuclearcells(PMBCs)anduterinemononuclearcells (UMCs)were isolatedbyHistopaque.PhenotypesofporcineTlymphocyteweredeterminatebytwo-colourFlowCytometricAnalysisusingspecificmonoclonalantibodiesagainstCD4andCD8surfaceantigens.FoursubpopulationsCD4+CD8-,CD4-CD8low+, CD4-CD8high+ T cells and CD4+CD8+double positive (DP) Tcellsweredetermined.Inaddition,PMBCsandUMCswerestimulatedseparatelywithP4orwithCon-A.InsupernatantsIL-10,INF-γandTGF-β2concentrationsweredeterminedbyELISA.AtthepreimplantationalperiodanincreaseofCD4+CD8+DPTcells(p<0.05)inperipheralbloodandaverysignificantincreaseofCD4-CD8high+Tcells(p<0.01)wereobserved.BeforeembryoimplantationCD4-CD8high+Tcellsincreasedinperipheralblood(p<0.01)anddiminishedsignificantlyatuterinelevel(p<0.05).CD4+CD8+DPTcellsremainedincreasedatperipheralbloodbutdecreasedatuterinelevel.DistributionchangesofCD4+CD8+DPTcellswerenotobserved.

Theabovementionedchangesareaccompaniedbyaspecialpat-ternofcytokineproductionwhen theUMCsareprogesterone-stimu-lated.At10dofpregnancyincreasedsignificantlyIL-10(p<0.05)andTGF-β2 (p<0.05)anddecreasedINF-γ (p<0.01). Inconclusion, thesefindingssuggest thatatuterine levelCD4-CD8high+Tcells function ismodulatedbyprogesteronepresentingaspecialcytokinepatterncoin-cidentallywithamodulatedcytolyticactivity.Key words: uterine mononuclear cells, pregnancy, IL-10, INF-γ ,TGF-β2Species:swine

er086. VITAMIN E MODULATES THE ExPRESSION OF TH2 CyTOKINES ON PORCINE PBMC

HERnánDEZJ1*,SoToE,REZEnDIZM1,PInELLI-AAVEDRAA1,KIRTCKLASInG2

1LaboratoriodeInmunología,CIAD,A.C.Hermosillo,Sonora,Mexico,2DepartmentofAnimalScience,UCDavis,USA.

Inadditionofitsantioxidantfunction,studieshaveshowedthatvita-minEcanmodulatetheimmunesystem.DifferentreportsdescribethatvitaminEcouldincreasethehumoralandcellularimmuneresponse,includingthecytokineproduction.TheaimofthisworkwastoevaluatetheeffectsofvitaminEonTh1andTh2cytokinesproduction.PBMCwere isolated from conventional pigs (n= 8) and supplementedwithdifferentconcentrationsofvitaminE(alpha-tocopheorl-AT,0,10,50,and100µM),andstimulatedwithPHAforeither,24htodetermine:a)the concentrationof tocopherol incorporated in themembrane cells,b)cytokineproduction(IL-2, IL-4, IL-10andIFN-γ)andTh1andTh2regulatorsgenes(TBX21andGATA-3),or72htodeterminetheprolif-erationofPBMCs.ATwasquantifiedbyHPLC,cytokineproductionbyintracellularstainingusingFACSanalysis,mRNAwassemi-quantifiedbyconventionalRT-PCR,TBX21andGATAwereanalyzedbyrealtimePCR,andproliferationwasevaluatedwithCFSEandFACSanalysis.Ourresultsshowedthatinvitrosupplementationincreasedthecontentof vitaminE inPBMCaccording the concentrationATwas suppliedintheculture.Theanalysisofproliferationdidnotshowedsignificantdifferencesinthepercentageofproliferation(p>0.05),butATinducedmultiplecyclesofproliferation incomparisonofcellswithoutsupple-ment.Theanalysisofcytokinesshowed that10µMofAT increasedthe mRNA expression and the percentage of cells producing IL-2(p<0.05).Theproduction of IFN-γ was inconsistent and no changeswereobservedwithanysupplementofvitaminE.ThemRNAexpres-sionofIL-4wasnotmodifiedbyvitaminE,howeverthepercentageofcellsproducingIL-4wasincreasedsignificantly(p<0.05)byvitaminE(10µM).mRNAexpressionofIL-10wasreducedwith10and50µMofAT,aswellasthepercentageofcellsproducingIL-10with10,50,and100µM.ATsupplementationinallconcentrationsincreasetherelativeexpressionofmRNAofTBX21vsGATA3.Theseresultsshowed forfirst time thatvitaminEmodulateTh1andTh2cytokines inpig,andsuggest that vitamin E down-modulate the expression of cytokinesassociatedwiththephenotypeTh2oranti-inflammatoryinthecaseofIL-10.Thesefunctionsappeartoberelatedwiththedown-modulationofGATA3,ageneresponsibleofregulateTh2cytokine.Key words:VitaminE,Th2cytokines,Species:swine

er087. NOREPINEPHRINE MODULATES PROLIFERATION AND IFN-α PRODUCTION By PORCINE IMMUNE CELLSELoDIEMERLoT1,2,FRIDAHASSLUnGWIKSTRöM1,SIRJE

TIMMUSK1,LISBETHFUxLER1,CARoLInEFoSSUM11DepartmentofBiomedicalSciencesandVeterinaryPublicHealth,SectionforImmunology,SwedishUniversityofAgriculturalSciences,SE-75123Uppsala,Sweden,2INRA,UMR1079Systèmesd’Elevage

NutritionAnimaleetHumaine,F-35590SaintGilles,France.elodie.merlot@rennes.inra.fr

Sympathetic system has been shown to play a major role inneuroendocrine-immune communication in rodents and humans.Norepinephrine(NE)modulatesTlymphocyteproliferation,productionofTh1andTh2cytokinesbyTlymphocytesanddendriticcells(DC),andstimulatesmigrationofDCtowardlymphnodes.Inthisstudy,weinvestigatedtheeffectofNEontheproliferationofporcineperipheralbloodmononuclearcells(PBMC)andontheirabilitytoproduceinter-feron-α (IFN-a).PBMCwere isolatedbydensitygradient centrifuga-tionfromheparinizedbloodfromconventionallyrearedYorkshirepigs(8-12weeks).Thedose-effectofNEwas investigatedbystimulatingPBMCwithvariousinducersandbyaddingNEsimultaneously(dosesrangingfrom0.01to100µM).Lymphocyteproliferationover96hourswasmeasuredbyincorporationoftritiatedthymidineduringthelast24hours.ForIFN-a,thesupernatantofPBMCwascollectedafter20hofincubationandporcineIFN-αwasquantifiedbydissociation-enhancedlanthanidefluoro-immunoassay(DELFIA).WhenNEalonewasaddedtoPBMC,proliferationwasstimulatedatconcentrationsrangingfrom0,1to100µM.WhenNEwasaddedincombinationwithT-lymphocyte(ConA,PHA)orB-lymphocyte(LPS)mitogens,thehighestdoses(10and100µM)ofNEdecreasedthemitogen-inducedproliferation.The

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productionofIFN-αwasstimulatedwhenPBMCwereexposedtoNEin combinationwitholigodeoxyribonucleotide (ODN)2216but itwasnotaffectedwhenNEwasco-administeredwithpolyriboinosinic-poly-ribocytidylicacid(polyI:C).Whenaddedincombinationwithcomplexmicrobialinducers,NEat100µMinhibitedIFN-a productionbyPBMCin response toheat inactivatedAujeszky’sdiseaseDNAvirus (ADV)andstimulateditinresponsetoliveSendaiVirus(SV).PorcineIFN-aproducingcellsthatresemblethehumanplasmacytoiddendriticcells(pDC)arebelievedtobethemainsourceofIFN-awhenporcinePBMCarestimulatedwithODN2216orADV(viaTLR-9activation),whileitisbelievedtobethemonocytesormonocyte-derivedDC(moDC)thatrespondtostimulationbydsRNA(viaTLR-3)e.g.,polyI:CorreplicatingSV.Thus,theregulatoryeffectofNEonIFN-aseemstodependonthecelltypeandonthepathwaybywhichtheyareactivatedbymicrobialcomponents. Inorder to investigate this interaction,wearecurrentlystudyinghowNEregulatesIFN-aproductioninporcinemoDC.Key words: Norepinephrine, T lymphocytes, Th1 cytokines, Th2cytokinesSpecies:swine

er088. THE INFLUENCE OF SEASONAL STRESS DURING GESTATION ON IMMUNE PARAMETERS IN POSTNATAL

STRESSED PIGLETSGABRIELARoDRIGUEZALonSo,noRAMAyER1,CECILIAR

GRECo1,nAnCyRoDRÍGUEZ1,ADRIAnAVIVASFacultaddeAgronomíayVeterinaria,1FacultaddeCienciasExactas.UniversidadNacionaldeRíoCuarto.5800RíoCuarto.Argentina.

cgreco@exa.unrc.edu.arNumerous infertilityproblemsarepresented inArgentinaduring

summer months. The most important clinical manifestations are theirregularzealandearlyabortions.StressactivationofHypothalamic-pituitary-Adrenalaxisgenerateschangesinimmunesystem.However,theeffectofdifferentstimulionsummerinfertilityonoffspringviabilityorits immuneresponseisnotwellknown.Theaimwastostudytheimmuneresponseofpigletstoprenatalstressduringsummertimewithaddition of postnatal stress like weaning. Male and females pigs of45-60daysofagewereused,LandracexYorkshire,mothers’offspringunderdifferentenvironmentaltemperaturesduringthepregnancy:hightemperatures (summerseason), stocking temperatures (autumnandspring) and low temperatures (winter). Forty five- sixty days of ageoffspringafterweaningwereseparatedfromtheirmothers, identifiedandleaveinspecialhousingduringthreedays.Bloodsamplesweretakenfrommalesandfemales,beforeandafterweaning.Thefollow-ing determinations weremade: Total blood leukocytes, in vitro lym-phocyteproliferation,serumgammaglobulinsandIgGconcentrations,serumglucoseandcortisolconcentrations.Theadditionoftwostresschangesimmuneresponsesthroughdecreasingtotalleukocytesnum-ber,adecrease inpercentageof lymphocytesaccompaniedwithanincreaseofpolymorphonuclearneutrophils.Inaddition,mitogen-stimu-latedproliferative lymphocytesweredisminished.An increase in IgGplasmaticconcentrationwasobserved.Inconclusion,stressmodifiestheimmuneresponsetryingtoavoidpossibleinfections.

er089. ONTOGENy AND ExPRESSION OF EQUINE FETAL AND NEONATAL IMMUNOGLOBULINS

REBECCALTALLMADGE,KRISTInEMCLAUGHLIn,MARyBMATyCHAK,MJULIABFFLAMInIo

CornellUniversity,CollegeofVeterinaryMedicine,Ithaca,NYUSAManyaspectsoftheimmunesystemofthehorsedevelopduring

fetallife,yetfoalsaredependentonthetransferofmaternally-derivedantibodiesduring theneonatal period forprotectionagainstenviron-mentalpathogens.Inaddition,studiesinvolvinginfectiousorganismshaveindicateddelayedproductionincertainimmunoglobulinisotypesinthefoal,whichcouldincreasetheirsusceptibilitytopathogens.Thisstudyexplores theprogressiveexpressionofB cellmarkers in lym-phoidtissuesfromfetal lifetoadulthood.Developmental,activation,andimmunoglobulinmarkerswereselectedforRT-PCRexperiments.WetestedmRNAexpressionusingatwostepRT-PCRinliver,bonemarrow, spleen, mesenteric lymph node, lung and peripheral bloodmononuclearcellsamplesfromequinefetus,neonate,youngfoaland

adulthorse(n=3).TocomparemRNAexpressionlevelstoproteinpro-duction,wequantified the concentrations of several immunoglobulinisotypesinpre-sucklefoal(n=11)andadulthorse(n=6)serausingcom-merciallyavailableradialimmunodiffusionassays(IgM,IgA,totalIgGandIgG3-5)andELISA(IgG1,IgG4-7).TheRT-PCRresultsrevealedthat theBcell specificmRNAexpressionpatternswere consistentlysimilaramongindividualswithinthesameagecategory(fetal,neonate,youngfoalandadulthorses).FetallymphoidtissuesexpressedmRNAformostgenesassayed,includingCD20,CD21,CD40,B220,CD79A,andCD79B.TheexpressionofRAG-1and-2,andTdtwastemporallyand spatially restricted. Expressions of particular IgG isotypes wereabsentorlimitedintheequineneonateandfoalupto3monthsofage.Insummary,ourdataindicatesanactiveBcellprogramduringequinegestation,but functionally, theadaptive immunesystemremains lim-itedintheproductionofcertainimmunoglobulinisotypesanddiversityinearlylife.Key words:Fetalimmunoglobulins,RAG-1,RAG-2,Tdt,CD20,CD21,CD40,B220,CD79A,CD79BSpecies:Equine

er090. THE CONTRIBUTION OF BODy CONDITION SCORE AND PERCENT BODy FAT TO THE INFLAMMATORy

RESPONSE IN AGED HORSESAMAnDAAADAMS,MADHUKATEPALLI,KATHARInAKoHLER,

STEPHAnIEREEDy,JPSTILZ,MAnDIMVICK,BARRyFITZGERALD,DAVIDWHoRoHoV

MaxwellH.GluckEquineResearchCenter,DepartmentofVeterinaryScience,UniversityofKentucky,Lexington,KY,

amanda.adams@uky.eduAdvancedageisassociatedwithalow-grade,systemicinflamma-

toryresponseinvivoandincreasedinflammatorycytokineproductioninvitro.Onepossiblesourceofthisincreasedinflammatorycytokineproduction are dysfunctional white blood cells associated with theaged immune system. In previous studies of the aged horse (≥20yrs), our results demonstrated a significant increase in the percentof lymphocytes fromoldhorsescompared toyoungproducing inter-feron-gamma(IFNγ)andtumornecrosisfactoralpha(TNFa).Itisalsoknown that increased white adipose tissue, associated with obesity,leadstoincreasedproductionofinflammatorycytokines.Todate,itisunknownwhether increased adiposity contributes to the age-relatedincreasedinflammatorystatus.Therefore,wetestedthehypothesisthatincreasedbodyconditionscores(BCS≥7)andpercentbodyfat(≥10)ofagedhorsesarepositivelyassociatedwithincreasedinvitroproduc-tionofinflammatorycytokines,IFNγandTNFa.Further,weproposedthat decreasing BCS (≤7) and percent body fat (≤10) will decreaseinflammatorycytokineproductionbothinvivoandinvitro.Bodycondi-tionscoresweredeterminedusing thestandardnine-pointHennekeScalingSystem.Bodyfatwasdeterminedbyultrasoundmeasurementofrumpfat.PBMCwerestimulatedwitheitherPMA/ionomyocin.Thecellswerefixed,permeabilizedandintracellularlystainedforIFNγandTNFa. SampleswereacquiredonaFACSCaliber. Percentpositiveweredeterminedusinglymphocytegates.TNFaproteinintheserumwasmeasuredusinganELISA.Therewasan increasedpercentageoflymphocytesstainingforTNFαinoldmareswithaBCS>7andper-centbodyfat>15.Likewise,IFNγproductiontendedtobeincreasedin the fatmares. Furthermore,decreasingadiposityandBCSscoredecreased the percent of IFNγ and TNFα positive lymphocytes.Decreasing adiposity in old horses also decreased TNFα protein inserum.Theseresultsindicatethatbodyconditionscoreand%rumpfatareconfoundingfactorswhenassessingage-associatedinflamma-toryresponses.DecreasingbodyfatandBCScouldimprovegeriatrichealthconditionsrelatedtoinflammatorymediators.Key words: aging, IFNγ, TNFα, lymphocytes, flow cytometry, bodyconditionscoresSpecies:Equine

er091. CHARACTERIZATION OF THE IMMUNOLOGICAL AND PHySIOLOGICAL RESPONSE OF AGED HORSES TO

EQUINE INFLUENZA INFECTION AMAnDAAADAMS,CoRMACCBREATHnACH,TRACy

STURGILL,ToMCHAMBERS,DAVIDWHoRoHoV

83

MaxwellH.GluckEquineResearchCenter,DepartmentofVeterinaryScience,UniversityofKentucky,Lexington,KY.

Amanda.Adams@uky.eduInfluenza is a serious health problem for the elderly population

and ranks as one of the top leading causes of death in the elderly.Therearealterationsinimmunefunctionthatoccurwithagethataffecttheabilityoftheelderlypopulationtorespondtovaccinationandresistinfection. In the horse population, equine influenza virus (EIV) is aleadingcauseofrespiratorydisease,howeverthesusceptibilityofoldhorses toEIV infection remainsunknown. Advancedage in horses(>20yrs)isassociatedwithage-relatedchangesinimmunefunction.Nevertheless, there are no specific recommendations regarding thevaccination of older horses even though a well characterized effectof aging inhorses is a reducedantibody response to standard vac-cination. Nor do we know if natural exposure to EIV is enough tosustainaprotectiveimmuneresponseintheolderhorse.Therefore,weevaluated the immunologicalandphysiological responseofagedhorses toEIVchallenge infection. NaïveyearlingswerechallengedwithEIVforcomparison.EIV-specificinterferon-gamma(IFN-γ)synthe-sisbyperipheralbloodmononuclearcells(PBMC)invitrowasusedameasureofcell-mediatedimmunity.Pro-inflammatorycytokinemRNAproductioninvivowasdeterminedusingRT-PCR.Clinicalsignsofthedisease (coughing, nasal discharge, dyspnea, depression, anorexia)aswell as rectal temperatureweremonitoredpost challenge.Therewasnosignificantdifferenceinclinicalsignsbetweennaïveyearlingsandoldhorsespostchallenge.However,therewasasignificantdif-ferenceinthefebrileresponsebetweennaïveandoldhorsesfollowingthechallenge.WhiletherewasnosignificantdifferenceinEIV-specificIFN-γsynthesisbetweenthenaiveandoldhorsespriortochallenge,thepercentofEIV-specificIFN-γ+lymphocyteswassignificantlyhigherinoldhorsescompared tonaïvehorsespostchallenge. BothnaïveandolderhorsesexhibitedsimilarincreasesintheexpressionofIL-6,IFN-γandIL-10mRNApostchallenge.However,therewassignificantincrease in IL-1βandTNF-α expression in theoldhorsescomparedto thenaïveyearlingspostchallenge. Insummary, theclinicaldatashowthatoldhorsesarejustassusceptibletoEIVinfectionasnaïveyearlings. The increased EIV-specific IFN-γ production may be anindication thatolderhorseshave immunologicalmemory toEIV,butnotenoughtoinsureprotectionfromEIVinfection.Key words:aged,equine,influenzaSpecies:equine

er092. INTERFERON-GAMMA ExPRESSION IN yOUNG FOALS WHEN TREATED WITH AN IMMUNE MODULATOR

DVMTRACyLSTURGILL,KRISTInAHAnDKE,DAVIDWHoRoHoV

GluckEquineResearchCenter,DepartmentofVeterinaryScience,UniversityofKentucky,Lexington,KY40546-0099.

tlstur2@uky.eduTheequineneonate, likeneonatesofotherspecies, isuniquely

susceptible to infectious disease. These diseases are a significantcause of neonatal mortality, thereby causing considerable economiclosstotheequineindustry.Neonatalimmuneresponsesareconsid-eredtobeimmature.Themechanismsinvolvedinthisimmaturityarenumerousandincompletelyunderstood.Immunemodulatorsthatcon-tainvariouspathogen-associatedmolecularpatternshavebeenusedextensively in the horse to combat respiratory and other infections.Whiletheuseofimmunomodulatorsinadultsresultsintheupregula-tionof IFN-γ, theireffecton IFN-γproduction in theneonatal foal isunknown. Thepurposeof this studywas todetermine theeffectoftreatmentwithanimmunemodulatoroncytokineproductioninfoals.Thirteen foalswere randomly divided into a treatment group receiv-ingEqStim®orthecontrolgroupreceivingnotreatment.Treatmentswereadministeredwithinthefirstfivedaysoflifeandrepeatedat30daysofage.EqStim®1ml,q48hrs,IV,wasadministeredforatotalofthree treatments,asrecommendedby themanufacturer. Startingatbirthandcontinuingweekly,peripheralbloodwasasepticallycollectedvia jugular venipuncture from each foal into heparinized tubes andPaxgene® tubes.Bronchoalveolar lavage(BAL)wasperformedninedayspostinitiationoftreatment.Heparinizedbloodwasusedforisola-tionofperipheralbloodmononuclearcells(PBMC).RNAwasisolated

from Paxgene® tubes according to the manufacturer’s protocol andreversetranscribedintocDNA.RT-PCRwasperformedfortheequinecytokinesIFN-γ,TNF-α,IL-4,IL-10,andIL-1withβ-GUSbeingusedasthehousekeepinggene.PBMCandBALcellswereculturedinvitroand intracellular staining for IFN-γwasperformed. ThereappearedtobeanincreaseinIFN-γproductioninBALcellsafterthefirsttreat-ment,howeverthiswasnotstatisticallysignificant.Additionally,whiletherewasanincreaseinIFN-γfollowingthesecondtreatmentinbothPBMCandmRNA,this,too,wasnotstatisticallysignificant.Thislackofstimulationmaybedueinparttoimpairedtoll-likereceptormediatedinnateimmunity.WhileEqStim®failedtostimulateIFN-γproductioninthefoalsinthisstudy,theroleofotherimmunemodulatorsforuseinfoalsneedstobeinvestigatedfurther.Key words: immune modulator, EqStim®, IFN-γ Bronchoalveolarlavagecells,PBMCsSpecies:Equine

er93. THE COMPARATIVE STUDy OF THE EFFECT OF VIRGINAMyCINE AND MANNAN- OLIGOSACCHARIDES

(MOS) ON HUMORAL IMMUNITy OF BROILER CHICKENSAZAKERI*1,MFADAEI2,SZAKERI3

1PostgraduatestudentofAvianDiseases,DepartmentofVeterinaryScience,FacultyofAgriculture,IslamicAzadUniversitybranch

Tabriz,Tabriz-IranandYoungResearchersClub(YRD),TabrizIran.E-mail:azakerii@yahoo.com,zakeri@iaut.ac.ir,2Studentofmaster(MS)courseofMathematics,Tehran-Iran,3StudentofVeterinary

Medicine,IslamicAzadUniversitybranchTabriz,Tabriz-IranMOS is a prebiotic and virginamycine is an antibiotic used as

growthpromoter(AGP). In thisstudy360Cobb500broilerchickensweredividedinthreesimilargroupswith120chickensineachgroup(withfourreplicatesof30chickensineachgroup).Onekg/tonMOSforexperimentalgroupΙand100g/tonvirginamycineforexperimentalgroupΙΙwereaddedtothebasicdietswhilethecontrolgroupchick-ens were fed only with basic diet. On days 9, 17 and 25 of growth(1 day before and 7, 14 days after first Newcastle B1 vaccination),fromeachgroup,eachtime40chickenswerechosenrandomlyandserumantibodiestitersweremeasuredagainstNewcastlevaccinebyHI test.After reviewing and analyzing the statistic data, the statisticresultsof theserumantibodiestitersbyHI test(5.40±0.44 incontrolgroup,5.87±0.47inexperimentalgroupΙand5.61±041inexperimen-talgroupΙΙ)indicatedastatisticaldifference(P<0.05)betweeneachthreegroups.MOSisanaturalsubstancethatdoesnothaveanydrugresidual inmeat of poultry and it is a suitable alternative for growthpromotersantibiotics.

Ref:Milner,JA.,M.Roberforid,1999.Nutritionalpropertiesofinulin

andoligofructose.JNutr:129,S1395–502.Roberfoid,MB.,2000.Healthbenefitsofnon–digestibleoligo-

saccharides.Adv,Exp,MedBull.427,211–219.Salminen.S. ,C.Bouley ,MC.Boutron–ruoult ,1998 . function

foodscienceandgastroinestinalphysiologyandfunction.BryNutr ,80(suppl),S147–71.

Vegad,JL. ,2004.Prebiotics,Probiotic ,AcidfiresandAntibioticgrowthPromotors.poultrydiseasesaguideforfarmersandPoultryProfessional.Firstedn.339-346.Key words:humoralimmunity,virginamycine,MOS,broilerchickensSpecies:avian

er094. EFFECT OF EARLy STIMULATIONS ON SOME IMMUNE PARAMETERS IN PRENATAL STRESS RATS

AnALIAUDAT,AURELIASARAnDón,nAnCyRoDRIGUEZ,CECILIARGRECo,ADRIAnABVIVAS1HECToRFGAUnA,noRA

MAyER1FacultaddeAgronomíayVeterinaria;FacultaddeCienciasExactas.UniversidadNacionaldeRíoCuarto;5800RíoCuarto.Argentina.

cgreco@exa.unrc.edu.arTheapplicationofstressorsduringpregnancyproducesanaltera-

tionofthehypothalamic-pituitary-adrenal(HPA)axisthatwouldinducea long-term alteration of the immune function in the offspring. Early

84

postnatal stimulations produce beneficial effect on the long- termemotionalresponseandHPAaxisactivitythatcouldrevert theeffectofprenatalstress.Theaimofthisstudywastoinvestigatetheeffectofearlystimulationsinprenatalstressedanimalsthroughthedistribu-tionofthesubpopulationsofleucocytesandtheinvitroproliferationsof lymphocytesT in response to acute stress in rats. For the studyweused:threemonthsoldmaleoffspringsfromimmobilization(IMO)stressedmothers(EP)andoffpringsfromnonstressedmothers.(CP).HalfoftheEPanimalsweremanipulated(M)duringthefirstweekoflife.Beforeextractionofbloodforbasaldeterminations,theanimalsofbothgroupswereunderacutestressIMO(20minutes).Then,bloodwasextractedat20,60,90,120,150and330minutespost-stresstocountwhitebloodcells,thesubpopulationsofleucocytesandthelev-

elsofcorticosterone.Thus,thespleensofallanimalswereremovedforlymphocytesTculture.Theprofileoftheleucocytes,lymphocytesandneutrophilswassimilarinEPandC,howeverresponsewasdepressedinManimalsunderpostnatalIMO.Theinvitroproliferationsoflympho-cytesTincreasedindevelopmentofManimals.Inconclusion,postna-talstimulationrevertstheeffectsofprenatalstressonthedistributionofthesubpopulationsofleucocytesandontheinvitrodevelopmentoflymphocytesTunderthesamepostnatalstress.Key words:prenatalstress,immuneparameters,ratsSpecies:other(Rat)

sm095. ROLE OF BASOPHILS IN THE RESISTANCE OF SENSITIZED GOATS TO AMBLyoMMA CAJENNENSE

FABRICIUS (1787) NyMPHSGERMonTEIRo1,2,GHBECHARA2

1DepartamentodeParaclinicas,FV-UniversidadeEduardoMondlane,Maputo,Mozambique,2DepartamentodePatologiaVeterinária,FCAV-UniversidadeEstadualPaulista,Jaboticabal-SP,Brazil.Lone-star ticks Amblyomma cajennense parasite primarily

horses,but itcan infestcows,deers,dogs,birdsandmenalso. It isthemainvectorofhorsebabesiosisandhumanspottedfeverinBrazil.AccordingtopreliminaryresultsofthelaboratorygoatsacquirepartialresistanceagainstA.cajennensenymphsafterrepeatedinfestations.Theaimofthisstudywastocharacterizethistick-hostinteractionbyexaminingmorphologicalfeaturesofthetickbitelesionduringrepeatedinfestations. Ten naive goats aged sixmonths, of both sexes, weredistributedintotwogroups:test(n=5),infestedwith15nymphsthriceat30daysintervalandcontrol(n=5).Skinfragmentsoftickbitesiteswerecollected24,48,72and120hourspostinfestationandprocessedaccordingtoroutinehistotechnology.Hematoxylin-eosinandGiemsa-stainedskinsectionsof4µmthicknesswereobserved,respectivelyforgeneralaspectsandinflammatorycells’countunderthecementconethroughan integratedeyepiece (10xmagnification)andanobjective100xmagnification (totalarea=0.0052mm2).At the tick feedingsite,itwasobservedepidermalfracturesuggestingtickmouthpart-inducedtissuedestruction,epidermalhyperplasiaandpustule-likeintra-epider-mal vesicles filled with polymorphonuclear cells, mainly neutrophils.Alimentary cavitywaspresent sometimesat thedermal layer, distaltothecementcone.Inaddition, itwasobservedinfiltratedinflamma-torycells into thedermis,mainlyneutrophils,eosinophils,basophils,mononuclearcellsandmastcells.The intensityof thesecellsvariedaccordingtotheexperimentalsituation.Infact,thefirstinfestationwasdominatedbyaneutrophils influx, itsnumbermaintainedhigh in thesubsequent infestations.On theother hand, a cutaneousbasophiliawasevidencedbyahighernumberofinfiltratingbasophilsincreasingsignificantlyfromthe48thto72ndhourafterboththe2ndand3rdinfesta-tions.Mononuclearcellsappearedintheinflammatoryfocus,buttheirnumbervariedlittleamonginfestations,withonlyasignificantincrease120hoursafter2ndinfestation.Eosinophilsandmastcellswerefoundoccasionallywithnostatisticaldifferencewhencomparedthe1standsubsequent infestations. It is concluded that basophils may play animportant role in the mechanism of resistance of goats against A.cajennensenymphs.Key words: cutaneousbasophilia,nymphs,amblyommacajennense,resistanceSpecies: ruminants

sm096. SKIN LESIONS INDUCED By TICKS RECRUIT DISTINCT CELLULAR POPULATIONS IN RESISTANT AND

SUSCEPTIBLE BOVINE HOSTS. FRAnZInAM1,MoRéDD1,CARVALHoWA1,ConTILHA2,

JoFPAULA2,AAMMAIA2,JSSILVA1,BRFERREIRA1,IKFDEMIRAnDASAnToS1

1Dept.ofBiochemistryandImmunology,RibeirãoPretoMedicalSchool,RibeirãoPreto,SP,UniversityofSãoPaulo,Brazil,2Dept.ofBasicSciences,SchoolofAnimalSciencesandFoodTechnology,

Pirassununga,SP,UniversityofSãoPaulo,Brazil.alefranzin@usp.br

Cattle present variable andheritable levels of resistance to thecattletick,Rhipicephalus(Boophilus)microplus.Inordertoobtainsomeof the immunecorrelatesof thesephenotypes,wecharacterized the

inflammatoryinfiltrateselicitedbytickbitesinskinofresistant(R)andsusceptible (S)bovines.RandScattleunderwent threesuccessiveinfestationsandbiopsiesweretakenatthefeedingsiteofnymphsandadults,fromnormalskinofthesamehostorfromskinofnaïvecontrols.Totalanddifferentialcellscountsweremadeonparaffin-fixedsectionsstainedbyMay-GrünwaldeGiemsa,ininfestedskintheywerelimitedtotheareaofthetickcementcone.Biteswithadultticksrecruitmoreinflammatorycellsthanthosebynymphs(P<0.05,one-wayANOVA).Neutrophilsaremorenumerousinskininfestedwithadultsthanwithnymphs(P<0.05).Conversely,mononuclearcellsaremoreabundantin skin infested with nymphs than with adults (P=0.001). Mast cellnumberswereequallydiminishedinadult-infestedskinofbothbreedswhencomparedwithnon-infestedskin(P<0.05).Nymph-infestedskinhadmoremastcellsthanRadult-infestedskin(P<0.05).Eosinophilswereabsent inskin fromnaïveanimals,butwerepresent innormalandinfestedskin,howevertheywerereducedininfestedskinofbothbreeds(P<0.05)andmoresignificantlyso inadult-infestedskinofRhosts(P<0.05).Basophilsweremoreabundant inRthan inSadult-infestedskin(P<0.05).Mastcellsaresourceofcytokinesandinflam-matorymediators thatplayeffectorsandmodulator roles in immuneresponses,theirreductionpossiblyduetodegranulationbyinflamma-torycytokines.The loweramountofneutrophils in infestedskinmayreflectthefactthatonlynymphsexpressRGD-containingdisintegrins,whicharepossibly neutrophil-specific.Eosinophils, aswell asbaso-phils,havebeenshowntobeimportantinresistancetoticksinexperi-mental models and their skin kinetics suggests a systemic effect oftickinfestations.ThegreaternumberofbasophilsininfestedskinofRhostssuggeststhattheyarethepivotalcellsthatimpairhematophagy.Ourresultsreflectthefactthat,whilethetickbiteinducesinflammation,ticksalivacontainsanti-adhesiveandimmunosuppressivemolecules,manyofwhicharestage-specific.

Supported:CNPq,CAPESandFAPESP.Key words:Ticks,Bovine,Basophil,Eosinophil,SkinSpecies:ruminants

sm097. SEQUENTIAL MORPHOLOGy AND GENE ExPRESSION PROFILES OF CUTANEOUS REACTIONS TO

TICK ANTIGENS IN BOVINESARRABATEPAULo1,JoÃoMoRELLI2,MATHIASPSZABó2,4,SSKASHIno3,CJnARDELLI3,GRGARCIA1,ERAMIRoDA

SILVAJR3,oBREGonETo3,JSSILVA1,GHBECHARA2,IKFDEMIRAnDASAnToS1

1RibeirãoPretoMedicalSchool,UniversityofSãoPaulo,RibeirãoPreto,SP,BRAZIL;2FacultyofAgriculturalandVeterinarySciences,StateUniversityofSãoPaulo,Jaboticabal,SP,BRAZIL;3Brazilian

EnterpriseforAgriculturalResearch,Brasília,DF,BRAZIL;4FacultyofVeterinaryMedicine,UniversidadeFederaldeUberlândia,Uberlândia,

MG,BRAZILIntroduction: In cattle the level of infestation with ticks varies

accordingtobreedandthedifferentphenotypesareheritable,geneti-cally susceptible animals harbour significantly more parasites thanresistantbreeds,evenafterrepeatedinfestations.

Objectives: To study themolecular and cellular components ofcutaneousinflammatoryreactionselicitedwithtickantigensindifferentphenotypesoftickinfestationsinbovines.

Methods:Animalsofa resistant (R,Bos indicus,Nelore,N=6)andsusceptible(S,B.taurus,Holstein,N=6)breedweremanagedin a pasture infested with the cattle tick, Rhipicephalus (Boophilus)microplus.Statusofresistancewasverifiedbycountingfemaletickslargerthan4mmoneachhost.Antigen(50µgin100µlofanextract

4. IMMUNOLOGy OF THE MUCOSAE AND SKIN AND OF THE MAMMARy GLAND: POSTERS SM095-SM118

86

ofunfedlarvae(UFLE)fromR.microplus)wasinjectedinthedermisoftheinternalsurfaceofoneearandthesamevolumeofthedilutionbufferwas injected in theoppositeearof theanimals.Skinbiopsieswere takenone, 72and96hours after inoculationofUFLEor buff-ered saline and processed for staining with haematoxylin/eosin andMay-GrünwaldandGiemsaorextractionof totalRNA.Totalanddif-ferentialcellscountsweremadeoftissuesectionsandquantificationofexpressionofcandidategenescodingforchemokinesandanti-andproinflammatorycytokineswasdonewithRealTimePCR.Student’st-testwasusedtoevaluatesignificanceamonggroupmediansandaP-value<0,05wasusedtoestablishthelevelofsignificance.

ResultsandDiscussion:Haematoxylin/eosinandGiemsa-stainedsectionsofskinbiopsiestakenonehourafterinjectionrevealedsimilartotalanddifferentialcellcountsatbothtestandcontrolsitesforbothbreeds,excepteosinophils,whichweresignificantlymoreabundantintheskinofSanimals.After72hours,inrelationtocontrolstheglobalnumbersofcellularinfiltrates,aswellasofeosinophils,increasedsig-nificantlyandequallyinSandRbovineskinstimulatedwithUFLE,how-ever,atthistimepointthenumbersofbasophilsincreasedsignificantlyinR,butnotSbovineskinstimulatedwithUFLE.Inrelationtogeneexpression,Rbovinespresentedhigherlevel,butnotsignificantly,ofexpressionofIFN-γinIHtestreactionsandsignificantlyhigherexpres-sionofIGF-1,IDO,TGFb,IL-16,SLURP-1andEndotelin-1indelayedreactionssignificantly.ThismolecularpatternsuggeststhatRbovineshaveagreatercapacity tomount inflammatory reactionscomprisingbasophilsthataredetrimentaltothetick.

Supportedby:FAPESPandCNPq.Key words: gene expression, cutaneous reactions, tick antigens,bovinesSpecies:ruminants

sm098. BOVINE γδ/WC1+ AND B LyMPHOCyTES, BUT NOT CD8+ CELLS, ARE REDUCED IN INFLAMMATION INDUCED

By TICKSALESSAnDRAMFRAnZIn1,DAnIELADMoRé1,WAnESSA

ACARVALHo1,LUIZHAConTI2,PAULAJoF2,AnTonIoAMMAIA2,MARKAJUTILA,JSSILVA1,BRFERREIRA1,IKFDE

MIRAnDASAnToS11Dept.ofBiochemistryandImmunology,RibeirãoPretoMedical

School,RibeirãoPreto,SP,UniversityofSãoPaulo,Brazil;2Dept.ofBasicSciences,SchoolofAnimalSciencesandFoodTechnology,

Pirassununga,SP,UniversityofSãoPaulo,Brazil.alefranzin@usp.br

Cattle present variable andheritable levels of resistance to thecattle tick, Rhipicephalus (Boophilus) microplus. In order to obtainsomeoftheimmunecorrelatesofthesephenotypes,wecharacterizedtheinflammatoryinfiltrateselicitedbytickbitesinskinofresistant(R)andsusceptible(S)bovines.RandScattleunderwentthreesucces-siveinfestationsandbiopsiesweretakenatthefeedingsiteofnymphsandadults, fromnormalskinof thesamehostor fromskinofnaïvecontrols.Lymphocytesurfaceantigensweredetectedwithspecificanti-bodiesreactingwithacetone-fixedcryostatsectionsofbiopsiesstainedusing the indirect immunoperoxidase technique and counter-stainedwithMay-GrünwaldandGiemsa, in infestedskin,sectionswere lim-itedtotheareaofthetickcementcone.Bitesbynymphsrecruitmoremononuclearcells thanthoseofadults(P=0.001,One-wayANOVA).The numbers of an important subset of mononuclear cells, CD3+ Tlymphocytes,werereducedinadult-infestedskinofbothbreedswhencomparedwiththosefoundinskinofnaïveandnon-infestedcontrols(P<0.05).NumbersofproinflammatoryγdWC1+/CD3+Tcellswerealsodiminished(P<0.05)innymphandadult-infestedskinofbothbreeds,whencomparedwiththosefoundincontrolskin.Moreover,numbersofγd+Tcellswereevenlower(P<0.05)innymph-infestedRskinwhencomparedwithadult-infestedRskin.B lymphocyteswerepresent inskin, but their numberswere reduced (P<0.05) innymphandadult-infestedskin,regardlessofthebreedofhost.Asthedistancefromthecementconeincreased,numbersofallthesecellsreachedthesameasnormalskin.NumbersofanotherCD3+Tcell subset,CD8+cyto-toxiclymphocytes,weresimilarininfested,normalornaïveskin.Skinimmuneresponsesinvolveresidentandinfiltratingsubsetsoflympho-cytesthataresourcesofimmuneregulatoryandeffectorsresponses.

TheobserveddepletionofCD3+Tcells,Bcellsandγd+/WC1+Tcells,butnotofCD8+Tcells,intickbitelesionsispossiblyduetocelldeath,emigrationand/orimpairedcellularmigrationtotickbitelesions.Itsug-geststhattheparasiteimpairstheacquiredimmuneresponseviaanti-adhesive and/or immunosuppressivemolecules.On the other hand,thegreaterreductionof inflammatoryγd+Tcellsseen in the infestedskinofresistantbovinesindicatesthatthesecellsmayplayaroleinresistancetoticks.Theexpressionofchemokinereceptorsassociatedwithmigrationofγd+Tlymphocytestoinflamedandnormalskinmustbeexamined.

SupportedbyCNPq,CAPESandFAPESP.Key words:lymphocytes,tick,bovine,skin,immunohistochemistrySpecies:ruminants

sm099. INFLAMMATORy CELL INFILTRATION AND INFLAMMATORy CyTOKINES: INDICATORS

OF STREPTOCOCCUS AGALACTIAE INFECTION IN ExPERIMENTAL MOUSE MASTITIS

GABRIELATRIGo1,2,AnGELAFRAnçA1,MáRCIADInIS1,2,RUIGILDACoSTA1,ELVAAnDRADE1,PAULAFERREIRA1,2,

DELFInATAVARES1,21InstitutodeCiênciasBiomédicasAbelSalazar(ICBAS),

UniversidadedoPorto,Portugal;2InstitutodeBiologiaMoleculareCelular(IBMC),Porto,Portugal

dcb03002@icbas.up.ptStreptococcusagalactiae isamajorcontagiouspathogencaus-

ingmastitishighlyadaptedtosurviveinbovinemammarygland.ThisstudyfocusesontheuseofamousemodelofStreptococcusagalac-tiae-inducedmastitisasapracticalapproachforregardingthepatho-genesisofthebacteria.BALB/cmicein10-15thdayoflactationwereintramammary (I.ma) challenge on both L4 (on the left) andR4 (ontheright)abdominalmammaryglandswith108cellsofS.agalactiaeisolated frombovinemastitis (infectedanimals)orwithPBS (controlanimals).Throughoutthestudythecolonizationwasevaluatedbybac-terialcounts(CFU)inthemammarygland,kidneys,spleenandliver.Mammary tissue alterations were evaluated by haematoxylin-eosinstainingofmammarysections.Cytokineproduction in themammarygland during infection was evaluated by ELISA. S. agalactiae I.mainfection showed that the bacteria replicates in the mammary glandandpeaked24hlater.Atthesametime,amassiveinfiltrationofpoly-morphonuclearcells(PMNs)andanincreaseinIL-1β,IL-6andTNF-a(inflammatorycytokines)levelsweredetectedinthemammarygland.Afterthis,agradualdecreaseonbacterialoadwasobservedwhichwasaccompaniedbyadecreaseinthenumberofPMNsandanincreaseofmacrophagesandlymphocytes,indicatinganevolutionintoachronicprocess in themammary tissue.Adecrease in the levelsofTNF-a,IL-1βandIL-6wereobservedinthemammarygland72hafterinfec-tion,whichwasaccompaniedbyanincreaseinthelevelsofIL-12andIL-10.Disseminationofthebacteriafromthemammaryglandstothekidneys,spleenandliverwasobserved6hoursafterinfection,withapeakoftheCFUafter12hoursforthekidneysandspleenandafter24hoursfortheliver.Thebacterialloadintheseorganswassignificantlylower than theoneobserved in themammarygland, throughout thestudy.Inconclusion,themousemodelofinfectiousmastitisproposedhereissuitableandlesscostlythancowsforthestudyofpathogenesisofS.agalactiae.

ThisworkwassupportedbytheFundaçãodaCiênciaeTecnologia(FCT)grantPOCI/CVT/57144/2004andFEDERKey words: Streptococcus agalactiae , mastitis, inflammation,cytokines,Specie:ruminants

sm100. ELUCIDATING THE BIOSyNTHETIC PATHWAy OF PAF PRODUCTION By MAMMARy ENDOTHELIAL CELLS

FOLLOWING ENDOTOxIN STIMULATIONJCGAnDy,CMCoRL,KBEGIn,LMSoRDILLo

DepartmentofLargeAnimalClinicalSciences,MichiganStateUniversity,EastLansing,MI

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Acutesymptomsassociatedwithcoliformmastitisareduetorapidgrowthoftheorganism,thereleaseofendotoxin,andthesubsequentdevelopment of an exacerbated inflammatory reaction. Endotoxinreleasepromotestheproductionofpotentpro-inflammatorymediators,suchasplatelet activating factor (PAF), that lead to thepathophysi-ological changes witnessed during gram-negative infections. WhilemuchattentionhasbeengiventotheproductionofPAFbyneutrophilsandmacrophages,verylittleresearchhasfocusedonPAFproductionbymammaryendothelialcellswhicharevitaltomaintainingvascularhomeostasis during the acute phase response. Previous researchhas shown increased lyso-PAF:acetyl coenzyme-A acetyltransferase(Lyso-PAF-AcT)activity, the rate limitingenzymeofPAFproduction,followingLPSstimulationinbovinemammaryendothelialcells(BMEC)occurspriortoincreasesintheacutephasecytokineresponse.Thebiosyntheticpathway leadingtothis increasedLyso-PAF-AcTactivityand subsequent PAF production following LPS stimulation has notbeendetermined.It ishypothesizedthatphosphatidicacid(PA)andphospholipaseD(PLD)arepreliminarymediatorsinthebiosynthesisof PAF by BMEC following endotoxin stimulation. Utilizing primaryBMECstimulatedwithLPS theproductionofPAandactivityofPLDwasmeasured, aswell as the activity of thePAF catabolic enzymePAF-acetylhydrolase (PAF-AH). Preliminarydatasuggests thatLPSstimulationcausesanincreaseinbothPAproductionandPLDactivityinBMEC. Endotoxin stimulation did not significantly alter the activ-ityofPAF-AH,furthersupportingour focusonthe initialsynthesisofPAF. Determining theearlymediatorsof theacutephase responseduring gram-negative infections could lead to the development ofnoveltherapeutictargetstodiminishtheuncontrolledinflammationandpathophysiologicaleffectsofcoliformmastitis.Key words:Endothelial cell, inflammation,Platelet activating factor,cytokineSpecies:ruminants

sm101. DyNAMICS OF B-CELL REPERTOIRE IN SHEEP JEJUNAL AND ILEAL PEyER’S PATCH SINGLE FOLLICLES

MASAHIRoyASUDA,CRAIGnJEnnE,LAURIEJKEnnEDy,JoHnDREynoLDS

ImmunologyResearchGroup,DepartmentofCellbiologyandAnatomy,UniversityofCalgary,DepartmentofVeterinaryAnatomy,

UniversityofMiyazakiIntheruminant’sintestine,therearetwotypesofgut-associated

lymphoidorgan:jejunalPeyer’spatch(PP)andilealPP.IlealPPisthoughttobetheprimarylymphoidorganofB-cell,whoserepertoireisdiversifiedbygeneconversionand/orsomatichypermutation.Onthe other hand, jejunal PP is thought to be the secondary lymphoidorgan for localmucosal immunityand the functionsof this lymphoidorgankeep throughout theanimal’s life. Theprenataldevelopmentof follicles in thePPbeginsfirst in the jejunumduring themiddleofgestationandthenintheileumduringlategestation.ThereforeitcanbeconsideredthatjejunalPPfolliclealsocontributesmakingprimaryB-cellrepertoireaswellasilealPPfollicleatfetaldevelopment.Thenafterbirth,jejunalPPmayformthecharacterforlocalmucosalimmu-nity. Weattempt toanalyzeB-cell repertoireof ilealand jejunalPPsingle folliclesduringontogeny. BothPPsingle folliclesatprenatalandpostnataldevelopmentwereisolatedunderstereoscopicmicros-copy. Igl light chain isdominant light chain in sheep.Vl-Jl1wasamplifiedbyPCRorPT-PCR.Atthepostnatalstage,ilealPPfolliclescontained oligoclonal B-cell, but jejunal PP follicles containedmuchmorepolyclonalB-cell.Similartendencyobservedatprenatalstage.HenceclonalityofbothPPfollicleswasmarkedlydifferent.Atprenatalstage,pointmutationaccumulatedinCDRregioninVlgeneofbothPPfollicularB-cell(14-19pointmutations/kbwereinilealPPand3-13mutations/kbwerein jejunalPP). B-celldiversity isobservedinnotonlyilealPPfolliclebutalsojejunalPPfollicle.ThedatashowthatbothPPfolliclescontributemakingB-cellrepertoireduringprenataldevelop-ment.Atthepostnataldevelopment,muchmoremutationsobservedin CDR regions in Vl gene of both PP follicular B-cell (32-64 pointmutations/kbwereinilealPPand39-54mutations/kbwereinjejunalPP).EspeciallymanyreplacementmutationsobserveinCDR3regionofilealPPfollicles.ThereforeilealPPfolliclescontributemakingverywidediversity ofB-cell after birth. This event probably causes theappearancemanyself-reactiveB-cell.

Key words:ilealPeyer’spatch,jejunalPeyer’spatch,B-cellrepertorie,prenatalandpostnatalSpecies:ruminants

sm102. STABILITy OF THE RECOMBINANT GM-CSF PRODUCED By BACULOVIRUS GENE ExPRESSION

SySTEMSHIGEKIInUMARU,HIDEyUKITAKAHASHI,SAToKoWATAnABE,

MASATooHTA,TAKAyUKIKUBoTANARONationalInstituteofAnimalHealth

inumaru@affrc.go.jpGM-CSFisknownasacytokinethataffectsthevarioushaema-

topoieticcells.BovineGM-CSFisexpectedtouseatherapeuticagentfordiseasescausedbycomplexandopportunistic infectionsuchasmastitisincows.SincenaturalGM-CSFisproducedonlytraceamountby particular cells, we established the efficient method to producerecombinantbovineGM-CSF(rboGM-CSF)bybaculovirus/cellculturegene expression system and reported that this rboGM-CSF was apotentialtherapeuticagentforsubclinicalmastitisofdairycowscausedby S. aureus infection.Though the information about stability of therboGM-CSFisimportanttodeveloprboGM-CSFagent,itisnotclearedup.Thereforewearestudying thestabilityof the rboGM-CSFunderseveralconditions.METHODS:BovineGM-CSFcDNAsequencewasinserted to a baculovirus (AcNPV) genome. The rboGM-CSF waspreparedwith this recombinantvirus infected insectcell(TN5cells).Theculturefluid,containingrboGM-CSFwasultra-filtratedtoremovevirusparticleanddilutedwithPBSwith10%FBS(PBS+)orwithoutFBS(PBS-). It is then stored at 4C or –20C.The biological activi-tiesweremeasuredwithrboGM-CSFadoptedTF-1cells.RESULTS&DISCUSSION:TostudythestabilityofrboGM-CSFat4C,rboGM-CSFinPBS+wasstoredat4C,andthebiologicalactivitywasmeasured.Theactivitywasnotreducedatleast5month.Similarly,theactivityofrboGM-CSFinPBS-wasnotreducedat least5month.TostudythestabilityofrboGM-CSFonthefreezeandthaw,rboGM-CSFinPBS+wasfrozenat–20Candthawedrepeatedly.Atleast3timesrepetition,thebiologicalactivitywasnotreduced.TheactivityofrboGM-CSFinPBS-wasalsonotdecreasedbythefreezeandthawatleast3timesrepetition. These results clearly showed that rboGM-CSF producedbybaculovirus/cell culture geneexpression system is very stable at4Candbyfreezeandthaw.It isanadvantageousfactortodeveloprboGM-CSF therapeutic agent formastitis etc.We are studying thestability under someother conditions.CONCLUSIONS:The rboGM-CSFproducedbybaculovirus/cellculturegeneexpressionsystemisstableforstorageat4Candfreezeandthaw.Key words:GM-CSF,stability,therapeutics,mastitisSpecies:ruminants

sm103. RELATIONSHIP OF T CELL PROLIFERATION AND THE UDDER TRANSCRIPTOME WITH MASTITIS

RESISTANCE TO STAPHLoCoCCUS AUREUSnICoLAHASTInGS1,FIonAyoUnG2,JoHnWILLIAMS3,JULIE

FITZPATRICK4,ELIZABETHJGLASS11RoslinInstitute,Roslin,Midlothian,EH259PS,U.K.;2AgricultureBranch,Agri-FoodandBiosciencesInstitute,Hillsborough,County

Down,NorthernIreland,BT266DR.;3ParcoTecnologicoPadano,ViaEinstein,PoloUniversitario,Lodi26900,Italy;4MoredunReseachInstitute,PentlandsSciencePark,BushLoan,Penicuik,Midlothian,

EH260PZ,U.K.Mastitisremainsoneofthemostimportantdiseasesindairycattle

particularlyinthewesternworld.ItcoststheEUalone€100-200million/yearwitheconomiclossesduetoreducedmilkproduction,lowermilkquality,veterinarytreatmentandalsoculling.Inaddition,asmastitisisanextremelypainfuldisease,thereareseriouswelfareimplications.Currentcontrolmeasuresarenotalwayseffectiveanddevelopingvac-cinestargetedatmucosalsurfacessuchastheudderareprovingdif-ficult.Breedingforresistancetomastitiscouldbeanalternativemeansofcontrolbutisnotstraightforwardforanumberofreasons.Althoughit is clear that there isageneticcomponentaccounting forvariationinmastitisresistance,itislikelytobemultigenicandthetraithaslowheritability,possiblybecausemastitisscoringforgeneticanalysisusu-

88

allydoesnottakeintoconsiderationthatmastitisiscausedbydifferentpathogenswhichelicitdifferenthostresponses.Additionallymastitisisbecomingmoreofanissuefordairyfarmersbecauseithasanegativecorrelationwith genetic selection for increasedproductivity.Wepro-posetotakeadifferentapproachbytakingadvantageofthegenomicresourcesnowavailableforcattletoidentifykeygenesandpathwaysthat couldultimately lead tonewgenetic testsandselectablemark-ers forbreeding formastitis resistance.Earlierstudies indicated thatmeasurementoftheproliferativeresponseofbovineperipheralbloodT cells to formalin-fixedStaphylococcus aureus might be used as apotentialmastitis-resistancepredictorwithhighresponderspotentiallypredictinggreaterresistancetoS.aureusmastitisthanthosewithalowproliferativeresponse.OurstudiesinacattlecrosspopulationshowedthatthelevelofproliferationtoS.aureuswasatleastpartiallygeneti-callydetermined(h2=0.2).WenowaimtodirectlyinvestigatewhetheryoungheifersselectedonthebasisoftheirTcellresponsetoS.aureusintotwogroupsofhighandlowresponders,dodifferintheirresponsetoexperimentalS.aureuschallengeinvivoat6-weeksoflactation.Inadditiontomeasuringclinicalparameters,wewillalsotranscriptionallyprofilethecellsenteringtheudderandtheuddertissueitselffollowinginfection.Thisprojectcouldprovidemoredirectevidenceofthepredic-tiveabilityof theTcell test,andhas thepotential to reveal relevantgeneandpathwaytargetsascandidatesfornewgenetictestsofmas-titisresistance.Key words:mastitis,resistance,cattle,microarraySpecies:ruminants

sm104. EVALUATION OF BRUCELLA ABoRTUS HyDROPHOBIC AND HyDROPHILIC FRACTIONS By

AVIDITy-IMMUNOBLOT FOR SERODIAGNOSIS OF BOVINE BRUCELLOSIS

AnACAMPAJUABA,DEISEAoSILVA,DÂMASoPRIBEIRo,JoSéRMInEo*

LaboratoryofImmunoparasitology,InstituteofBiomedicalSciences,FederalUniversityofUberlândia,Av.Pará1720,38400-902

Uberlândia,MG,Braziljrmineo@ufu.br

Brucellosis is a major zoonosis and has been considered anemerging or re-emerging disease worldwide, particularly leading toabortion and infertility in domestic herds. Diagnosis of brucellosis incattle ismainlybasedonserologicalmethods thathaveusedwholecellpreparations,sonicatedcellextractsor lipopolysaccharide (LPS)enrichedfractions.Asspecificityofthesetestsislow,alternativeanti-genshavebeencharacterizedaspotentiallyusefultoolsindiagnostictestsforbrucellosis.Inthiscontext,theTritonX-114nonionicdetergenthas been successfully used for extraction of membrane-associatedproteins,butnodataareavailableonitsuseforBrucellaabortusanti-genextraction.Smooth lipopolysaccharide (S-LPS)andTritonX-114fractions from B. abortus were analyzed in immunoblot and avidity-immunoblot to identify differences between antigenic markers fromS19vaccinated fromnon-vaccinatedseropositivecows.Fourgroupswith15cattleseraeachwereanalyzed:(I)non-vaccinatedseropositivecows from Brucella-endemic areas, (II) non-vaccinated seropositivecows fromBrucellanon-endemicareas, (III)S19vaccinatedheifers,and (IV) non-vaccinated seronegative cows. Classical agglutinationtestswereusedtoselectthegroupsofcattlesera.S-LPSimmunoblotshowedawideclusterofbands(22to105kDa)recognizedbyserafromnon-vaccinatedcows(groupsIandII)whileamorerestrictcluster(43to58kDa)wasseeninserafromvaccinatedheifers(groupIII).Inavidity-immunoblotusingS-LPS,nodistinctreactivityprofilecouldbeidentified.TritonX-114hydrophobicfractionrevealedawideclusterofantigenicbandsincontrasttohydrophilicfractionthatshowedamoreclearlydefinedreactivityprofile.Immunoblotusingthehydrophilicfrac-tionrevealedsomeimmunodominantantigens(30,35,43,57and73kDa) in all three seropositive groups, whereas a significantly lowerreactivitywasseenforthe69and65kDabandsonlyinvaccinatedheif-ers(groupIII).Avidity-immunoblotwiththehydrophilicfractionshowedsignificant reactivity impairment for the immunodominant antigenicbands(57,43and35kDa)recognizedbyseraofgroupIII,reflectingin lowervaccinalantibodyavidityforsuchantigenicmarkers.Incon-clusion,B.abortus-specificIgGreactivityprofileinavidity-immunoblotdemonstratedthatvaccinalantibodiesshowedaloweravidityforsome

antigeniccomponents(57,43and35kDa),thusrepresentingpotentialantigenicmarkerstodiscriminatevaccinatedfrominfectedcattle.Key words:Brucellaabortus,Avidity-Immunoblot,TritonX-114,Cattle,LPS.Specie:ruminants

sm105. IDENTIFICATION OF ADAPTIVE TRAITS OF BOVINE MASTITIS ESCHERICHIA COLI STRAINS

SBLUM1,SSELA2,oHAMMER-MUnTZ2,oKRIFUCKS1,LWEISBELITH1,DHELLER3,GLEITnER1*

1NationalMastitisReferenceCenter,KimronVeterinaryInstitute,MinistryofAgriculture&RuralDevelopment,BetDagan,Israel,

2MicrobialFood-SafetyResearchUnit,DepartmentofFoodSciences,AgriculturalResearchOrganization(ARO),TheVolcaniCenter,BetDagan,Israel,3FacultyofAgriculture,TheHebrewUniversityof

Jerusalem,Rehovot,Israel.leitnerg@moag.gov.il

Escherichiacoliisamajoragentofacutebovinemastitis.Inspiteofextensiveresearch,thebacterialpathogenicfactorsassociatedwithE.colibovinemastitisarestillunclearand,todate,noE.colistrainssubsetorvirulencefactorshavebeenparticularlyassociatedwiththedisease.Inthiscontext,thisstudyaimedtoassessifbovinemastitisE.colistrainshaveuniqueadaptivetraits.Forthispurpose,E.coliisolatesfromacutebovinemastitisandfromtheenvironmentwerecomparedregardingphenotypicandphysiological traits, resistance tokillingbybovine leukocytes and genetically, byPulse-field gel-electrophoresis(PFGE).Mastitisandenvironmentalstrainscouldnotbephenotypicallydistinguishedbymeansoftheconventionallystudiedtraits,namelyOantigenandantibioticsensitivity.Thephysiologictraitsevaluatedwerebacterialgrowthinmilk,nutrientbrothandlactosefermentationrates.Whilenodifferencesinbacterialgrowthrateswereobservedinnutrientbroth, inmilk,however,bacterialgrowth ratesofallmastitis isolatesweresignificantlyhigherthanmostofenvironmentalstrains.Similarly,the rate of lactose fermentation was higher in mastitis isolates thanenvironmental ones, beingpositively correlated to growth inmilk. Inaddition,themeanresistancetokillingbybovineleukocyteswashigherin mastitis isolates than in environmental strains. PFGE revealed ahighergeneticvariabilityamongenvironmentalstrains than inmasti-tis isolates,~40%ofmastitis isolates formedarelativelygeneticallysimilarcluster.Sinceonlypartoftheenvironmentalstrainsphysiologi-callyandgeneticallyresembledmastitisisolates,ourresultsreinforcetheideaofanE.colisubsetofstrainsmoreadaptedtocauseacutebovinemastitis.This subset is apparently characterizedbyadaptivetraitsrelatedtoahigherabilitytomultiplyandevadethehostimmuneresponsewithinthebovineudderandgenetichomogeneity.Key words: Escherichia coli, mastitis, adaptive traits, lactosefermentationSpecies:ruminants

sm106. INFLUENCE OF SEMINAL PLASMA ON TGF BETA1 MRNA ExPRESSION IN THE PIG OVIDUCT

JJIWAKAnon1,MBERG2,CFoSSUM2,EPERSSon3,AMDALIn1

1Dept.ofClinicalSciences,2Dept.ofBiomedicalScienceandVeterinaryPublicHealth,3Dept.ofAnatomy,Physiologyand

Biochemistry,SLU,Uppsala,SwedenIthasbeenshownthatsemeninducesuterineimmuneresponse

and that seminal plasma may regulate uterine cytokine expression.Theaimofthepresentstudywastoevaluateifseminalplasmaperseaffectstheexpressionofthepotentimmunoregulatorycytokine,trans-forminggrowthfactorbeta1(TGFβ1) intheporcineoviduct.Sixteengiltswere inseminatedonce,15-20hafterstanding reflex,with freshsemendilutedinBTS(extender)(n=4),seminalplasma(n=4),spermsisolated by colloidal centrifugation and diluted in BTS (n=4) or BTSalone(n=4).Thegiltswereslaughtered5–6hafterinseminationandoviductalsamplesweretakenimmediately,plungedintoliquidnitrogenand storedat -80 °Cuntil analyzed.TGFβ1mRNAexpression levelinoviductaltissues,isthmusandinfundibulum,wasquantifiedbyrealtimePCRusingTaqMan-probenormalizedtothegeometricmeanoftwo housekeeping genes, hypoxanthine-guanine-phosphoribosyl-transferase(HPRT)andcyclophilin.ThemRNAexpressionofTGFβ1

89

wassimilarininfundibulumcomparedtoinisthmus,regardlessifthegiltswereinseminatedwithfreshsemen,purifiedspermortheextenderalone.Thus, the resultssuggest thatseminalplasmapersehasnoearly effect on the level ofTGFβ1mRNA in the oviduct and similarexpressionlevelofTGFβ1mRNAwereobservedbetweentheupperpartandthelowerpartoftheoviduct.Key words:gilt,TGFbeta,oviduct,andseminalplasmaSpecies:swine

sm107. THE IN VIVO EARLy TRANSCRIPTIONAL INTESTINAL RESPONSE TO ROTAVIRUS INFECTION IN

GERM-FREE PIGLETSTHEonIEWoLD1,MARCELHULST,HInRIKERSTEnS,AGnES

DEWIT,MARISMITSAnDJAnVAnDERMEULEnAnimalSciencesGroupofWageningenUniversityandResearch,P.O.Box65,Lelystad,TheNetherlands,1NutritionandHealthUnit,FacultyofBioscienceEngineering,KatholiekeUniversiteitLeuven,

B-3001Heverlee,Belgium(presentingauthor)Rotavirusesareamajorcauseofseverediarrheainyoungchil-

dren worldwide. Most studies regarding the molecular mechanismunderlyingrotavirusinduceddiseasefocusonisolatedenterocytesorenterocyte cell-lines. In vivo,matureenterocytes lining the intestinalepithelial layer are the primarily target cells for rotavirus replication,however,thedifferenttypesofcellsthatcomposetheintestinalmuco-sallayerareprobablyalsoinvolvedinanti-viralresponses.Theinvivosystemisalsomuchmorecomplexforthepresenceoftheintestinalmicrobiota. Inordertoseparaterotavirusspecificeffectsfrommicro-biotaassociatedeffects,weusedgerm-freepiglets.PigletshousedinsterileincubatorswereorallyinfectedwithvirulentgroupArotavirusat3weeksandwholemucosalgeneexpressionvsuninfectedcontrolswasanalyzedbycDNAmicroarrayon12and18hourspostinfection.IFN-γmRNAlevelswere10to50-foldhigherininfectedpiglets.Microarrayanalysis identified 13 down-, and 19 up-regulated genes in infectedpiglets.Microarray data were validated by Northern blot analysis ofnineselectedgenes.Regulatedgeneswere functionallyclustered ininterferon-regulatedgenes,signal transductionandapoptosisgenes,(enterocyte)metabolismandcellmaintenancegenes,andgeneswith-outaknownfunction.Up-regulationwasobservedforseveralgenesassociated with the innate defense against viral infections, such asIFN-γinducedguanylatebindingprotein2(GBP-2),aproteinthatwasdescribedearliertoeffectivelyinhibitVSVandEMCVvirusreplicationinvitro.Furthermore,agenecodingforanuncharacterizedhypotheticalproteinwasupregulated,carryingaphospholipaseA2inhibitordomain,suggestinginvolvementin(anti-)inflammatorypathways.Wehypoth-esizethatboththeseproteinsparticipateincellularmechanism(s)thatprovide the intestinalmucosaprotection to theeffectsof rotavirus inthe jejunum. Histological analysis showed a significant reduction ofvilluslengthduetorotavirusinfection.It isconcludedthatdifferentialexpressionreflectsinpartashiftintherelativecontributionofcertaincelltypes(e.g.lossofmatureepithelialcells),andinanotherpartrep-resents inductionbyrotavirus itself.Finally,amoststrikingfinding isthelimitednumberofdifferentiallyexpressedgenesinintactmucosainvivocomparedtothoseobtainedininvitroenterocytecultures.Dilutionofexpressionmayplayaroleinthat,butourfindingsareprobablyalsoconsistentwithmodulatoryeffectsoftheheterogenouscellpopulationinvivo.Key words:intestinalgenomics,rotavirus,pigletsSpecies:swine

sm108. COLIFORM MASTITIS IN SOWS ENHANCES TNF-ALPHA PROTEIN AND MRNA ExPRESSION IN DIFFERENT

COMPARTMENTSyAoHonGZHU1,3,MIKAELBERG2,CARoLInEFoSSUM2,

ULFMAGnUSSon1,3,*1DepartmentofClinicalSciences,FacultyofVeterinaryMedicineandAnimalScience,SwedishUniversityofAgriculturalSciences,SE-

75007Uppsala,Sweden;2DepartmentofBiomedicalSciencesandVeterinaryPublicHealth,SwedishUniversityofAgriculturalSciences,SE-75123Uppsala,Sweden;3CentreforReproductiveBiologyin

Uppsala,Uppsala,Sweden

∗Correspondingauthor:UlfMagnusson,DepartmentofClinicalSciences,FacultyofVeterinaryMedicineandAnimalScience,SwedishUniversityofAgriculturalSciences,P.O.Box7054,SE-

75007Uppsala,Swedenulf.magnusson@kv.slu.se

Twelvehealthyprimiparoussowsreceivedintramammaryinocula-tionwithEscherichiacoli(E.coli,serotypeO127)duringthe24-hourperiod preceding parturition. Eight of the sows remained clinicallyhealthy, whereas four developed clinical signs of mastitis such asfever, mammary swelling, and lethargy. Jugular blood samples andbiopsysamplesfromtheinoculatedglandswerecollectedimmediatelybefore inoculation,and24hoursafter inoculation.Twenty fourhoursafterinoculation,theserumconcentrationsofTNFawerehigher(P<0.001)insowsthatdevelopedclinicalsignsofmastitiscomparedwiththose that remainedclinicallyhealthy. In themammaryglandbiopsyspecimens,analysisofTNFabyimmunohistochemistryrevealedthattheproductionofTNFα24hoursafterinoculationwashigher(p<0.05)insowsthatdevelopedclinicalsignsofmastitiscomparedwiththosethatremainedclinicallyhealthy.Reversetranscription-PCR(RT-PCR)analysisofthebiopsyspecimensshowedthatTNF-amRNAexpres-sion increased in the inoculatedmammaryglandsofbothsows thatdevelopedclinicalsignsofmastitis(P<0.01)andthosethatremainedclinicallyhealthy(P<0.05)24hoursafterinoculation,howevertherewerenodifferences inmRNAexpressionbetweenthetwogroupsofsows. Hereweshow that thehigherserumconcentrationsofTNF-α in sows that developed systemic clinical signs ofmastitiswere inaccordancewithahigherproteinproduction in themammarygland.However,suchadifferencebetweenhealthyandnothealthysowswasnotseenatthegeneexpressionlevel.Thesedataillustratesthedis-crepancybetweendifferentread-outswhenrelatingTNF-a toclinicaldisease.Key words:cytokine,mammarygland,mastitis,E.coliSpecies:swine

sm109. MODULATION OF PERIPHERAL DENDRITIC CELLS TOWARDS MUCOSA-TyPE DENDRITIC CELLS By ALL-

TRANS RETINOIC ACIDLESLIESAURER,KEnnETHMCCULLoUGH,ARTUR

SUMMERFIELDInstituteofVirologyandImmunoprophylaxis,Sensemattstrasse293,

CH-3147Mittelhäusern,Switzerland.Efficientinductionofmucosalimmunitymostoftenemploysnasal

ororalvaccinationwhileparenteralimmunizationgenerallyisineffectiveatgeneratingmucosalimmuneresponses.Thisrelatestotheuniqueabilityof residentmucosal dendritic cells (DC) to induce IgAswitch-ingandtoimprintmucosa-specifichomingreceptorsonlymphocytes.Basedonthewell-knownplasticityoftheDCsystem,thisstudysoughtto investigate whether peripheral DC could be modulated towards“mucosa-type”DCbytreatmentwithimmunomodulatoryandthereforepotentiallyadjuvant-likefactors.Here,weshowthatmonocyte-deriveddendriticcells(MoDC)pre-treatedwiththevitaminAderivativeall-transretinoicacid(RA) indeedacquiredseveralattributescharacteristicofmucosalDC:secretionofTGFβand IL-6and thecapacity to induceIgAresponsesandtheexpressionofmucosalhomingreceptorsinco-culturedlymphocytes.AdditionofaTGFβneutralizingAbsignificantlyinhibited inductionofa4β7 integrin,butnotofCCR9mRNAexpres-sionby theRA-treatedMoDC.Bothα4β7 integrinandCCR9mRNAexpression,butnotIgAproductionweresuppressedinthepresenceofaretinoicacidreceptorantagonist.Collectively,ourfindingsidentifiedanovelroleforRAasmucosalimmunemodulatortargetingDC.SucharolewouldbeconsistentwiththevicinityofmucosalDCtoRA-pro-ducingintestinalepithelialcellsandtheautocrineproductionofRAbyintestinalDC.Importantly,ourresultsfurtherdemonstratethatDCcanactasefficientcarriersofRAatleastinvitro.DCtargetingwithRAmaythus hold promise for promoting vaccine-induced mucosal immuneresponsesviatheparenteralrouteofimmunization.Key words:dendriticcells,mucosalimprinting,alltransretinoicacidSpecies:swine

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sm110. THE MUCOSAL IMMUNOGENICITy IN PIGS OF F4(K88) FIMBRIAE IS DECREASED By REDUCING ITS

POLyMERIC STABILITyFVERDonCK1,JDEMEyER1,JJoEnSUU2,MMUILU2,B

GoDDEERIS1,3,VnIKLAnDER-TEERI2,ECox11LaboratoryofImmunology,FacultyofVeterinaryMedicine,GhentUniversity,Belgium;2DepartmentofAppliedBiology,Universityof

Helsinki,Finland;3DepartmentofBiosystems,KatholiekeUniversiteitLeuven,Belgium

ETECarean importantcauseofdiarrhoea inmanandanimalsand there is need for the development of vaccines against thesepathogens.WehaveshownthatpurifiedfimbriaefromF4+ETECcaninduceaprotectiveF4-specificintestinalimmuneresponseagainstanoralchallengewithF4+ETECwhengivenoraltoF4receptor-positive(F4R+) piglets. Adhesion to the F4R is necessary for this mucosalimmunogenicity. Binding of other soluble antigens such as F18 fim-briae to enterocytes does not lead to a mucosal immune response,soF4must have interesting properties that can explain itsmucosalimmunogenicity.Oneof the importantdifferences lays in their struc-ture.F4fimbriaearemainlycomposedofadhesiveFaeGsubunitsandsomelessfrequentminorsubunits,whereas inF18theadhesinFedF is only present at the tip of the fimbriae and themajor subunit isnon-adhesive.Furthermore,wedemonstratedthatF4formsastablepolymericstructure,whereasFedFinF18hasaweakinteractionwiththemajorsubunitFedA.Toanalysetheimportanceoftheseobserva-tions,twopointmutationsweremadeinFaeGresultinginareducedstabilityandpolymericnatureofF4fimbriaebutretainingitscapacitytobindtotheF4R.ComparisonofwildtypeF4(wtF4)andmutantF4(mF4) inanoral immunizationexperiment inpigletsshowedthat theinductionofanintestinalF4-specificimmuneresponseismoreeffec-tivewithwtF4thanwiththemF4.withhighernumbersofF4-specificIgAantibodysecretingcells in the jejunaland ilealPeyer’spatches,mesentericlymphnodesandlaminapropriaofwtF4(meansof20,9,27and9per5.106monomorphonuclearcellsrespectively)thanofmF4immunizedanimals(3,4,16and4respectively).TheseresultsshowthatthestablepolymericappearanceoftheF4adhesinesisimportantforitsmucosalimmunogenicity.Key words: enterotoxigenic E. coli, F4 fimbriae, oral immunisation,intestinalimmunitySpecies:swine

sm111. COLOSTRUM FROM SOWS VACCINATED WITH AN INACTIVATED PCV2 VACCINE CONTAINS ANTIGEN

SPECIFIC LEUKOCyTESAGoUBIER1,FPIRAS2,MGnUDI3,LCHAPAT1,HELGARCH1,

FJoISEL3,CCHARREyRE4,SRICHARD1,LFoREST1,CAnDREonI1AnDVJUILLARD1

1MerialS.A.S.,DiscoveryResearch,254rueMarcelMérieux,69342Cedex07Lyon,France;2Sanofipasteur,Research,1541avenueMarcelMerieux,69280,Marcyl’Etoile;3MerialS.A.S.,TechnicalSupport,254rueMarcelMérieux,69342Cedex07Lyon,France,

4MerialS.A.S.,R&D,254rueMarcelMérieux,69342Cedex07Lyon,France

Inspecieshavinganepitheliochorialplacenta, likeswine, ithasbeen largely demonstrated that colostrum from vaccinated motherscontains antigen-specific maternal immunoglobulins which have animportantroleinthepassiveprotectionofnewbornanimals.However,ithasbeendemonstratedthatcolostrumalsocontainsTandBlympho-cytes.Little isknownabout thefunctionalityof thesecolostral leuko-cytesandtheirimportanceinprotectionofnewbornsagainstinfection.Theaimofthisstudywastoevaluateifsowvaccinationwouldleadtothepresencenotonlyofspecific immunoglobulinbutalsoofspecificimmunecellsincolostrum.Twogroupsofspecificpathogenfreesowswere included in this study. ThefirstgroupwasvaccinatedwithaninactivatedPCV2vaccine,whereastheothergroupdidnotreceivethevaccine.Asexpected,PCV2-specificIgG1andIgG2antibodiesweredetectedincolostrumofvaccinatedsowsbyELISA.Interestingly,theIgG1/IgG2ratiodifferedbetweenbloodandcolostrumimmunoglobulinswithalowerratiointhecolostrumthanintheblood.PhenotypicanalysisofcolostralleukocytesdemonstratedanincreaseinCD8+TcellsandadiminutionofCD4+Tcellsinvaccinatedsows’colostrumascompared

tonon-vaccinatedsows.Thesecolostralleukocyteswereassessedbyflowcytometryforintra-cellularcytokinesandbyIFNγ ELIspotsassayfortheirabilitytoproduceIFNγandTNF auponantigenicre-stimula-tion.ThesefunctionalexperimentsclearlyshowedthatcolostrumfromvaccinatedsowscontainedIFNγ-andTNFa-producingPCV2-specificCD4+andCD8+Tcells,whereasnoIFNγorTNFaproductioncouldbedetectedinthecolostrumofnon-vaccinatedsows.Ourdata,togetherwith previous reports demonstrating that leukocytes isolated fromcolostrumcanpass through the intestinalbarrierofnewbornpiglets,strongly suggest that maternal antigen-specific leukocytes may betransferredtothepigletsviathecolostrumandconstituteanotherlineofactivedefenseagainstinfectionsinneonatepiglets.Key words:Colostrum,PCV2,cellularimmuneresponse,inactivatedvaccine,pigSpecies:swine

sm112. CPG ODN ACTIVITy IN SWINE: FROM Ex VIVO TO IN VIVO ACTIVITy

AGoUBIER1,FPIRAS2,HELGARCH,SRICHARD1,LFoREST1,CAnDREonI1,JCAUDonnET1,RnoRDGREn3,VJUILLARD1*1MerialS.A.S.,DiscoveryResearch,254rueMarcelMérieux,69342Cedex07Lyon,France;2sanofipasteur,Research,1541avenue

MarcelMérieux,69280,Marcyl’Etoile,France;3MerialLimited,3239SatelliteBlvd.,Duluth,GA,USA

CpGODNssignalthroughTLR9andtriggeracascadeofeventsthatleadtoactivationofinnateandadaptiveimmuneresponses.Thepost-TLR9 signalling pathways and the biological activities resultingfromtheactivationdependontheclassofCpGODNused.Interestingly,ithasbeenshownthatsyntheticCpGODNsmayenhancethequalityof the immuneresponse tovaccination,aneffect thatcouldbe rein-forcedbyusingappropriateformulationagents.

This study aimed at selecting an effective combination of CpGODNandformulationagentswhichcouldbeusedtoenhancethepor-cineimmuneresponsetovaccination.

DifferentCpGODNsequenceswereanalyzed for theircapacitytoactivateporcinePBMCsexvivo,especiallytheirinductionofIFN-asecretionandpromotionofBcellproliferation.Twosequencesshowingpromisingfeatureswereretainedandassessedinvivoincombinationwithtwodifferentoil-in-wateremulsions.Varioussystemicparameterswere followed up at different times after injection, and the resultswereanalyzedasacombinationoflinearparametersinadiscrimina-toryanalysis.The resultsof thatanalysisallowedus todiscriminateboth sequences and to select one of the emulsions for its ability toincrease the biological activity of the CpGODNs tested. Themorepotent sequence, in combinationwith theoil emulsion showingbesteffects,wasfurtherassessedasadjuvantofarecombinantproteininpigs.WeshowedthatCpGODNenhancedIFN-γ+antigen-specificTcell responses. Interestingly, analysis of cellular immune responsesafterre-stimulationwithanoverlappingpeptidelibraryclearlyshowedthatCpGODNincreasedthenumberofTcellepitopesrecognizedbyantigenspecific-Tcells.ThisCpG-mediatedTcellepitopespreadingemphasizesthatCpGODNnotonlyimprovethemagnitudeofthecel-lularimmuneresponsebutalsoimpactonthequalityofthisresponse.Collectively,ourdatasuggestthatCpGODNcouldbeemployedasaneffectiveimmuno-adjuvanttooptimizeimmuneresponsesinducedbyvaccinationinpigs.Key words: CpG, swine, TLR9, IFNγ, immunomodulation, epitopespreadingSpecies:swine

sm113. A WEST NILE VIRUS (WNV) RECOMBINANT CANARyPOx VIRUS (ALVAC) VACCINE ELICITS WNV SPECIFIC NEUTRALISING ANTIBODIES AND T-CELL

MEDIATED IMMUNE RESPONSES IN THE HORSE: ABSENCE OF INHIBITING ANTI-VECTOR IMMUNITy FOLLOWING

REPEATED INJECTIONSHELGARCH1,JMMInKE1,JREHDER2,SRICHARD1,

CToULEMonDE3,SDInIC3,CAnDREonI1,JCAUDonnET1,RnoRDGREn4,VJUILLARD1*

91

1MerialSAS,DiscoveryResearch,254rueMarcelMérieux,69342Cedex07Lyon,France,2MerialLimited,115TranstechDrive,Athens,

GA,USA,3MerialSAS,ClinicalOperationsSaintVulbas,France,4MerialLimited,3239SatelliteBlvd.,Duluth,GA,USA

SuccessfulvaccinationagainstWestNileVirus(WNV)isthoughttorequire inductionofbothneutralizingantibodiesandcell-mediatedimmune responses. In this study,wehaveassessed theability of arecombinant ALVAC®-WNV vaccine (RECOMBITEK WNV) to elicitneutralizing antibodies and virus specific T cell-mediated immuneresponsesinhorses.Inaddition,weexaminedwhetherpriorexposuretoALVAC®-WNVvaccinewould inhibitBandTcell specific immuneresponses against the transgene product upon subsequent boosterimmunizationswiththesamevaccine.Theresultsdemonstratedthatthe recombinantALVAC-WNV vaccine induced neutralising antibod-iesandprM/E insert-specific IFNγ+Tcell responsesagainstWNV invaccinated horses. Prior exposure toALVAC®-WNV vaccine did notimpair the ability of horses to respond to two subsequent boosterinjectionswith thesamevaccine,althoughanti-vector-specificTandBcell responseswere induced invaccinatedhorses.Weshowed inthisstudythatALVACvectorallowsforastrongimmunogenicityoftherecombinantantigenintheabsenceofaninhibitoryanti-vectorimmuneresponseevenaftermultipleinjections.Inthisregard,ALVACvectorssuccessfullycombinethesafetyofaninactivatedvaccinewiththeeffi-cacyofamodifiedlivevaccine.

sm114. CD134+ T AND B LyMPHOCyTES FROM EFFECTOR SITES OF THE UPPER GASTROINTESTINAL TRACT

CONSTITUTE A SITE OF ACTIVE REPLICATION OF FIV IN CHRONICALLy INFECTED CATS

HELGARCH1,AGoUBIER1,HPoULET1,yMUELLE1,SRICHARD1,LFoREST1,LCHAPAT1,CAnDREonI1,VJUILLARD11MerialS.A.S.,DiscoveryResearch,254rueMarcelMérieux,69342

Cedex07Lyon,FranceFelineImmunodeficiencyVirus(FIV)isamajorpathogenofcats,

responsible for an acquired immuno-deficiency syndrome (AIDS),comparable to Human Immunodeficiency Virus (HIV)-associatedAIDSinhumans.Therefore,notonlyisFIVamajorissueinveterinarymedicine,butitalsoprovidesanaturalmodeltostudylentiviruspatho-genesis.EntryofHIVandFIVinhostcellsfollowsatwo-stepmodelin which binding to a primary receptor induces conformational rear-rangements exposing the co-receptor binding site.Both viruses useCXCR4asaco-receptorbutdiffer in theirprimaryreceptor(CD4forHIVandCD134forFIV)andintheirtropism,restrictedtoCD4+TcellsforHIVandbroaderforFIV.Gut-associatedlymphoidtissue(GALT)isareservoirforthevirusandasiteofCD4+Tcellsdepletioninchroni-callyHIV-infectedhumans,butitsroleinFIVpathogenesisneedstobeinvestigatedfurther.

Our objectiveswere to characterizeFIV-induced immunedisor-derandFIVtargetpopulationinGALTofchronicallyinfectedcats.WethereforedevelopedamethodtoisolateGALTimmunecellsandana-lyzed immunologicalchangesandvirusmultiplication throughdetec-tionofp24.WefirstobservedadepletionofmucosalCD4+TcellsandB cells.As inHIV infection, this depletion is preferentially observedin GALT effector sites (intestinal epithelium and lamina propria), ascompared to the inductive compartments (mesenteric lymph nodesandPeyer’sPatches).ByfocusingonCXCR4andCD134expressingcells,weobservedapreferentialdepletionofmucosalCD134+BcellsandCD4+CD134+Tcells,especiallyinthoseco-expressingCD134andCXCR4.Whetherthesedepletionswereassociatedwiththeinductionofapoptosiswasnotinvestigated.

To further characterize FIV replication in the gut of chronicallyinfectedcats,theexpressionofp24wasanalyzedbyflowcytometryinmucosalimmunecells.Weobservedthatp24positivecellswerewithintheCD134+cellpopulationandwerepreferentiallyobservedinGALTeffectorcompartments,aT-cellrichsite.

AltogetherourdatashowedthatmucosalCD4+TcellsandBcellsconstituteasiteofactivereplicationofthevirusinchronicallyinfectedcats. Our results emphasize the role of GALT in FIV pathogenesis,further reinforcing the homologies observed between FIV and HIVinfection.

Key words: FIV Gut-associated lymphoid tissue, CXCR4, CD134,CD134+Bcells,CD4+CD134+TcellsSpecies:Feline

sm115. CHARACTERIZATION OF INFLAMMATORy CELLS POPULATION IN MAMMARy TUMOR IN FEMALE DOGSAnACARoLInATRoMPIERISILVEIRA1,AnTonIoCARLoS

ALESSI1,RoSAnGELAZACARIASMACHADo1,FELIPEAUGUSToRUIZSUEIRo2

1UniversidadeEstadualPaulista(UNESP),Jaboticabal,SP,Brazil,2UniversidadedeFranca(UNIFRAN),Franca,SP,Brazil

actrompieri@yahoo.com.brMammaryneoplasiasarethemostfrequentlyobservedtumortype

in the femaledogs.The incidence is threefoldhigher in comparisontowomanbreasttumors.Caninemammaryneoplasiasareadequatemodels forbiologicaland therapeuticstudies.Studiesevaluating thespontaneous tumor type in domestic animals havebeen consideredagoodchoice toelucidate important issues related tooncologyandprobablymaybeusefulonstudiesapproachinghumanneoplasia.Dataobtainedfromhumantumorhavesuggestedthatrelationshipbetweentumorandhostcellsmaystimulateandalsoinhibitanti-tumorimmu-nity,according toheterogeneouscell infiltratecomposition,and thendifferenttumorcellsinteraction,whichinturnmayleadtovariableprog-nosis.Specimensusedinthisstudywereobtainedfrom1995to2006fromtheHistopathologyLaboratoryfilesattheVeterinaryPathologyDepartment.Suchsamplesweredividedintothreegroups:malignantmixed type tumor,adenocarcinomaandsolidcarcinoma.The immu-nohistochemical stainings were performed using the avidin-biotin-peroxidasecomplexmethod(ABC).Cellularinfiltratecharacterizationwasperformedbyusingmonoclonalantibodies:TCD3,BCD79aandHLA-DR,allantibodieswereproducedinmouseagainsthumananti-gen,andalsotheTGF-βcytokinepolyclonalantibodywereused.Theanti-CD4andanti-CD8,ThelperandTcitotoxiclymphocyteandprog-nosticmarkersKi-67,Estrogenreceptor(ER),Progesteronereceptor(PR)andp53arebeingstandardized.Firstresultshaddemonstratedthatcellularinflammatoryinfiltratehadpositivecellsmarkedwithanti-CD3,anti-CD79a,anti-HLA-DRandanti-TGF-βatdifferentproportionsondifferent tumorsstudied. Intensestainingwereobservedoncellsmarked byCD3 andHLA-DR, but weak stainingwere observed oncells marked by TGF-β and CD79a. Inflammatory cells distributionwereinterstitialpredominantly.Key words: Immunohistochemistry, Mammary neoplasias, dog,antibodySpecies:canine

sm116. IMMUNOHISTOCHEMICAL ExPRESSION OF TGF-β, LyMPHOCyTES T, B, MACROPHAGES, AND MHC CLASS II

OVER CANINE TRANSMISSIBLE VENERAL TUMORAnACARoLInATRoMPIERISILVEIRA1,MIRELATInUCCI

CoSTA1,DAnIELGERARDI1,JULIAnAMoRo11UniversidadeEstadualPaulista(UNESP),Jaboticabal,SP,Brazil,

actrompieri@yahoo.com.brCanine transmissible veneral tumor (TVT) is a experimentally

transplantabletumorandhasbeenusedasanexperimentalmodeloftherelationtumorversushost.Naturally,TVTistransmittedbysexualcontact,throughdedepositoftumoralcellsonthemucosascaredduringcoitus.ThisstudyhadthegoaltoevaluatetheinfiltratingTlymphocytes(CD3,CD4,CD8),B(CD79-a),macrophages(MAC-3),totheexpres-sionof themajorhistocompatibilitycomplexclass II (MHCII)andofTGF-βonTVT,bymeansofimmunohistochemistry(ABCmethod).Theexperimentalgroupswerecomposedoftumorsofnaturaloccurrence(Group1)(n=8),andofothersfromdogpupsthatdevelopedTVTaftertransplantationinthephasesoftumorprogression(n=8)(Group2a),latency(n=8)(Group2b),andregression(n=7)(Group2c).Thelym-phocytesTCD3+prevailedintheprogressionandregressionphasesincomparisontolatencyphaseandnatural.ThelymphocytesTCD4+,CD8+prevailedintheprogressionphasefollowedbynaturalTVTandshowedasmallerexpressivenessforTVTinregressionphase.MHCIIprevailedinnaturalTVTfollowedbyTVTinprogressionphaseandasmallerconcentrationintheregressionphase.LymphocytesBwerein

92

greateramountintheregressionphase,followedbynaturalTVT,andwith little expression in the progression phase. TGF-β was similarlyexpressedinthephasesofthetransplantedTVTinanyphasesofthetransplantedTVTandofthenaturalTVT.TVTcellsreactedtomacro-phagemarker,TGF-βandMHCII,however,theydidnotreacttoanyoftheleucocytemarkersused.Theresultsfromthisexperimentshowedthattherewasnoprevalenceofaspecificcelltypeinanyofthetrans-plantedTVT development phases. The reactivity of tumoral cells tohistiocytic/macrophagicmarkers(MAC-3)andtoMHCIIcorroboratesthehistiocyticoriginofTVT.TheexpressionofTGF-βcytokinebythetumorcellsandleucocytessuggeststhatthiscytokinemaybeoneoftheresponsibleelements for the immunoregulationobserved inTVT.Apparently,thetumoraltissuedonorinterferesonthetumorxhostrela-tion,atleastintermsoftheamountofinflammatorycellsinfiltratedinthetransplatedTVT.Key words: canine transmissible veneral tumor, infiltrating Tlymphocytes,macrophages,immunohistochemistrySpecies:canine

sm117. PERSISTENCE OF RECOMBINANT FOWLPOx VIRUSES IN CHICKEN TISSUES AND THE LOCAL IMMUNE

RESPONSE IELDAGHAyES1,2,LRoTHWELL2,MASKInnER3AnDP

KAISER2

1DepartmentofMicrobiologyandParasitology,FacultyofVeterinaryMedicine,Al-FatehUniversity,P.O.Box13662,Tripoli,Libya.;

2InstituteforAnimalHealth,Compton,BerkshireRG207NN,U.K.;3DeptofVirology,FacultyofMedicine,ImperialCollegeLondon,St.

Mary’sCampus,NorfolkPlace,London,W21PG,U.K.ibrahim1971@hotmail.com

Fowlpoxvirus(FPV)hasbeenunderdevelopmentasarecombi-nantvaccinevectorfor20years.Todate,surprisingly,veryfewdataexistonthepersistenceoffowlpoxvaccinevirusinchickentissues,ortheimmunecellsthatrespondtothevaccinationatthesiteofinocula-tion.Althoughbothhumoralandcellularmediatedimmunity(CMI)playapart in overall immunity against FPV, little is known regarding thecell-mediatedimmuneresponsestoFPVinfection.

Themainaimofthisstudywastomeasurepersistenceofrecom-binantfowlpoxvaccinevirusinskintissuesfollowingvaccination.TherecombinantFPVsdidnotpersistforlong.Viruswasdetectedinskintissueaftervaccinationatveryhighconcentrations2dayspost-vac-cination(dpv),toalesserdegreeat4dpvandwasalmostclearedfrom6dpv.Wealsoinvestigatedthekineticsofresponseofimmunecells

(macrophages,Bcells,CD4+andCD8+Tcells) in infiltratingthesiteofvaccination.Key words:Fowlpoxvirus,recombinantvaccineSpecies:avian

sm118. EFFECT OF TRANSFERRINS ON CHLAMyDOPHILA PSITTACI

DELPHInEBEECKMAnCARoLInEVAnDRooGEnBRoECKAnDDAISYVANROMPAY

GhentUniversity,DepartmentofMolecularBiotechnology,CoupureLinks653,9000Ghent,Belgium.

Daisy.Vanrompay@ugent.beObjectives: the effect of ovotransferrin (ovoTF), human lacto-

ferrin (hLF) and bovine lactoferrin (bLF) on the obligate intracellularGram-negativebacteriumChlamydophila(Cp.)psittaciwasevaluatedusingAfricanGreenMonkey(BGM)kidneyepithelialcellsandchickenmacrophages (HD11 cells). Subsequently, transferrins were used topreventaCp.psittaci infection inspecifiedpathogen free (SPF) tur-keys.Results:firstly,theeffectoftransferrinsonextracellularbacteriawasevaluated.Pre-incubationofCp.psittaciwith0.5to5mg/mlovoTFpriortoinfectingBGMcellssignificantlyloweredtheinfectionrate.Forbothlactoferrins,theinfectionratecouldonlybereducedwith5mg/ml,albeitnotsignificantlyascomparedtotheinfectionratecreatedbytheuntreatedbacteria.Secondly, transferrinswere tested for theirabilitytoinfluencebacterialadhesionandentryinHD11cells.Maximalnon-cytotoxicandnon-bactericidalconcentrationsof0.05mg/mlovoTFand0.5mg/mlhLFandbLFwereused.Overall,ovoTFwasmoreeffectivethanhumanandbovineLFininhibitingbacterialattachmentandcellentryandthelatterwasaccompaniedbyadose-dependentreductionofactin recruitmentat thebacterialentrysite.However,oncebacte-riahadenteredHD11cells, transferrinshadapparentlynoeffectonintracellularreplication.Thirdly,ovoTFbeingthemosteffectiveanti-Cp.psittaci transferrin invitro,wasevaluated forprotectingSPF turkeysagainstanaerogenicchlamydialinfection.Resultsoftheinvivostudywillbepresented.Conclusion:presentfindingssuggestapossiblerolefortransferrinsandespeciallyovoTF,inpreventingavianCp.psittaciinfections.Key words:transferrins,Chlamydophilapsittaci,respiratorydisease.Species:avian

AP119. GENERATION OF B CELLS IN BOVINE FETUSESAnnAEKMAn,AnTTIIIVAnAInEn

DepartmentofBasicVeterinarySciences,UniversityofHelsinki,Helsinki,Finland

antti.iivanainen@helsinki.fiThe human and murine bone marrow generates naïve B cells

throughoutpostnatallife.Inmanyotherspecies,however,Bcellontog-enyisapparentlylimitedtothefetalperiod,founderBcellsarefewinnumberandtherecombinationalrepertoireislimited.Atlatefetalandearly postnatal age, the preimmune repertoire is diversified throughpost-recombinationalmechanisms.TheB cell population is dramati-callyexpandedinvariousgutassociatedlymphoidtissues,i.e.inavianbursa,rabbitappendixandruminantilealPeyer’spatch(IPP).DespitethewellestablishedroleofruminantIPPinthegenerationofBcellmassandpreimmunerepertoire,verylittlespecificinformationisavailableontheontogenyof ruminantB cells.Wehave systematically assessedhematopoietictissuesofbovinefetusesfromdifferentdevelopmentalstages for signs of B lymphopoiesis. The material consisted of 29fetusesrangingfrom85to280daysofembryonicdevelopment.ByacombinationofimmunohistochemistryandquantitativeRT-PCR,asetofmarkerscharacterizingBcelldifferentiationwasanalysed.SurfaceIgMpositiveimmatureBcellsweredetectedduringthethirdtrimersterin spleen and in lymph nodes. CD21-positive, CD79a-positive andsurface IgM-lowor IgM-negativeserialsections inspleenand lymphnodessuggestthepresenceofpre-Bcellsduringthesecondtrimester.QuantitationofRAG-1andRAG-2expressionrevealed low levelsofrecombinationactivationgeneactivityinspleen,lymphnodesandthebonemarrowbutnot intheterminalsmall intestine.EventhoughwedidnotpurifytheIPPinthiswork,theresultssuggestthatmassiveBlymphoiddifferentiationcharacterizedbyRAGmediatedimmunoglobu-lin gene recombinationdoesnot takeplace in fetal bovine IPP. It ispossible,however,thatlowlevelsofBlymphoiddifferentiationoccursinfetalspleen,bonemarrowandlymphnodes.Key words:Bcells,ontogeny,CD79a,CD21,RAG-1,RAG-2Species:ruminants

AP120. TUMOR NECROSIS FACTOR RECEPTORS ON LyMPHOMA CELLS IN ENZOOTIC BOVINE LEUKOSIS

KoSUKEoKADA1,2,MAnABUIKEDA1,2,SAToRUKonnAI3,MISAoonUMA3,nAoTAKAISHIGURo1,MASAnoBUGoRyo1,21DepartmentofPathogenicVeterinaryScience,TheUnitedGraduate

SchoolofVeterinarySciences,GifuUniversity;2DepartmentofVeterinaryPathology,FacultyofAgriculture,IwateUniversity,and3LaboratoryofInfectiousDiseases,DepartmentofDiseaseControl,

GraduateSchoolofVeterinaryMedicine,HokkaidoUniversityEnzootic bovine leukosis (EBL) is a complex disease of cattle

associatedwithB lymphocytotropic retrovirus,bovine leukemiavirus(BLV).Sincethediseasetakesalongperiodtodevelop,itisbelievedthatBLVandthehostimmuneresponsesarecloselyrelated.Arecentstudyontherelationshipbetweentumornecrosisfactor(TNF)andBLVinfectionsuggestedthatTNFisakeyfactorinthediseaseprogression.ThepurposeofthisstudywastoreporttheexpressionsofTNFrecep-tors(TNF-Rs)ontumorcellsobtainedfromcattlewithEBL,discusstherelationshipamongTNF-Rsexpression,phenotypeoftumorcellsandcellularmorphology,andattempttoclarifythepathogenesisofEBL.

Obtained lymphomas in 29 animals with EBL were histopatho-logically classified into three types: Diffusemixed type (10 cases) ,diffuselargetype(9cases),anddiffuselargecleavedtype(10cases).Immunohistochemicallyusingamonoclonalantibodytoabovinelym-phocyte surface antigen, the lymphomas were classified into three

phenotypes:B-1a(CD5+/CD11b+)(13cases),B-1b(CD5-/CD11b+)(8cases)andB-2(conventionalB)(CD5-/CD11b-)(8cases).TheresultsofTNF-RsexpressionwerealltumorcellsinEBLuniformlyexpressedtheTNF-RII,butnotTNF-RI.

TNF-a activity ismediatedby two functionallydifferent cell sur-face receptors,TNF-RI (55kDa)andTNF-RII (75kDa).MostbiologicresponsesofTNF-α,suchastheinductionandsuppressionofapop-tosis,areconsideredtobemediatedbythesetwodifferentreceptors.In this study, EBL tumor cells expressed TNF-RII, but not TNF-RI,suggestingthatTNF-RIIcarriesoutimportantfunctionsinBLV-inducedleukemogenesis.

We conclude that TNF-Rs play the most important role in themalignantproliferationofBcellsandformationoflymphomasinEBL.However, in future studies, theobservationsofTNF-a expression inneoplastic tissues inEBLandTNF-Rsexpression inaleukemic (AL)andpersistentlymphocyotsis(PL)willbenecessarytoclarifythepro-cessbywhichEBLarisesfromBLVinfection.Key words:TumorNecrosisFactorReceptorI,TumorNecrosisFactorReceptorII,enzooticbovineleukosis,cellapoptosisSpecies:Ruminants

AP121. IDENTIFICATION OF BOVINE CyTOTOxIC T LyMPHOCyTE EPITOPES ON THE INTRACELLULAR

PARASITE THEILERIA PARVA SIMonPGRAHAM1,RoGERPELLé1,ETIEnnEPDE

VILLIERS1,MATyAMAGE1,DUnCAnMMWAnGI1,yoSHIKAZUHonDA1,SHIRLEyAELLIS2,nIALLDMACHUGH3,WIVAn

MoRRISon3,EVAnSLnTARACHA11InternationalLivestockResearchInstitute,P.O.Box30709,Nairobi00100,Kenya;2ImmunologyDivision,InstituteforAnimalHealth,Compton,RG207NN,UK;3DepartmentforAnimalHealthandWelfare,Royal(Dick)SchoolofVeterinaryStudies,Universityof

Edinburgh,EasterBush,Roslin,EH259RG,UKs.graham@vla.defra.gsi.gov.uk

Immunity against the bovine intracellular protozoan parasiteTheileriaparvahasbeenshowntobemediatedthroughthedestruc-tionofparasiteinfectedlymphocytesbyMHCclassIrestrictedCD8+CTL.SixparasiteproteinstargetedbyCTLfromT.parvaimmunecattlehavebeenidentifiedandraisedtheprospectofasubunitvaccine.Inthisstudy,weidentifiednineCTLantigenicpeptidesonthesesixpro-teinswithminimalpeptidelengthsofbetween9to11aminoacidslong.ThebovineMHC(BoLA)classIallelesthatrestricttheseepitopeswereidentified by functional screening of BoLA class I cDNA clones andshowedthatindividualallelesrestrictedfiveofthesepeptidesandtwoadditionalalleleswereeachfoundtorestricttwopeptides.ThesedatawereusedtoevaluatetheabilityofavailablebioinformaticssoftwaretosuccessfullyidentifybovineCTLepitopes.Ahighdegreeofagree-mentwasobservedbetweenthepositionsofepitopespredictedtobindhumanormouseMHCclassImoleculesandthoseactuallyrecognizedbybovineT cells.These results support theapplicationof availablesoftware to assist in the screening of CD8+ T cell epitopes recog-nised by veterinary species, although the efficiency of this processwillundoubtedlybe improvedasmorespecies-specificMHC-bindingmotifsaredeveloped.Key words:Theileriaparva,CTLantigenicpeptidesSpecies:ruminants

AP122. IDENTIFICATION OF CIRCULATING LINEAGE-NEGATIVE TyPE-I IFN PRODUCING PLASMACyTOID

DENDRITIC CELL-LIKE CELLS IN THE BOVINE BLOOD

5. ANTIGEN PRESENTATION AND DENDRITIC CELLS, EFFECTOR CELLS, B AND T CELLS, NK AND NK T CELLS, IMMUNOREGULATORy CELLS: POSTERS AP119-AP140

94

ASTALKER1,JBRoWnLIE1,SMIAH3,PGRIEBEL2,DWERLInG11RoyalVeterinaryCollege,Dept.ofPathologyandInfectious

Diseases,HawksheadLane,Hatfield,UK;2VeterinaryInfectiousDiseaseOrganization(VIDO),120-VeterinaryRoad,U.of

Saskatchewan,Saskatoon,Canada;3UniversityCollegeLondon,InstituteofOrthopaedics&MusculoskeletalScience,Stanmore,UK

Theclearanceofaviral infectionby thehostoftendependsonthehost-abilitytomountaninterferonresponse.Thus,severalviruseshaveevolvedtointerferewiththisearlyinnateimmuneresponse.Overtherecentyears,ithasbecomeevidentthatnaturalinterferonproduc-ingcells/plasmacytoiddendriticcells(pDC)areinthecentreofinducingsuchatypeIinterferonresponse.However,thiscelltypehasnotyetbeendefinedinthebovinesystem.AsubsetofimmunecellspotentiallyresemblingbovinepDChavebeenisolatedthroughnegativeselectionfromwholeblood.Thesecellsweresubsequentlycharacterisedforsur-faceantigenexpressionandtype-IIFNproduction.Thesecellsexpressdonotexpresslineage-specificsurfaceantigen,butdoexpressmark-ersexpressedbymyeloidaswelllymphoidcells,assimilarlydescribedforpDCintheporcine,humanandmurinesystem.Inaddition,activa-tion of these cells by typeA CpG ODN, but not type B or poly(I:C)induced a cell-specific IFNαβ response. In addition, these cells hadmRNAforTLRsnormallypresentwithpDCofotherspecies.SEMandTEMofthesecellsrevealedasizesimilarto lymphocyteswithsmalldendritesprotrudingfromthesurface.Thus,ourdatadescribeforthefirsttimethepresenceofacirculatingcell-typewithbovineperipheralbloodpotentialresemblingplasmacytoiddendriticcells.Key words:plasmacytoiddendriticcells,naturalinterferonSpecies:ruminants

AP123. TyPE I INTERFERON PRODUCING CELLS (IPC) CAN GAIN LyMPH NODES VIA THE AFFERENT LyMPHATIC

ROUTEFLoREnTInAPASCALE1,VAnESSAConTRERAS1,

ALExAnDRECoURBET1,MICHELBonnEAU2,STEFAnCHILMonCZyK1,MATHIEUEPARDAUD1,CLAUDIABEVILACqUA3,AnnE-MARIEBALAZUC4,ARTUR

SUMMERFIELD5,BéATRICERITEAU1,JAynEHoPE6,BERnARDCHARLEy1*,AnDISABELLESCHWARTZ-CoRnIL1*

1VirologieetImmunologieMoléculaires,UR892INRA,DomainedeVilvert,78352Jouy-en-JosasCedex,France2CentredeRechercheenImagerieInterventionnelle,DomainedeVilvert,78352Jouy-en-JosasCedex,France3PICT,DomainedeVilvert,78352Jouy-en-JosasCedex,France4Plate-formedecytométrie,InstitutPasteur,Paris5InstituteofVirologyandImmunoprophylaxis,Mittelhausern,Switzerland6InstituteforAnimalHealth,Compton,UnitedKingdom

*isabelle.schwartz@jouy.inra.frWhilemostdendriticcells(DC)enterlymphnodes(LN)bymigrat-

ingfromperipheraltissues,plasmacytoidDC(pDC),alsoreferredasinterferonproducingcells(IPC),areknowtogainLNdirectlyfromthebloodbycrossinghighendotheliumvenules.WedemonstrateherethatbonafideIPCaremigratinginafferentlymphintwolargemammals,sheepandmini-pigs.Insheep,lymphcellsproducedtypeIIFN/invivo/after type-ACpG oligonucleotide (ODN) injection and /in vitro/ afterstimulationwithtype-ACpG-ODNandenvelopedviruses.SheeplymphIPCwerefoundwithinanonBnonclassicalDC(cDC)minorsubset(<1%)expressingCD45RB.TheCD11cnegBnegCD45RBposcellsshowedhighlevelsofTLR-3,7and9mRNA,theydisplayedconstitutiveIRF-7mRNA expression, they presented a plasmacytoid morphology, theyinducedIFN-γproductioninallogeneicCD4Tcellsandtheydifferenti-atedintoDC-likecellsunderviralstimulation.Furthermoreinmini-pig,aCD4posSIRPpossubsetinafferentlymphcells,correspondingtopDChomologues,producedtypeIIFNaftertype-ACpG-ODNtriggering.

Altogether,thisworkdemonstratesthatcellswithIPC/pDCchar-acteristicsmigrateinafferentlymph,thusbeingideallylocatedtopos-sibly transportantigen fromtissueandrapidly interactwith incomingcDCinlymphnodes.Key words: plasmacytoid dendritic cells; CpG oligonucleotide; typeIIFNSpecies:ruminants

AP124. CHARACTERIZATION OF BOVINE REGULATORy CELLS

AADHoEK*1,VICToRRUTTEn1,JoLAnDAKooL1,ILDIKoVAnRHIJn1,AnDKoETS2

1DepartmentsofInfectiousDiseasesandImmunologyandFarmAnimalHealth;2FacultyofVeterinaryMedicine,UtrechtUniversity,

TheNetherlands.a.hoek@vet.uu.nl

Regulatory cells, especially the natural regulatoryT cell (CD4+/CD25+High), are regarded as essential for controlling the immunereactivity.Cowssuffering fromclinical paratuberculosis, an intestinalinflammatorydiseasecausedby infectionwithMycobacteriumaviumsubspeciesparatuberculosis (MAP)suffer froma lossofTcell func-tion,andshowincreasedIL10production,whichmay indicatearolefor (increased) regulatoryT cell activity in the pathogenesis duringprogressivestagesofdisease.

Toenableidentificationofregulatory(T)cells,cell(sub)populationswere isolated fromperipheral bloodand laminapropria cell suspen-sions of cowswith known paratuberculosis status by flowcytometricsorting.ThesesubpopulationswerecharacterizedbyquantitativerealtimePCR (qRT-PCR), intracellular staining (ICS) specific for bovineFoxp3, IL-10,TGF-β and IFN-γ. Inaddition IL-10, and IFN-γElispotassayswere performed. Lymfocyte stimulation assays (LST) and invitroco-cultureassays(IVCA)incombinationwithPBMCdepletedforspecificsubpopulations,blockingbymAbneutralizingbovineIL-10orγdTCRandCFSEstainingwereapplied to test cellular functional-ityof the isolatedpopulations.ResultsofqRT-PCR,Elispotand ICSassaysperformedonpurifiedbovinecell(sub)populationsmeasuringbovineFoxp3,IL-10,TGF-βandIFN-γmRNA/protein,wereusedtoclassifypotential regulatorycells.Theseshowed tobeCD3+Tcells,CD4+/CD25+High/Low T cells, CD14+monocytes and γd T cell subsets.Intracellularstainingbycross reactiveanti-Foxp3mAb incowCD4+/CD25+High cellswas found.CowCD4+/CD25+HighTcells isolated fromperipheralbloodorlaminapropriashowednosuppressiveactivityinabovine invitroco-cultureassay,whilehumanCD4+/CD25+HighTcellstestedinaparallelinvitroco-cultureassaydisplayedstrongfunctionalsuppressioninadosedependentmanner.

BasedonphenotypingandFoxp3measurementsbovineCD4+/CD25+High population was considered comparable to human CD4+/CD25+HighnaturalTreg,howeverthebovinecellsdidnotdisplayfunc-tionalsuppressioninan invitroco-cultureassay.FurthermorebasedonIL-10measurementscowCD14+(monocytes)andγdTcellsubsetscouldbeclassifiedaspotentialregulatorycellsandpreliminaryinvitroco-culture assays showed indications for functional suppression bythesecellsindicatingthatimmuneregulationincattlemaybeofadif-ferentnatureascomparedtohumanandrodentsystems.

This investigation received financial support from the DutchTechnologyFoundationSTW/NWO.Key words: T regulatory cells, Foxp3, IL-10, TGF-β, IFN-γ, IL-10,IFN-γSpecies:ruminants

AP125. DRAINING LyMPH NODES APCS IN THE RESISTANCE OF GOATS TO AMBLyOMMA CAJENNENSE

FABRICIUS 1787 NyMPHSGERMonTEIRo1,2,GHBECHARA2,AMFRAnZIn3,IKDE

MIRAnDASAnToS31DepartamentodeParaclínicas,FV-UEM,Maputo,

Moçambique;2DepartamentodePatologiaVeterinária,FCAV-UNESP,Jaboticabal-SP,Brazil;3LaboratóriodeImunologia,FMRP-USP,

RibeirãoPreto-SP,Brazil.Preliminaryresultsofthelaboratoryshowedthatgoatsacquirepar-

tialresistanceagainstnymphsofthelone-startickAmblyommacajen-nenseafter repeated infestations.On theotherhand it iswell-knownthatantigen-presentingcellsdevelopanimportantroleintheimmunereaction to tick infestation.Themostpotentwell-definedantigen-pre-sentingcells forThcellsaredendriticcells,mononuclearphagocytesandB-lymphocytes.Langerhancellsareoneofthefirsttobeexposedtotickimmunogensinskinforwhichtheymigratetodraininglymphnodes.IntheparacorticalareaoflymphnodesLangerhancellstransforminto

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dendriticcellsandfunctionthereasantigen-presentingcellsforT-cells.Immunohistochemistryanalysisofdraininglymphnodesofgoatsrepeat-edlyinfestedwiththeixodidtickA.cajennensewasperformedtosearchforantigen-presentingcells.Threegoatsagedsixmonthofbothsexeswereusedthroughouttheexperiment.TwoofthemwereinfestedthricewithA.cajennense nymphsat30days intervals.The thirdgoatwasusedascontrolwithnoinfestation.Pre-scapularlymphnodesdrainingthetickinfestationsiteswerecollected15daysafterboththefirstandthethirdinfestations.Lymphnodesofthenon-infestedgoatwereusedascontrol.TheimmunohistochemicalanalysiswasperformedinlymphnodescryostatsectionsbyusingtheABCmethod. Itwasusedthreecellular surface markers, Cd11b (CC126), CD11c (CC21) and CD21(BAC153A)formacrophages,dendriticcellsandBcells,respectively.Pre-scapular lymphnodesfromtick infestedgoatsshowedincreasedCD11b,CD11candCD21cellularinfiltrationwhencomparedtothosefromnon-infestedanimals.However,therewerenosignificantchangesinthenumberofcellsinfiltratinglymphnodesofgoatsinfestedonceorthrice.Itisconcludedthatantigen-presentingcellsmayplayanimpor-tantroleinacquiredresistancemechanismofgoatsagainstnymphsofthelone-startickA.cajennense.Key words: Amblyomma cajennense, goats, lymph nodes,immunohistochemistry,APCs.Species:ruminants

AP126. QUANTIFyING THE CONTRIBUTION OF BOLA-DRβ3 TO IMMUNE RESPONSES IN A CATTLE CROSS-POPULATIONREBECCABAxTER1,nICoLAHASTInGS1,yLAW1,ELIZABETHJ

GLASS11RoslinInstitute,Roslin,Midlothian,EH549PS

Oneapproachtoreducingtheimpactthatpathogenshaveonlive-stockhealthandwelfareistobreedforenhanceddiseaseresistance.Suitableselectablemarkersmaybe found ingeneswithin theMajorHistocompatibilityComplex(MHC),inparticularthehighlypolymorphicMHCclassIIDRgenes.Thepolymorphismsresideinexon2ofthedrβ3genewhichencodesthepeptidebindingcleft.Asthepolymor-phismscanaltertheconformationofthePBC,theyaffectthebindingefficacyofthepathogen-derivedpeptides,andconsequentlytheleveloftheensuingimmuneresponse.Howeverotherregionsofthegenomearelikelytoaccountforasignificantproportionofthevariabilityseenindiseaseresistance. Inorder toassess therelativecontributionoftheMHCtodiseaseresistanceandimmuneresponsivenessincattle,wehavemeasureda rangeof immune traits ina500F2Charolais-Holsteincrosspopulation.Wehaveimprovedasequencebasedtyping(SBT)methodtoreliablydeterminebovineMHC(BoLa)classIIdrβ3exon2polymorphisms,andhavetypedallof thesecattle. Although104alleleshavebeenidentifiedforBoLA,only22differentalleleswerepresent in this population. Preliminary evidence from 117 animalshas identified significant associations between some of the immunetraitsandBoLaclassIIdrβ3types,andfurtheranalysisison-going.Thispopulationhasalsobeengenotypedfor139microsatellitemark-ersacrossthegenome.Thisinformationwillenableustodeterminethe relative importanceof theBoLadrβ3genescompared tootherregionsofthegenomeindeterminingimmuneresponsiveness.Key words:BoLA,diseaseresistanceSpecies:ruminants

AP127. IN VITRo MAINTENANCE OF BOVINE NK CELLS By CULTURE WITH THEILERIA-INFECTED LyMPHOCyTES

TConnELLEy,nMACHUGH1,ASToRSET2,WIMoRRISon11DivisionofClinicalVeterinarySciences,UniversityofEdinburgh,Edinburgh,United-Kingdom;2Dept.ofFoodSafetyandInfectionBiology,NorwegianSchoolofVeterinaryScience,Oslo,Norway.

WereportamethodofgeneratingandmaintaininginvitrobovineNKclonesusingco-cultureofPBMCfromnaïvecattlewithautologousTheileria-infectedlymphocytes.FollowingdepletionofCD4+andγd T-cells fromculturesofnaïvePBMCstimulatedwitheitherT.parvaorT.annulata-infectedlymphocytes,NK-enrichedpopulationshavebeenidentifiedfromwhichindividualNKclonescouldbederived.NineNKclonesgeneratedfromoneanimalhavebeenmaintainedincultureinIL-2supplementedmediaforaperiodof2monthsbyre-stimulationwith

autologousT.annulata-infectedlymphocytesevery10-14days.ThesecellshaveaCD2+,CD3-,CD8lo,NKp46+,CD16+phenotypeconsistentwithpreviousdescriptionsofbovineNKcells.Initialobservationsindi-cate thatbyday10post-stimulation there isa4-10 fold increase inNKcellnumbers.Studies toexamine themechanismsbywhichco-culturewithTheileria-infectedcellsmaintainsbovineNKproliferationinvitroandtodefinethefunctionoftheNKclonesobtainedarecurrentlybeingundertaken.TheabilitytopropagatebovineNKclonesinvitroforprolongedperiodsandachievesignificantexpansionofcellnumbersprovidesavaluablemethodwhichwillfacilitateindepthcharacterisa-tionofbovineNKcellsattheclonallevel.Key words:NK,bovine,cultureSpecies:ruminants

AP128. ExPRESSION OF TOLL-LIKE RECEPTOR 2 (TLR2) IN PORCINE LEUKOCyTE SUBSETS AND TISSUES

BELEnALVAREZ1,ConCEPCIónREVILLA1,nIEVESDoMEnECH2,CARLoSPEREZ1,PALoMAMARTÍnEZDELARIVA1,FERnAnDoALonSo1,AnGELEZqUERRA1,JAVIER

DoMÍnGUEZ11DptodeBiotecnología,INIA,Madrid,Spain;2UnidaddeInvestigación,HospitalJuanCanalejo,ACoruña,Spain

juncal@inia.esToll-likereceptors(TLR)areagroupofpatternrecognitionrecep-

torsthatplayacrucialroleininnateimmunity.TLR2recognizesavarietyofmicrobial components includingbacterial lipopeptides, lipoteichoicacid, lipoarabinomannan,peptidoglycanandzymosan,leadingtothedevelopmentofinflammatoryandimmuneresponses.Tocharacterizetheexpressionand functionalpropertiesofporcineTLR2,we raisedapanelofmAbagainst thismolecule.Mouse3T3cell transfectantsexpressingpTLR2wereusedforimmunizationofmice.Thespecificityof theseantibodieswasconfirmedby their reactivitywithCHOcellstransfected with pTLR2 but not with pTLR4 or with non-transfectedcells.UsingoneofthesemAbs,named1H11,TLR2wasfoundoncellsoftheinnateimmunesystem,includingmonocytes,macrophagesandgranulocytes, but not on peripheral blood lymphocytes. Staining oftissuesectionsshowedthatTLR2isalsoexpressedonepithelialcellsliningthetracheobronchialandintestinaltracts,bileductsintheliverandrenaltubulesandonthebasallayeroftheepidermis.Thisdistribu-tionisconsistentwithasurveillancefunctionatentrysitesallowingforearlydetectionofmicrobialinvasion.Key words:Toll-likereceptors,monoclonalantibodiesSpecies:swine

AP129. CD2 AND CD21 ExPRESSION CAN BE USED TO DESCRIBE MATURATION PATHWAy FOR PORCINE B CELLS

MAREKŠInKoRA1,JAnAŠInKoRoVá2,JoHnEBUTLER31AcademyofScienceoftheCzechRepublic,InstituteofMicrobiology,

SectorofImmunologyandGnotobiology,NovýHrádek,CzechRepublic;2A.R.T.,NovýHrádek,CzechRepublic;3UniversityofIowa,

DepartmentofMicrobiology,IowaCity,IA,USA,marek.sinkora@worldonline.cz

Analysis of CD2 and CD21 expression on porcine B cells dur-ing ontogeny showed that the vast majority of µHC+ B cells in thefetusareCD2+CD21+.ThiscontrastswiththepostnatalperiodwheremanyBcellsfromdifferentlymphoidorgansareCD2—and/orCD21—.Moreover,invivostudiesofgerm-freepigletsinfectedwithPRRSV,SIVorPCV-2developedvariousproportionsofCD2—and/orCD21—Bcellsalwaysgreaterthansham-infectedcontrols.Thesefindingspromptedin vitro studies using different CD2/CD21 subpopulations of B cellssortedaspurepopulationsbyflowcytometry.ThesestudiessuggestedthatCD2+CD21+BcellsrepresentinexperiencedmatureBcells.Thesemay proliferate and down-regulate CD21 expression spontaneouslyduringcultureorafter invitroactivation.TheresultantCD2+CD21—BcellscansubsequentlygeneratelargeproliferatingμHC—plasmablastsandsmallnon-dividingµHC—plasmacells.Sortingexperimentrevealedthatoncedown-regulated,CD21moleculecannotbere-expressedandtherefore isvaluableasadevelopmentalmarker forBcell inswine.Ontheotherhand,CD2,whichactsasfunctionaladhesinduringinter-cellularBcells interactions, isdown-regulatedbycell-to-cellcontact.

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OnceBcells recover fromsuch intereactions,CD2expressiononBcellsisre-establishedinashortperiodoftime.DuringtheCD2—CD21+stage,Bcells cannotbeactivatedandcannotgenerateproliferatingµHC—plasmablastornon-dividingµHC—plasmacells.However,oncetheyre-expressCD2andbecomeCD2+CD21+Bcells,atleastasub-population can progress to the CD2+ CD21—stage of differentiation.ThisworkwassupportedGrant524/07/0087and523/07/0088fromtheGrantAgencyoftheCzechRepublicandbyGrantA5020303fromtheGrantAgencyoftheAcademyofSciencesoftheCzechRepublic.Key words: B cells, development, maturation, differentiation,ontogeny,Species:swine

AP130. THE FINAL CUT: CD1+CD2+CD8— γδ THyMOCyTES ARE PRECURSORS OF ALL PERIPHERAL γδ T CELLS

MAREKŠInKoRA1,JAnAŠInKoRoVá2,WoLFGAnGHoLTMEIER3

1AcademyofScienceoftheCzechRepublic,InstituteofMicrobiology,SectorofImmunologyandGnotobiology,NovýHrádek,Czech

Republic;2A.R.T.,NovýHrádek,CzechRepublic;3Johann-WolfgangGoetheUniversitätFrankfurt,FrankfurtamMain,Germany,

marek.sinkora@worldonline.czUsingflowcytometrysorting,invitrocultures,analysisofcellcycle

andlymphocyte-specifictranscriptsandinvivostudiesofindividualγdthymocyte subsets during ontogeny, we have shown that CD1 andCD45RC expression on porcine γd thymocytes define subpopula-tions with developmentally dependent changes in their phenotype.Proliferating CD2+CD8—CD1+CD45RC— γd thymocytes are the com-monprecursorgivingrisetoallγdTcellsfoundintheperiphery.TheseprecursorsdifferentiateintoCD2+CD8aa+,CD2+CD8—andCD2—CD8—

γd thymocytes, which subsequently mature by loss of CD1 and byeventual gain ofCD45RCexpression. γd thymocytes that lose CD1expression canbeexported to theperiphery so the finalmaturationstepfromCD1—CD45RC—intoCD1—CD45RC+cellsmayoccurthere.ExtrathymicmaturationofCD2+CD8—intoCD2+CD8aa+Tcellwasalsoshowntooccuruponactivation.However,theoriginofCD2+CD8αα+T-cellscanbemonitoredbyMHCclassIIexpressionsincethosewhichareMHC-II— appear to originate in the thymus, while MHC-II+ cellsariseintheperipheryafterstimulationoftheirCD2+CD8—counterparts.These results indicate that the maturation of γd thymocytes occursaftersuccessfulexpressionofTCRγdandthatalthoughγdTcellsdonot followαβTcellprogenitorsthroughMHCdependentpositiveandnegativeselection,αβandγdcelllineagesrequireaboutthesametimeforfullmaturation.ApartfromtheabovementionedCD4—γdTcells,asmall fractionofγd thymocytesareCD4+CD8αβ+CD1+.ThesecellshavenocounterpartintheperipheryandwereshowntobeatransientandindependentpopulationofthymocytesthatareabletoextinguishtheirTCRγdexpressionanddifferentiatealong theaβTcell lineageprogram.

This work was supported Grant 524/07/0087 and 523/07/0088fromtheGrantAgencyoftheCzechRepublicandbyGrantA5020303from the Grant Agency of the Academy of Sciences of the CzechRepublic.Key words: γd T cells, development, maturation, differentiation,ontogeny,Species:swine

AP131. CHARACTERIZATION OF CD79α+CD21– B CELLS IN SWINE

WILHELMGERnER,SABInEHAMMER,ToBIASKäSER,nICoLADIny,MAŠAPInTARIC,ARMInSAALMÜLLER

ClinicalImmunology,UniversityofVeterinaryMedicineVienna,Vienna,Austria

wilhelm.gerner@vu-wien.ac.atTheidentificationofBcellsbytheirexpressionofCD21isacom-

monapproachinswineimmunology.However,inmicetheexpressionof CD21 is restricted to mature and late transitional (T2) B cells. Incontrast,theexpressionofCD79aandCD79βoccursalreadyfromthepro-Bcellstageonwards.BycombiningantibodiesagainstCD79aandCD21inflowcytometrywithPBMCfromadultandsixmontholdswine

weidentifiedapopulationofCD79a+CD21–cellswhichaccountedfor3to8%oftotallymphocytes.Withthisphenotype,thecellsresembleearlytransitional(T1)Bcellswhichshouldbeinregardtotheirimmu-noglobulinclassIgD–andIgMhigh.ForfurtherstudiesonthisminorB-cellsubsetweisolatedCD79a+CD21–cellsbyfluorescenceactivatedcellsortingandperformedreversetranscriptionPCR,specificallytargetingporcine IgD transcripts. Interestingly, the CD79a+CD21– subset con-tainedcellspositiveforIgDtranscripts.Also,analysesofIgMsurfaceexpressionbyflowcytometry indicatedIgM-expressingBcellswithinthat subpopulationwithmoderate IgMsurfaceexpression.However,there was no difference in the IgM expression pattern between thetwo CD21-defined B cell subpopulations. Further analyses of theCD79a+CD21– subpopulation within PBMC showed that these cellsweremainlynegativeforCD5butthemajorityexpressedCD2(∼70%)withahighantigendensitycomparabletotheCD2antigenexpressiononTlymphocytes.

By analysing further lymphatic organs of adult and six monthold swine we detected only low frequencies (3% and below) ofCD79+CD21–cellsinspleen,lymphnodesandbonemarrow.

Takentogether,thesedataindicatethattheidentifiedpopulationofCD79+CD21–cellsinswinedespitethisphenotype,whichpointstoanimmaturedevelopmentalstage,showanumberofphenotypicaldif-ferencestotransitional-1Bcellsdescribedformice.Thisclearlyraisesthequestionofthefunctionofthesecellswhichwillbeaddressedinfurtherstudies.Key words:Bcells,swine,CD79a,IgD,IgMSpecies:swine

AP132. SyNERGISTIC EFFECTS OF IL-2, IL-12 AND IL-18 ON CyTOLITIC ACTIVITy AND IFN-γ PRODUCTION OF PORCINE

NATURAL KILLER CELLSMaŠaPIntarIČ,WILHELMGERnER,ARMInSAALMÜLLERClinicalImmunology,UniversityofVeterinaryMedicineVienna,

Vienna,Austriaarmin.saalmueller@vu-wien.ac.at

Naturalkiller(NK)cellsareoneofthemaincellularcomponentsoftheinnateimmunesystem.Theyplayanimportantroleintheimmuneresponseagainstinfectionsaswellastumourcellsandthereforehavetwomajorproperties:productionofimmuneregulatorycytokinesandchemokinesaswellascytolyticdestructionofparticulartargetcells.

TheexistenceofNKcells inswine iswellknownaswellasthephenotype of resting NK cells, but their response following activa-tionbycytokines isstill poorlyunderstood.Therefore,we tested theinfluenceoftheimmuneregulatorycytokinesIL-2,IL-12andIL-18oncytolyticactivity,phenotype,IFN-γproductionandtheaccumulationofperforinincytoplasmofperipheralbloodmononuclearcells(PBMC)aswellaspurifiedNKcells.NKcellswereenrichedfromPBMCusingamagneticcellseparation(MACS)strategywithmonoclonalantibodiesagainstCD3,CD21andSWC3,therebyremovingT-,B-andmyeloidcells.Respectivefractionswereusedinflowcytometry(FCM)basedcytolyticassayswiththehumantumourcelllineK562astarget.Afterstimulationwith thecytokinesdescribedabove, theNKcellenrichedCD3–CD21–SWC3– fraction showed an evident increase in the cyto-lyticactivitycomparedtoPBMC.ThisenhancedcytolyticactivitywasaccompaniedbyastrongenrichmentofIFN-γproducingcellswhenacombinationofallthreecytokines(IL-2/IL-12/IL-18)wasused,asdeter-minedinELISPOTassaysandintracellularstainingofIFN-γinFCM.Also,thecombinationofthesethreecytokinesledtoanaccumulationofperforininthecytoplasmandanup-regulationofCD25comparedtocontrolculturesincubatedinmediumwithoutcytokines.

Theexperimentsperformedso farclearly indicateastimulatoryroleandstrongsynergisticeffectsoftheinvestigatedcytokinesintheactivationofporcineNKcellsinvitro,inducingIFN-γ,perforinproduc-tionandcytotoxicityagainst targetcells.FurtherstudieswilladdresstheroleofNKcellsandlymphokineactivatedkillercellsagainstvirus-infectedtargetcellsandinvivoduringviralinfections.Key words:NaturalKillercells,IFN-γ,perforin,IL-2,IL-12,IL-18Species:swine

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AP133. CLASSICAL SWINE FEVER VIRUS INDUCES DENDRITIC CELL ACTIVATION IN BLOOD AND SECONDARy

LyMPHOID ORGANS AGnèSJAMIn1,STéPHAnEGoRIn1,RoLAnDCARIoLET2,MARIE-FRéDéRIqUELEPoTIER1,GAëLLEKUnTZ-SIMon11AFSSA-LERAPP,SwineVirologyImmunologyUnit,Ploufragan,France;2AFSSA-LERAPP,SectionofHealthyPigProductionand

Experimentation,Ploufragan,Franceg.kuntz.simon@afssa.fr

Classical swine fever virus (CSFV)causes indirect leucopoeniaanddisruptionof invitroTcell stimulationcapacity. It canefficientlyreplicateinmonocyte-deriveddendriticcellsandblood-isolatednaturalinterferonproducingcellswithout interferingwith their immune reac-tivity (1, 2), but noevidencehasbeen foundof a roleof stimulateddendritic cells (DC) in immunosuppression. Intending to understandinteractionsbetweenCSFVandDCin infectedpigs,weinvestigatedactivationofconventionalDC(cDC)andplasmacytoidDC(pDC),twoDCsubstetswepreviouslycharacterizedinswinesecondarylymphoidorgansandbloodat thesteadystate (3).Changes in thesepopula-tionswerestudiedintheearlytimecoursepost-inoculation, togetherwithviralcomponentsdisseminationinhostandcytokineproductioninserum.Whereas CD11R1+CD172a+ cDC frequencies were markedlyreducedinbloodandspleenafterinfection,analysisofCD4+CD172a+pDCnumbers revealeda rapid turn-over of thisDC type in tissues.Both DC subsetsmatured and were activated, as demonstrated bydown-regulationofCD1a,up-regulationoftheco-stimulationmoleculeCD80/86andexpressionof cytokines. cDCmainlyexpressed tumornecrosis factor alpha (TNF-α) and interleukin (IL)-10, whereas pDCproducedalphainterferon(IFN-α)andIL-12.IFN-αandTNF-αproduc-tionsrevealedenhancementofinnateanti-viralimmuneresponses.IL-10expressionindicatedinitiationofhumoralresponse,alsoevidencedbydetectionofantigenactivatedBlymphocytesintonsilT-cellareasat72h,subsequentlytothetransienttranslocationoftheviralE2proteinwithingerminalcentresat48h.IL-12expression,aswellastransientdetectionofIL-18andIFN-γinserum,wouldreflectinitiationofcellularresponses.However,theuncommonlyhighlevelsofIFN-αandTNF-αproducedbyDCandmeasuredinserumwouldplayaroleindisrup-tionofimmunesystemcells,eitherinducingapoptosisorimpairingDCfunctionalities.

(1) CarrascoC.P.,RigdenR.C.,VincentI.E.,BalmelliC.,CeppiM.,BauhoferO.,TacheV.,HjertnerB.,McNeillyF.,vanGennipH.G.,McCulloughK.C.,SummerfieldA.(2004)Interactionofclassicalswinefeverviruswithdendriticcells.J.Gen.Virol.85,1633-1641.

(2)BalmelliC.,VincentI.E.,RauH.,Guzylack-PiriouL.,McCulloughK.,SummerfieldA. (2005)FcgammaRII-dependent sensitisationofnaturalinterferon-producingcellsforviralinfectionandinterferon-alpharesponses.Eur.J.Immunol.35,2406-2415.

(3) JaminA.,GorinS., LePotierM.-F., Kuntz-SimonG. (2006)Characterization of conventional and plasmacytoid dendritic cellsin swine secondary lymphoid organs and blood. Vet. Immunol.Immunopathol.114,224-237.Key words:Classicalswinefevervirus,dendriticcells,plasmacytoiddendriticcells,TNF-α,IFN-a,IL-10,IL-12Species:swine

AP134. PRRSV MODULATE CyTOKINE RESPONSE OF PORCINE DENDRITIC CELLS AND COMPROMISE THE

ACTIVATION OF T CELLS ESILVA-CAMPA,LFLoRES-MEnDoZA,MRESénDIZ-SAnDoVA,

JHERnánDEZ*1LaboratoriodeInmunología,CIAD,A.C.Hermosillo,Sonora,Mexico.

Porcinereproductiveandrespiratorysyndrome(PRRS)infectionischaracterizedbyanunconventionalimmuneresponse,ourhypoth-esis is that thePRRSvs.dendriticcells (DCs) interactioncontributeto this typeof response.Previous reportshaveshowed thatPRRSvinfectandreplicateon immature(iDCs)andmatureDCs(mDCs). InthisworkweusediDCandskin-derivedDCstoinvestigatethemodula-toryproprietiesofPRRSv.iDCsweregeneratedbyculturingadherentcellswith IL-4 andGM-CSF for 5 days, and skin-derivedDCswereobtainedfromthesupernatantofculturingskinfor24h.Theexpres-

sion of C80/86, MHC-II, CD-172a, CD1 y CD14 was analyzed byFACS.DCswereinfectedfor1hatm.o.i.of0.1,washedandcultur-ing for 24 h.Theexpression ofmRNA IFN-a, IL-10 and IL-12wereanalyzed by qPCR.Apoptosis was evaluated with FITC-conjugatedAnnexinVandpropidium iodide,andendocytosisandphagocytosiswas evaluated with FITC-latex particles and –dextran, respectively.ThehomologousandheterologousstimulationofTcellwasanalyzedwithCFSE.Tregulatorscells(Tregs)wasanalyzedbytheexpressionofCD25+andFoxp3+.OurresultsshowedthatiDCsandskin-deriveDCswereinfectedwithPRRSv.Infectedskin-derivedDCsexpressedlevelsofmRNAINF-αandlowexpressionofmRNAIL-10.InfectionofiDCsrevealedanmRNAincrementof1,000foldcomparedwithmocktreatediDCs,andIFN-anotwasexpressed.ApoptosiswasdetectediniDCsat24hpost-infection,increasingat48hand72hpost-infection.TheendocytosisandphagocytosiswasreducedoninfectediDCs,andthecapacitytostimulateheterologouswasreduced30%comparedtomock-treatedcells.Theanalysisofhomologousstimulationwasalsoreduced,andshowedthepresenceofCD25+Foxp3+cells(9±4%)inno-adherent cells stimulatedwith infected iDCs, this phenotypewasduetovirusreplication,becauseheatinactivatedvirusdidnotproduceCD25+Foxp3+cells.Also,therewasnotchangeofCD25+Foxp3+yieldwheninfectedDCsweretreatedwith IFN-a.Preliminaryresults test-ing porcine circovirus showed thatCD25+Foxp3+ was not produced.Inconclusion,themodulationofcytokineproductionandtheinductionofcellswithphenotypeofTregscellscouldbeamechanismusedbyPRRSvtoevadetheimmuneresponse.Key words:Porcinereproductiveandrespiratorysyndrome,dendriticcells,CD25+Foxp3+TregulatorycellsSpecies:swine

AP135. DOG LEUKOCyTE ANTIGENS IDENTIFIED By A CDNA LIBRARy

PAULoHPAGUIAREscoladeMedicinaVeterinária–UniversidadeFederaldaBahiaTheidentificationofantigensrecognizedbymonoclonalantibod-

ies (MoAbs) directed against dog’s Peripheral Blood MononuclearCells(PBMCs)wasperformedbyusingacomplementaryDNA(cDNA)library, produced by following the manufacturer protocol (picoBlueImmunoscreeningkit,StratageneCat.).Thisprocedurewasusedasan alternative to immunoprecipitation because it has the advantageof allowing the subcloning of the genes identified and the produc-tion of recombinant proteins in the amount necessary for furtherassays.Twelvemonoclonalantibodieswereproducedand testedby(1) recognizing different dog’s leukocytes populations in an IndirectImmunofluorescence(IIF)usingcryopreservedtissuesections(lymphnode, liver,skinandkidney)and/orhealthydog’sPBMCsand(2)byrecognizingantigenswithdistinctmolecularweightsonwesternblot-tingassays.OnlyoneMoAb,calledasAB9(IgG2a,34KDaidentifyingmembraneand/orcytoplasmicproteins),recognizedaphagecloneonthelibrary.Thispositiveclonewasfurtherbi-directionallysequenced,twice. The evaluation of the other elevenMoAbs resulted negative,possiblybyinherentpeculiaritiesassociatedtotheuseofthegeneticmaterialforinvitroproteinproduction.TheproductionofacDNAlibrarystartsbytheisolationoftotalRNAfromthetargettissueandthefurtherselection of the poliA+ RNA, which accounts for the majority of themessagesonagivencell.The isolationofgoodqualitypoliA+RNAis essential, as any degradation will affect the library sequences asrepresentative.Although cDNA libraries are standard tools for geneexpression studies and theoretically have complete copies of eachmessenger RNA obtained from the initial sample, losses may occurduringtheprocedureofcloningtechniques.Hence,librariesmaycon-taingene fragmentswhichwouldbedifferent than the realgenome,whatwouldinterferewiththefurtheranalysis.Key words:DLA,cDNAlibrariesSpecies:canine

AP136. IGM SELECTIVE DEFICIENCy AND TOLL-LIKE RECEPTORS – A BRAZILIAN STUDy

CMASSoCo,MZMELEIRo,AyAHoGE,MMCAMARGoSelective IgMdeficiency (SIgMD) isa rareprimary immunodefi-

ciencywhosemolecularmechanismsremainunclear.Hereinwepres-

98

enttenadulthorsesofseveralbreedswhoseSIgMDwasdiagnosedon the basis of clinical history of recurrent bacterial infections andreducedserumIgMconcentrations(morethan2standarddeviationsbelowthenormalmean).Alltenhorseshadahistoryoflatedevelop-ment and respiratory, gastrointestinal and/or reproductive disorders.Immunoglobulin quantification was carried by radial immunodifusionand revealed normal IgG, IgG(T) and IgA concentrations, but verylowIgMconcentration.Invitroproliferationstudiesrevealedahetero-geneous inter-individual profile of proliferation in response to PHA,flagelinandPam3Cys.Incontrast,alloftheseindividualshadskewedresponses to LPS, while proliferative responses to ConA and polyI:Cwere comparable to healthy controls.TLR expression patterns ofseveralindividualswillbeshown.Asthesespecificdefectsleadingtofailuresintheimmuneresponseareidentifiedandcorrelatedwiththepathogenesisofthisdiseasewewillextendourunderstandingoftheworkingoftheequineimmunesystem.Key words:IgMdeficiency,Toll-likereceptorsSpecies:Equine

AP137. GENERATION AND CHARACTERISATION OF EQUINE DENDRITIC CELLS FROM HIGHLy ENRICHED

PERIPHERAL BLOOD MONOCyTESECSIFFRIn,JRoHWER,WLEIBoLD

Dendritic cells play a pivotal role in initiating a wide range ofimmunereactionsagainstmostantigens.Peripheralbloodmonocytesare a suitable source to generate dendritic cells (DC) in vitro underappropriate conditions. Aiming for a high yield of peripheral bloodmonocyteswhichhaveneitherbeenpreselectednorpreactivatedbytheharvestingprocedurewescrutinizeddifferentpublishedmethods.Subsequently,theenrichedmonocyteswereusedtogeneratemono-cytes-derived-DC(MoDC)whichwerecharacterisedphenotypicallybymeansof theirCD83andCD86expressionand functionallyby theirantigenpresentingcapacity.

Enrichmentofequinemonocytesduetotheiradherenceonplasticsurfacesturnedouttobeineffective,becauseitcomprisedaconsider-ablelossofmonocytesandaninsufficientpurityofmaximally30%ofthemononuclearcell(MNC)fraction.DepletingMNCfromCD4-andCD8-positivecellsbymagneticcellsorting(MACS)resultedinahigherpurityofmonocytes(upto40%ofMNC)butwithanunacceptablelowgainofonly less than7%of theMNCtostartwith,plusaselectivelossofCD4+andCD8+monocytes.Theenrichmentof IgEpositivemonocytesviaMACS resulted inan impressivepurityofmonocytes(up to 70% of MNC), but led to a monocyte subset selection (IgE+monocytes)andavery lowyieldofmonocytes (less than2%of themonocytesavailable).Thebestmethod toenrichequinemonocytesfromwholebloodprovedtobeacentrifugationonahyperosmoticden-sitygradient,resultinginapurityofupto90%monocytesandayieldof70%ofthemonocytesavailable.Bythismethodneitherapreselectionnoradetectablepreactivationofmonocytestookplace.

ToexaminewhetherthesemonocytesareareasonablesourceforthegenerationofDC,theywereculturedfor7daysinmediumsupple-mented with rec.eq.IL4 and rec.hu.GM-CSF. Phenotypical charac-terisationwithmonoclonalantibodiesagainsthumanCD83andCD86revealedup to30%CD83+andup to60%CD86+DC.TheantigenpresentingcapacityofthegeneratedMoDCsweretestedinallogeneicandautologousmixedleucocytereactions(MLR).UnpulsedallogeneicDC(withoutexogenousantigenloading)ledtoasignificantlyincreasedT-cellproliferationbutdidnotstimulateautologousT-cellstoasignifi-cantproliferation.Exogenous loadingofDCwithantigen24hbeforeapplicationoftheDCintheMLRcausedclearstimulationofallogeneicaswellasautologousT-cells.Key words:Dendriticcells,IgEpositivemonocytes,mixedleucocytereactionsSpecies:Equine

AP138. TICK SALIVA SUPPRESSES DENDRITIC CELL MIGRATION By REGULATING CHEMOKINE ACTIVITy AND

CHEMOKINE RECEPTOR ExPRESSIONCJFoLIVEIRA1,KACAVASSAnI1,JCSALIBERTI2,JSSILVA1,BR

FERREIRA3

1DepartmentofBiochemistryandImmunology,SchoolofMedicineofRibeirãoPreto,USP,SP,Brazil;2DivisionofMolecularImmunology,

CincinnatiChildren’sHospitalMedicalCenterandUniversityofCincinnatiCollegeofMedicine,Cincinnati,OH,USA;3DepartmentofMaternal-ChildNursingandPublicHealthoftheRibeiraoPreto,

SchoolofNursingoftheUniversityofSaoPaulocarlo@usp.br

Ticksareblood-feedingarthropodsofvastmedicalandveterinarypublichealthimportanceduetodirectdamagecausedbyfeedingandtheirrolesintransmittingwellknownandemerginginfectiousagents.Manystudieshaveshownthatticksalivacontainsavarietyofactivemoleculesthatantagonizehostinflammatoryandimmuneresponses.Asdendriticcells(DCs)playamajorroleinhostimmuneresponses,westudiedtheeffectsofRhipicephalussanguineusticksalivaonDCmigration and function. To evaluate the effect of tick saliva on DCmigration, a Boyden microchamber migration assay was performed.The percentage of cells expressing chemokine receptors was mea-sured by flow cytometry analysis. To investigate the effect of salivaon the functionofDCsacelladoptive transferassaywasdoneandtheTcellproliferationandcytokineproductionwasevaluated.Bonemarrow-derived immature DCs pre-exposed to tick saliva showedreducedmigration towardsMacrophage InflammatoryProtein (MIP)-1α,MIP-1βandRegulateduponActivation,NormalT-cellExpressedandSecreted(RANTES)byapproximately60%.Thisinhibitionmedi-atedby saliva significantly reduced thepercentage (59.8%)and theaveragecell-surfaceexpressionofCCchemokinereceptorCCR5.Incontrast,salivadidnotaltermigrationofDCstowardsMIP-3βnotevenifthecellswereinducedformaturation.Whenevaluatedtheeffectofticksalivaon theactivityofchemokinesrelated toDCmigration,weshowed,inaBoydenmicrochamberassay,thatticksalivaperseinhib-itsthechemotacticfunctionofMIP-1α(40.7%),whileitdidnotaffectRANTES,MIP-1βandMIP-3β.ThesesalivaeffectscouldreducethepoolofimmatureDCspresentatthetick-feedingsite.Finally,totestthebiologicalactivityofthesaliva-exposedDCs,wetransferredDCspre-culturedwithsalivaandloadedwiththeKeyholeLimpetHemocyanin(KLH)antigentomiceandmeasuredtheircapacitytoinducespecificT-cellcytokines.DatashowedthatsalivareducedthesynthesisofThelper (Th)1andTh2cytokines, suggesting the inductionofa “non-responsive”T-cell.Thesefindingspropose that the inhibitionofDCsmigratoryabilityandfunctionmaybepotentmechanismsusedbytickstoparalyzetheimmuneresponseofthehost.

FinancialSupport:FAPESP,PRODETABandCNPq.Key words: Ticks, Dendritic cells, Chemokines, Rhipicephalussanguineus.Species:other

AP139. RAPID IMMUNIZATION AGAINST SCORPION TOxIN By IN VIVO TARGETING MICE DENDRITIC CELLS

GPESPIno-SoLIS1,AFLICEA-nAVARRo2,GAGURRoLA-BRIonES,LDPoSSAnI

1UniversidadNacionalAutónomadeMéxico–InstitutodeBiotecnología–DepartamentodeMedicinaMolecularyBioprocesos,AvenidaUniversidad,2001,ApartadoPostal510-3.Cuernavaca,Morelos62210MEXICO;2CentrodeInvestigaciónyEducacionSuperiordeEnsenada.DepartamentodeBiotecnologiaMarina,

Esenada,BajaCalifornia,MEXICO.Scorpionism(accidentscausedbyscorpionstings) inMexico is

a public health problem with an excess of 200,000 cases, annually.Theneedofanti-venomproductionismandatory.Herewedescribearapidprotocoltoproduceneutralizingantibodies.ToxinCn2,whichismostabundantcomponent(6.8%)ofthemostdangerousscorpionofMexico(Centruroidesnoxius)wascovalentlycoupledtoamonoclonalantibodycapableofbindingdirectly to the integrinCD11c,aspecificmarkerofthedendriticcellsofmice.TheantibodyusedwasN418andthetoxinwasattachedbyaSchiff’sbasetothecarbohydrate

moietyofthisantibody,viaNaIO4oxidation.AgroupoffivemicewasimmunizedoncebyintradermicinjectionofthecomplexN418-Cn2dissolvedinphosphatesalinebuffer,pH7.2.Anantiseracapableofrec-ognizingCn2(titer1:6000)wasobtainedaftersevendays.ThisserumwasshowntoneutralizetoxinCn2witha50%efficiencycomparedto100%obtainedwithtraditional immunizationprotocols,whichinclude

99

several immunizationsduringa90daysperiodoftime.Thisstrategyopensthewayforaquickproductionofantitoxinserainmice.

Acknowledgements:This work was supported in part by grantsfrom:DGAPA-UNAMNo.IN227507andInstitutoBioclónS.A.deC.V.GPEShaveafellowshipfromCONACYTNo.169946.Key words:Scorpiontoxin,anti-venom,dendriticcellsSpecies:other

AP140. EFFECTS OF PTERIDIUM AqUILINUM ON MICE ARE NOT ASSOCIATED WITH THE ELICITATION STAGE IN

DELAyED-TyPE HyPERSENSITIVITy RESPONSEAnDREIAoLAToRRE1,MonICASAKAI1,MITSUEHARAGUCHI2,

SILVAnALGóRnIAK11DepartmentofPathology,FacultyofVeterinaryMedicineandAnimalSciences,UniversityofSãoPaulo,Brazil;2BiologicalInstitute,São

Paulo,Brazilalatorre@usp.br

Pteridiumaquilinum(Pa)isoneofthefivemostcommonplantsontheplanetandisknowntocausecancerinanimalsandhumans.OurearlierstudieshaveshownthatPacausesinjuryinlymphoidorgans,decreasesbonemarrowcellularity,reducesdelayed-typehypersensi-tivity (DTH) response andNK cytotoxicity inmice. In this study,wehaveevaluatedtheactivityofTlymphocytesofspleenandperitonealmacrophagesinordertoseewhetherthereisanycorrelationoftheseeffectswithreductioninDTHresponse.ForTlymphocytesproliferationassay,nineC57BL/6malemicewereusedpergroupandtreatedbygavageupto14daysasfollows:control(Co)0.0,Pa(P)30.0gPa/Kg

BW.Atsacrifice,thespleenwasremovedtopreparecellsuspension.Fromthecellsobtainedineachmouse,1x107cells/mLwereincubatedwith1µLofCFSEfor20mininthedark.Afterthis,cellswerewashedandsuspendedat2x106cells/mL inRPMIand100µLaliquotswereadded toa96-wellmicrotiter plate. UntreatedandPHA (10µL/well)treatedwere set up in triplicates and incubated for 96h at 37°C inahumidifiedatmosphereat5%CO2.Thesampleswereanalysedbyflowcytometry.Inanotherstudy,fortheanalysisofmacrophages,eightC57BL/6malemicewereusedpergroupandthesameprotocolwasutilizedfortreatment,buton10thdayallmicewereinjectedipwith1mLofthioglycollate10%.Atsacrifice,theperitonealmacrophageswerecollectedwithPBS. For the oxidative burst analysis, from the cellsobtainedineachmouse,1x106cells/tubewereincubatedduring30minat37ºCwitheitherDCFH-DAorDCFH-DAandPMAorDCFH-DAandStaphylococcusaureusconjugatedwithpropidiumiodide(SAPI).Forthe phagocytosis analysis, from the cells obtained in each mouse,1x106 cells/tube were incubated 30min at 37ºC with either SAPI orDCFH-DAandSAPI.Thesampleswereanalysedbyflowcytometry.NochangeintheproliferationofTlymphocytesandmacrophageactivi-tieswasobservedafterPatreatment,suggestingthatthereductioninDTHresponseisprobablyduetoinefficiencyonthesensitizationstage(activationandexpansionofantigen-specificmemoryTh1cells)andnotontheelicitationstage(Th1andmacrophageactivation).Furtherexperimentsare inprogresstoseethe immunomodulatoryeffectsofPa.Key words:toxicplants,immunomodulationSpecies:other

Pr141. DEVELOPMENT OF AN ANTI-TICK VACCINE TO PROTECT CATTLE AGAINST TICK-BORNE TRANSMISSION

OF THEILERIA PARVASAIKIIMAMURA,SAToRUKonnAI,CHIEnAKAJIMA,SHInJI

yAMADA,yUKoITo,KAZUHIKooHASHI,MISAoonUMAGraduateSchoolofVeterinaryMedicine;HokkaidoUniversity,Kita18,

Nishi9,Sapporo060-0818,Hokkaido,Japan.saiki-i@vetmed.hokudai.ac.jp

Ticksarefacultativebloodsuckingectoparasitesfoundinallter-restrialregionsoftheworld,wheretheyarethemajorvectorstransmit-tingquitealargenumberofpathogens.Therefore,suppressionofthetickvectorpopulationisconsideredtocontrolspecificdiseasestrans-mittedbyticks. Theapplicationofanti-tickvaccinehasbeenshowntobethemostpromisingalternativetickcontrolstrategycomparedtothecurrentuseofacaricides,becauseacaricidessufferfromanumberofdisadvantagessuchaschemicalpollutionofthefoodchainandtheenvironmentaswell as the rapiddevelopmentof resistanceagainstacaricidesinticks.

Rhipicephalusappendiculutusserpin-3(RAS-3),R.appendiculu-tusserpin-4(RAS-4)andRIM36,a36kDaimmuno-dominantproteinof R. appendiculatus, were reported as anti-tick vaccine candidatesfortheixodidtick.Amongmanycandidateantigensconsideredsofar,serpins (e.g. RAS-3 and -4) and cements (e.g. RIM36)may be themostinterestingantigensforthedevelopmentofananti-tickvaccine,becauseoftheirimportantrolesintickphysiology.

Inthecurrentstudy,wegeneratedrecombinantproteinsofRAS-3,-4, and RIM36 and assessed their potency as an anti-tick cocktailvaccine in cattlemodel. Immunization of cattle with a combinationof rRAS-3, -4 and rRIM36 raised antibody against all recombinantsandtickwholeextractasdeterminedbyWesternblot.Tickchallengeinfestationdemonstratedsignificantprotectiveimmunityagainstticks,resulting in41.3and12.8%ofmortality rate for thevaccinatedandcontrolgroup,respectively.InordertoevaluatethelevelsofpathogentransmissioncapacitybyTheileriaparva-infected ticks fedon immu-nizedanimal,anappearanceofT.parvaintheparotidlymphnodeandperipheralbloodwasalsodeterminedandquantifiedbyreal-timePCR.T.parvaDNAloadinthelymphnodewaslowerintheimmunizedgroupthaninthecontrolgroup.Anappearanceofthepathogeninthebloodwasdelayed1-to2-dayposttickchallengeinthevaccinatedgroup.

Ourfindingssuggestedabeneficialeffectoftheanti-tickvaccineforthepreventionofbothtickandinfectiousdiseasestransmittedbyticks.However,itisnecessarytoidentifyothertickantigensthatcanbeused inacocktail to induceanti-tick immunitymorepotentlypre-ventingthetransmissionofpathogensoftick-bornediseases.Key words: anti-tick vaccine, Theileria parva, RhipicephalusappendiculatusSpecies:ruminants

Pr142. EVALUATION OF IMMUNOGENICITy IN BOVINES OF RIPHICEPHALUS (BOOPHILUS) MICROPLUS AND

HAEMAPHySALIS LONGICORNIS RECOMBINANT CALRETICULINS

LUÍSFERnAnDoPARIZI1,2,HERBERTRECH1,2,SAIKIIMAMURA4MISAoonUMA4,AoIMASUDA1,3,ITABAJARADA

SILVAVAZJR1,21CentrodeBiotecnologia,UniversidadeFederaldoRioGrandedoSul,AvenidaBentoGonçalves,9500,PortoAlegre-RS91501-970,Brazil;2FaculdadedeVeterinária,UniversidadeFederaldoRio

GrandedoSul,PortoAlegre-RS,Brazil;3DepartamentodeBiologiaMoleculareBiotecnologia,UFRGS,RS;4HokkaidoUniversity,

Sapporo,Japanluisfparizi@cbiot.ufrgs.br

TheticksRiphicephalus(Boophilus)microplusandHaemaphysalislongicornisareblood-suckingectoparasitiesofbovines,causingseri-ousdamagestothelivestockproduction.Themainmethodofcontrolis basedon theacaricides.However, theuseof vaccineshasbeenstudied as a promising control method. The calreticulin (CRT) is amultifunctionalproteinpresentinalmostallcellsofanimals.Thesecre-tionofCRTduring feedingmight be linked to themodulationof theparasite-host interaction. In the present study, recombinantCRTs ofR.microplus(rBmCRT)clonedinpET-5bandH.longicornis(rHlCRT)clonedinpET-43awereexpressedinEscherichiacoliandpurifiedbyionexchangechromatographyandusedforimmunizationofbovines.Thefractionusedfortests invitrowaspurifiedby ionexchangeandgelfiltrationchromatography.ByELISA,itwasdemonstratedthatbothCRTsare recognizedby immunizedbovines.The immunogenicandantigenic capacities of rBmCRT and rHlCRT were analyzed by twomethods. In silico, despite the difference in amino acid sequences,antigenicindexanalysisofrHlCRTandrBmCRTwiththeJameson-Wolfalgorithmindicatedthatbothproteinswereverysimilarintheantigenic-ity index.Althoughsixdifferent regionsbetween the tickCRTshavebeendetermined.Invitro,thisdatawerecorroboratedbycompetitiveELISAthatsuggeststhepresenceofdifferentepitopesbetweenpro-teins.ByWesternblot,anti-native rBmCRTand rHlCRTbovineseraalso recognized thenativeproteins in larvaeextracts.These resultsdemonstrate the presence of shared epitopes between recombinantandnativeproteins.Inconclusion,theresultssuggestthattherBmCRTandrHlCRTcouldhaveasimilarimmunogenicityforbovines.

Supported by CNPq, FAPERGS, FINEP, PRONEX (Brazil) andJSPS(Japan)Key words:calreticulin,vaccine,Riphicephalus(Boophilus)micropluseHaemaphysalislongicornisSpecies:ruminants

Pr143. EVALUATION OF DNA IMMUNIZATION WITH PLASMIDS ExPRESSING RByC (BOOPHILUS yOLK PRO-

CATHEPSIN)MARIALúCIASMEDEIRoS1,ALExAnDRETLEAL1,CARLoS

LoGULLo5,SAnDRAEFARIAS1,4,AoIMASUDA1,3,ITABAJARADASILVAVAZJR1,2

1C.Biotecnologia-UFRGS;2Fac.Veterinária-UFRGS;3DeptoBiol.MoleculareBiotecnologia-UFRGS;4DeptoFisiologia-UFRGS;

5LQFPP-CBB-UENF,CamposdosGoytacazes,RJ.malu@cbiot.ufrgs.br

TheBoophilusmicroplustickisthemajorbovineectoparasiteandcausesimportanteconomicallossesoncattlebreeding.Theimmuno-logic control has been studied as an alternativemethod for the tickcontrol.BYC(BoophilusYolkProCathepsin)isanasparticproteinasefound ineggs that is involved in theembryogenesisofB.microplus,andithasbeenproposedasaprobableantigeninvaccinedevelop-ment. The purpose of this study was to evaluate whether the DNAimmunizationcontainingBYCcDNAcouldelicitthespecificanti-BYCimmune response invivo.ThecDNAofBYCwasamplifiedbyPCRand it was cloned into two eukaryotic expression vectors (pcDNA3and pME18-Neo). XL1-Blue E.coli were transformed with clones,BYC-pcDNA3 or BYC-pME18-Neo, and the plasmids were purifiedbyalkalinelysismethod.InordertoevaluateimmunogenicityofBYC,BALB/c mice were immunized with DNA vaccines by intramuscularinjection.Themicereceivedtwo intramuscular inoculationsof100μgplasmidsDNA(BYC-pcDNA3orBYC-pME18-Neo)and thenegativecontrolsreceivedonlyPBS,pcDNA3orpME-18-Neo.Theproduction

6. IMMUNOPARASITOLOGy: IMMUNE RESPONSES TO PROTOZOA, HELMINTHS AND ECTOPARASITES; CANINE VISCERAL LEISHMANIASIS: POSTERS PR141-PR196

of antibody after the immunizations was evaluated byWestern BlotandELISA.AntibodiesagainstBYCweredetectedinmiceinoculatedwithBYC-pcDNA3.TheseresultsshowthatDNAvaccinationproducesspecificanti-BYCantibodiesandsuggestthatDNAcouldproveusefulforvaccinedevelopment.

SupportedbyFAPERGS,FINEP,PRONEXKey words: B.microplus,vaccineDNA,BoophilusYolkProCathepsin,embryogenesisSpecies: ruminants

Pr144. CLONING AND ExPRESSION OF THE RECOMBINANT TRIOSEPHOSPHATE ISOMERASE (TIM), AN ENZyME INVOLVED IN METABOLISM OF TICK BOOPHILUS

MICROPLUSFERnAnDAEKSILVA1,JoRGELDACMoRAES3,CARLoS

LoGULLo3,AoIMASUDA1,4,ITABAJARADASILVAVAZJR1,21CentrodeBiotecnologia-UFRGS;2Fac.Veterinária-UFRGS;3UniversidadeEstadualdoNorteFluminense;4DeptoBiologia

MoleculareBiotecnologia-UFRGSfeklein@cbiot.ufrgs.br

Cattle raising is a very expressive economical activity inBrazil.Nevertheless,B.microplusisresponsibleforlossesintheorderofonebilliondollarsayear.Consequently, it isnecessary todesignnew control strategies that are technically and economically viable.Thehomodimericenzymetriosephosphateisomerase(TIM)convertsglyceraldehyde-3-phosphate to dehydroxyacetone phosphate, a keyreactioninglycolysis.Previousstudiesofthisenzymeinotherparasite-hostmodelhaveindicatedthatTIMisapromisinganti-parasitevaccineantigen.Therefore, thestudyof theenzymeB.microplusTIMcouldcontributetodevelopanewvaccineforimmuneprotectionagainstthisparasite.Inordertostudythisenzymeatthemolecularlevel,wehaveisolated,cloned,sequencedandexpressedonecDNAcloneencodingTIM from tick eggs usingRT-PCRwith specific primers. The ampli-conwith750pbwasdigestedwithrestrictionenzymesandligatedinpET-43aexpressionvector.Thecloningwasconfirmed for cleavagewithrestrictionenzymes,PCRandDNAsequencing.EscherichiacoliAD494 (DE3) pLysS was transformed with TIM-pET-43a vector forexpression of recombinant protein.Optimal rTIM-Bm (a proteinwithtwosubunitsofthe27kDaandwithHis-tag)productionwasachievedby testingdifferentgrowth temperatures, timesand IPTGconcentra-tions.TheexpressionwasanalyzedforSDS-PAGE12%andthepres-enceoftherecombinantproteinwasconfirmedforWesternblot,usingmonoclonalantibodyanti-histidine.ThepurificationoftheproteinhasbeendonebyNi2+affinitychromatography.Immunizationwillbedoneinmiceandcattlestoassessitscapacitytoinduceimmuneresponseintheanimalsandimmuneprotectioninthehost.

Supportedby:CNPq,FAPERGS,FINEP,PRONEX,CAPES.Key words:triosephosphateisomerase,boophilusmicroplus,cloning,vaccineSpecies:ruminants

Pr145. RECOMBINANT ExPRESSION AND PARTIAL CHARACTERIZATION OF AN ASPARTIC PROTEINASE FROM

BOOPHILUS MICROPLUSPAULACPoHL1,MARCoSSoRGInE2,ITABAJARADASILVAVAZ

JUnIoR1,3,AoIMASUDA1,41CentrodeBiotecnologia-UFRGS;2DeptoBioquímicaMédica-

UFRJ;3DeptoBiologiaMoleculareBiotecnologia-UFRGS;4Fac.Veterinaria-UFRGS

paula@cbiot.ufrgs.brThetickB.microplusisahematophagousectoparasiteofbovine.

Theactualmethodforthecontrolofcattletickistheuseofchemicalpesticides,which causes damage to environment and contaminatesthemeatandmilk.Analternativeandpromisingmethodforcontrolistheuseofvaccines.Theresearchesareguidedtowardstheidentifica-tionofnewproteinsthathaveimmunoprotectivepotential.THAP(tickheme-bindingasparticproteinase)isanasparticproteinasepresentineggsofB.microplusinvolvedintheembryogenesis,bydegradingvitel-linaccordingwiththenecessityofembryonicdevelopment.ThecDNAencodingfullsequenceofTHAPwasclonedinthepET43aexpression

vectorandninestrainsofE.coliweretransformedwithresultantplas-mid.Thebestconditionsestablishedforproductionoftherecombinantprotein (rTHAP with fusion protein NusTag) in the soluble form wastheexpressioninE.coliBL21(DE3)RILat23ºCand1mMofIPTGfor4hours.AnalysisoftheexpressionwasperformedbySDS-PAGEand immunoblotting with a rabbit anti-THAP serum. rTHAP-NusTagwas purified by affinity chromatographywith sepharose-Ni2+ resin.Apartiallypurifiedfractionwasobtainedandsubmittedtohydrolysisforremoval the fusion protein (NusTag). The rTHAP enzymatic activitywasassayedwith fluorogenic substrateAbz-AIAFFSRQ-EDDnpandwasmonitoredfor1hourwithF-MAXfluorometer.Thespecificactivityobtainedwas9.55RFU/min/mgofproteinandtheactivitywasblockedby 20 µM of pepstatinA. In order to characterize the imunogenicityoftheprotein,twofragmentsofthecDNAencodingtheproteinwerecloned.OnecDNA fragmentencoding thefirst170aminoacidsandanotherencoding the last185aminoacids.TheencodingsequenceofaminoacidsoftheTHAPwereobtainedbyPCRandclonedinthepET23aexpressionvector.Therecombinantproteinswereproducedininsolubleformbytheexpressionat37ºCand1mMofIPTGfor4hours.Thefullproteinandcarboxi-endfragmentwereimmuno-reactiveagainstserafromrabbitandbovinepreviouslyimmunizedwithnativeTHAP.FurtherstudiesontheTHAPenzymaticactivityanditspotentialimmunoprotectiveroleareinprogresstodefinetheimportanceofthisproteintotickcontrol.

ThisworkwassupportedbygrantsfromCNPq-PIBIC,CAPES,PRONEX-FAPERJ.Key words:thap,tick,vaccine,embryogenesisSpecies:ruminants

Pr146. CLONING AND CHARACTERIZATION OF THE RECOMBINANT GLyCOGEN SyNTHASE KINASE (GSK-3β) FROM BooPHILUS MICRoPLUS ExPRESSED IN E. CoLI

CARoLInEPDEAnDRADE1,JoSIAnAGDEAnDRADE3,SAIKIIMAMURA2,MISAoonUMA2,AoIMASUDA1,5,CARLoS

LoGULLo3,ITABAJARADASILVAVAZJUnIoR1,41C.Biotecnologia-UFRGS;2HokkaidoUniversity;3LQFPP-CBB-UENF;4Fac.Veterinária-UFRGS;5DeptoBiologiaMoleculare

Biotecnologia-UFRGScarolandrade@cbiot.ufrgs.br

Boophilus microplus is a hematophagous parasite that infestsanimalsofeconomic importance,causing losses incattleproductionworld-wide.Theconventionalmethodfor thecontrol isbasedontheuse of synthetic chemical products.However, these products causetheselectionofresistantticktotheacaricidesbeingusedandcausepollution in the environment. A vaccine is a reasonable alternativeapproachandseveralstepstowardsitsdevelopmenthavebeentaken.Thesuccessofthisstrategyisdependentonthecloningandcharac-terizationofthephysiologicalfunctionoftickmolecules.Acombinationofantigensmayberequiredbothtoimprovevaccineefficacyandtocir-cumventpossibleparasiteevasionmethods.TheGlycogenSynthaseKinase (GSK-3β) isaserine/threoninekinase that is involved ingly-cogensynthasephosphorylation inglycogenmetabolism,during tickembryogenesis.DegeneratePCRprimersweredesignedtoannealtohighlyconservednucleotidesequencesbasedontheseaurchinGSK-3βcDNAsequenceandutilizedinPCRreactionstoamplifyapartialGSK-3βsequencefromtheB.micropluscDNA.ThePCRproducedafragmentofapproximately600bp,corresponding to thepartialGSK-3βcDNA.Toobtain the full-lengthcDNAencodingGSK-3β,5´RACEand3´RACEofcDNAendswereproducedusingthesequenceofthe600bpamplifiedfragment.Theproductsof5´RACEand3´RACEwerecloned in pGEM-TEasy vector and sequenced. To confirm that the5´RACEand3´RACEproductscorrespondtotheauthentictranscripts,the full-length cDNAwasamplifiedbyPCR, from totalRNA isolatedfromegg.Theamplifiedof1,230bp(full-lengthcDNA)wasclonedintopGEM-TEasy and sequenced. Thereafter to confirm the full-lengthcDNAsequence,thefragmentwasclonedintoexpressionvectorpET-5b,toexpressionandpurificationoftherecombinantproteinandevalu-ationofimmunogenicity.

Supported by CNPq FAPERGS, FINEP, PRONEX, FAPERJ(Brazil)andJSPS(Japan).Key words:Boophilusmicroplus,glycogensynthasekinase,cloning,

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embryogenesisSpecies:ruminants

Pr147. GSK ACTIVITy IS INVOLVED TO PEPCK GENE ExPRESSION DURING TICK EMBRyO DEVELOPMENT

CARLoSLoGULLo1,2,WILLIAnWIToLA2,CARoLInEAnDRADE3,JoSIAnAGoMES1,ITABAJARADASILVAVAZJR3,

SAToRoKonnAI2,KAZUHIKooHASH2,SAIKIIMAMURA2,MISAoonUMA2

1LaboratóriodequímicaeFunçãodeProteínasePeptídeos-CBB-UEnF,CamposdosGoytacazes,RJ,Brazil;2LaboratoryofInfectious

Diseases,DepartmentofDiseaseControl,GraduateSchoolofVeterinaryMedicine,HokkaidoUniversity,Sapporo,Hokkaido,Japan;

3CentrodeBiotecnologia-UFRGS,PortoAlegre,RS,Brazil.logullo@uenf.br

Ticks are the major ectoparasite that causes vast economicallossesincattleproductionontheworld.Thestudyoftheembryogen-esisoftickshasseesfundamentalpaperthat,inagreementwiththedrawingof thenewmethodologiesproposed for thecontrolof thesevectors. Inmanycases,genesthatparticipate indifferentmetabolicpathwaysorgenesregulatorsofdifferentaspectsoftheadultorganismarealsoexpressedduring theembryogenesisandyourmodificationcan result indecreaseof theviabilityoreven inprecocious lethality.InourpreviousworkwedemonstratedthattheembryogenesisofthehardtickBoophilusmicropluswasmetabolicallyseparatedintwospe-cialphases: An initialphase,until thecellularblastodermformation,characterizedbytheconsumptionofthematernalglycogenandasec-ondphasecharacterizedbyanintenseaminoacidsdegradationthatpromotesinaccumulationofglycogenandglucoseinthisstage.WealsoobservedthatthespecificactivityofPEPCK,akeygluconeogen-esisenzyme,issurprisinglyincreasingaftertheembryocellularizationanditsactivityismajorityinthemitochondrialcompartment.Glycogensynthasekinase3(GSK3)hasbeenclassicallydescribedasinvolvedtophosphorylateglycogensynthase.TheactivityofGSK3isdepen-dentof insulincascade involvingaproteinkinaseB,calledAkt. AktmediatestheinhibitionofGSK3andcontributestoactivationofglyco-genandproteinsynthesis.GSK3βhasbeenshownbyhomologueoftheshaggy(orzestewhite3), inDrosophilamelanogasterandGSK3in xenopus. In both cases this gene is implicated in dorsoventralpatterningoftheearlyembryos.GSK3activitywasrequiredforbothPEPCKandG6Pasepromoteractivityandwhichselectiveinhibitorsofreducethegenetranscriptionoftheseenzymesinmammalianscells.However,thereareevidencesthatinsulinregulatesageneexpressionofG6PaseandPEPCK inmammals’ cells. In thepresent study,wehaveclonedandstudiedtheexpressionofGSK3andPEPCKduringtickembryodevelopment. On theother handwehave investigatedthe influence of GSK activity in the PEPCK expression during tickembryogenesis. We also demonstrated that lithium chloride, insulinandSB216763,theinhibitorsofGSK3activity,canreducedrasticallyanexpressionofPEPCKinembryonictickcellsculture.WeattributedthatregulationactivityofGSKprobablyoccursbytheinsulincascadeduringthisdevelopmentprocess.

SupportedbyCNPq,FAPERJ,JSPSandCAPES.

Pr148. IMMUNOBLOTTING OF AMBLyOMMA CAJENNENSE ANTIGENS USING SERA FROM SENSITIZED GOATS. CROSS

REACTIVITy WITH A. HEBRAEUMGERMonTEIRo1,2,RZMACHADo2,GHBECHARA2

1DepartamentodeParaclínicas,FV-UEM,Maputo,Moçambique;2DepartamentodePatologiaVeterinária,FCAV-UNESP,Jaboticabal-

SP,BrazilThecontroloftickshasbeenamajorconcernbecauseofthevery

highcostsof thechemicalmethodsof control usuallypracticed, theenvironmentalcontaminationtheyproduce,andthedevelopmentoftickresistanceproblems.Antigenicextractsderivedfromwholetickbodyhomogenatesofbothadultandimmatureinstarsandofinternalorgansas salivary gland and gut of partially fed female ticks were alreadytestedanddemonstrated to inducevariousdegreesof resistance toticks in several hosts. This approach is promising as an alternativemethod for tickcontrol. Inaddition,preliminary resultsof the labora-

toryshowed thatgoatsacquirepartial resistanceagainstnymphsofthelone-startickAmblyommacajennenseafterrepeatedinfestations.With the aim to search for antigens fromA. cajennense recognizedbyserafromrepeatedlyinfestedgoats,fiveanimalsagedsixmonths,of both sexes, were infested thrice with 100A. cajennense nymphsat30days interval.Serafromthesegoatswerecollectedbeforetheinfestation and 30 days after both the 1st and 3rd infestations andusedinwesternblotanalysistoidentifypotentialantigensfromunfednymphal extract homogenate. Sera collected from goats infested atsameconditionswithA.hebraeumtickswereusedtosearchforcross-reactivitybetween tickantigens from the ixodidspecies.These laterserarevealedninepolypeptidesof12,19,24,34,46,52,66,83and160 kDa while those collected from goats infested once and thricewithA.cajennensenymphsrevealedeightcommonpolypeptidesof26,36,5,37,5,55,72,86,98,and170kDa.Atnon-infestedanimals’blots,itwasobservedreactivepolypeptidesaswell,revealingtheexistenceof cross reactivitywith antigens fromectoparasites other than ticks.However,itshouldbestressedthatthreepolypeptidesof14,11and7kDarelatedtoA.cajennensenymphsobservedonlywhenusedserawerefromanimalsinfestedthricecouldplayanimportantroleininduc-ing resistance ingoats tosuch tickspecies.Finally, thepresenceofclose polypeptides reactive toA.hebraeum serum probably revealstheexistenceofcrossreactivitybetween the two ixodidspecies,butforwardstudiesareneededtoconfirmit.Key words:Immunoblotting,Amblyommacajennense,A.hebraeum,goatsSpecies: ruminants

Pr149. TRANSCRIPTOMES OF TICKS FED ON RESISTANT AND SUSCEPTIBLE CATTLE: GENES AFFECTED By HOST

IMMUNE RESPONSESSRCMARUyAMA1,GRGARCIA1,LGBRAnDÃo1,JMCRIBEIRo2,

BRFERREIRA1,IKFDEMIRAnDASAnToS11DepartamentodeBioquímicaeImunologia,FMRP-USP,Ribeirão

Preto,Brasil.2NIAID-NIH,Rockville-MD,USAIntroduction and Objectives: Rhipicephalus microplus causes

enormous losses for animal production and health. Ticks induceimmune response in their hosts, indicating that its immunobiologicalcontrol is possible. Bovines present different phenotypes related tointensity of tick infestations and those phenotypes are mediated byqualitativelydistinctimmuneresponse.Ticksfedonresistantbovinesdonotfeedwellanddisplaylowreproductiveefficiency.Ourhypothesisisthatdifferentlevelsofhostanti-tickimmunityaffectgeneexpressionincattle ticks.Theobjective is todiscover larvaeandsalivaryglandgeneswhoseexpressionisaffectedbyimmuneresponsesofsuscep-tibleandresistanthosts.

MethodsandResults:ThecDNAlibrarieswereconstructedwithSMART (Clontech-BD) technology and mRNA from unfed larvae ofunfed larvae proceeding by female fed on susceptible or resistantbovines;salivaryglandsofnymphs,malesandfemalesfedonsuscep-tibleorresistantbovines.ThecloneswererandomlyselectedforPCRamplificationandtheDNAinsertsweresequencedandanalyzedwithbioinformatictools.Wegenerated7,923ESTs,whichweretrimmedofprimerandvectorsequences,clusterizedinto3,342contigsandcom-paredwithpublicdatabasessuchasNR,GO,KOG,P-fam,SMART,rRNA,MIT-PLA,andprivateonescontainingsequencesforAcariandESTs of R. microplus and submitted in batch to theSignalP server.Manyclusterspresenteddifferentialexpressionaccordingtotheoriginoftheticksbloodmeal–susceptible(RmS)andresistant(RmR)hosts.The most of high expression clusters are in RmH, like clusters withsimilarityhistaminebindingproteinsandoxidantmetabolismproteins.Noteworthy,clusterspresentingwithsimilaritiestoanticoagulantproteins(ACs) that areessential successblood feedandmetalloproteinases(MMPs)canberelatedwithscapeimmuneresponseandextracellu-larmatrixdegeneration.RmSpresents63ESTs(expected43),whileRmRexhibits14ESTs(expected33)similartoACs(P<0.001),beingthatexpressionwasmorenotableinnymphsandmalesstages.SimilartoMMPs,RmHpresents26ESTs(expected18),whileRmRexhibits7ESTs(expected14)P=0.016andthiscasethedifferentialexpressionappearanceonlybetweenmales(RmSxRmN).

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Conclusion: Our data suggest that the host immune responseaffectsgeneexpressioninR.microplusandthestudyofticktranscrip-tomescanbearelevantsourceofantigensforvaccinesanti-ticks.

FinancialSupport:CNPq,FAPESPandValéeSA.Key words:Rhipicephalusmicroplus,Transcriptome,salivaryglands,hostimmuneresponseSpecies:ruminants

Pr150. COMPARATIVE IGG RECOGNITION OF ADULT TICK ExTRACTS By SERA OF ExPERIMENTALLy INFESTED

BOVINESAPRCRUZ1,2,RTMATToS1,2,PRMDELISA3,SSSILVA3,IDA

SILVAVAZJR2,AMASUDA2,CASFERREIRA11LaboratóriodeImunologia,Fabio,PUCRS;2Centrode

Biotecnologia,UFRGS;3DepartamentodeVeterináriaPreventiva,UFPel

The tick Boophilus microplus is a hematophagous ectoparasiteofbovines,widelydistributedinherdsfromAmerica,Asia,AfricaandOceania. The use of acaricides is the main method for tick control,howeveritmaybecomeunfeasibleduetothecostofdrugsandlaborrequiredtoapplythetreatment,aswellastheincreasinglyappearanceofresistanttickstovariousacaricides.Also,chemicalresiduesinfoodandenvironmentalpollutionaremajorconcernsnowadays.Thedevel-opmentofanimmunologicalcontrolmethodasanalternativeforthechemicalcontroldependsonfindingouttickantigenicmoleculesthatinduceaprotectiveimmuneresponseinthehost.Asbovinesdevelopresistanceto ticksduringsuccessive infestations, theanalysisof theimmune responses developed by infested bovines may become ofgreatimportanceinthesearchforprotectiveantigens.Enzyme-linkedimmunosorbentassay(ELISA)andwestern-blotanalyseswereusedto investigate the antibody responses of six bovines experimentallyinfestedtwelvetimes,during18months,withB.microplus againstsali-varygland,gutandlarvaeproteinextracts.ThelevelsofIgGagainstallthreeextractspresentedanincreasefollowingtheinitialinfestationswhereasasignificantdecreasewasshownfollowingthefinalinfesta-tions.IndividualvariationswereobservedinELISA,aswellasandinthe pattern ofmolecules recognized bywestern-blot, which showedthatagreaternumberofantigenswererecognizedbytheinitialinfesta-tionsserawiththepresenceofdifferentprofiles.Althoughtheprofilesofthefinalinfestationsshowascarcernumberandlessintensemol-ecules,newmoleculeswererecognizedandsomeincreaseditsrela-tiveintensity.HigherIgGlevelsandamajornumberofmoleculeswereobservedagainstsalivaryglandextract,whichcorroboratethatsalivaisthegreaternaturalsourceofimmunogenicmoleculesofB.microplus.

Support:CAPES,FAPERGS,CNPqeCNPq/PRONEX-FAPERJ.Key words:Boophilusmicroplus,tickantigens,immunoglobulinGSpecies:ruminants

Pr151. INFESTATIONS WITH RHIPICEPHALUS (BOOPHILUS) MICRoPLUS TICKS INCREASE LOCAL AND SySTEMIC BLOOD CLOTTING TIME IN TICK RESISTANT

AND SUSCEPTIBLE CATTLE WAnESSAACARVALHo1,DAnIELADMoRé1,AnTônIoABATEPAULo1,GUSTAVoRGARCIA1,ALESSAnDRAM

FRAnZIn1,LUÍSHEnRIqUEAConTI2,AnTônIoAMAIA2,GERVASIoHBECHARA3,ISABELKFDEMIRAnDASAnToS1

1Dept.BiochemistryandImmunology,RibeirãoPretoMedicalSchool,UniversityofSãoPaulo,RibeirãoPreto,Brazil;2Dept.Zootenics,PirassunungaVeterinaryandZootecnicSchool,UniversityofSão

Paulo,Pirassununga,Brazil;3Dept.VeterinaryPathology,SchoolofVeterinaryandAgronomicalSciences,SãoPauloStateUniversity,

Jaboticabal,BrazilE-mail:wanac30@hotmail.com

Tick saliva containsmanymolecules described as inhibitors ofbloodcoagulationinvitro.Itisbelievedthatthesemoleculespromoteefficiencyofbloodfeedingbyticks,butthiseffecthasnotbeenevalu-atedinvivo.Thisworkwasperformedtoevaluate,invivo,thesystemicand local clotting time (CT) in tick-resistant (Bos indicus, Nelore; n= 4) and -susceptible cattle (B. taurus, Holstein; N = 4) undergoing

artificialinfestationswith15.000larvaeofR.microplus.Skinbiopsies,withorwithouta feeding tickattached,werecollectedwithan8mmpunch.Blood fromthebiopsy lesionwerecollect immediatelywithacapillaryforcountingCT.Theseexperimentswereperformedwithticksatdifferenttimesintheirlifecycle.PeripheralbloodwasalsocollectedfrominfestedanimalstocomparethelocalandsystemicCT.ResultswereanalyzedwithOneWayANOVAtest.Theresultsshowedthatinsusceptible, but not in resistant bovines the localCT is significantly(P<0.05)increasedinskininfestedwithlarvaeoradultswhencom-paredtotheCTofbleedingfromuninfestedskin.Therewasnodif-ference in the systemicCTs of resistant and susceptible bovines orbetweeninfestedbovinesanduninfestedanimals.Thisdatasuggeststhatticksalivainhibitsthecoagulationcascadeonlyatthesiteoftickattachment and themechanism involved does not affect the animalsystemically.The fact that the localCT time isnot increased in tick-infestedskinofresistantbovinesindicatesthattheirimmuneresponsemaybe targetingandneutralizing tickanticoagulantmolecules.Thispossibilityisfurthersupportedbythepoorfeedingefficiencydisplayedbyfemalesfeedingonresistantbovines.Key words:Rhipicephalusmicroplus,clottingtime,Zebuine,TaurineSpecies:ruminants

Pr152. IMMUNOBLOT ANALySIS OF IGG ANTIBODy RESPONSES TO RHIPICEPHALUS (BOOPHILUS)

MICRoPLUS IN RESISTANT AND SUSCEPTIBLE BOVINES.WAnESSAACARVALHo1,ALExAnDREFIRMIno2,DAnIELADMoRé1,GERVASIoHBECHARA3,BEATRIZFDEMIRAnDA

SAnToSRoSSETTI,KFISABEL11Dept.BiochemistryandImmunology,RibeirãoPretoMedicalSchool,UniversityofSãoPaulo,RibeirãoPreto,Brazil;2EMBRAPAGeneticsResources,Brasília,Brazil;3Dept.VeterinaryPathology,SchoolofVeterinaryandAgronomicalSciences,SãoPauloStateUniversity,

Jaboticabal,Brazilwanac30@hotmail.com

Bovines express breed-specific, heritable, contrasting pheno-types when exposed to the cattle tick, R. microplus. Breeds of Bosindicus(R)aresignificantlymoreresistantthanthoseofB.Taurus(S).Differentbreedsdevelopqualitativeandquantitativedifferentimmuneresponsesagainstticksincludingproductionimmunoglobulinandanti-bodiesspecificforticksaliva.Thisstudywasperformedtodeterminethe frequenciesandspecificitiesofbovine IgGantibodiesbinding tocomponents of saliva, egg extracts (EE) and unfed larvae extracts(UFLE)ofR.microplusandalso todeterminewhether therearedif-ferent immunological patterns of recognition of proteins from thesematerialsbyR(n=5;B.indicus,Nelorebreed)andS(n=5;B.Taurus,Holsteinbreed)bovinesundergoinghighandlownaturaltickinfesta-tions.InordertodeterminethelevelofinfestationineachbreedtickswerecountedononesideofScattleatweeklyintervalsforthedurationof the experiment and serum was collected at different time points.Proteinsfromticksaliva,EEandUFLEwereseparatedbySDS-PAGEandwereelectrophoreticallytransferredontonitrocellulose(NC)paper.IgG-bindingproteinsweredetectedinindividualserafrominfestedani-malsandasacontrolweusedtheserafromtheseanimalsbeforetheyhaveanycontactwiththeectoparasite.Atotaloffifteenproteinbandswithmolecularweightsrangingfrom200to10KDawererecognizedinticksaliva,butresistantcattlerecognizedlessproteinbandswhetherexposedtohighorlownumbersofticks.EEandUFLEarerecognizedwiththesamepatternbyRandSbovinesandthispatternisconsistentacrossall levelsof infestation.Salivaryproteinswereseparated ina2-Dgeland transferred toNCpaper.Aspotdifferentially recognizedbyRserawaselutedfromareplicategelanditsaminoacidsequencerevealed100%homologywithbovinehemoglobin,howeveritspIwasnotasexpected for thenativeprotein,suggesting ithadbeenmodi-fied in the ticksalivarygland. Immunoblotsofsalivawith lectinsandanti-Lewisx antibody indicate that themodification is due to fucosyl-atedcarbohydrates.Our results indicate that there isadifference inthereactivitytoantigenicproteinsinsalivafromR.microplusandthatthelevelofinfestationisrelatedtotheantibodyresponseofthehost.Inaddition,antigensthataredifferentiallyrecognizedbyresistanthostsmaytargetcomponentsofticksalivathatareessentialforbloodfeed-ingandconstituteusefulantigensforavaccine.

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Key words: Rhipicephalus microplus, antibody, lectin, saliva,immunoblotSpecies:ruminants

Pr153. KINETICS OF LEUKOCyTE RESPONSES OF BOVINES IMMUNIZED WITH SBBO23290 (BABESIA BoVIS)

AND SBM7462 (RIPHICEPHALUS (BooPHILUS) MICRoPLUS)DIoGoCoELHoDEPADUAoLIVEIRA,HUGoGUIEIRoRIBEIRoRoCHA,BRUnAALVESDEVénS,MARILIAnoGUEIRADAGAMA,AnnAPAULABRIBEIRoFERREIRA,CARLALEITEMEDEIRoS,JoAqUÍnHERnAnPATARRoyo,MARLEnEISABELVARGAS

VILoRIALaboratoryofBiologyandControlofHaematozoaandVectors,

InstituteofBiotechnologyAppliedtoAgricultureandAnimalScience(BIOAGRO/VeterinaryDepartament),FederalUniversityofViçosa,

36571-000,Viçosa,MG,Braziljpatarro@ufv.br

ThetickRiphicephalus(Boophilus)microplusisresponsibleforthegreateconomicaldamages in thecattlebreeding,because itcausesreductionintheproductionofmilkandmeatanditprovokesdamagestotheleather.ThisparasiteisthetransmitteroftheprotozoaBabesiabovisandBabesiabigeminaandoftherickettsiaeAnaplasmamargi-nale,whichcause theBovineTickFever.Nowadays theseparasitespresenthighincidenceandprevalenceinBrazilandinotherscountriesoftropicalclimate.ThepresentworkhadasobjectivetoanalyzetheleukocytekineticofbovineimmunizedwiththesyntheticimmunogensSBm7462andSBbo23290, in themonovalentandpolyvalent forms.These peptides were prepared using saponina as adjuvant, beingapplied via the subcutaneous route, individually in the left and rightcervicalarea(Treatment1)andinassociateddosesinthesamesideofthecervicalarea(Treatment2).Eachtreatmentwasappliedthreetimeswith intervalsof30days.Thechallengewasmade30and34daysafterthethirdinoculation,beingput±2000larvaesofR.(B.)micro-plus (strain free from haemoparasites) and inoculating the B. bovis(strainUFV1-9thpassageintheconcentrationof1x106babesias/mL),respectively.Throughtheanalysisoftheresults,wasobservedthatallthevaccinatedanimals, independentlyof the inoculation formof theimmunogen(monovalentandpolyvalent),presentedalterationsinthepopulationoflymphocytespresentinthecirculatoryblood.Amongthesub-populationsoflymphocytesanalyzed,therewasmoreprominencefortheBcellsCD21+andTcellsWC1+,whichhaveimportantfunctionsinthecontrolofthebabesioses.Itwasalsoverifiedbytheanalysisofthesamples,accomplishedfivedaysafter thesecondvaccination,asignificantincreaseinthenumberofTcellsCD4+.Throughtheevalu-ationofthecellularimmuneresponsepresentedbytheanimals,itcanbeconcluded that thesyntheticpeptidesSBbo23290andSBm7462in thetreatmentsIandII,usingsaponinaasadjuvant,werecapabletostimulatetheimmunesystemofthebovineand,besides,occurredahighassociationbetweentheconferredprotectionbythesyntheticpeptidesandgreatlevelsofTcellsWC1+andCD4+.Key words: Leukocyte kinetic, Babesia bovis, Riphicephalus(Boophilus)microplus,VaccineSpecies:ruminants

Pr154. HUMORAL IMMUNE RESPONSES TO THE SyNTHETIC IMMUNOGENS SBBO23290 (BABESIA BOVIS)

AND SBM7462 (RIPHICEPHALUS (BOOPHILUS) MICROPLUS)JAVIERAnTônIoBEnEVIDESMonTAño,HUGoGUIEIRoRIBEIRoRoCHA,VInICIUSEBCAMPoS,AnnAPAULAB

RIBEIRoFERREIRA,CARLoSHEnRyqUESoUZAESILVA,GABRIELDoMInGoSCARVALHo,LARISSATAVARESCyRIno,SIDIMARSoSSAI,JoAqUÍnHERnAnPATARRoyo,MARLEnE

ISABELVARGASVILoRIALaboratoryofBiologyandControlofHaematozoaandVectors,

InstituteofBiotechnologyAppliedtoAgricultureandAnimalScience(BIoAGRo/VeterinaryDepartament),FederalUniversityofViçosa,

36571-000,Viçosa,MG,Braziljpatarro@ufv.br

The bovine babesioses caused by a protozoa obligatory intra-cellular parasite from the generous Babesia. This illness occasions

greateconomicaldamages in tropicalandsubtropicalcountries.Thetransmissionoccursduetothehaematophagyofthearthropodsfromthe family Ixodidae,being thebiologicalvector inAmerica is the tickRiphicephalus(Boophilus)microplus.Inthepresentworkthehumoralimmune responseof theHolsteinbreed7/8 (HolsteinxGir)animalswasevaluated.Theanimalswereimmunizedwiththesyntheticimmu-nogensSBbo23290andSBm7462forthecontrolofBabesiabovisandR.(B.)microplus,respectively.Theyweredividedinfourexperimentalgroups, each one with four animals that received three immuniza-tionsevery30days,via thesubcutaneous route. In theGroup I theinoculationwereapplied in thecervicalarea, rightand leftsides, foreach one of the immunogens individually (monovalent form), usingsaponinaasadjuvant. In theGroup II thesepeptideswerepreparedin association doses (polyvalent form), inoculated in the same sideof thecervicalarea,usingsaponinaasadjuvant.Theanimalsof theGroupIIIreceived2mLofMilli-QwaterandtheanimalsoftheGroupIVreceived1,5mgofsaponina.Theanimalswerechallenged30and34daysafterthethirdinoculation,beingput±2000larvaesofR.(B.)microplus (haemoparasites free) and inoculating theB.bovis (strainUFV1-9thpassage-1x106babesias/mL),respectively.Theevaluationof the humoral responsewasmade by ELISA test (l= 492nm) andthedataweresubmitted to theanalysisofvarianceandcomparisonamongaveragesapplying theDunnetTest.Theresultsshowedhighlevelsofspecific total IgG for thepeptideSBbo23290since thefirstweekaftertheinoculation,withtitlesof1,14± 0,2968formonovalentand1,06±0,2486 forpolyvalent form.The titlesofspecific total IgGforSBm7462werepresentinhighlevelsintheeighthweekwith1,75±0,1849 for the monovalent and 1,13 ±0,2819 for polyvalent form.ThehumoralresponseforthepeptideSBbo23290hadhighlevelsofIgG1onIgG2.Itcanbeconcludedthatcrossedresponsesconferredby intraclonal competitions could be determining the immunologicaleffect in theGroups Iand II.The immunologicalprotectiveresponseconferredbythepeptideSBm7462inthepolyvalentformdidnothaveadirecteffect intheproductionofspecificIgG1for thispeptide,andspecific IgG1 for thepeptideSBbo23290showedcrossedprotectionwiththepeptideSBm7462.Key words: Humoral immune response, synthetic immunogens,Babesiabovis,Riphicephalus(Boophilus)microplusSpecies:ruminants

Pr155. BIOLOGICAL PARAMETERS OF TICKS COLLECTED FROM ANIMALS IMMUNIZED WITH PEPTIDES SBBO23290

AND SBM7462 JAVIERAnTônIoBEnEVIDESMonTAño,HUGoGUIEIRoRIBEIRoRoCHA,MARILIAnoGUEIRADAGAMA,VInICIUS

EBCAMPoS,AnnAPAULABRIBEIRoFERREIRA,GABRIELDoMInGoSCARVALHo,FABRÍCIoLUCIAnIVALEnTE,JULIAnA

DELGIúDICEPAnIAGo,JoAqUÍnHERnAnPATARRoyoLaboratoryofBiologyandControlofHaematozoaandVectors,

InstituteofBiotechnologyAppliedtoAgricultureandAnimalScience(BIOAGRO/VeterinaryDepartament),FederalUniversityofViçosa,

36571-000,Viçosa,MG,Brazil

jpatarro@ufv.brThe tick Riphicephalus (Boophilus) microplus, belonging to the

family Ixodidae, is considered the main ectoparasite with economi-cal importance, due to its capacity to causedamages to theanimalhost, through the haematophagy, inoculation of toxins, depreciationoftheleatherandtransmissionofmultiplepathogensasthehaema-tozoa Babesia bovis and Babesia bigemina. In countries of tropicaland subtropical climate the babesioses constitutes a limitant factorfor thedevelopment of the cattle breeding, causingproblems to theanimal health, with high mortality and morbosity, losses in the meatandmilkproduction,andhighcostswith theprophylaxiaandcontrolofthediseases.Theobjectiveofthepresentworkwastoevaluatethebiological parametersof ticks collected frombovinesof theHolsteinbreed 7/8 (Holstein x Gir) which were immunized with the syntheticpeptides SBbo23290 and SBm7462, against B. bovis and R. (B.)microplus,respectively,inthemonovalentandpolyvalentforms.Thesepeptideswerepreparedusingsaponinaasadjuvant,beingappliedviathesubcutaneousroute,individuallyintheleftandrightcervicalarea(Treatment1)andinassociateddosesinthesamesideofthecervical

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area(Treatment2).Eachtreatmentwasappliedthreetimeswithinter-valsof30days.Thechallengewasmade30and34daysafterthethirdinoculation,beingput±2000 larvaesofR. (B.)microplus (strain freefromhaemoparasites)and inoculatingtheB.bovis (strainUFV1-9th

passageintheconcentrationof1x106babesias/mL),respectively.Theengorgedfemaleswerecollectedafterthe21stdayafterthechallenge,weighed individually in precision scale, identified and conditioned incultureplatesof24wells.Theywere incubatedat28ºCandrelativehumidityof80%,and theeggswerecollectedandweigheduntil 15daysafterthelaying.Thedataweresubmittedtotheanalysisofvari-anceandcomparisonamongaveragesapplyingtheDunnetTest.Theresults,inrelationtothereductionoftheeggsweightwere2.5%and8.8%intheTreatments1and2,respectively.TheTreatment2showeda great reduction in the number of the engorged females (32.98%),decrease in the fertility (14.24%) anda efficacy of the immunogens(46.80%). Itcanbeconcluded that theassociated immunogens rep-resent an important tool for the control of thepopulations ofR. (B.)microplus,reducingtheeconomiclossescausedbythisparasite.Key words:Biologicalparameters,tick,peptides,vaccineSpecies:ruminants

Pr156. ExPRESSION OF PROINFLAMMATORy CyTOKINES AND CHEMOKINES IS INCREASED IN TICK-INFESTED SKIN

OF RESISTANT BOVINESARRABATEPAULo,WACARVALHo,DDMoRé,BRFERREIRA,JS

DASILVA,IKFDEMIRAnDASAnToSDept.ofBiochemistryandImmunology,RibeirãoPretoMedical

School,UniversityofSãoPaulo,RibeirãoPreto,SPIntroductionandObjectivesTicksarehematophagousarthropods

thatcauseseriouslossestoanimalandpublichealthanddemandnewcontrolmethods.Vaccinesareonealternativebecausehostsproduceimmuneresponsesagainsttheparasite.Itisnecessary,however,tofirstunderstand themechanisms that participate in protective responsesagainstticks.Bovinehostsexpressheritable,contrastingphenotypesofinfestations,therebeingsusceptible(S)orresistantbreeds(R).Thisworkseekstocomparetheexpressionprofileofgenesthatcodeformoleculesthatarecandidatestomediatetheanti-R.microplusimmuneresponsesthatmayresultinthesephenotypes.

Methods and Results: Genetically susceptible (Holstein; N=5)and resistant (Nelore; N = 5) breeds of bovines were managedin a pasture naturally infested with the cattle tick, Rhipicephalus(Boophilus) microplus, and weekly tick counts were done during 16months.Biopsiesofnormalandinfested(withaningurgitatingfemaletick)skinwerecollectedfromeachanimalatdistincttimepointsandRNA was extracted. Expression was measured by quantitative RT-PCR for the following candidategenes: TGF-β, IFN-γ,TNF-a, IL-4,IL-8,IL-10,IL-18,iNOS,IFN-a,MIP1-aIP-10,IGF-1,MCP-1,IDOandIL5.ExpressionofIGF-1,achemokinethatisselectiveforbasophils,wassignificantlyhigher in the infestedskinofRbovineswhencom-paredwith itsexpression inSbovinesundergoinghighandmediumtickinfestationsbutnot.ExpressionofIL-8,andIDOwassignificantly(P=0,05, t test)higher intheinfestedskinofRbovineswhencom-paredwithexpressioninSbovinesduringperiodsofhighinfestations.ExpressionofMCP-1,MIP1-a and iNOSwashigher in the infestedskinofRbovineswhencomparedwithexpression inSbovinesdur-ingperiodsofhighinfestations(P=0,06ttest),ExpressionoftheotherproinflammatorycytokineswashigherintheinfestedskinofRbovineswhencomparedwithnormalskinofsameanimalandwithinfestedskinofSbovines,althoughthedifferenceswerenotsignificant.

Conclusion:Ourfindingsshowthattickinfestationsmodulatetheexpression of various molecules that may participate in the distinctoutcomesofinfestationsinRandSbovinehosts.Takentogethertheysuggestthatbasophilsarepivotaleffectorcells,andthattheirrecruit-mentismoreefficientinRhosts,inwhichaTH1profiledominatesthemicroenvironmentofthetick’sfeedingsite.

SupportedbyFAPESP,CNPqandValléeSA.Key words: Resistence, Tick, Chemokines, PROINFLAMMATORYCYTOKINESSpecies:ruminants

Pr157. TICK INFESTATIONS AFFECT SUBPOPULATIONS OF PERIPHERAL BLOOD LyMPHOCyTES OF BOTH

SUSCEPTIBLE AND RESISTANT BOVINE HOSTSDDMoRé1,IKDEMIRAnDASAnToS1,AMFRAnZIn1,WACARVALHo1,AAMMAIA2,JSSILVA1,AKSToRSET3,MA

JUTILA4,BRFERREIRA51DepartmentofBiochemistryandImmunology,SchoolofMedicineofRibeirãoPreto,UniversityofSãoPaulo,Brazil;2DepartmentofBasicSciences,SchoolofAnimalScienceandFoodEngineering,UniversityofSãoPaulo,Brazil;3DepartmentofFoodSafetyandInfectionBiology,NorwegianSchoolofVeterinaryScience,Oslo,

Norway;4DepartmentofVeterinaryMolecularBiology,MontanaStateUniversity,Bozeman,USA;5DepartmentofMaternal-ChildNursingandPublicHealth,SchoolofNursingofRibeirãoPreto,Universityof

SãoPaulo,Brazilddmore@usp.br

Successoftickinfestationdependsontheoutcomeofhostdefenseresponses versus parasite escape mechanisms. Bovines expressbreed-specific,heritable,contrastingphenotypeswhenexposedtothecattletick,Rhipicephalusmicroplus.BreedsofBosindicus(i.e.Nelore)aresignificantlymoreresistantthanthoseofBostaurus(i.e.Holstein),whileanimals frompopulationsderived fromcrossesbetween thesegroups showvarying levelsof resistance.Thisoffersagoodoppor-tunity to identify specific mechanisms of response associated withresistance to ticks, once that different breeds develop qualitativelydifferent immuneresponsesagainst ticks. Inorder to identifycellulardifferences between these breeds, 20,000 larvae of Rhipicephalusmicroplustickwereplacedonthebackofsusceptible(n=4)andresis-tant(n=4)animalsandthetickinfestationwasaccompaniedforthreeweeks.Thebloodsampleswerecollectedineachstageofinfestation(larvae, nymph and adult), at one week interval, for flow cytometeranalysis for CD3, CD4, CD8, NKp46, TCR γd and WC1 moleculesexpressionintwoconsecutiveinfestations.Atthelastinfestation,thebrachiallymphnodesalsowerecollectedforthesameanalysis.Theresultsofperipheralbloodshowedthat,duringthefirstinfestation,thepercentage of CD4+ cells in adult-infested susceptible animals wasaugmented two fold relative to larvae- and nymph-infested bovines.Moreover,CD8+cellsweresignificantlyincreasedduringtheinfestationwithnymphsinbothbreedswhencomparedwithotherstagesofthelifecycle(P=0.004inHolsteins;P<0.001inNelores).Additionally,thepercentageofp46NK+cellswashigherinlarvae-infestedsusceptibleanimalsthaninlarvae-infestedresistantanimals(P=0.004).Duringthelarval and nymph stages, two time tick-infestedHolsteins presenteda significant reduction (P<0.001) on thepercentageof p46NK+ cellswhencomparedtothefirst infestation,whileduringthenymphstagetwotimetick-infestedNelorespresentedadecreaseinCD8+lympho-cytes(P=0.004).Nodifferencesinlymphnodescellpopulationswereobserved.Takentogether, theseresults indicate that tick infestationsaffecttheblood-lymphocytepopulationsinbothresistantandsuscep-tiblecattlebreeds.Support:CNPq,FAPESP,Valleé.Key words:Tickinfestation,Cattle,LymphocytesSpecies:ruminants

Pr158. TLR ExPRESSION IN BOVINE MONOCyTES DERIVED FROM CATTLE BREEDS WITH DIFFERING

SUSCEPTIBILITy TO TROPICAL THEILERIOSISLARoWAn,GDMAKInS,LSMITH,KJEnSEn,EJGLASS

RoslinInstitute,Roslin,Midlothian,EH259PS,UKTheileriaannulataisanapicomplexanparasiteofglobaleconomic

importance. It is thecausativeagentof tropical theileriosis,which isadebilitatingdiseaseofcattle.It isspreadbyticksoftheHyalommaspecies and covers areas from the Mediterranean basin to China.EuropeancattlesuchasHolstein-Friesians(Bostaurus)areverysus-ceptible to tropical theileriosisand themortality ratesamongst thesebreeds is between 40 and 90%.However, several breeds native toendemicareas,suchastheSahiwalcattle(Bosindicus)fromPakistan,aretoleranttothedisease.

T. annulata primarily infects bovine macrophages, which arereversiblytransformedcausingthemacrophageandparasitetopro-liferate insynchrony.Asa resultof this transformationex-vivo cell

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lines,derivedfromexperimentalinfectionofcattle,havebeengrownin culture. Infection with T. annulata alters the function and phe-notype of the macrophage, for example, the pathogen recognitionreceptor (PRR)CD14,which is involved inmacrophageactivation,isdown-regulated. InordertobetterunderstandtherolesofPRRsduring infectionwithT.annulatawehave investigated theexpres-sionofToll-like receptors (TLRs) in resting,activatedand infectedmonocytes.

Using ex-vivo cell lines we have shown that all 10 TLRs areexpressedduringT.annulatainfectionandacorrelationbetweenpro-inflammatorycytokinesandTLRexpressionhasalsobeenobserved.Abovinemacrophage-specificcDNAmicroarrayhasbeenconstructedtoinvestigatebreed-specificdifferencesinSahiwalandHolsteinmono-cytesduringinfectionwithT.annulataandafteractivationwithlipopoly-saccharideand interferon-γ.Analysisof themicroarraydatashoweda breed-specific difference in TLR10 expression during monocyteinfectionandactivation. InSahiwalmacrophagesTLR10expressionisup-regulatedafterinfectionandactivation,whiletheHolsteinmacro-phagesslightlydown-regulateTLR10atthesametimepoint.

Additionalstudiesareinprogresstoinvestigatefurthertheimpor-tanceofTLRsandpro-inflammatorycytokinesandtoelucidatefurthertherolestheyplayduringT.annulatainfection.Species:ruminantsKey words:macrophage,protozoan,toll-likereceptor,cytokine

Pr159. MODULATION OF THEILERIA PARVA POPULATIONS By THE BOVINE IMMUNE RESPONSE

FRAnKKATZER1,2,DAnIELnGUGI2,ALAnRWALKER2,DECLAnJMCKEEVER1,2

1TheMoredunResearchInstitute,Pentlands,SciencePark,BushLoan,Penicuik,Midlothian,EH260PZ,UK;2CentreforTropicalVeterinaryMedicine,EasterBushVeterinaryCentre,Universityof

Edinburgh,EasterBush,Midlothian,EH259RG,UKTheileria parva is a tick-borne intracellular protozoan parasite

that causes a severe and often fatal lymphoproliferative diseaseknown as East Coast fever in cattle in sub-Saharan Africa. Theprotective immunity of the bovine host against T. parva is medi-ated by parasite-specific class I MHC restricted CD8+ cytotoxic Tlymphocytes. Thisresponse is tightly focused in individualanimalsrecognising only a few antigenic determinants. So far ten parasiteantigens, seen by the bovine CTL response, have been identifiedandalmostallof theseproteinsarepolymorphic.Thetightfocusoftheimmuneresponsecouldpermittheparasitetoevaderecognitionthrough antigenic diversity and by shuffling of antigen variants viarecombination.Hereweprovideevidencethattheimmuneresponsecanspecificallyeliminateapredominantparasitegenotype,whichitwasprimedagainst,duringan infectionwhilepermittingminorandantigenicallydistinctgenotypestoestablishinfectionandtotransmitto the tick host. Experimental evidence for breakthrough parasitepopulationswasobtainedbyimmunisingcattlewithclone72-01andchallengingthemwithstabilate72.Thisparticularstabilatecontains72-01 as a dominant parasite genotype, which makes up 75% ofparasite numbers. All calves showed mild levels of infection andself curedalthoughpiroplasmscouldbedetected.Characterisationofbreakthroughparasitepopulationsrevealedthatclone72-01waseliminatedduringinfectionanditsgenotypewasnolongerpresentinthedaughter stabilate.Wealso foundevidence that recombinationfrequentlyoccursinthetickhost,whichallowsshufflingofantigenicdeterminants. The resulting recombinant parasites, with reshuffledantigens,willhaveagreaterchanceofsurvivalinsubsequentroundsofinfectionofcattlewhichhadbeeninfectedpreviouslywithdifferentT.parvagenotypes.Key words:Theileria,CTLresponse,Antigenicity,RecombinationSpecies:ruminants

Pr160. THE BOVINE CD8+ T-CELL RESPONSE TO THEILERIA PARVA DISPLAyS EPITOPE IMMUNODOMINANCE

AND OLIGOCLONALITy TConnELLEy1,nMACHUGH1,SGRAHAM2,ETARACHA2,A

BURRELLS1,DnGUGI1,WIMoRRISon1

1DivisionofClinicalVeterinarySciences,UniversityofEdinburgh,Edinburgh,United-Kingdom;2InternationalLivestockResearch

Institute,P,O,Box30709,Nairobi00100,KenyaTheileriaparvaisanintracellularprotozoalparasitewhichcauses

EastCoastFever(ECF),amajorconstrainttocattleproductionineast-ernandsouthernAfrica.Characteristicsof theprotectiveCD8+T-cellresponseagainstT.parvasuggestitisfocusedonalimitednumberofimmunodominantepitopesthatexhibitpolymorphismbetweenparasitestrains.The recent identificationofantigens recognisedbyT.parva-specificCD8+T-cellshascreatednewopportunitiesinthedevelopmentofasubunitvaccine,andpermittedthespecificityoftheCD8+responsetobeexamined.Cyotoxicityassayswithpeptide-loaded targets,andanalysesofTCRBVgeneusagehavebeenusedtostudytheantigenspecificityandclonalcompositionoftheCD8+memorypopulationinT.parva immuneanimals.Analysesof largepanelsofT.parva-specificCD8+ T-cell clones from memory T-cells of immune animals homo-zygous for MHC haplotypes expressing either theA10 orA18 classI specificities, revealed that over 65%of the responsewasdirectedagainstasingle immunodominantepitope(Tp1.1 forA18andTp2.2.forA10).TCRB-chainsequenceanalysisshowedthatforeachimmu-nodominant epitope the antigen-specific T-cells were polyclonal butdominatedbylargeoligo-clonalexpansions.TheseclonalexpansionsutilizeddifferentVBgenesegments indifferentanimals. Importantly,usinganewlydevelopedbovineTCRB-CDR3heteroduplexassay, ithasbeenpossibletoidentifythesamelargeT-cellclonalexpansionsinvivo followingchallengewithT.parva, indicatingthat the immuno-dominantpopulationsofCD8+T-cellsobservedininvitroculturesarerepresentativeof thememorypopulationpresent in vivo. Thisworkprovides the first definitivedemonstrationof immunodominance in aCD8+T-cellresponsetoaprotozoanparasite,providinganexplanationfortheincompletecross-protectionbetweenT.parvastrainsandhasimportantimplicationsforthedesignofasubunitvaccine.Key words:Theileria,Immunodominance,T-cellsSpecies:ruminants

Pr161. TISSUE ESTABLISHMENT OF L3 HAEMONCHUS CONTORTUS LARVAE IS PREVENTED IN HIGHLy

SENSITISED SHEEPJMKEMP,nRoBInSon,EnTMEEUSEn,DMPIEDRAFITAAnimalBiotechnologyResearchLaboratories,MonashUniversity,

Clayton,AustraliaHaemonchuscontortus(Hc)isabloodsuckingnematodeofrumi-

nants,residingintheabomasum(fourthstomach).Itisresponsibleforsignificant losses inanimalproductionanddeathof stock. ImmunityagainstHcdoesestablishovertime,howevertheimmunemechanismsbehindnematodedeathand/orrejectionarenotyetfullyelucidated.Inorder toexplore thesemechanisms,weareusinganexvivo larvalestablishment assay (LEA) which we have adapted for Hc. SheepweresensitisedwithhighorlowdosesofHc,givenoneinfectingdoseofHcorleftnaïve.Theanimalswereeuthanizedandabomasalfoldsremovedandused in theLEA.The tissueexplantswerechallengedwithL3larvaeandthenumberoflarvaethatpenetratedthetissuewascounted.Theabilitytopreventlarvalestablishmentinthetissuewasdependent on previous exposure to the parasite.Thus those sheepthat had been repeatedly sensitised (either high or low dose) hadsignificantly lower larvalestablishment than those thathadonlyonechallenge.Thelowerlarvalestablishmentwasassociatedwithasignifi-cantlygreaternumberofglobuleleukocytesandmastcells,andtheiractivityiscurrentlyunderinvestigation.Key words:Haemonchuscontortus,larvalestablishment,mastcells,globuleleukocytes.Species:ruminants

Pr162. EFFECTS OF HAEMoNCHUS CoNToRTUS On THE HUMORAL AND CELLULAR IMMUNE RESPONSE OF

PARASITE-RESISTANT HAIR SHEEPKATHRynMMACKInnon,ISISKMULLARKy,DAVIDRnoTTER

VirginiaTech,Blacksburg,Virginia,USAkmackinn@vt.edu

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Among sheep producers, the parasitic nematode Haemonchuscontortusisamajoranimalhealthconcern.Caribbeanhairsheepareknown to bemore resistant to this abomasal parasite than conven-tionalwoolsheep.Ourobjectivewastodeterminedifferencesingeneexpressionassociatedwithparasiteresistancebetweenresistanthairandsusceptiblewoollambs.Toaddressthisobjective,12hairand12wool lambswereeach infectedwith10,000H.contortus larvaeand14animalsofeachbreedwereleftasuninfectedcontrols.Fecaleggcounts(FEC)weremeasuredatday0,16,21and27inallanimalstoassesswormburden.SusceptiblewoollambshadhigherFECcom-pared to resistant hair lambs throughout the study. Total RNA wasextracted fromabomasumandabomasal lymphnode tissues. Afterreversetranscription,geneexpressionofTh1andTh2cytokines,cyto-kine receptors andantibodieswereevaluatedby real-timeRT-PCR.Geneexpressionpatternswererelativelyconsistentbetweenaboma-sumandlymphnodetissues,indicatingpotentialcoordinationoflocalandhumoralimmuneresponsesduringinfection.Comparedtounin-fected control animals, infected sheephaddecreasedexpressionofIFN-γ in lymph node tissue and increased expression of IL-5, IL-13and IgE in both tissues. However, even though Th2-type mecha-nisms seemed to be in place by this time, there was no differencein expression level of the cross-regulatory cytokine IL-4. At 3 dayspost-infection,resistanthairsheephadgreater(P<0.01)expressionofIgEinlymphnodetissuewhencomparedtowoollambs.By27dayspost-infection,resistanthairlambshadlower(P<0.10)expressionofIFN-γ,andhigher(P<0.10)expressionofIgEandTh2cytokineIL-13in both tissues as compared to susceptible wool lambs. The meanlevelofexpressionof IL-4 receptorαdidnotdifferbetweenbreeds,tissuesanddays,butindividualexpressionlevelsofthisreceptorwerehighly correlated (-0.98)withFECat 27dayspost-infection. Theseresultssuggestthatgastrointestinalnematodeinfectioninresistantascompared to susceptible sheep elicits a modified Th2-type immuneresponse,characterizedbydecreasedIFN-γ,steady IL-4expressionandincreasedIL-13andIgE.DifferentialregulationofTh2cytokinesbetweenbreedsmaybepartiallyresponsiblefordifferencesinparasiteresistance.KEy WORDS: Haemonchus contortus, cytokine, abomasum, lymphnodeSpecies:ruminants(sheep)

Pr163. CyTOKINE QUANTIFICATION IN ABOMASAL LyMPH NODE OF BOVINE INFECTED WITH HAEMoNCHUS SPP

PARASITES ADRIAnAMGIBELLI1,LILIAnECnAKATA,RoGéRIoAnDRéo,

MáRCIACSoLIVEIRA2,LUIZLCoUTInHo3,LUCIAnACAREGITAno2

1ProgramadePós-GraduaçãoemGenéticaeEvolução,UniversidadeFederaldeSãoCarlos,SãoCarlos,SP,Brasil;2

EmpresaBrasileiradePesquisaAgropecuária,CentrodePesquisaPecuáriadoSudeste,SãoCarlos,SP,Brasil;3EscolaSuperior

AgrícolaLuizdeQueiroz,UniversidadedeSãoPaulo,Piracicaba,SP,Brasil

adriana.ibelli@gmail.comInfections caused by gastrointestinal parasites represent great

losses inanimalproductionall over theworld, affectingbothbovineandovine. In theBraziliansoutheast,wormswithhigherprevalenceinbovinearefromHaemonchussppandCooperiaspp.Theseendo-parasites are responsible for several immune responses, innate aswellasacquiredby thehost.Thecytokinespresentacentralpaperin the modulation of the immune response, including lymphocytesactivation,proliferation,differentiation,cellularapoptosisandTh1/Th2polarization. Some of them, as for instance, IL-12, IL-8 andMCP-1mediate innate immunity recruiting lymphocytes to the inflammationsites.Another aremore relatedwith theacquired immunity, as IL-2,theprincipalfactorofTcellsgrowthandIL-4,thatstimulatesIgEpro-duction. The objective of this project was to verifymessenger RNA(mRNA)abundanceofthecytokinesIL-2,IL-4,IL-8,IL-12,IL-13,IFN-γ,MCP-1andTNF-ainabomasallymphnodesofBosindicussubmit-ted tofirst infectionwithHaemonchusspp.Tencalvesof theNelorebreed were maintained without parasitic infections since they wereborn.Thoseanimalsweresplit in twogroups:5calvessubmitted totheartificial infectionwithHaemonchusspp larvae(challengegroup)

and5animalswithoutinfection(controlgroup).Abomasallymphnodeswerecollectedsevendaysaftertheinfection,totalRNAwasisolatedusingTrizolreagentandcDNAwassynthesizedbyreversetranscrip-tion.Cytokinequantificationwasevaluatedby theRT-PCRreal timetechnique,usingthehousekeepinggeneRPL-19ascontrolandSYBRGreenasflorescentlabel.ResultswereanalyzedbythesoftwareREST(RelativeExpressionSoftwareTool).DifferencesofmRNAabundancewereobservedfortheinterleukingenesIL-4andIL-13(P<0,05).IL-4was15timesup-regulatedinchallengegroupincomparisontocontrolgroupandIL-13wasup-regulated inchallengegroupby26times incomparisontocontrolgroup.Fortheothergenes,nodifferenceswereobservedbetweenthechallengeandcontrolgroupsanalyzed.Itcanbeconcludedthat, for theanalyzedexperimentalconditions, thefirstinterleukins tobestimulatedandproducedafterprimary infectionbyHaemonchussppareIL-4andIL-13.TheseresultscorroborateseveralstudiesthatindicatecytokinepolarizationTh2ininfectionscausedbyendoparasites.Inordertoobtainabroaderviewofthebovineimmuneresponseofthisparasite,othergenesrelatedtotheimmunesystemofthehostwillbeselectedand,later,analyzed.Key words:Haemonchusspp,geneexpression, cytokine, real timePCRSpecies:ruminants

Pr164. EFFECT OF ANTHELMINTHIC COMBINED WITH IMMUNOSTIMULANT ON GASTROINTESTINAL SHEEP

NEMATODESPDEnAPoLI1,SCHEn1,CFGARCIA1,FETonIAZZI1,JSMUSSALEM2,CCSqUAIELLA2,ILonGo-MAUGERI2,RJ

SHAW3,LCJABEL11CursodeMedicinaVeterinária-UniversidadePaulista-UNIP.RuaDrBacelar1212,SãoPaulo-SP05726-100;2Disciplinade

ImunologiadoDeptodeMicrobiologia,ImunologiaeParasitologiadaUniversidadeFederaldeSãoPaulo-SãoPaulo-Brasil3;

3WallacevilleResearchCentreWardStreet,POBox40-063,UpperHutt,NewZealand

luciaabel@uol.com.brThefailureofanthelminticstocontrolnematodeparasitesin

sheep isaworldwidephenomenonof rapidly increasingdimensions.Development of alternative strategies and immunological methodstocontrol theparasites isneeded.Thepurposeof thisstudywas todetermine the effectiveness of an immunostimulant combined withanthelmintic on experimental helminthiasis through IgE analysis, Tcell proliferation assay and fecal egg counts (FEC). Crossbred 12-month-oldmalesheepweregroupedinto4groups:I-animalstreatedwithLPSandPropionibacteriumacnesandanthelminthic;II-animalstreated with Propionibacterium acnes andanthelminthic; III- animalstreatedwith only anthelmintic; and IV- animals treatedwith a sterilesalinesolution0.9%,asuntreatedcontrols.All theanimals received10,000 infective larvae (L3) orally on day 0 and were treated withwhenthefecaleggcountspeakedat35days;thatis,thetreatmentwasadministeredonthe35th,63rdand67thdaysafterinfection.Fecalandbloodsampleswerecollectedweeklyforfecaleggcounts(FEC),IgE antibodies detection and T cell proliferation assay. The resultsshowedthatPBMCfromgroupI,II,IIIdisplayedenhancedproliferationresponse toConcanavalina -Awithmaximal responseobservedonday28and35withstimulationindicessignificantlyhigheringroupIIthangroupIV(p=0.0121).

Duringtheinfection,theanimalsfromgroupIandIIhadhigherIgE antibody levels in serum than other groups (p<0.05). Significantreduction in the fecaleggcounts,determined42daysafter infection,wereobserved ingroups I, II and III comparedwithgroup IV (I x IVp=0.0083;IIxIVp=0.0081andIIIxIVp=0.0013).TheseresultssuggestthattheanthelminthiccombinedwithimmunostimulantcanbeeffectiveforthetreatmentofgastrointestinalsheepnematodesbystimulationofIgEantibodiesandunspecificimmuneresponse.Key words:helminths,sheep,Propionibacteriumacnes,anthelminthicSpecies:ruminants(sheep)

Pr165. ADJUVANT EFFECT OF LPS AND PRoPIoNIBACTERIUM ACNES ON ExPERIMENTAL

GASTROINTESTINAL NEMATODE INFESTATION IN SHEEP

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SCHEn1,LGRICCA1,MFMARTInS1,MGARCIA1,RZAnAnIAS2,JSMUSSALEM2,CCSqUAIELLA2,ILonGo-

MAUGERI2,LCJABEL1CursodeMedicinaVeterinária-UniversidadePaulista-UNIPRuaDrBacelar1212,SãoPaulo-SP05726-100;2Disciplinade

ImunologiadoDeptodeMicrobiologia,ImunologiaeParasitologiadaUniversidadeFederaldeSãoPaulo-SãoPaulo-Brasil

luciaabel@uol.com.br Gastrointestinalhelminthic infection isoneof themost

important sheep diseases worldwide which can cause anemia,anorexia, depression, progressive weight loss and eventual death.Theemergenceof anthelmintic resistance in sheephas led to seeknewtherapeuticalternativestocontrolhelminthiasisinsheep.Recentreports demonstrated that LPS and Propionibacterium acnes haveanadjuvanteffect intheinnateandacquiredimmuneresponse.TheoverallaimofthisstudywastoevaluatetheadjuvanteffectofLPSandPropionibacteriumacnesintheinductionofexperimentalhelminthiasisinsheep.

Crossbredmalesheep,12-18monthsold,weredividedintotwogroups.GroupI,receivedLPSandPropionibacteriumacnesandgroup II receivedsterilesaline0.9%.Bothgroupswere infectedwith10,000infectivelarvae(L3)orallyonday0.Parasitological,hae-matologicalexaminationsandTcellproliferationassayweremadeondays0,14,21,28,35after infection.The results showed that fecaleggcountsweresignificantlyloweringroupIthangroupII(p<0.05).Themeanfecaleggcountreachedapeakat35days.Thehemato-logicalparametersshowedanincreaseintheleucocytes,eosinophils,lymphocytesatthe14th,21thand35thdayintheanimalsfromGroupIItreatedwithimmunostimulantwhencomparedtoGroupI(p<0,05)andtheresponseofPBMCtomitogen(Conc-A)werehigherintheanimalsfromGroupII(p<0,05).

Our findings suggest that immunostimulants can be used asa strategy to control helminthiasis in sheep.A significant correlationbetween theLPSandPacnesadministrationandnumberofwormswasalsoobserved.Key words:helminthiasis,lymphocytes,adjuvant,sheepSpecies:ruminants(sheep)

Pr166. INHIBITION OF HAEMoNCHUS CoNToRTUS HEMOGLOBINOLyTIC ACTIVITIES By N-ALKyL FERULATES

FROM MAPRoUMEA GUIANENSISSÍLVIACoSAnToS1,JUCEnIPDAVID1,JoRGEMDAVID2,

JoRGEALóPEZ1,FARoUKZACHARIAS3,MARIATBGUEDES3,FERnAnDAWDEMEnDonçALIMA1

1FaculdadedeFarmácia-UFBa;2InstitutodeQuímica-UFBA;3EscoladeMedicinaVeterinária-UFBa4EmpresaBaianade

DesenvolvimentoAgrícola-EBDAIntroduction and objectives:Haemonchus contortus is a blood-

suckingnematodeoccurringinthefourthstomachofsheepandotherruminants.TheH.contortusL4larvaeandadultscauseconsiderabledamagetothemucosalliningoftheinfectedsheepabomasum,result-inginextensivehemorrhagesandseverechronicanemia.ProteinasesfromadultH.Contortusdegradehemoglobin(Hb)andthisdegradationis, inpart,due tocysteine-andasparticproteinases, but,probably,alsobymetallo-andserineproteinases (McKerrow,2000;Redmond&Windham,2005).Inthepresentstudy,weinvestigatedtheinhibitoryeffectonhemoglobinolyticactivityofH.Contortusproteasesindifferentproductsobtainedfromsemi-aridnativeplantsofBahia,Brazil.

Materialandmethods:Forstandardizationofthenematodehemo-globinolyticactivities,lysates(8mg/mLofprotein)wereincubatedwithasolutionofgoatHb10.5mg/mLat37°Covernightinacitratephos-phatebufferpH5.0with2-ME2mM,inafinalvolumeof500uL.ControlofHbwasdonebyincubatingtheproteinwithoutlysate,inthesameconditions. The products of hemoglobin hydrolysis were electropho-resedon15%SDS-PAGEandgelswerestainedwithcomassieblue(Lewis,1999).Inhibitionassayswereperformedbymixingtheparasitelysate (72ug/mLprotein)withdifferentproducts isolated fromnativeplants.Positivecontrolofinhibitionwasachievedusingastandardcys-teineproteaseinhibitorE64.AlysatesampleatthesameconcentrationwasalsoanalysedbySDS-PAGEundersameconditions.

Resultsandconclusions:ProteasesofH.contortuslysatedigestcompletely polypeptide chain of goat Hb. This effect is abrogate byE64, which indicate that probably the involved proteolytic enzymesbelongtotheclassofcysteineproteases.Amongthetestedplantprod-uctsonlyaninseparablemixtureofN-alkylferulatesfromMaproumeaGuianensis(Euphorbiaceae)showedaninhibitoryeffectonhemoglobi-naseactivities,inadosedependentmanner,sugestingaspecificityforcysteineproteinases.Supportedby:BNB-FUNDECI;FAPESB-PIBIC;UFBA.Key words:Proteases,Haemonchus,hemoglobinolytic,Maproumea,n-alkilferulates.Speciez:ruminants

Pr167. PROTECTIVE EFFECTS OF BRAZILIAN NATIVE PLANTS 0N GOATS NATURALLy INFECTED WITH

HAEMoNCHUS CoNToRTUSRoBERToRBDoSSAnToS2,JUCEnIPDAVID1,JoRGEMDAVID4,MARCUSVBAHIA4,SÍLVIACoSAnToS1,FARoUK

ZACHARIAS3,MARIATEREZABGUEDES2,FERnAnDAWDEMEnDonçALIMA1

1FaculdadedeFarmácia-UFBA;2EscoladeMedicinaVeterinária-UFBA;3EmpresaBaianadeDesenvolvimentoAgrícola-EBDA;

4InstitutodeQuímica-UFBAIntroductionandObjectives:Plantproductsareknowntoexhibit

medicinalvalueagainstdiseases(Desaietal,2002).Ourearlierstud-ies showed that crude extract of two Leguminosae plants (CratyliamollisandCaesalpiniapyramidalis)wereeffectiveon inhibitionofH.contortusparasitism innatural infectedgoats. Inorder to investigatea possible immunomodulator role, the humoral immune responsewasevaluated in treatedanduntreatedanimalswith theseaqueousextracts.

AnimalsandMethods:Sixtyanimalsweresubdividedinfivegroupsoftwelveeach,being:Group1(G1)negativecontrol;G2positivecon-trol for anti-helminthic action (Doramectin 1mL /50kg, Dectomax®,Pfizer);G3receivedextractfromC.mollisintheconcentrationof2.5mg for kg/bodyweight,G4andG5 receivedC.pyramidalis extracts(2,5mg/kgand5mg/kgbodyweight,respectively).Theanimalshadbeenkeptonthesamebreedingconditions,inanextensiveregimenof pasturing.Blood samples of 5mL of each animalwere collectedfromthejugularvein,andtheirserumweretestedforconcentrationofspecificIgGanti-H.contortus.TotalIgAandIgGwasalsoquantifiedbyusingacommercialKitofsandwichELISA(Bethyl,Inc,U.S.).

Results and conclusions: G5 animals showed the best resultsof specific humoral immune response,with a significant variation of33.3%overthebaselineconcentrationofanti-H.contortusantibodies.ThehighestconcentrationsofIgAweredetectedinsamplesfromG4andG5.TheconcentrationsofG5were581ng/mLon thebaselinemoment,615ng/mLon30daysand575ng/mL60days later.WiththeexceptionofvariationobservedforG4,thatshowedabiggestIgAconcentrationafter60daysoftreatment,being367ng/mLbeforetreat-ment,400ng/mLon30daysand411ng/mLon60daysaftertreatment,remaininggroupshadaincreasingconcentrationvaluesfrombaselineuntil30daysanddecreasingconcentrationson60days.TheobservedIgGconcentrationswererisingonlyinG4,whichvaluesrangedfrom412ng/mLbefore treatment to451ng/mLafter60dayof treatment.Weightgain in the treatmentgroupswassuperior to thecontrol thatreceivedonlysaline,beingG290%,G310%,G450%andG520%,respectively. Although the need of evaluating others immunologicalaspectstoexplaintheimprovedresistancetoH.contortusontreatedgoatsofG4andG5,inthisworkitwasdemonstratedsomeprotectiveeffectexhibitbythetestedplantsonthecontrolofthisinfection.

Supported by: BNB-FUNDECI; FAPESB; EBDA-SEAGRI-BA;UFBA.Key words:Cratyliamollis eCaesalpiniapiramidalis,Haemonchuscontortus,goats.Specie:ruminants

Pr168. TRANSFER OF MATERNAL HUMORAL PASSIVE IMMUNITy TO KIDS IN GOAT HERD AGAINST HAEMoNCHUS

CoNToRTUS INFECTION

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MARIATEREZABGUEDES2,FARoUKZACHARIAS3,KELyCPEDRoZA1,SILVIACARoLInEoSAnToS1,RoBERToRBDoS

SAnToS2,FERnAnDAWDEMEnDonçALIMA11FaculdadeDeFarmácia-UFBA;2EscolaDeMedicinaVeterinária-UFBA;3EmpresaBaianaDeDesenvolvimentoAgrícola-EBDAIntroductionandobjectives:InfectionwithHaemonchuscontortus

represents the main cause of economic loss in caprine breeding intropicalandsubtropicalareasoftheworld(Meloetal,2003).Theaimof thisstudywas toevaluate thehumoral immune responseofgoatkids,beforeandafternaturalcolostrumsingestion,againstH.contor-tus.Theefficiencyofmaternal immunitytransferandits influenceonkids’healthwasalsoinvestigated.

Animalsandmethods:Thirtynewbornkidswereweightedandhadbloodsamplecollectedbeforecolostrum ingestionandafter30,60,120,180and260daysafteradlibitumcolostrumandmilkinges-tion.Thetwentyfourdamswerealsoclinicalexaminedandhadsampleofbloodandfecescollected.TheIgGclassantibodiestoherd-homolo-gous strains of H. contortus were estimated using ELISA of serasamplesofdamsandseraofkids.Thefaecaleggcountandcultureforhelminthesinkidsanddamsfaecalsampleweredone.

Resultsandconclusion:Hereweareshowingforthefirsttimethenatureofmaternal transferof immunityagainstH.contortus ingoat.Firstofall,everyserumsamplefromkidsbeforecolostrumsingestionhadnegativeresultsforantibodiestoH.contortus.TheELISAoftheserumsamples collectedon thatmoment gaveoptical density (DO)alwaysunder0.04nm.However,resultsobservedwithserumsamplecollectedonsecondandthirdmomentaftercolostrumandmilkinges-tion, were superior to 0.867 + 0.735 DO. None of sample collectedaftercollostrumingestionwasinferiorto0.17DO.Thefaecalexamina-tionsofthekidshadbeenpresentednegativeforStrongiloydea,whatit iscompatiblewiththemomentofthiscollection,theseanimalsarefedonlysuckling theirdams, thereforehadnothadcontactwith thegrass.The results of thematernal faecal culture identified1.85%ofHaemonchus, but in faecal sample kids it was of 0%.On averageuntil the present moment, the weight among in the studied animalsvariedfrom2.7kgofnewbornupto7.3kg.Accordingtotheresultsofthepresentwork,wecanpredictthebestmomentformanagementofprophylacticsoranthelmintic intervention.Earlystudieshavealreadypointedthepassivetransferofimmunitytroughthecolostrumingoats,buttheydidnotspecifiedtheimmuneresponseagainstH.contortusinfection(Simõesetal,2005)Supportedby:BNB-FUNDECI;EBDA-SEAGRI-BA;UFBA.Key words:Haemonchus,passiveimmunity,colostrums,antibodies.Species:ruminants

Pr169. INTERFERON GAMMA PRODUCTION AND PROTECTIVE EFFECTS AFTER NEoSPoRA CANINUM TACHyZOITE ACTIVATION OF BOVINE SPLEEN CELLS

DIAnABACIGALUPE,MARÍACVEnTURInI,GASTónMoRé,JUAnMUnZAGA,ALEJAnDRALARSEn,WALTERBASSo,LUCILA

VEnTURInILaboratoriodeInmunoparasitología,FacultaddeCienciasVeterinarias,UniversidadNacionaldeLaPlata,Argentina.

dianab@fcv.unlp.edu.arneospora caninum is a major cause of abortion in bovines

throughout the world. Protection from disease is primarily given bycellular immunity, and interferon gamma (IFNγ) is one of the mostrelevant cytokines for protection. Inorder todetect apossible IFNγ-mediatedprotectiveeffect,bovinecellswerestimulatedwithdifferentneosporacaninumantigens.Bovinespleencellsfromcowsserologi-callynegativeton.caninumwereculturedandstimulatedwithliven.caninumtachyzoites(groupI),formalin-heat-inactivatedtachyzoites(groupFI),andatachyzoitelysateobtainedthroughsonication(groupSI), leavinguninfectedcellsasanegativecontrol.AnELISA test forIFNγ(Bovigamtm)wasperformedonsupernatantsfromtreatedcellscollected at days 3, 7 and 10 after inoculationwith negative resultsinalltreatments.Inaddition,stimulatedandcontrolspleencellswerestainedwithGiemsa.Typicalactivationmorphologywasobserved incells treated with live tachyzoites and lysate, while those that weretreatedwith inactivated tachyzoites remainedsimilar to thenegative

control. In addition, supernatants from each treatment were placedonmonocytebovinecells(BM)thatwerelaterinoculatedwithliven.caninum tachyzoites. Lysis plaque formation was evaluated on theBMcellmonolayerusingImageProplussoftware.Significativediffer-enceswereobservedamonggroups(x2p<0.001).SupernatantfromcellsgroupI,FIandSIhadaprotectiveeffectonBMcells.Also,INF-γmRNAexpressionwasevaluatedbyRT-PCR,revealingINF-γexpres-sionincellsfromgroupI,aswellasingroupSI.It isconcludedthattachyzoitelysatecombinedwithanappropriateadjuvantmayprovideavaluabletoolfordesigningprotectionstrategiesforneosporosis.Key words: Neospora caninum, gamma interferon, antigen, spleencellsSpecie:ruminants

Pr170. BOVINE NATURAL KILLER CELLS ACT AS PRIMARy RESPONDERS IN THE EARLy STAGES OF NEOSPORA

CANINUM INFECTED CATTLESKLEVAR1,SKULBERG1,PBoySEn2,ASToRSET2,T

MoLDAL1,CBJØRKMAn3,IoLSEn11DepartmentofAnimalHealth,NationalVeterinaryInstitute,P.O.

Box,8156Dep.,N-0033Oslo,Norway;2DepartmentofFoodSafetyandInfectionBiology,NorwegianSchoolofVeterinaryScience,P.O.Box,8146Dep.,N-0033Oslo,Norway;3DepartmentofClinicalSciences,SwedishUniversityofAgriculturalSciences,P.O.Box

7054,SE-75007Uppsala,SwedenThe intracellular protozoan parasite n. caninum is a cause

of abortion and congenital diseases in cattle world wide. We havepreviouslyshown thatNKcellscanproduce IFN-γ in response ton.caninumtachyzoitesinvitro.Thisstudyaimedtoinvestigatecellularimmune responses in an experimental infection model in cattle andinparticulartheroleofNKcellsinearlyinnateimmuneresponses.Ininfectedcalves,thepercentageofNKcellsinblooddroppedatday4-6after inoculation followedbyan increase in thepercentageofCD8+Tcellsonday7andthenanincreaseofNKcellsaroundday12.Byusingawholebloodflow-cytometricassaytoidentifyIFN-γproducingcellsweobservedthatNKcellsandCD8+T-cellswhereanimportantsourceofIFN-γintheearlystagesofinfectionwhiletheCD4+T-cellsdominated lateron.Wealsocompared theabilityof twodifferentn.caninum antigen preparations, i.e., sonicated soluble antigens andintactheat-inactivatedparasites,toinduceproliferationandIFN-γpro-ductioninvariouscelltypes.Theheatinactivatedtachyzoitesinduceda3.7timeshigherincreaseinthenumberofIFN-γproducingNKcellscomparedtothesonicatedsolublefraction.ThisindicatesthepresenceofNKcellstimulatingantigensintheintacttachyzoite.WealsofoundthatheatinactivatedwholetachyzoitesclearlyinhibitedγdT-cellsprolif-erationwhilethesolubleantigensfromn.caninumdidnot.ForthefirsttimewedemonstratetheroleofbovineNKcellsasprimaryrespondersinancaninuminfectionincalves,andadditionallyadistinctdifferencein the effect of heat-inactivatedn. caninum tachyzoites and solubleantigensontheIFN-γproductionfromNKcellsandproliferationofγdT-cells.Key words:NEOSPORACANINUM,NKcells,gdT-cellsSpecies:ruminants

Pr171. INTERLEUKIN-4 DOWNREGULATES THE GOAT BETA-DEFENSIN-2 GENE IN CAPRINE INTESTINAL EPITHELIAL CELLS INFECTED WITH EIMERIA SPPFRoyLAnIBARRA-VELARDE,yAZMInALCALA-CAnTo

DepartamentodeParasitología.FacultaddeMedicinaVeterinariayZootecnia.UniversidadNacionalAutónomadeMéxico

yazmin@servidor.unam.mxDefensinsareantimicrobialpeptidesproducedbyleukocytesand

epithelialcells.Thesepeptideshavebeenshowntoplayanimportantrole in innate immune responses.However, the role of defensins ingoateimeriosis remainsunknown.Therefore, this study investigatedthe expression of the goat beta-defensin named GBD-2 in caprineintestinal epithelial cells (CIEC) stimulated with recombinant bovineinterferon-gamma(IFN-gamma)inthepresenceorabsenceofrecom-binantbovineinterleukin-4(IL-4)byareversetranscriptase-polymerasechainreaction(RT-PCR)assay.GBD-2mRNAwasclearlyexpressed

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in IFN-gamma-stimulatedCIEC.On the other hand, the direct addi-tionofIL-4showednosignificanteffectonGBD-2expressioninCIEC.However,whensupernatantsfromperipheralbloodmononuclearcells(PBMC) cultured with IL-4 were added to CIEC, the expression ofGBD-2decreased.ToelucidateifIFN-gammafunctionsasasignalingmoleculethatfacilitatesthegenerationofGBD-2againstEimeriaspp.ingoats,anti- IL-4wasadded toPBMCfromEimeria-infectedgoatsandlevelsofIFN-gammainculturesupernatantsweredeterminedbyan enzyme-linked immunosorbent assay test. Results showed thatIFN-gammasecretionincreasedwhenanti-IL-4wasaddedtoPBMC.Hence it issuggested that IL-4maybea further factor in thepatho-genesisofgoatcoccidiosisanditsinductionmaybepartofanevasionstrategyoftheparasitetoavoidpro-inflammatoryresponses.Key words:defensins,eimerosis,caprine,cytokinesSpecies:ruminants

Pr172. FEEDING PROBIOTIC BACTERIA TO SWINE ENHANCES IMMUNITy TO ASCARIS SUUM

GLoRIASoLAno-AGUILAR1,TEREZSHEA-DonoHUE2,KATHLEEnMADDEn3,HARRyDAWSon1,ETHIoPIA

BESHAH1yoLAnDAJonES1,MARTARESTREPo1,JoSEPHURBAnJR1

1NutrientRequirementsandFunctionsLaboratory,BeltsvilleHumanNutritionResearchCenter,ARS-USDA,Beltsville,MD

20705,2MucosalBiologyResearchCenter,UniversityofMaryland,Baltimore,3UniformedServicesUniversity,Bethesda,MD

gloria.solanoaguilar@ars.usda.govProbiotic bacterial species are included in the diet to promote

health.Probioticspurportedlyprotecttheintestineagainstpathogenicmicroorganismsandcanreduceinflammation,however,quantitativemeasurement of probiotic growth and related effects on intestinalfunctionareoftenlacking.AscarissuumcommonlyinfectspigsandinducesaTh2-derivedresponseintheintestinethatisassociatedwithexpulsionofthefourth-stagelarvae(L4)fromthejejunum.Thephysi-ologicalaspectofexpulsionrepresenta“weepandsweep”responsecharacterized by smooth muscle hyper-contractility and increasedluminalfluidassociatedwithreducedsodium-linkedglucoseabsorp-tion. We investigated the effect of feeding Bifidobacterium lactissubspeciesanimalis(Bb12)ontheimmuneandintestinalfunctionofyoungpigssubsequentlyinfectedwithA.suum.PregnantsowswereorallyinoculatedwithadailydoseofBb12(3.5x1010cfu)orapla-ceboduringthelasttrimesterofpregnancy,andtotheiroffspringfrombirthuntilweaning.Sixweeksafterweaning,pigletswereinoculatedwithA.suumandthejejunalmucosaewasstrippedandmountedinUssingchamberstodeterminechangesinpermeabilityandglucoseabsorptionat21daysafterinfection.Pigjejunum,mesentericlymphnodes, and proximal colon were collected and assayed for geneexpressionbyrealtimePCR.Bb12wasquantitativelydetectedusingasinglecopytufgeneandfoundatthehighestconcentrationinthecolon of probiotic-treated piglets. Probiotic treatment did not affectintestinal permeability, but significantly attenuated the reduction inglucose absorption and the hyper-secretory response to histamineinducedbyA.suuminfection;suggestingaselectiveeffectofthepro-bioticonnutrientabsorptionandmastcellresponsesagainstparasiteinfection. Probiotic treatment of A. suum-infected pigs significantlyincreased mRNA expression of genes associated with enhancedprotectionagainstparasitic infection, including IL-25,RETNLB,andSOCS3,anddidnot interferewithnormalexpulsionofL4 from thejejunum. The results show that probiotic bacteria can selectivelyenhancelocalimmunitytoA.suumwithoutaffectingnutrientabsorp-tion.ThisexperimentalsystemcouldbeusedtoevaluatetheeffectoffeedingBb12andotherprobioticsonresponsestodifferentinfectiousagentstoreduceantibioticuse.Italsomodelstheeffectoffeedingprobioticstomothersandtheirnewbornchildrenonreducedexpres-sionofallergicdiseaseinhuman.Key words: probiotic, Ascaris suum, immunity, gastrointestinalfunctionSpecies:swine

Pr173. RHIPICEPHALUS SANGUINEUS TICKS FED ON RESISTANT HOST: HISTOLOGy OF LESIONS ASSOCIATED

WITH IMMUNITyVIVIAnEAPARECIDAVERonEZ1,MáRCIoBoTELHoDE

CASTRo2,GERVáSIoHEnRIqUEBECHARA1,MATIASPABLoJUAnSZABó1,3

1DepartamentodePatologiaVeterinária,UniversidadeEstadualPaulista,Jaboticabal,SP,Brasil;2FaculdadedeAgronomiae

MedicinaVeterinária,UniversidadedeBrasília,Brasília,DF,Brasil;3FaculdadedeMedicinaVeterinária,UniversidadeFederalde

Uberlândia,Uberlândia,MG,Brasilszabo@famev.ufu.br

Dog is thenatural,non resistanthost,of the tickRhipicephalussanguineuswhereasguineapigsdevelopastrong resistance to thesametickspeciesfollowingrepeatedinfestations.Inthisworkhistologi-calfeaturesofticksfedondogsandonguineapigswerecomparedtoevaluatecausedbytheimmuneresponseoftheresistanthostlesionsintargettissues.AdditionallyhostcomplementfractionC3andIgG1andIgG2antibodiesweresearchedinticktissuesbyimmunohistochemistrytoassociateeffectormechanismswith lesions.Ticks fromeachhostspecieswerecollectedduringthefirstandthethirdinfestationandpro-cessedaccording to routinehistological techniques. Itwasobservedthat many ticks from guinea pigs, especially during third infestationwhereunattached,dehydratedandsmallerwhencompared to thosefromdogs.Gutof13.6%and38%oftheticksfedonguineapigsduringfirstandthirdinfestation,respectively,hadhostinflammatorycellswiththe features of eosinophils and basophils,whereas no inflammatorycellwasseeninthegutofticksfedondogs.Vacuolizationofgutcellswereobservedinall ticksexcept inthosefedondogsformorethan96hours.Itwasalsoobservedthat85.7%ofticksfedonguineapigsduringfirstinfestationhadvacuolatedtracheaeandalldisplayedswell-ingofMalpighiantubules.Duringthirdinfestation,allticksfromguineapigshadvacuolatedtracheaeandswellingofMalpighiantubulesandin25%oftheseticksvacuolizationofoocyteswasalsoobserved.Atthesametimesuchalterationswerelackinginticksfromdogs.Anotherinterestingfeaturewasthepresenceofanincreasednumberofgua-ninegranulesinticksfedonguineapigs,especiallyduringthirdinfesta-tion.Noassociationbetweenhostcomplementandantibodieswithticklesionscouldbedonebecauseofthediffusestainingofseveraltissuesonticksfrombothdogsandguineapigsduringthefirstandthirdinfes-tation.OverallresultsshowthatthenaturalresistanceofguineapigstoR.sanguineusticksisassociatedwithlesionsinmanydifferenttissuesanddehydrationofthetick.Eosinophilsandbasophilsinthegutmightbeinterferingwithtickfeeding.Finally irrespectiveofhostresistancehost complement and antibodies seem to reach several tick tissuesduringinfestationsandthusfunctionalactivitiesoftheseproteinsinsidetheticksshouldbeevaluated.

Financialsupport:FAPESPandFAPEMIGKey words: Rhipicephalus sanguineus, lesions, immune response,dog,guineapigSpecies:dog,guineapig

Pr174. SEROLOGICAL, HISTOLOGICAL AND IMMUNOHISTOCHEMICAL TESTS FOR CANINE VISCERAL

LEISHMANIASIS DIAGNOSISnInAMARIGPDEqUEIRoZ1,WILMAASTARKE-BUZETTI1,RITADECASSIASVIVEIRoS1,SILVAnACPAULAn1,KAREn

ITASCA1,FLáVIALLIMA1,MICHELy,STEnoRIo1,MARIAFRAnCISCAnEVES1,RoSAnGELAZACARIASMACHADo2,

TRICIAMARIADEoLIVEIRA2,AnTonIoCARLoSFDEnoRonHAJúnIoR3

1UniversityofSãoPauloState,FEIS/UNESP-CampusofIlhaSolteira,SP,Brazil;2UniversityofSãoPauloState,FCAVJ/UNESP-CampusofJaboticabal,SP,Brazil;3CentreofZoonosisControl,Ilha

Solteira,SP,BrazilCaninevisceral leishmaniasis (CVL) is causedbyaparasiteof

thespecieLeishmania (L.) chagasi, endemic forhumansanddogsin many regions of Brazil.The purpose of the present study was toevaluate the serological testes (RIFI and ELISA) in asymptomatic,oligosymptomatic and symptomatic Leishmania-infected dogs from

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Solteira, SP, Brazil. These methods were also compared with othermethodsusedfordiagnosisof thisdiseaseindogssuchashistological(H&E)andimmunohistochemical(IMHC)methodsfordemonstrationofLeishmaniainskin.Whenserologicaltestsweredone,24(70.6%)dogswerepositivebyELISAorIFAT,butthenumbersofdogsposi-tivebyELISAwasslightlyhigherthanIFAT(56%forIFATand65%forELISA),andin7(21%)dogstherewasnoagreementbetweenELISAand IFAT. There was also no agreement between serological testsandH&EorIMHCin32.4%,particularlyinasymptomatic(8dogs)andoligosymptomatic(17dogs)groups.Inasymptomaticgroup,5(62.5%)werenegativebybothserologicaltests,but3(37.5%)ofthesedogswerepositivesbythepresenceoftheparasiteintheirskinsconfirmedby H&E and IMHC methods. On the other hand, one asymptomaticdogwaspositive inall tests(serological,H&EandIMHC), indicatingthatevendogswithoutanyclinicalsignscanbeinfected.Insymptom-aticgroup(9dogs),theagreementamongtestswas100%,but1dog(11.1%)ofthisgroupwasnegativeand8(88.8%)werepositives.Theskin biopsies from asymptomatic dogs had negligible if any lesions,andparasitedirectexaminationshowedthat themostof thesedogs(62.5%)werenegativeorsuspect,but3(37.5%)werepositiveinskinswithoutanydermatologicalalterations.Inoligosymptomaticdogs,14(82.3%)showeddiscreteparasiteload,particularlyinlesionskins.Insymptomatic group, 100% of dogs had several forms of cutaneousalterationswithstrongparasiteloudintheirlesionskins.Incompari-sonwithserologicaltests(IFATorELISA),theimmunohistochemistrymethodshowedasensitivityof65%andspecificityof100%thatwashigherinlesionthaninnormalskins.Inconclusion,becauseofthecomplexityofthisdisease,theresultsofthepresentstudysupporttheobservationsthatonlyonetest(serologicalorparasitological)maybenotsufficient forLVCdiagnosis. Inaddition, IMHCwasconsideredavaluablemethodtosupportthediagnosisofhisdiseaseinadditiontoserologicalandparasitologicaltests.Key words: Dogs; Diagnosis, Histochemical, /Leishmania chagasi,/LeishmanioseVisceralCanina.Species:canine

Pr175. TRyPANOSOMA CRUZI NATURAL INFECTION OF HOUSEHOLD DOGS DIAGNOSED By PCR AND ELISA METHODS IN AN ENDEMIC AREA FOR CANINE VISCERAL LEISHMANIOSIS IN BARRA DO POJUCA, BAHIA, BRAZILCSAGUIAR,ACDEALCAnTARA,DVVBITTEnCoURT,Ro

GGAMA,ACCSILVEIRA,RSLIMA,CMBGoMES-nETo,CRFRAnKE,SMBARRoUIn-MELo,PHPAGUIARUniversidadeFederaldaBahia,salvador,BA,Brazil

aguiarcs@yahoo.com.brChagas disease or American trypanosomiasis is restricted to

theAmerican continent and is considered a major tropical infectionoftheworld.Characterizedbyendemicnatureandchronicevolution,thediseaseiscausedbyaflagellateprotozoannamedTrypanosomacruzi,which is transmittedtohumansandothermammalsmostlybyhematophagousbugsofthesubfamilyTriatominae.Duetothevector’spredominantly domiciliary and anthropophilic behaviour, the parasit-ismwas transformed into zoonoses, includingdomesticanimals likecatsanddogs,aswellaswildanimalslikerodents,monkeys,groundsquirrelsandothers,whichcouldalsoserveasimportantparasiteres-ervoirs.Dogsareconsidered important reservoirsofT.cruzi inLatinAmerica countries.There are reports of clinical disease in domesticdogsinsomeareasoftheUnitedStatesandinendemicareasattheNortheast regionofBrazil.Thisstudyaimed to investigate thepres-enceofnaturalcanineinfectionbyT.cruzi intheDistrictofBarradoPojuca, Bahia, Brazil, where human infection by T. cruzi has beenreportedandzoonoticvisceralleishmaniosisisendemic.MaterialandMethods:274randomlycollecteddogserumsampleswereanalyzedbyindirectELISAforseropositivitytoanti-T.cruziandanti-Leishmaniaspp. antibodies. The ELISA test was performed with crude solubleantigensfromepimastigoteformsidentifiedasbeingColombianstrainbycomparisontothereferencestrain.Allseropositivedogsforanti-Tcruziantibodies,aswellasdoubleseropositivedogs foranti-Tcruziandanti-Leishmaniaspp.antibodiesinELISAtestswereevaluatedbyPCRwithprimersdesignedtoamplifyspecificT.cruziminicircleDNAsequence.Results:The seroprevalence forT. cruzi infection was of11.31%(31/274dogs),being29.03%(9/31dogs)doubleseropositive

forT.cruziandLeishmaniaantibodiesbyELISA tests.Fourteenoutof31T.cruziseropositivedogsweretestedforpresenceofparasiteDNAbyPCRtechnique,whichresultedin50%ofpositivity(7/14dogs).Conclusions:CaninenaturalinfectionforT.cruziwasconfirmedinthestudiedareabyELISAandPCRtechniques.Duetothehighfrequencyof double positivity forT. cruzi and Leishmania antibodies in ELISAtestsforserologicaldiagnosisoftheseinfections,theuseofmorespe-cific and accurate techniques, such asPCR, become indispensablefordistinguishinginfectionsinendemicareas.Sincetheefficiencyandthe reliabilityofdiagnosticmethodsare important for thesuccessofdisease control measures, more accurate serological methods needtobestandardized.Key words:Trypanosomacruzi,ELISA,PCRSpecies:canine

Pr176. STUDy OF THE CANINE ExPERIMENTAL MODEL OF THE INFECTIVITy AND IMMUNOGENICITy OF LEISHMANIA

INFANTUM NEW VARIANTS ISOLATED FROM HIV-LEISHMANIA CO INFECTED PATIENTS

JoRGEMIRET1,JAVIERnIETo1,CARMEnCHICHARRo1,CARMEnCAñAVATE1,EUGEnIACARRILLo2,FERnAnDo

GonZáLEZ3,JAVIERMoREno21CentroColaboradordelaOMSparaLeishmaniasis.CentroNacionaldeMicrobiología,InstitutodeSaludCarlosIII,Majadahonda,Madrid,Spain;2CentrodeInvestigacionesBiológicas,ConsejoSuperiordeInvestigacionesCientíficas,Madrid,Spain;3DepartamentodeToxicologíayFarmacología,FacultaddeVeterinaria,Universidad

Complutense,Madrid,Spainjavier.moreno@cib.csic.es

L. infantum/L. chagasi is the causative agent of zoonotic vis-ceral leishmaniasis (ZVL) in the Mediterranean basin, China andSouth America. Enzymatic characterization of L. infantum isolatesfromHIVpatientshasshowedthatsomestrainsarenewvariantthathavenotbeendescribedtocauseVLinthedog,themainreservoiroftheparasite.Atthepresentworkwehaveevaluatedonthedog,theinfectivity, pathogenicity, immune response and the possible protec-tivecapacityof twonewvariants isolated from immunocompromisedpatients.TheL.infantumstrainsLLM-759(zymodemeMON-190)andLLM-480(zymodemeMON-228).both isolatedfromimmunodeficientpatients,and thehighlyvirulentstrainLLM-724 (zymodemeMON-1)wereused in this study.Dogswereexperimentally infectedwith108

promastigotes, and the clinical, immunological, and parasitologicalstatuswasmonitoredmonthlyduring1 year.Dogs infectedwith thestrainsLLM-759andLLM-480didnotshowedclinical,immunologicalandparasitologicalstatusofdiseasealongthefollowup.HoweverthedogsinoculatedwiththestrainLLM-724showedalterationsinalltheparametersstudiedandaparasitologicalpositivestatusoneyearafterinfection. Inordertotest theprotectivecapabilityof thesenewvari-antsofL.infantum,thosedogsinfectedwiththestrainsLLM-759andLLM-480 were challenged intravenously with the virulent strain andmonitoredduring8months.Leishmania-specificserumantibodiesandpresenceof theparasitewas confirmed in thoseanimals previouslyinfectedwithLLM-4806monthsafterchallengewiththevirulentstrain,whiledogsinfectedwithLLM-759remainedparasitologicallynegative.TheseresultsconfirmthatnewL.infantumvariantsLLM-759andLLM-480arenotableto inducetheoutcomeof thediseaseinthecaninehostandthepossibleprotectivecapabilityofthestrainLLM-759.Key words: canine visceral leishmaniasis, experimental infection,virulence,protectionSpecies:canine

Pr177. CHARACTERIZATION OF CLINICAL, IMMUNOLOGICAL AND PARASITOLOGICAL PARAMETERS DURING A STEADy STATE OF IMPROVEMENT OF AFTER CHEMOTHERAPy OF LEISHMANIA CHAGASI NATURALLy

INFECTED DOGSDAnIELAFARIASLARAnGEIRA1,PAULoHEnRIqUEPALIS

AGUIAR1,2,GERALDoGILEnoDESáoLIVEIRA1,WASHInGTonLUÍSConRADoDoS-SAnToS1,LAInPonTES-DE-CARVALHo1,

STELLAMARIABARRoUIn-MELo1,2

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1CentrodePesquisasGonçaloMoniz-FIOCRUZ,R.WaldemarFalcão,121,Salvador,BA,CEP:40295-001;2DepartamentodePatologiaeClínicas;EscoladeMedicinaVeterinária-UFBA,Av.

AdhemardeBarros,500,Salvador,BA,CEP:40170-000;Canineleishmaniosis(CL)isinanexpandingzoonosisinthestate

ofBahia,Brazil,despitethegovernmentalprogramsforthecontroloftheinfection.Thereisaconsensusabouttheneedfornewmethodsto control the disease, like vaccines and immunotherapy, since thehostimmuneresponseplaysanimportantroleinthedevelopmentofresistancetoparasiteinfection.Resistantdogswouldbedesirableforlivinginendemicareas,sincetheywouldbepoorlyornon-parasitized,being, therefore,bad reservoirs for transmitting theparasite tophle-botominevectors. In thisstudy,weaimed tocharacterizeaconjunctof parameters of infected dogs before and after treatment with ananti-leishmaniadrug,inordertomakefeasibletheperceivanceofanyadditionaleffectofimmunotherapyappliedfromapost-chemotherapysteadystate.Materialandmethod:Ninedogsnaturally infectedwithLeishmaniachagasiwerestudied incontrolledkennels, located inanon-endemicalarea forCL,at theResearchCenter’spremises.Theanimalswereclassifiedaccordingtotheintensityoftheclinicalsymp-tomsasasymptomatic(AS),symptomatic(SY)andoligosymptomatic(OL).Eachanimalwasevaluatedbymeansofparasitologicaldiagnosisbycultureofspleenaspirates,serologybyELISA,invitroLeishmaniaantigen-induced lymphoproliferation using PBMC, hemogram andserum biochemistry (BUN, bilirubin, proteins, albumin and globulin)beforeand threeandsixmonthsafterhaving received treatmentbyashortcourseofmeglumineantimoniate.Thedogsweredividedintogroupsfor3differenttreatments,havingeachgrouponeanimalwitheachprofile,AS,SYandOL.Thedogsofgroups1and2weretreatedwith100mg/Kgofmeglumineantimoniatesubcutaneously,twicedailyfor10days.Thedogsofgroup2receivedtwoadditionaldosesofimu-notherapicpreparation(hidroxideofaluminum+recombinantproteinsofLeishmania)afterthecourseofantimonyandthedogsofgroup3didnotsufferanyintervention.Results:Beforetreatment,allanimalspresentedvariablealterationsinserumbiochemistryandhemograms,positiveparasitologyandhighOpticalDensity(OD)readingsinELISAfordetectinganti-Leishmaniaantibodiesinsera.OnlythesymptomaticanimalspresentedabsenceofPBMCproliferationinthepresenceofLeishmania antigen as stimulus.After 3 months all treated animals(groups1and2)presented remissionofclinicalsigns, improvementoflaboratoryparametersofhemogramandbiochemistryandnegativeparasitologicaltests.PBMCfromallanimalsfromthesegroups,includ-ingthetwosymptomaticdogs,presentedpositiveLeishmaniaantigen-induced proliferation.At this time point, the animals of group 3 hadtobe treatedduetheworseningof thesymptoms.After6monthsoftreatment,3dogs,twofromthechemotherapygroupandonefromthechemotherapy+immunotherapygrouppresentedpositiveparasitologyandbeguntohaveclinicalsigns.Allanimalshadnormalhemogramsandserumbiochemistry,aswell aspositive in vitro lymphoprolifera-tion.TheELISAtestsofalldogsshowedhighlevelsofODduringthewholetimeofstudy.Conclusions:The6-monthperiodofremissionofclinicalsignsandimprovementoflaboratoryparameters,includingaswelltherecoveryofantigen-specificlymphoproliferationandreductionofsplenicparasitismtoundetectablelevels,wasachievedwithashortcourseofchemotherapywithananti-leishmaniadrug.Thesefindings,althoughcarriedout in controlledconditions,are inaccordancewiththemajority of the reported results of CL chemotherapy, in special-izedliterature.Afteracourseofchemotherapy,naturallyinfecteddogscome toasteadystate inwhich immunologicalmodulatorscouldbeintroducedinexperimentalstudiesforthedevelopmentofefficientpro-tocolsforcontrollingthetransmissionfromdogstovectors.SerologybyELISAwasnotagoodtoolfortheevaluationoftheanimalsduringthestudiedtimesinceitdidnotshowanymodificationalongwiththeotherparameters.Key words:canineleishmaniasis,immunotherapyechemotherapy.Species:canine

Pr178. TRyPANoSoMA CRUzI: CORRELATION OF IMMUNOGLOBULINS PROFILES WITH PARASITEMIA IN

DOGS IMMUNOSSUPRESSED AND IMMUNOCOMPENTENT WEnDELCoURAVITAL1,HELEnRoDRIGESMARTInS1,oLInDoASSISMARTInS-FILHo3,MARIATEREZInHABAHIA1,MARTADE

LAnA2,WASHInGTonLUIZTAFURI1,VAnJAMARIAVELoSo1,CLáUDIAMARTInSCARnEIRo2,ALExAnDREBARBoSAREIS21UFOP-LaboratóriodeImunopatolgia,NúcleodePesquisasemCiênciasBiológicas;2UFOP-EscoladeFarmácia;3CPqRR-

LaboratóriodeDoençadeChagas,CentrodePesquisaRenéRachoualexreis@nupeb.ufop.com.br

Previousstudies inour laboratoryhavedemonstrated thatdogsexperimentally infected with Berenice-78T. cruzi-strain presented alongerpatent-parasitemia,andmyocarditisofvariabledegree.Despitethese parasitological and pathological findings, little is known aboutthehumoral immuneresponseduringacutephaseoftheinfectionindogs. Herein we have performed a detailed follow-up investigationofserologicalprofilebyELISAusingspecificmonoclonalanti-canineisotypes (IgG, IgG1, IgG2, IgM, IgAe IgE)employingasolubleepi-mastigotes T. cruzi antigen. Twenty-four dogs were subdivided insix experimental groups with four animals in each one: uninfectedcontrol, infectedwithblood trypomastigotes, infectedwithmetacyclictrypomastigotes, uninfected immunossupressed, infected with bloodtrypomastigotes immunossupressedandinfectedwithmetacyclictry-pomastigotesimmunossupressed.Wereused2,000metacyclictrypo-mastigotes(MT)orbloodtrypomastigotes(BT)ofBE-78T.cruzistrain,perKg/body/weight,typifyingvectorialandtransfusionaltransmissionofhumanChagasdisease,during35daysoftheacutephaseoftheinfection.Thekineticsofimmunoglobulinlevelswasevaluatedinzero,seven,14,21,28and35daysafter infections including thecorrela-tionswiththeparasitemiacurve.DogsinfectedwithBTpresentedanincreaseof IgGand IgM levels,maintenanceof IgG1, IgG2and IgAlevelsalongthetimeandanincreaseofIgEfollowedbyadecrease.Ontheotherhand,dogsinfectedwithformsMTpresentedanincreaseof IgG, IgG2, IgMand IgAandmaintenanceof IgG1and IgE levels.Independent of the infective forms, the profile of humoral responsepresentedreducedlevelsofIgG,IgG1,IgG2,IgAandIgEinallanimalsimmunossupressed.However, the IgMshowed theelevations levelsduringthekineticsinimmunossupressedgroups.Takentogether,thesefindingsemphasizetheimportanceoftheinoculumsource,suggestingthatvectorialortransfusionalroutesofT.cruziinfectionmaytriggerdis-tinctparasite-hostinteractionduringacuteChagasdisease.Duringtheacutephaseoftheinfectionitwasnotfoundacorrelationbetweentheprofilesofimmunoglobulinsandparasitemiainbothimmunocompetentandimmunossupresseddogs.OursresultsstillsuggestalsothattheearlyproductionofIgGandIgG2seemstoplayapivotalrolecontrolof the immunopathology observed in acute canineChagas disease.Inaddiction, thesedatasuggested that the routeof infectioncanbean importantelementdriving the immuneresponse in infecteddogs.Thereforethedifferentprofileofclassandsubclassofimmunoglobu-linscouldbecontributeforthedevelopmentofimmunophatogenesisofcanineexperimentalinfections.

Supported by: CNPq, CAPES, FAPEMIG, CPqRR-FIOCRUZ &UFOPKey words:Chagasdisease,immunossupressed,humoralresponse,cellresponseSpecies:canine

Pr179. KINETICS OF DERMAL CELLULAR INFILTRATES IN DOGS VACCINATED WITH LEISHMANIA-VACCINE, SAPONIN

AND SALIVA FROM LUTzoMyIA LoNGIPALPISJULIAnAVIToRIAnoDESoUZA1,náDIADASDoRES

MoREIRA1,HEnRIqUEGAMAKER1,RoDRIGoCoRRêA-oLIVEIRA2,RoDoLFoCoRDEIRoGIUnCHETTI1,ALExAnDRE

BARBoSAREIS1,CLáUDIAMARTInSCARnEIRo11LaboratóriodeImunopatologia,NUPEB/UFOP;2Laboratóriode

ImunologiaCelulareMolecular,IRR,FIOCRUZ/MGEfforts have focused on understanding the early events that

influenceeffectivenessofvaccineantigens. In thepresentstudy,weinvestigated kinetics of dermal cellular infiltrates in dogs vaccinatedwith Leishmania-vaccine, Saponin and saliva ofLutzomyia longipal-pis.Basedoncellmigrations,wehavedevelopedanewexperimentalmodeltostudysomeaspectsoftheinflammatoryimmuneresponse.Inthissense,intradermalinjectionsintothedorsalregionofdogswereperformedatdifferenttimes(1,12,24,48,96hours).Stimuliwithdis-tinct vaccine antigen components were evaluated, such as saponin

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(Sap),sandflysaliva(Sv),wholeparasiteantigenagainstVL(WPA),andWPAassociatedwithSap.Thosestimuliwerecomparedwithcon-trolsgroups:salinainoculum(Sn)andwithoutsalina(Ns).Macroscopicobservations (local swelling,hyperemiaandnecrosis)were reportedduringthecourseoftheexperiment.Skinsampleswerefixedin10%neutral buffered formalin for routine histopathological examinationof sections by subsequent haematoxylin and eosin (HE) staining.The dermal cellular infiltrate was graded in absent (–); discreet (+);moderate(++)andintense(+++).Indogsinoculatedwithsaponinorsaponin plusWPA,we observed local swelling and hyperemia after12hs. However, no macroscopic alterations were observed in dogsinoculatedwithsandflysaliva.Allgroupspresentedcellularinfiltratescomposedmainlyofneutrophils.Themajorfindingsoccurredat12,24and48hoursafterinoculation:increasedcellularinfiltratesinSapandWPAplusSapwhencomparedtoSnandNsgroups.Moreover,after48hours,thegroupinoculatedwithSvpresentedanincreasedcellularinfiltrationwhencomparedwithSn.Additionally,after96hours,onlySapandWPAplusSapinoculateddogsshowedenhanceddermalcel-lularinfiltrates.Ourdatapointtothepotentialearlystimulatoryeffectsoninnateimmuneresponses,particularlyneutrophilrecruitment,aftersaponininoculation.Furthermore,weobservedadelayincellinfiltra-tionintheSvgroup,whichmayreflecttheimmunomodulatoryproper-tiesofsaliva.Eventhough,edematousreactionshavefrequentlybeenobservedaftersaponininjectionorincombinationwithWPA,theinduc-tionofastrongcellularresponse,simpleformulation,safety,andthelowcosts,allowitsuseasalternativeadjuvantinveterinarymedicine.Key words:Leishmania,dogsandKineticsSpecies:canine

Pr180. SELECTION OF RECOMBINANT PEPTIDES FOR CANINE LEISHMANIASIS DETECTION By PHAGE DISPLAy

JULIANAFRANCOALMEIDA,GUILHERMEROCHALINOSOUZA,FAUSTOEMÍLLIOCAPPARELLI,CARLOSROBERTOPRUDÊNCIO,

ANAPAULAPERESFRESCHI,RONECARDOSO,FLÁVIAFIGUEIRAMESSIAS,LUIZRICARDOGOULART

1InstitutodeGenéticaeBioquímica-UniversidadeFederaldeUberlândia-MG

almeidajf@hotmail.comHuman visceral leishmaniasis (HVL) is a canine zoonose lead-

ingto500,000newcases/year,inwhichnovaccineisavailable,andchemotherapyishighlytoxic.Canineleishmaniasis(CL)isasystemicdiseasecausedbydifferentspeciesof thegenusLeishmania that istransmittedbybloodsuckingphlebotominesandflies.Dogsareconsid-eredthemaindomesticreservoiroftheparasite,constitutingpartoftheepidemiologicalcycleofhumantransmission,accountingformorethan90% of visceral leishmaniasis cases described in the world. Phagedisplayhasbeenutilized fornumerouspurposes, includingmappingprotein-ligandinteractions,identifyingbindingantagonistsandenzymeinhibitorsthroughthedesignofmimotopes.Thistechnologywasusedto identifypeptidesagainstHVLantibodiesexpanding itspurpose toCLepitopesscreeningintheperspectiveofusingthemasvaccineordiagnostics’ antigens. IgGwas purified from sera of human visceralleishmaniasispatients,andusedforscreeningagainstarandompep-tidephagelibrary.Theselectionwasperformedinliquidphasethroughagarosebeads(Gproteinagarose).After four roundsofbiopanning,phage clones were isolated, sequenced, translated, and submittedto bioinformatic analyses. Peptide analysis revealed a high degreeofsequencesimilaritiesbetweenimportantparasiteproteinsinvolvedin thediseasedevelopment,suchasgp46,gp63,kinesinK39,DNAtopoisomerase II,LACKantigen,amongothers.Twohighly frequentepitopes were identified, and the phagotopes were used in ELISAassaysagainst positive andnegative canine sera, showingdifferen-tial reactivity,with83%ofsensitivity.Thehighly reactiveclonesmaybe useful in diagnostics and therapeutics ofCanine Leishmaniases.Financialsupport:FINEP,CNPq,UFU,DiaMed,ImunoScan.Key words:CanineLeishmaniasis,epitopes,PhageDisplay.Species:canine

Pr181. DETECTION OF ANTI-LEISHMANIA CHAGASI IGG SUBCLASS ANTIBODy IN INFECTED AND VACCINATED

DOGS

TRÍCIAMARIAFERREIRADESoUSAoLIVEIRA1,MELInABASon1,TIAGoWILSonPATRIARCAMInEo1,RoSAnGELA

ZACARIASMACHADo11UniversidadeEstadualPaulista(UNESP),Jaboticabal,SP,Brazil

triciaoliveira@yahoo.com.brVisceralleishmaniasisisazoonoticdisease,causedbyLeishmania

chagasiandtransmittedbythebiteofsandfliesfromLutzomiyalongi-palpis species inBrazil. In thiscountry, it isanemergingdisease inurbanareas, anddogsare themaindomestic reservoir.Theaimofthis studywas toanalyze thepresenceofanti-Leishmania IgGsub-classantibodiesbyELISAin121seraofseropositivedogsfromBeloHorizonte(MG)endemicarea,and40seraofvaccinatedanimalswithvaccineLeishmune(FortDodge®).ThisstudyalsoaimstoverifythediferenceofIgGsubclassamongthegroups.Theproportionofdogswitch produced detectable levels of anti-Leishmania IgG subclassantibodiesfromBeloHorizontewas80,17%(97/121)forIgG1;0,83%(1/121) for IgG2;39,67%(48/121) for IgG3and55,37%(67/121) forIgG4.TheresultsshowedsignificanthigherpercentageinIgG1anti-bodies (p<0,01).Theproportionof vaccinateddogswhichproduceddetectablelevelsofanti-LeishmaniaIgGsubclassantibodieswas22%(9/40)forIgG1;87,5%(35/40)forIgG2;2,50%(1/40)forIgG3and5%(2/40)forIgG4.ResultanalysisdisclosedsignificantincreaseinIgG2antibodies(p<0,01)invaccinateddogs,andthattheIgG2responseofvaccinateddogswassignificantlyhigherthanininfecteddog(p<0,01).TheseresultssuggestthattherewasgreatvariationinlevelsofIgG1andIgG2betweenthetwogroups,butitneedsmorestudiestousethemarkersofresistanceorsusceptibilityofindividualdogs.25,4mmKey words:leishmania,subclass,IgGantibodies,ELISASpecies:canine

Pr182. COMPARISON BETWEEN ANTIGEN TOTAL ELISA AND FML ELISA IN DIAGNOSIS OF VISCERAL CANINE

LEISHMANIASISTERESInHACRISTInACÂnDIDo,TATIAnADEoLIVEIRA

GERZoSCHKWITZ,MARIACECÍLIARUILUVIZoTTo,VALéRIAMARçALFELIxDELIMA

DepartamentodeApoioProduçãoeSaudeAnimal,FaculdadedeOdontologiadeAraçatuba,UniversidadeEstadualPaulistaJúliode

MesquitaFilhoVisceralcanineleishmaniasisorcalazarisBraziliananendemic

antropozoonosis caused by Leishmania L. chagasi.The immunoen-zymaticassayinsolidphasetest(ELISA)hasbeenusedinroutineofepidemiologicalstudiesanddiagnosticofdogs.TheELISAassayusingLeishmaniaL.chagasitotalantigen(AgT-ELISA)andFucoseManoseLigand-ELISA(FML-ELISA)hasbeenusedtodetectedleishmaniasisindogs.ThepresentpaperhasthegoaltocomparetheefficiencyoftwoantigensbyELISAassayindogsfromAraçatuba,SP-Brazil,anendemic leishmaniasis area. Thirty dogs were selected by positiveimprints and divided into two groups: oligosymptomatic (increase oflymphonodesandagoodcondition)andsymptomaticpresenceofatleastthreeclinicalsignsassociatedwithactivevisceralleishmaniasis(fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss,cachexia,locomotorydifficulty,conjunctivitis,epistaxis,hepatospleno-megaly,edema,andapathy).Controlgroupwasconstitutedofdogsfrom Leishmania free. Dogs oligosymptomatics presented 86,7% ofsensibility inAgT-ELISAand90%atFML-ELISA.Thespecificitywas100%atAgT-ELISAand96.7%forFML-ELISA.AtsymptomaticsdogsthesensibilityandspecificityforAgT-ELISAwererespectively90%and93.3%and,theFML-ELISAshowed86.7%and96.7%.OnELISA-AgTthepositivepredictivevalorwas93.1%atsymptomaticand100%atoligosypmtomatic,moreoverinthedogstestedbyFML-ELISAthevalorwas96.3%forthesymptomaticand96.4%foroligosymptomatics.TheKappa statistics was used for measure the real concordance gradebetweenthetwoELISAassayandthedirectparasitologicalexamandshowedagoodconcordancebetweenbothtestedmethods.Inconclu-sion,bothmethodsshowedgoodsensibilityandspecificityfordetec-tiondiseaseinasymptomaticandoligosymptomaticandcanbeusefultodiagnosisofvisceralcanineleishmaniosis.Key words:Diagnosis,ELISA,VisceralLeishmaniasisSpecies:canine

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Pr183. MyELOPOIESIS AND ERyTHROPOIESIS IN CANINE VISCERAL LEISHMANIASIS

RAqUELTRóPIADEABREU1,MARIADASGRAçASCARVALHo2,RoDoLFoCoRDEIRoGIUnCHETTI11,CLáUDIAMARTInS

CARnEIRo1,BRUnoMEnDESRoATT1,RoDRIGoDIAnDEoLIVEIRAAGUIAR1,ALExAnDREBARBoSAREIS1

1LaboratóriodeImunopatologia,NUPEB/UFOP;2LaboratóriodeHematologiaClínicaFaculdadedeFarmácia/UFMG

Caninevisceral leishmaniasis (CVL)manifests itselfasabroadclinicalspectrumrangingfromasymptomaticinfectiontopatentseveredisease.Herein,bonemarrow(BM)smearsstainedbyGiemsawereevaluatedconsideringthreeclinicalgroupsofdogsnaturallyinfectedbyLeishmaniachagasi[i.e.asymptomatic(AD,n=54),oligosymptomatic(OD,n=48)andsymptomatic(SD,n=73)]comparedwithnon-infecteddogs(NID,n=14).Analysisusingpreliminarydataonerythropoiesisshowednosignificantdifferencesinproerythroblast,basophilic,poly-chromaticandorthochromaticerythroblastswhenalldifferentclinicalgroups were compared to control and among themselves. After aminuciousobservationofthebonemarrowsmearswehavefoundthatleucopoiesishaspresentedsomealterationsininfecteddogs.Todate,eosinophiliccellsnumberhasshownasignificantdecreaseinthedif-ferentclinicalgroupscompared tocontrol.Neutrophiliccellsnumbershow slight fluctuations in the different clinical groups, however, nodifference was found among them. Related to mononuclear cells, itwasobservedforlimphocytesnumberasignificantincreaseinODandSDgroupswhencomparedtoADgroup.Similarresultswerefoundforplasmacellnumbershowingaclear tendence toagradual increaseaccordingtotheseverityoftheinfection.Byotherhand,monocytescellnumberhassignificantlydecreasedinallclinicalgroupscomparedtocontrol.Thebonemarrowchangesobservedininfecteddogs,mainlythose inmononuclearcells, suggest thatdefencemechanismsweretriggeredinordertocombattheinfectiousagentbycellularresponseandantibodyproduction.Inconclusion,ourstudyshowsthattheclinicalevolutionofCVLinnaturallyinfecteddogspromotesclearalterationsin bone marrow cells. Since these alterations are directly correlatedwithCVLclinicalstatuswouldbetakenintoaccountwhendealingwithdiagnosisandprognosisfeatures.

Financialsupport:CNPq,FAPEMIG,UFOPandUFMG.Key words:leishmania,bonemarrow,myelopoiesis,erythropoiesisSpecies:canine

Pr184. PHENOTyPIC CHANGES IN POLyMORPHONUCLEAR CELLS OF BRAZILIAN DOGS NATURALLy INFECTED By LEISHMANIA (LEISHMANIA)

CHAGASILUAnDALGUERRA1,AnDRéATEIxEIRA-CARVALHo1,3,oLInDo

AMARTInS-FILHo3,RoDoLFoCGIUnCHETTI1,2,RoDRIGoCoRRêA-oLIVEIRA1,ALExAnDREBREIS1,2

1LaboratóriodeImunologiaCelulareMolecular,IRR-FIOCRUZ-BeloHorizonte/MG;2LaboratóriodeImunopatologia,ICEBII-NUPEB/UFOP-OuroPreto/MG;3LaboratóriodeDoençadeChagas,IRR-

FIOCRUZ-BeloHorizonte/MGluanda@cpqrr.fiocruz.br

CanineVisceralLeishmaniasis(CVL)isoneofthemostimportantemergingdiseaseswithhighprevalenceinLatinAmericancountries.Consideringtheputativeroleofdogsinthetransmissionofthediseaseand the importance of polymorphonuclear leukocytes in Leishmaniainfections, we proposed to evaluate the frequency of different cellmarkers (CD4, CD14, CD45RA, CD45RB and MHC-II) in systemicneutrophils of dogs naturally infected by Leishmania (Leishmania)chagasi.Herein,30dogsweresubdivided into threegroupsaccord-ingtospleenorbonemarrowparasitism:LowParasitism(LP;n=10),Medium Parasitism (MP; n=10) and High Parasitism (HP; n=10).The criteria used for parasite loadwas the LDU values (“LeishmanDonovan Units”), defined such as number of amastigotes per 1000nucleatedcells.Twentynon-infecteddogs(NI),usedascontrolgroup,were serologically and parasitologically negative forL. (L.) chagasi.Ourdatademonstratedadecrease in the frequencyofCD14+neu-trophils indogswithhighsplenicparasitismincomparisontotheLPgroup.TheresultsofbonemarrowparasitismshowedthatMPdogspresentedanincreaseinthefrequencyofMHC-II+neutrophilswhen

comparedtoNIandHPgroups.Inbothparasitizedcompartments,wedidn’t find any statistically significant differences in the frequency ofCD4+,CD45RA+andCD45RB+neutrophilsbetweenthegroups.ThedecreaseinthefrequencyofCD14+neutrophilsinHPgroupandtheincrease in the frequencyofMHC-II+neutrophils inMPgroup, bothshowingpatternsofactivatedcells,suggesttheparticipationofinnatecellstryingtoeliminatetheparasitesthroughphagocyticmechanismsandthroughanoxidativeburst.OurnextaimistoevaluatethedifferentphenotypicmarkersbyfrequencyandMediumFluorescenceChannelineosinophilsandmonocytespopulationsofdogspresentingdifferenttissueL.(L.)chagasidensities.

Supported by: FAPEMIG, PAPES IVb, UFOP andIRR/FIOCRUZ/MG.Key words: caninevisceral leishmaniasis,Leishmania (Leishmania)chagasi,tissueparasiteload,polymorphonuclearleukocytesSpecies:canine

Pr185. CHARACTERIzATIoN oF PoTENTIAL SECRETED/ExCRETED ANTIGENS oF LEISHMANIA CHAGASI AND

LEISHMANIA BRAzILIENSISSoLAnGECUBUSEK1,DAnIELMSoUZA1,AnDREARSC

GUIMARÃES1,SAMUELLBRAGA2,BRUnoMRoATT2,RoDRIGoDoASoARES2,REnATAGUERRASA3,RoDRIGoCoRREA-

oLIVEIRA1,ALExAnDREBREIS1,21LaboratóriodeImunologiaCelulareMolecular-IRR/FIOCRUZ;2LaboratóriodeImunopatologia-NUPEB/UFOP;3Laboratóriode

BioquímicaeBiologiaMolecular-NUPEB/UFOPdanielsouza@cpqrr.fiocruz.br

The Leishmaniasis ranges from asymptomatic infection to self-limitingcutaneous lesion(s)or fatalvisceral forms,dependingontheLeishmania species involvedandon the immunological responseofthevertebratehost.Previousreportsstressedthatsecreted/excreted(SE)proteins from theseveral intracellularpathogenscontainhighlyimmunogenicandprotectiveantigensinvaccinemodels.Similarly,SEantigensofL.majormayconstituteputativevirulencefactors.Inaddi-tion,afteritsphagocytosisbymacrophages,moleculesreleasedbypro-mastigotesofLeishmaniacouldrepresentanimmuneevasionstrategytoavoid cellular immune response.To characterizeSEproteins,weobtainedculturesupernatantsofstationary-phasepromastigotescol-lected12hafterincubation,withthepHandtemperaturesimilarofthephagolysosomalvacuoleconditions.OurresultsshowedthatL.cha-gasiincubatedinnormalcultureconditions(pH7.2and26ºC)releasedmoreproteinsthanL.braziliensis.Ontheotherhand,inconditionsthatpartiallymimicmacrophagevacuoleenvironment(pH5.5and35ºC),thereleaseofproteinsinbothspecieswasdiminished.However,theprofileofproteinsofbothspecieswassimilarinthetwopHconditions.Westernblotwascarriedoutto identifySEantigens.ThemajorityofproteinsfromsupernatantofL.chagasiinthebothconditionsofculturestronglyreactedwithserumofnaturallyinfecteddogswithL.chagasi.Incontrast,onlyfiveproteinsofL.braziliensiswereidentifiedasanti-genicbyserumofnaturallyinfecteddogswithL.braziliensis.Thetimecourseof thestudyshowed thatsecretion/excretionofantigenswasqualitativelyandquantitativelydifferentamongthespeciesstudiedandthe repertoire of these secretedproteinswill helpus select relevantantigensforthedevelopmentofanti-Leishmaniavaccine.Key words:Leishmaniachagasi;Leishmaniabraziliensis;antigensSpecies:canine

Pr186. NEoSPoRA CANINUM MODULATES CANINE SySTEMIC CELLULAR IMMUNE RESPONSES DURING

ACUTE ORAL INFECTIONTIAGoWILSonPATRIARCAMInEo,RoSAnGELAZACARIAS

MACHADoDepartmentofVeterinaryPathology,FCAV/UNESP,Jaboticabal,SP

neospora caninum is a protozoan parasite which has beenpointedoutasamajorabortioninducingdiseaseincattle.Dogs,coy-otes and, probably, other canids are the parasite’s definitive hosts,responsibleforthefecaleliminationofresistantforms(oocysts),whichare disseminated through the environment. Canine cellular immuneresponsesaftern.caninuminfectionhavenotbeenevaluatedtodate.

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Cattle and mice respond to infection by the protozoa in a predomi-nantlyTh1manner,controllingparasitereplication.Theknownexcep-tions are gestational periods, when female hormone production isshiftedtowardsprogesterone,whichpossesspotentanti-inflammatoryactionatthefetal-maternalinterface.Takentogether,thisworkaimedtoobserve theevents triggered incaninesystemiccellular immunityby oral infection with n. caninum. Clinically healthy dogs, serologi-callynegativeforn.caninumandcorrelatedparasites,wereselectedanddividedintothreemaingroups:Inoculatedadultdogs,inoculatedpuppies,andcontrolgroup.Inoculatedanimalsweresubmittedtooralintakeof500g/dayoffreshbovinemeat,nervoustissuesandoffal,forthreeconsecutivedays.Bloodsampleswerecollectedbyveinpunc-tionatclosetimeintervalstoobserveCD4+andCD8+TcellkineticsbyFACStill200thdayspost-infection(dpi),andpopliteallymphnodeswere removed surgically at 30dpi for immunohistochemical assays.Additionally, acute phase (90 dpi) cytokine mRNA expression wasmeasuredinPBMCfromthepuppygroupbyReal-timeRT-PCR.Theresultshereinobtainedindicatethatdogspresentaprotractedacutephase,withoocystsheddingbeingcorrelatedtoadropinCD4+andCD8+Tcellbloodlevels,andlowMHCclassIIexpressioninthelymphnodes.CytokinemRNAprofilerevealedthatdogspresenthighmodu-latorycytokineexpressiontillthe60thdpi,followedbyasharpincreasein inflammatorycytokines,as IFNγ.Basedon theseresults,wemayconcludethatn.caninummodulatesthedogcellularimmuneresponseduringitssexualcycle.Key words:Neosporacaninum,CD4+andCD8+Tcells,MHCclassII,CytokinemRNAexpressionSpecies:canine

Pr187. CANINE HUMORAL IMMUNE RESPONSES ELICITED IN ORAL INFECTIONS WITH NEoSPoRA CANINUM TIAGoWILSonPATRIARCAMInEo,MICHAELJDAy*,

RoSAnGELAZACARIASMACHADoDepartmentofVeterinaryPathology,FCAV/UNESP,Jaboticabal,SP;*DivisionofVeterinaryPathology,InfectionandImmunity,Schoolof

ClinicalVeterinaryScience,UniversityofBristol,Bristol,UKneosporacaninum isanApicomplexanparasitefirstlydescribed

as cause of encephalomyelitis in puppies serologically negative toToxoplasmagondii.Previous reportson theparasite’sdefinitivehostindicatealateIgGantibodyresponseandthatclinicaldiseaseisdifficulttobeinduced.Theaimofthisstudywastoinvestigatecaninehumoralimmunityduringn.caninumoralinfection.Forthatpurpose,clinicallyhealthy dogs, serologically negative for n. caninum and correlatedparasiteswereselectedanddividedintothreemaingroups:inoculatedadultdogs,inoculatedpuppies,andcontrolgroup.Inoculatedanimalsweresubmittedtooral intakeof500g/dayof freshbovinemeat,ner-vous tissues and offal, for three consecutive days. Blood sampleswerecollectedbyveinpunctionatclosetimeintervalstoobservetotalandspecificantibodykinetics for250dayspost-infection (dpi).Totalantibody productionwas followed up by commercial ELISA kits andspecificimmunoenzimaticassayswerestandardizedforIgM,IgA,IgE,IgGandsubclasses(IgG1, IgG2, IgG3, IgG4).Additionally,detectionofspecificIgMandIgGwererealizedthroughindirectimmunofluores-cenceantibody tests (IFAT),and IgGantigen recognitionprofilewasvisualizedthroughonedimensionalwesternblotting(WB).Theresultsobtainedindicatethatdogspresentalowspecificantibodyproductionduringacutephaseofinfection,showinganunstableseroconversionpattern,withonlyIgG1andIgG3beingdetectedinadultdogsandpup-pies,respectively,betweenthesecondandthirdmonthsof infection.Corroboratingwiththoseresults,WBanalysisdemonstratedabsenceofantigenrecognitionwithinthefirst30dpiinalmostallanimals.IFATshowedsimilarpattern,withlateIgMandIgGseroconversion.Duringthe experimental period, five IgG2and IgEpeakswere observed inan associated manner, and were also correlated to lower Th1 typeantibody production.Analyzing total antibody production, it could beobservedthatyoungeranimalspresentacrescentcurveinallanalyzedantibodies,whereasadultanimalsdemonstratestableserumantibodyconcentrations.After60dpi,specificantibodylevelsshowedanincre-ment,especially IgG1and IgG4 inpuppies.Taken together,wemayconcludethatantibodyproductionisinitiallyimpairedindogsafteroralintakeofparasitecontaminatedtissues,andthatTh1andTh2profileantibodies are present concomitantly after infection. Moreover, the

highersusceptibilityofpuppieston.caninummaybeattributedtotheontogenyoftheimmunesystem.Key words: Neospora caninum, Antibody kinetics, Delayedseroconversion,OntogenyofimmunesystemSpecies:canine

Pr188. FREQUENCIES OF ANTI-TRyPANOSOMA CRUZI ANTIBODIES IN DOGS FROM SOUTHERN OF RIO GRANDE

DO SUL STATECHARLEnEnSTRInDADE,FABIoPLLEITE,nARARFARIAS

DepartamentodeMicrobiologiaeParasitologia-UFPelfabio_leite@ufpel.tche.br

Chagas’ disease is a trypanosomiasis found in the AmericaswhoseetiologicalagentisTrypanosomacruzi.Thedifferentepidemio-logicalandclinicalmanifestationsoftheinfectionandthemechanismsand/or factors that determine the development of morbidity are stillunclear. Several important points remain to be clarified with regardtothenaturalhistoryofChagas’disease.Oneoftheseistheroleofdomestic’s animals, principally the dog, as potential element for thetransmissionandmaintenanceof theparasite in thehumanpopula-tion. The prevalence of infection among dogs from endemic zones,the close relationship between human and the identity of parasitesinfectinghumansanddogs,suggestthatthisdiseasepossiblyinvolvestheseanimalsasreservoirs.Theaimofthisstudywastocharacterizethe frequencyofantibodiesanti-T.cruzi inadogpopulation fromaChagas’diseaseinanendemicarealocatedatthesouthofRioGrandedoSulstate.Serum from99dogswascollected, in ruralandurbanarea,andanalyzedbyacommercial indirect immunofluorescent test(IFI)usingananti-dogIgGlabeledwithFITC.Theserawerediluted1:32andanalyzedmicroscopicallyinduplicate.Fromthe99serastudied90.8%(89)werepositivetoT.cruzi.ThemajorityofthepositivedogstoT.cruziliveinhouseswithpeople,independently,ofruralorurbanarea.Theseresultssuggestthatthedogmightplayanimportantroleinthetransmissionand/ormaintenanceofT.cruziinendemicareasinthesouthofBrazil.Thisworkisrelevanttothequestionwhetherdogsact as reservoirs, disseminating the infection or are just susceptibletoT.cruziwithouthavingamajor role in thespreadof thedisease.Thus,betterdiagnosticsandepidemiologicalsurveyshouldbedoneindomestic’sanimals inorder toprevent thisdisease.Thisobserva-tionssuggestthatpopulationinthisareasshouldbealertedaswelltheauthoritiesresponsibleforthecontrolofthisimportantzoonose.Key words:Trypanosomacruzi,dogs,antibodiesSpecies:canine

Pr189. LEISHIMUNE VACCINE-INDUCED IMMUNE RESPONSE IN DOGS FROM AN ENDEMIC AREA OF

VISCERAL LEIHMANIASISVMFLIMA1,FAIKEDA2,CnRoSSI2,MMFEIToSA1,Ro

VASConCELoS2,DPMUnARI,2,HGoTo31Dept.ofClinic,SurgeryandAnimalReproduction-FO-UNESP-Araçatuba-S.P-Brazil;2UNESP-Jaboticabal-Brazil;3Tropical

MedicineInstitute-USP-SãoPaulo-S.P-BrazilDogswithvisceralleishmaniasis(VL)constitutesapublichealth

problemastheyarethepotentialsourcesoftheparasitestransmittedtohumans through thevector.Tocontrol the transmissionavaccinenamed Leishmune (Fort Dodge) based on Fucose-mannose Ligand(FML)ofL.chagasihasbeenwidelyused,but the immunereponsein vaccinated dogs has not been extensively studied. Twenty dogsfromAraçatuba,endemicareaforVLintheSoutheastofBrazil,with-outpreviousLeishmaniainfectionscreenedbyserologyandnegativeparasitologicalexamsforleishmaniasis,withnormalhemogram,werevaccinatedsubcutaneouslywithLeishmuneandcellularandhumoralimmune responses evaluated before (control) and ten and 30 daysafter vaccination.After vaccination, the lymphoproliferative responseofperipheralbloodmononuclearcells(PBMCs)waspositivein80%toLeishmaniawholeantigenandin85%toFMLantigen.TheexpressionofCD4/CD25+onTcells(bycytometry)decreasedsignificantlyafterimmunization(p<0.05).TheIFN-γlevelwashigherinsupernatantofLeishmania antigen- or FML-induced culture cells after vaccination(P<0,05),butIL-4andTNF-alevelsweresimilarintheseperiods,and

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noIL-10wasdetected.ThevaccinateddogsproducedhighlevelsofantibodyagainstLeishmaniawholeantigenandFMLantigen(ELISA)that remainded high after one year (P <0.05) although the animalswereparasitologicallynegative.PositivelymphoproliferativeresponsetoFMLandLeishmaniaantigen,decreasedfrequencyofCD4/CD25+T cell, an increaseof IFN-γ level andnodetectable IL-10 in culturesupernatantmay be considered a counterpart of protective immuneresponseagainstLeishmaniaparasite.Key words: vaccine, leishmania, cellular immunity, visceralleishmaniasisSpecies:canine

Pr190. ALTERNATIVE METHODS TO DIFFERENTIATE DOGS VACCINATED WITH LEISHMMUNE AND INFECTED DOGS IN AN ENDEMIC AREA OF CANINE VISCERAL LEISHMANIASIS

VMFLIMA,JPCoRREA,KRFATToRIDeptoClínica,CirurgiaeReproduçãoAnimal–UNESPAraçatuba

vmflima@fmva.unesp.brZoonotic visceral leishmaniasis is caused by Leishmania

(Leishmania) chagasi. It is endemic inAmerica, Europe, andAsiancountrieswithnewdisseminationareasbeingidentified.TransmissionbetweenvertebratehostsisbythephlebotominehematophagousbiteofLutzomyialongipalpis.Dogs(Canisfamiliares)wereconsideredthemost importanturban reservoirsofL. (L.)chagasi,asshowa largerprevalencetoinfectionandfromwhereitgetstransmittedtohumansDogshavebeenshowntobehighlysusceptible to infectionand thecurrenttreatmentshavelimitedefficiency.Therefore,theuseofapre-ventivevaccineisfundamental.SeveralstudieshavebeenshownthatthevaccinationwithLeishimmunevaccine inducesastronghumoralresponsewithproductionofantibodiestoFMLantigenandantibodiesthat reactswith total antigen from Leishmania sp.The antigen totalis frequentlyused inRIFIandELISAassay todetectdisease, so inendemicareastheinfectioninvaccinateddogsshouldbemonitoringby othermethods.The aims of this studywas investigated byPCRinperipherical bloodof infecteddogsand the sororeactivity of dogsvaccinatedtoantigenk39asaalternativemethodstodiagnosisanddifferenciationofinfectedandvaccinateddogs.ToPCRonehundredanimalsinfectedwereused,DNAfromperiphericalbloodwasobtainedand the PCR with primer 13A and 13B was performed. The PCRshowed91%sensibility. InsororeactivityagainstK39 twentyhealthyanimalswerevaccinatedandtendaysaftertheserawastested,only10%ofgroupshowedpositivereaction to thisantigen, in theendofvaccinationthebonemarrowexamswasnegative,suggestingthatthepositivityobservedcanbedue tocrossreaction. Inconclusion,bothPCRandserumreactivityagainstk39canbeuseful todiagnosisofcaninevisceral leishmaniosiswithasecurityof90% inareaswhereLeishimmunevacccinehavebeenused.

Financialsupport:FAPESPKey words:LEISHIMMUNEVACCINE,DIAGNOSIS,PCR,K-39Species:canine

Pr191. AMERICAN VISCERAL LEISHMANIASIS IN SPEoTHoS VENATICUS

KARInAREInALDoFATToRI1,VALéRIAMARçALFELIxDELIMA21UNESP-FCAV-CampusJaboticabal,ProgramadeMicrobiologiaAgropecuária;2Depto.Clínica,CirurgiaeReproduçãoAnimal-

UNESPARAÇATUBAVisceralLeishmaniasis(VL)causedbyLeishmaniaLchagasi,isa

diseaseofhumanandcanids.Thedomesticdogisthemainsourceofhumaninfection,withperidomesticanddomestictransmissioneffectedprincipally by sandfly vector Lutzomya longipalpis. Control measureshave included thecontroversialexterminationofserologicallyorpara-sitologicallypositivedog,but theexistenceofwildanimalhostclearlycomplicatesthesituation.The“wild”canidsasadditionalreservoirshavebeenstudied.Theaimsof thepresentstudieswereto investigatethepresenceofantibodiesanti-LeishmaniabyELISAinseraandthepres-enceofDNAfromLeishmaniabyPCRinbiopsiesfromlymphonodes,spleen,skinandliverfromtwoSpeothosvenaticus(maintainedinCenterofsylvesteranimals-CESP-IlhaSolteira-S.P-Brazil).ELISAassaytheantigenusedwasatotalantigenfromLeishmaniaandtodetectionof

antibodieswasusedproteinAperoxidaseconjugated,thecut-offwasthesameusedtodomesticdog.TheDNAwasisolatedfollowingthemethodphenol/clorophormio/isoamílic (25:24:1)andPCRwasperformedwith13A,13Bprimer.IntwoanimalinvestigatedbyELISApositivereactionwasobserved.ThebiopsysanalyzedbyPCRshowedpositivein liverandlymphonodessamples.ThisisthefirstreportthatindicatethatthisspeciesasapossiblereservoirofLeishmaniaspKey words:LEISHMANIA,Speothosvenaticus,ELISA,PCRSpecies:other

Pr192. NEoSPoRA CANINUM TACHyZOITES, ATTENUATED By PASSAGE IN TISSUE CULTURE, INDUCE PROTECTIVE

IMMUNITyELISABETHAInnES,PAULMBARTLEy,STEPHEnEWRIGHT,

FRAnCESCACHIAnInI,DAVIDBUxTonMoredunResearchInstitute,PentlandsSciencePark,BushLoan,

Midlothian,EH26OPZlee.innes@moredun.ac.uk

neosporacaninumisanapicomplexanparasitethatisrecognisedasamajorcauseofreproductivefailureincattleworldwide.Cell-medi-atedimmuneresponsesinvolvingCD4+T-cellsandinterferongamma(IFNγ)areknowntobeimportantinhostprotection.Vaccinedevelop-menttohelpcontrolbovineneosporosisislikelytorequireinductionofspecificCMIresponsesandthis isofteneasier toachieveusing livepreparations.

LivevaccinesusingattenuatedorganismshavebeensuccessfulininducingprotectiveimmunityagainstotherprotozoanparasitessuchasLeishmania,ToxoplasmaandTheileriawhere inductionofCMI isimportant.neosporacaninumtachyzoiteswerepassagedwithinVerocellsfordifferentlengthsoftimeinvitroandcomparedfortheirabilitytocausediseasefollowinginoculationintomice.Miceinoculatedwiththehigh-passageparasitessurvivedsignificantlylonger(P<0.05)andshowedfewerclinicalsymptomscomparedtomicereceivingasimilardoseofthelow-passageparasites.Thehighpassageparasitesmul-tipliedmorerapidly(P<0.001)invitrothanthelowpassageparasites.Similarresultswereachievedinthreeseparateexperimentsindicatingthatitispossibletoattenuatethevirulenceofn.caninumtachyzoitesinmicethroughprolongedinvitropassage.

Miceinoculatedwithattenuatedn.caninumtachyzoiteswerefullyprotectedagainstachallengedoseofparasitesthatprovedtobelethalinnaïvecontrolmice.Braintissuewasexaminedat28daysafterchal-lenge,inallmice,usingaquantitativePCRtestforneospora-specificDNA.Inmiceinitiallyinoculatedwiththeattenuatedparasitesandthenchallenged,56%hadneospora-specificDNAinbraintissue,whereas100%ofthecontrolmicehadneospora-specificDNAfollowingchal-lengeand insignificantlyhigherquantity (P<0.001)compared to theimmunisedgroup.

Attenuationofvirulencewasachievedthroughprolongedpassageofn.caninumtachyzoitesintissuecultureandinoculationoftheattenu-atedparasitesintomiceinducedprotectionagainstalethalchallenge.Key words :Neosporacaninum,attenuation,virulence,protectionSpecies:other

Pr193. PARTICIPATION OF THE MACROPHAGE INFLAMMATORy PROTEIN-CCL3 IN THE IMMUNE

RESPONSE INDUCED By FASCIOLA HEPATICA INFECTION IN C57BL/6J MICE

REnATACRISTInADEPAULA1,ADRIAnoLUISSoARESSoUZA2,GIoVAnICASSALI3,MARCoPEZZIGUIMARÃES1,

MAURoMARTInSTEIxEIRA2,DéBoRAHnEGRÃo-CoRRêA11DepartmentofParasitology22BiochemistryandImmunologyandPathology;3BiologicalScienceInstitute-UFMG,BeloHorizonte,MG

Fasciolahepatica is a trematodeparasite that inhabits the liverand gallbladder ducts of hosts, including bovine, ovine and human.The hepatic injuries are associated with direct lesion as well as theintensity of the inflammatory reaction induced by the parasite dur-ing the migration of the immature forms and establishment of adultworms. Macrophage inflammatory protein (CCL3), produced andsecretedmainlybyactivatedmacrophages,hasacrucial role in therecruitmentofpro-inflammatorycells to the lesionsite.Therefore, in

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thepresentworkweevaluated the immune response inducedbyF.hepatica infection (10metacercariae/mice) inC57Bl/6Jmice geneti-callydeficientinCCL3production(CCL3KO)versusthewildtypemice(WT).AsignificantlyhighernumberofimmaturewormswererecoveredfromCCL3KOmicecomparedtoWTmice.Althoughinhighernumber,theliverlesioninducedbyF.hepaticawaslessextensiveinCCL3KOinfectedmice. In addition, antigen-stimulated spleen cells recoveredfrom20day-infectedCCL3KOmiceproducedsignificantlylowerlevelof IL-4, IL-13, IL-10and IFN-γcytokinesand the liverof thesemiceshowed significantly lower eosinophil peroxidase and mieloperoxi-daseactivitythantheWT-infectedmice.ThediminishedinflammatoryresponseofCCL3KOinfected-micewasfollowedbysignificantlylowerF.hepatica-inducedmortalityratebetween20and23daysofinfection,however, the mortality increases in CCL3KO infected-mice after 25daysofinfection.TheresultsindicatedthatCCL3productioniscrucialtocellularactivationandmigrationtoinfectionsiteduringF.hepaticainmice.Key words: chemokines, macrophages migration, CCL3, FasciolahepaticaSpecies:other

Pr194. INDUCTION OF ExPERIMENTAL AUTOIMMUNE MyOCARDITIS WITH AUTOANTIGEN IMMUNIZATION

ASSOCIATED WITH LOW NUMBERS OF TRyPANoSoMA CRUzI

LSDEARAGÃo1,2,RRSFEIToSA3,2,RSLIMA2,VMGSILVA1,2,PSoLIVEIRA4,2,TBAqUEIRo5,MBPSoARES2,RRIBEIRoDoSSAnToS2,WLCDoS-SAnToS2,LPonTESDE

CARVALHo21InstitutodeCiênciasdaSaúde,UFBA,Salvador,Brazil;2CentrodePesquisaGonçaloMoniz,FIOCRUZ,Salvador,Brazil;3EscoladeMedicinaVeterinária,UFBA,Salvador,Brazil;4InstitutodeBiologia,UFBA,Salvador,Brazil;5InstitutomultidisciplinardeSaúde,UFBA,

Salvador,BrazilMyocarditiscanbeinducedinmicebyimmunizationwithmyosin-

enriched heart antigen (myosin-enrichedAg) emulsified in completeFreund´sadjuvant(CFA)(1,2).Thisprocedure,however,didnotcon-sistentlyinducemyocaditisindifferentmousestrainsinourlaboratory.InthisworkaprotocolinvolvingtheimmunizationofBALB/cmicewithmyosin-enrichedAg,emulsifiedinCFA,followedbyinfectionwithalownumberofY-strainT.cruzitrypomastigotes,wasevaluated.Themyo-sin-enrichedAgusedinthisprotocolwasproducedinourlaboratoryasdescribedbyMargossianetal(3).Thisprotocolinducedmyocarditisinalltreatedmice,whichwassignificantlymoreseverethaninmiceonlyinfectedwithT.cruzi.Immunizationwithmyosin-enrichedAgalonedidnot inducedmyocarditis.Preliminaryresults indicate thatareductionof Mycobacterium tuberculosis concentration in themyosin-enrichedAgemulsion increases the severity of the experimentalmyocarditis.ExperimentstoinvestigateiftheuseofthelowM.tuberculosisamountimprovestheefficacyofthetwo-stepprotocolareunderway.

1.PontesdeCarvalhoL,Santana,C.C.,Soares,M.B.P.,Oliveira,G.G.S.,Cunha-Neto,E.,SantosRR.ExperimentalchronicChagasdiseasemyocarditisisanautoimmunediseasepreventablebyinduc-tionofimmunologicaltolerancetomyocardialantigens.J.Autoimmun.,(2002)18,131–138

2.Godsel L.M.,WangK.,SchodinB.A., Leon J.S.,MillerS.D.,Engman D.M. Prevention of autoimmune myocarditis through theinduction of antigen-specific peripheral immune tolerance. (2001)Circulation103:1709–1714

3.MargossianSS,LoweyS.Preparationofmyosinanditssub-fragmentsfromrabbitskeletalmuscle.MethEnzymol85:55-71,1982Key words:autoimmunity;Inflammation;disease;myocarditisSpecies:other

Pr195. SUPPRESSION OF PROLIFERATION AND CyTOKINE ExPRESSION By HELIS, A TICK SALIVARy GLAND-DERIVED

PROTEIN OF HAEMAPHySALIS LoNGICoRNISSAToRUKonnAI,CHIEnAKAJIMA,SAIKIIMAMURA,MICHIKoDAMA,SHInJIyAMADA,HIDETonISHIKADo,WIToLA

WILLIAMHARoLD,KAZUHIKooHASHI,MISAoonUMA

DepartmentofDiseaseControl,GraduateSchoolofVeterinaryMedicine,HokkaidoUniversity,Sapporo,Hokkaido060-0818,Japan

konnai@vetmed.hokudai.ac.jpPreviously,anovelimmunosuppressiveproteincodinggenewas

identifiedfromacDNAlibraryderivedfromsalivaryglandofpartially-fedHaemaphysalis longicornis (hard tick). By real-timePCRassay,the gene was expressed during blood feeding and suggested to beexpressedmainly in the salivary gland. In order to investigate thefunctionofthisnovelprotein,weexaminedtheproliferativeresponseand cytokine expressions of mitogen-stimulated peripheral bloodmononuclear cells (PBMCs) and splenocytes from cattle and micebyadditionwiththisrecombinantprotein(recombinantH.longicornisimmunosuppressor:rHeLIS).AdditionofrHeLISandcultivationfor72hclearlyshowedtheinhibitionoftheproliferationofmitogen-stimulatedcells inadose-dependentmanner,anddownregulationofthemeaninterleukin-2mRNAexpressionlevel.Additionofanti-HeLISantibodywith rHeLSandcultivation for72h clearlyenhancedproliferationofcells,indicatingadirectinvolvementofHeLISinthecellproliferation.Furthermore, in in vivo experiment, proliferative responseof spleno-cytesisolatedfromrHeLIS-inoculatedmice(150µgx3)wassignificantlylowerthanthatofcontrolmice.Interestingly,microarrayanalysisofthesplenocytesderived fromrHeLIS inoculatedmiceshowedsignificantdownregulationofseveral immunomodulatinggenes,suchasMHC,CD2andCD8.Inconclusion,theseresultssuggestthatHeLISisanimmunosuppressor,whichmightplayanimportantroleinthemodula-tionofhostimmuneresponses.Key Words:Tick,salivarygland,immunosuppressor,cellproliferationSpecies:other

Pr196. DETECTION OF ANTIBODIES AGAINST EHRLICHIA CANIS AND BABESIA CANIS IN BRAZILIAN WILD CAPTIVE

FELIDSMARCoSRAnDRé1,RoSAnGELAZMACHADo1,SILMARAMALLEGRETTI2,CRISTInAHADAnIA3,PAULoAnFELIPPE2,

KETTyFSILVA1,AnDRéACHnAKAGHI11UniversidadeEstadualPaulista(UNESP),Jaboticabal,SP,Brazil;2UniversidadeEstadualdeCampinas(UNICAMP),Campinas,SP,Brazil;3CentroBrasileirodeConservaçãodeFelídeosNeotropicais,

Jundiaí,SP,Brazilaguaron@fcav.unesp.br

Ehrlichiosisisanemergentzoonosiscausedbyanobligateintra-cellularbacteriabelongingtoAnaplasma,Ehrlichiaandneorickettsiagenera, present in the environment through complex interactionsamong invertebrates and vertebrates hosts. Babesiosis is causedby infection with intraerythrocytic parasites of the genus Babesia.Bothparasitesaretransmittedbyticks.BraziliandogsarecommonlyinfectedbyE.canisandB.canis,whosevectoristhedogbrowntick,theRhipicephalussanguineus.Littleisknownabouttheepidemiologyofehrlichiosisandbabesiosisinwildfelids,whichcanbeseenasgoodsentinelsforthesediseases,sincetheyarehostsforbothhemopara-sitesandvectors.ThepresentworkaimedtodetectantibodiesagainstE.canisandB.canisvogeli in72BrazilianwildcaptivefelidsbyIFA(IndirectImmunofluorescentAssay),tryingtoverifyiftheseanimalsareexposedtotheseparasites.BloodsampleswerecollectedfromPumaconcolor (cougar), Leopardus pardalis (ocelot), Puma yagouaroundi(aguarondi),Leoparduswiedii(margay),Leopardustigrinus(littlespot-tedcat),oncifeliscolocolo(pampascat)andPantheraonca(jaguar)maintainedincaptivityinBrasilia,RibeirãoPreto,Pedreira,CampinasZoos and inAssociaçãoMataCiliar de Jundiaí (Brazilian Center ofNeotropic Felids Conservation). Forty-four (70.96%) of 62 animalswereseroreagentsforB.canisand24%forE.canis.Antibodiestitersfoundrangedfrom1:20(cutoff)to1:2,560forE.canisandfrom1:40(cutoff)to1:1,280forB.canis.Fourteenfelidswereseropositivesforbothantigens,whileseventeenanimalswereseronegatives forbothagents.Toauthors´sknowledge,thisisthefirstreportofseroreactivityofBrazilianwildfelidsforbothE.canisandB.canisvogeli,showingthat theseanimals are exposed to vector andagents of ehrlichiosisandbabesiosis.Key words:wildfelids,Ehrlichiacanis,Babesiacanis,antibodiesSpecies:feline

Ci197. SIMILARITIES AND DIFFERENCES OF MICROSCOPIC STRUCTURE OF SPLEEN IN BELUGA HUSo HUSo WITH

TERRESTRIAL ANIMALS MoHAMMADTSHEIBAnI

DepartmentofBasicSciences,FacultyofVeterinaryMedicine,UniversityofTehranTehran,Iran

tsheibani@yahoo.comOneofthelargestandthemostimportantsturgeonsintheCaspian

Sea from theaspectof caviarproduction isbeluga.For this investi-gationatotalnumberofthreebelugasfishedfromsouthernCaspianSea,weresubjectedtostudy.Byanincisionontheabdominalwallthespleenwasremovedandafterfixationin10%bufferedformalin,routinehistologicalprocessesweredone,paraffinblocksweresectionedat6micronsandstainedwithHematoxylinandEosinandstudiedunderlightmicroscope.Theresultsshowedthatthespleen,unliketothoseofmammals,wassurroundedwithacapsulecomposedofthreelayersincludinganepitheliumofcuboidaltocolumnarcellswithsomesmallroundsecretorycellswitheosinophilicgranules.Underwhichawavinglayerof elastic fibersand thena condensedconnective tissuewerelocated.Underthecapsuleadenselymphatictissuecontainingsomemelanomacrophages was observed. The parenchyma of the spleenwascomposedofwhiteandredpulpsasinothervertebrates.Whitepulps consisted of a lymphatic nodule containing a small branch ofsplenicartery,as thecentralartery, like thatofmammals,butalsoavenousbranch,unlikemammalsthatdifferfromthisaspect.Thelym-phaticnoduleisfollowedbyalymphaticcordconsistingofanaggre-gation of lymphocytes around an arteriole, as periarterial lymphaticsheath.Theredpulpcontainedtoomanysinusoidsfilledwithredbloodcells surroundedby some trabeculaeanddiffused lymphatic tissuesthroughouttheredpulpofspleen.Key Words:Spleen,Beluga,Terrestrialanimals,HistologySpecies:fish

Ci198. GROSS AND MICROSCOPICAL CHARACTERIZATION OF THE THyMUS AND SPLEEN OF PIARACTUS

MESOPOTAMICUS (PACU)GMCoSTA,CEBARRoSo,ASoLIVEIRA,CBIASI,RSIUnES,

ARLIMA,JMMonTEIRo,RVBoSCH,PFAVARon,MAIVo,JRKFoURyJR

MedicalSurgicalDepartmentofVeterinaryMedicineandZootenicCollegeoftheUniversityofSãoPaulo

Piaractusmesopotamicus (Pacu) isafish fromtheCharacidaeFamily, intensively bred in Brazil because of its rusticity, easy rais-ing and adaptation, besides its excellent flavor. In order to produceahealthyfish, informationon its immunologicalsystem including thehistologyofthelymphoidorgansisneeded.Therefore,thisprojectper-formedagrossandhistologicalanalysisofthethymusandspleenoffiveyoung(about25cminlength)andfiveadultpacus(about38cminlength).Thethymusappearedasapairedorgan,placedonthecaudalportion of the head, dorsally to the gills in the opercular cavity andinvolved by amembranous capsule. The thymuswasmore evidentin fisholder thanfivemonths; it presentedagelatinousconsistencyvaryingfromvividtodarkredcolor.Histologically,itshowedmedullarand cortex regions consisted by conjunctive tissue and colagenousfibersandthepresenceof thymocytesandlymphocytes.Thespleenwas locatedcaudally to the liver, ventrally to theswimbladder,dor-sallytothemediumcaudalportionofthebiliaryvesicle.Itwasroundedshaped,dorsoventrallyflattenedanditpresentedanirregularsurfaceinyoungandlobedinadultfish.Itwasdelimitedbyamembraneand,histologically,itwascomposedbyawhitepulp,richinmelanomacro-phagesandextensiveredpulpareas.

Key words:Piaractusmesopotamicus,anatomy,histology,spleenandthymus.Species:fish

Ci199. IMUNOLOCALIZATION OF HEAT SHOCK PROTEIN (HSP) 70 IN THE LIVER OF DIPLOID AND TRIPLOID

RAINBOW TROUT RSIUnES1,JMMonTEIRo1,GMCoSTA,ARLIMA,RVBoSCH1

JRKFoURyJR11MedicalSurgicalDepartmentofVeterinaryMedicineandZootenic

CollegeoftheUniversityofSãoPauloHeatshockproteins(HSPs)belongtoahighlyconservedfamily

ofcellularproteinspresentinallorganisms.HSPsareexpressedindif-ferenttissuesundernormalconditions;howeveritincreasessubstan-tiallywhenstressispresent.Fishshowtwotypesofstressresponse:one is physiological and the other cellular. HSPs play a role at thelatter,whereafishmaypresentphysiologicalresponsetoastressoragentwithoutshowinganychangeofthecellularHSPsprofile.AmongtheHSPs,HSP70 is themost studied in fish,andweattempted tocompareitsexpressionintheliverofdiploidandtriploidrainbowtrout(oncorhynchusmykiss).Liverfromeightdiploidandtentriploidtroutswerefixedin10%formalinandprocessedforimmunohistochemistry,usingamonoclonalantibodyspecific toHSP70 (Stressgen®mouseanti-HSP70monoclonalSPA-810).TheimmunohistochemistryresultsshowedacytoplasmaticexpressionofHSP70inhepatocytesandinthe epithelial cells of the biliary duct. Differences on the intensity ofstaining were observed in both ploidy, where triploid fish presentedamoreevidentvariation, fromintensetonegativestaining.Negativecontrolsshowednostaininginbothdiploidandtriploidfish.OurresultssuggestedtriploidrainbowtroutshowdifferencesonHSP70expres-sioncharacteristicsfromitsdiploidcounterpart.Key words:Trout,HSP,stress,liverSpecies:fish

Co200. CELLULAR FUNCTION EVALUATION OF SANGUINEOUS LEUKOCyTES IN PHRyNoPS GEoFFRoANUS USING FLOW CyTOMETRIC

METHODOLOGyAGEnoy-PUERTo1,SRoSSI1,BoFERRonATo2,VMSá-RoCHA3,CSLISBoA4,LCSá-RoCHA3,ERMATUSHIMA31AcademicProgramofExperimentalandComparedPathology-

FMVZ/USP;2AcademicPrograminAppliedEcology-ESALQ/USP;3DepartamentofPathology-FMVZ-USP;4SectorofReptilesofthe

FoundationZoologicalParkofSãoPauloalexandergenoy@usp.br

AlthoughPhrynopsgeoffroanusdisplaysamplegeographicaldis-tributioninSouthAmerica,fewstudieshadbeendevelopedinrelationto thenaturalhistoryof this freshwater turtle.Thisspecies frequentlyappears in contaminated water sources, butthe effect of adversesituations (anthrophiceffects)on theanimals ‘cellular immuneactiv-ityareunknown.Inthisworkamethodemployingflowcytometrywasdeveloped inorder toevaluate the cellular functionsof phagocytosisandspontaneousandinducedoxidativeburstinleukocytesfrombloodsamplesofPhrynopsgeoffroanusfoundincontaminatedenvironmentsof thePiracicaba river andPiracicamirim brook, inSãoPauloState.Bloodsampleswereobtainedfrom8animalsfromPiracicabariver,9fromPiracicamirimbrook,and7fromZoologicalParkFoundationofSãoPaulo; thesamplesfromzoologicalparkwereusedascontrolgroup.ThesampleshadbeenkeptincultureinRPMImediumunderrefrigera-tionanddirectedtotheNeuro-immunologyLaboratoryofDepartmentofPathologyinFMVZ/USPtobeprocessed.Thefactthattheredblood

7. COMPARATIVE IMMUNOLOGy; IMMUNOECOLOGy: POSTERS CI197-CI206

cellsarenucleatedintheseanimals,madenecessarytheuseofFicoll-PaqueTMPLUSatdifferentdensities(1,085;1,070and1,055g/mL),inordertopre-separatethesecells.Thenthesamplewascentrifugedtoobtaincellsofinterest,at18°C,450xg,acceleration9andbrake0,resultinginawhiteringofleukocyteslocatedbetweenthesuperiorlayersof theFicoll.Thesamplesweresubmitted to twowasheswithRPMItoremovetheexcessesofFicoll.Afterthewashes,thesamplesweresuspendedagainin500µLRPMIformakingaviabilitycountingwithTrypanBlue;sampleswithinferiorpercentagesto94%werenotused in thestimulates:ZymosanA(Sacaromycescerevisiae) theBioParticles®,AlexaFluor®594conjugatetoinducephagocytosisand12-myristate13-aceticacid(PMA)andSacaromycescerevisiae(Zymosan)intheinductionofoxidativeburst.Theanimal’sleukocytesofthezoopresented greater phagocytosis intensity induced by Zymosan A, inrelationwithtothecellsoftheanimalsofthePiracicabariver.Theinten-sityofoxidativeburstinducedbyPMAwassmallerintheanimalsofthePiracicamirimbrookwhencomparedwithturtlesofPiracicabariverandthezoo.Financialsupport:CNPq,CAPESandFAPESPKey words:CellularfunctionEvaluation,Leukocytes,FlowCytometric,PhrynopsgeoffroanusSpecie:reptile(turtle)

Ci201. CHARACTERIZATION OF BOVINE ACTIVATION-INDUCED CyTIDINE DEAMINASE

SUBHASHVERMA,ToMGoLDAMMER1,RoBAITKEn2DivisionofInfectionandImmunity,GlasgowBiomedicalResearch

Centre,UniversityofGlasgow,G128TA,UnitedKingdom;1ResearchUnitMolecularBiology,ResearchInstitutefortheBiologyofFarmAnimals,Dummerstorf,D-18296,Germany;2Correspondingauthor

Activationinducedcytidinedeaminase(AID)playspivotalroleinthegenerationofantibodydiversity,beingessentialforsomatichyper-mutation(SHM),geneconversion(GC)andclassswitchrecombination(CSR).Here,wereportthesequencing,mapping,expressionpatternandmutatorphenotypeofAIDfromBostaurus(BoAID).

Atthegenelevel,exonsequenceswerefoundtobeverysimilartotheircounterpartsfromhumansandmice, implyingahighlevelofstability throughout vertebrate evolution. In contrast, little sequencesimilaritycouldbedetectedbetweenintronicsequencesofbovineAIDand its counterparts from othermammals. Bovine intron sequenceswerealsoconsistentlylongerthanthoseofhumansandmice.MappingusingaradiationhybridpanelsuccessfullyplacedtheBoAIDgeneonBostauruschromosome5(BTA5)inaregionsynthenicwithHSA12.

BoAID cDNA had an open reading frame of 199 amino acidresidues.Acomparisonwithitscounterpartinmammalsandchickenidentifiedmostlyconservedaminoacidresiduesespeciallywithinthecytidine deaminase motif. BoAID carried an extra codon in exon 3,whichcodesforlysine,thesignificanceofwhichisunknown.

TheleveloftranscriptionofBoAIDwascomparableinyoungandoldanimals.Interestingly,BoAIDtranscriptswerepresentinadayoldanimaleventhoughgerminalcentrescouldnotbedetectedinthelym-phoid tissue by histology. BoAID mRNA in these animals was beenshown tobepresent inboth lymphoidandnon-lymphoid tissuesbuttranscriptswereobservedparticularlyabundantinthespleenandmes-entericlymphnodes.Muchlowerabundancewasevidentintheliver,andBoAIDmRNAwasundetectableinmuscletissue.

TheBoAIDcDNAwascloned intoabacterialexpressionvectorandwheninducedinEscherichiacoli,itconferredamutatorphenotype.Thiswasevidentthroughdisruptionofthe lacZreadingframeandinseparateexperiments,throughtheappearanceofrifampicinresistantcolonies,aresultofhighfrequencymutationinrpoB.

CloningofBoAIDanddefinitionof its chromosomal localizationandfunctionalpropertieswillfacilitateourunderstandingoftheroleofthisenzymeintheimmunobiologyofB.taurus.Key words:Bovine,cytidinedeaminase,BTA5Specie:ruminants

Ci202. THE CARCINOEMBRyONIC ANTIGEN (CEA) FAMILy IN PLACENTAL MAMMALS OF THE SUPERORDINAL CLADE

LAURASIATHERIA

RoBERTKAMMERER1,2,TAnJAPoPP1,STEFAnHAERTLE3,BERnHARDBSInGER4,KATHRynVHoLMES5,,WoLFGAnG

ZIMMERMAnn11Tumor Immunology Laboratory, LIFE Center, Klinikum

Grosshadern, LMU, Munich, Germany; 2Institute for MolecularImmunology, GSF National Research Center for the EnvironmentandHealth,Munich,Germany;3InstituteforAnimalPhysiology,LMU,Munich,Germany;4Institute forAnatomy,UniversityHospitalEssen,Essen,Germany;5UniversityofColorado,HealthSciencesCenteratFitzsimons,DepartmentofMicrobiology,Aurora,USA

TheprimordialCEAgenefamily,whichislocatedintheexpandedleukocytereceptorcomplex,wascomposedoffivegenes(CEACAM1,CEACAM16, CEACAM18, CEACAM19, CEACAM20).The CEACAM1genegaverise toexpansionsof theCEAfamiliesbygeneduplicationand exon shuffling. In rodents and primates, the CEACAM1-relatedmembers of the CEA family can be divided into the CEA-related celladhesion molecules (CEACAM) and the pregnancy-specific glycopro-teins(PSG).WhilevariousCEACAMarepivotal forawell-coordinatedimmune response, thePSGare supposed tobe instrumental for suc-cessfulpregnancies.ThecharacterizationoftheCEAfamiliesinprimatesand rodents revealed profound species-specific differences. In themouse three CEACAM (CEACAM1, CEACAM2 and CEACAM17) aretransmembraneboundwhile theothersevenaresecreted. InhumansCEACAM1,CEACAM3andCEACAM4aretransmembraneboundwhiletheotherfourareGPIanchored.CEACAM1andCEACAM2harborITIMandCEACAM3andCEACAM4ITAMintheircytoplasmictails.SincelittleisknownabouttheCEAgenefamiliesinotherspecies,wehaveanalyzedCEA-relatedgenes incattle,dogandhorse.We identifiedorthologousgenesforCEACAM1andtheotherancestralmembersinallthreespe-cies.ExpansionofCEACAM1-relatedgenesoccurredinthesespecies,leadingto3,7and11differentCEACAM1-relatedgenesincattle,dogandhorse,respectively.MoststrikingisthecompleteabsenceofPSGinbothcattleanddog.InthehorsefivepossiblysecretedCEACAMswereidentified.BasedonthreeESTclones,oneofthesegenesisexpressedin placenta.Therefore, theexistenceofPSG in thehorse is possible.TheCEACAM1-relatedmoleculesincattleanddogsaretransmembranemolecules,whichharborITAMoreITAM-likemotifsandareexpressedbyimmunecells.Inthehorse,therearetwogenes,whichmaybeortholo-gous to CEACAM1 (CEACAM1, CEACAM43). The closest relative toCEACAM1isanITAM-bearingmolecule,similartothefindingsincattleanddogs.TheclosestrelativetoCEACAM43isapossiblysecretedpro-tein,similartothesituationpreviouslyfoundinmice.Takenintoconsider-ationthatCEACAM1representsavirusreceptorinmiceandabacterialreceptorinhumanswehypothesizethatthestructureoftheCEAfamilymirrorsthehistoryofthearms’racebetweenCEACAM1-bindingpatho-gensandthehost.Therefore,wegeneratedanti-bovineCEACAM1mAbandbovineCEACAM1-FcfusionproteinsforthescreeningofpathogensbindingbovineCEACAM1.Indeed,wehaverecently identifiedaviruswhichinfectscellsbybindingtobovineCEACAM1.Key words:carcinoembryonicantigen,pathogenreceptor, leukocytereceptorcomplex,pathogen-hostcoevolutionSpecies:other

Ci203. MHC POPULATION STRUCTURE IN THE NEW ZEALAND BRUSHTAIL POSSUM

oLIVIAJHoLLAnD1,2,4,PHILIPECoWAn3,LAWREnCEWCHAMLEy4,DIAnnEMGLEESon2

1NationalResearchCentreforPossumBiocontrol,LandcareResearch,Auckland,NewZealand;2EcologicalGeneticsLaboratory,

LandcareResearch,Auckland,NewZealand;3PestControlTechnologies,PalmerstonNorth,LandcareResearch,NewZealand;4ObstetricsandGynaecology,UniversityofAuckland,Auckland,New

Zealandhollando@landcareresearch.co.nz

Thebrushtailpossum(Trichosurusvulpecula) isamajor invasivepestinNewZealand,causingseveredamagetonativeecosystemsandactingasavectorofbovinetuberculosis.Oneoptionforcontrollingthispestmaybetheuseofimmunocontraceptivevaccines,amethodoffer-tilitycontrolthatemploystheimmunesystemtoattackreproductivecellsorproteins.Trialshaveshownthereisvariabilityinimmuneresponsesamongindividualpossumstothevariousimmunocontraceptivevaccinescurrentlyundergoinglaboratorytrials.Ifthisvariabilityisundergenetic

120

control, then the application of immunocontraception as a populationcontrolmechanismwillhavetobecarefullymanagedtoavoidpreferen-tialselectionofanimalsthatfailtomountasignificantimmuneresponseandremain fertile. Themajorhistocompatibilitycomplex(MHC) isanimportantcomponentoftheimmunesystem,whichinfluencesthenatureof immune responses and has been extensively studied in eutherianmammalsandbirds,howevertodateonly limiteddata isavailableforthe marsupial MHC loci. This study aims to characterize alleles anddocumentgeneticvariationintheMHClociofNewZealandpossums,andinvestigatewhetherthereisarelationshipbetweenMHChaplotypesandindividualimmuneresponsestofertilitycontrolvaccines.Weusedknownmarsupial(possum,red-neckedwallaby,tammarwallaby,opos-sum) MHC sequences to design PCR primers for possum MHC loci.The variability of these lociwas screened in populations of possumsfrom locations throughoutNewZealand,aswellasbetween individu-alswithknownresponsestoimmunocontraceptivevaccines.AlphaandbetachainsfromtwoclassIIfamilies,whicharespecifictomarsupials,(DAandDB)havebeenconfirmedtobepresentinpossumsthroughoutNewZealand. Over20newMHCalleleshavebeen identified in thepossumandpreliminarypopulationsurveyshaveshownevidence forvariation in thedistributionofMHCalleles in geographically separateNewZealandpossumpopulations.ThelevelsofvariabilityintheMHCofthismarsupialappeartobecomparabletothoseofeutherianspecies.Inthisstudy,wehaveidentifiedalargenumberofnovelpossumMHCallelesandfoundevidenceforMHCclustering/variabilityindifferentpos-sumpopulationsinNewZealand.Suchextensivevariationmaygreatlyimpacttheeffectivenessofimmunocontraceptivecontrolmeasures.Key words:Immunocontraception,MHC,PCR,marsupialSpecies:other(marsupial)

Ci204. ENZOOTIC CALCINOSIS: EFFECTS ON ORGANS AND CELLS OF THE IMMUNE SySTEM IN ExPERIMENTALLy

INTOxICATED RABBITSPAULAAFonTAnA1,2,CARoLInAnZAnUZZI1,2,LEonARDo

ACHInCHILLA1,CLAUDIoGBARBEITo1,2,EDUARDoJGIMEno1,2,EnRIqUELPoRTIAnSKy1,2

1InstitutodePatología.FacultaddeCienciasVeterinarias.UniversidadNacionaldeLaPlata.60y118(1900)LaPlata.BuenosAires.Argentina;2CONICET(ConsejoNacionaldeInvestigaciones

CientíficasyTécnicas)pfontana@fcv.unlp.edu.ar

Solanumglaucophyllum(Sg) isacalcinogenicplantthatcauses“Enzooticcalcinosis”incattle.Itsmaintoxicprincipleisthe1,25-dihy-droxyvitaminD.VitaminDacts incalciumhomeostasisandhaspro-nouncedimmunomodulatoryproperties.ThegoalofthepresentworkwastotestimmunesystemmodificationsduetoVitD3inSg-intoxicatedanimals.Rabbitswereselectedasexperimentalmodelsdue to theirhighsusceptibilitytotheSgintoxication.NewZealandmalesreceived125mg/kgofSgleavespowdertwiceaweekduring15and30days.Agroupwas intoxicatedduring15daysbutsacrificedat30dayspi(recoverygroup).Controlanimalsweresacrificedat0and30days.Phagocytosistests, immunohistochemistryforCD4,CD8andBcellson frozen isolated lymphocytes from lymph nodes, and histopathol-ogy of thymus, appendix, popliteal lymph nodes and spleen wereperformed. The phagocytic activity in peritoneal macrophages wasprogressivelyreducedintheintoxicatedanimalswithtime.Asignificantdecrease in thenumberofCD4+,CD8+andBcellswereobservedaswell.Histopathologyshowedagradual increaseof thedegreeofthymic and appendix atrophy. However, no changeswere observedin the analyzed lymph nodes and in the spleen. Recovery groupshowedsimilar results thancontrols.Theseresultsdemonstrate thatSgprinciplesinduceimmunesystemalterationsinthisanimalmodel.ThechangesobservedintheSg-intoxicatedanimalsresembledthoseproducedunderphysiologicalagingprocess.Key words: Enzootic Calcinosis, Solanum glaucophyllum,hypervitaminosisD,immunesystem,rabbitsSpecie:other(rabbits)

Ci205. A COMPARATIVE ANALySIS OF THE PORCINE, MURINE, AND HUMAN IMMUNE SySTEMS

HARRyDDAWSon,JoSHUAJREECE,JoSEPHFURBAnJR

UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,BeltsvilleHumanNutritionResearchCenter,Nutrient

RequirementsandFunctionsLaboratory,Beltsville,Maryland,20705USA

A literature and laboratory-based analysis compared selectedfeaturesofgenotype,phenotype,andfunctionalexpressionofthepor-cine,murine,andhumanimmunesystems.Atotalof147parameterswereexamined.Post-genomicanalysisfoundabout300uniquemRNAcodingsequencesbetweenmiceandhumanswithapproximately100relatedtoimmunity.Todate,weorothershaveidentified43porcineimmune-relatedgenesnot found in rodents.Furtheranalysis identi-fiedalimitednumberofgenespresentinrodentsandpigsbutnotinhumans,andgenesabsentinpigsbutfoundinrodentsandhumans.Thephenotypeofmanyimmunecells,includingalternativelyactivatedmacrophagesandTregulatorycells,aremoresimilarbetweenpigsandhumanscomparedtorodents.Pigsarenaturallysusceptibletoinfectionwithspeciesofhelminthsthatarecloselyrelatedoridenticaltothoseinfectinghumans(Ascaris,Taenia,Trichuris,Trichinella,Shistosoma,Strongyloides) indicating functionally similar host characteristics.Additionally, pigs are excellent models for bacterial (Campylobacter,E.coli,Helicobacter,neisseria,Mycoplasma,Salmonella),protozoan(Toxoplasma)andviralinfections(Coronavirus,HepatitisE,Influenza,nipah,Reovirus,Rotavirus)infections.Overall,approximately80%ofthe 147 parameters examined were more similar between pigs andhumans,suggestingthatevaluatingimmunefunctioninpigsprovidesdatathatismorephysiologicallyrelevanttohumans. Key words:review,human,mouseSpecies:other

Ci206. GENE ExPRESSION OF CHICKEN INTERLEUKINE-4 By BACULOVIRUS

TAKAyUKIKUBoTA,MAKIKoyoKAyAMA,MASAToKISHIMA,SAToKoWATAnABE,MASATooHoTA,HIRoKAZUHIKono,

CHARLESoAoMWAnDHo,SHIGEKIInUMARUNationalInstituteofAnimalHealth

tkubota@affrc.go.jpINTRODUCTION: Mammalian T cells are classified into Th1

andTh2according to thecytokineproduction.AlthoughchickenTh2cytokineshavenotbeenidentifieduntilrecentyears,existenceofTh2cytokinewassuspectedtemporarily.FollowingthecloningofTh2cyto-kinessuchas interleukin4(IL-4), IL-5,andIL-13in2004, itbecameclear that thesecytokinesandtheir lociareconserved inchicken. Inthisstudy,weusedabaculovirusgeneexpressionsystemtoexpresschickenIL-4andinvestigateitsfunction.METHODS:mRNAwasiso-latedfromchickenperipheralbloodorthymusandRT-PCRwasusedto synthesis cDNA. IL-4 cDNA was inserted into pFastBac1 vectorand transferred intoAcNPVbaculovirusbyusingBac-to-Bacsystem(Invitrogen).RecombinantviruswasinfectedintoTn5insectcells.TheculturesupernatantcontainingrecombinantchickenIL-4(rIL-4)proteinwasharvestedandpartiallypurifiedbyammoniumsulfatesedimenta-tionmethod.Biologicalactivitywasexaminedby 3H-tymidneuptake.RESULT & DICUSSION: After the recombinant baculovirus wasinfectedintoTn5insectcells,theproteinsecretedinthemediumwasaccumulated intotheculturesupernatant.Supernatantwascollectedon the 5th day when protein concentration was highest. When par-tialrefiningofthisproteinwascarriedoutwiththeammoniumsulfatesedimentationmethod,itprecipitatedbetween80%to90%saturation.Expression was checked by tricine polyacrylamide gel electrophore-sis(PAGE).Twouniquebandswerefound.Themolecularmassesofthe two expressed proteins were about 18.6kDa and 15.5kDa. ThemolecularmassofmaturechickenIL-4calculatedfromtheaminoacidsequenceis12.4kDaandtheprecursorwithsignalpeptideis14.8kDa.Wepresumelargersizeoftheseproteinsonthegelwereduetogly-cosylation,becausethesequenceofchickenIL-4contains5N-linkedglycosylationsitesandthosediffusedbandsaretypicalofglycoprotein.Theseprecipitatedproteinsfacilitatedproliferationactivityofperipheralwhitebloodcell indosedependentmannersuggestingchickenpos-siblyhaveTh2function.Key words:“intereukin4”,“chicken”,“protein”,“activity”Species:avian

mi207. IDENTIFICATION OF A MHC CLASS I-RESTRICTED CD8+ IMMUNE RESPONSE TO FOOT-AND-MOUTH DISEASE

VIRUS IN CATTLEEFRAInGUZMAn,GERALDInETAyLoR,BRyAn

CHARLESTon,SHIRLEyELLISInstituteforAnimalHealth.DivisionofImmunology.Compton,

NewburyRG207NN.UnitedKingdomefrain.guzman@bbsrc.ac.uk

Footandmouthdiseasevirus(FMDV),anapthovirus,isamemberofthegenuspicornaviridaeandcanbeclassifiedintosevenserotypes.Itcausesahighlycontagiousviralinfectioninruminants,pigsandclo-ven-hoovedanimalswithveryimportanteconomicconsequences.Thedisease is characterised by the formation of vesicles on themouth,tongue, noseand feet.Control of thedisease is achievedby vacci-nation with a chemically inactivated whole virus vaccine emulsifiedwithadjuvant,providingonlyshort-term,serotypespecificprotection.A better understanding of protective immunemechanismsmayhelpin development of novel vaccines. While much attention has beendevotedtohumoralresponsestoFMDV,lessisknownabouttheroleofcell-mediatedresponsesinprotectiveimmunity.Aninvitroassayforthedetectionofantigen-specificinterferon-gamma(IFN-γ)releasebyCD8+TcellswasusedtodeterminethelevelofCD8+TcellactivityinvaccinatedandinfectedcattleofknownMHCtype.MHC-restrictedCD8+Tcell recognitionof thestructuralproteins (P1)ofFMDVwasdetectedinvaccinatedcattle.AsignificantcellularimmuneresponsetobothliveandUV-inactivatedFMDVandtotheP1regionofFMDVwasalsodetectedinFMDV-infectedanimals.UsingmousecellsexpressingindividualcattleMHCclassIgenes,wehaveidentifiedspecificallelesresponsibleforthepresentationofFMDVP1peptidestoCD8+Tcells.WearecurrentlymappingMHCclassI-restrictedCD8+epitopeswhichwillenableustocarryoutadetailedstudyoftheroleofCD8+Tcellsfollowingvaccinationorinfectionincattle.Key words:Footandmouthdiseasevirus;CD8+Tcells;aβCD4+Tcells;interferon-γ;MHCclassI-restrictedCD8+epitopesSpecies:ruminants

mi208. IDENTIFICATION OF NOVEL RECEPTORS FOR IGG FC IN BOVINE LyMPHOCyTES

MATTAFIRTH,KULDEEPSCHATTHA,DoUGLASCHoDGInS,PATRICIAESHEWEn

UniversityofGuelph,DepartmentofPathobiology,OntarioVeterinaryCollege.Guelph,Ontario,Canada

mfirth@uoguelph.caReceptors for the Fc portion of immunoglobin molecules (FcR)

providean important and vital link between circulatingantibodyandcellulareffectorfunctions.CellsurfaceFcRforIgG,IgE,IgAandIgMhavebeenidentifiedonmanycelltypes.MuchofthecurrentliteraturefocusesonFcRsequenceandfunctioninthemurineandhumanspe-cies.Todate,onlyahandfuloftheseFcRshavebeensequencedandcharacterized in livestockspecies. Fc[gamma]RII (CD32) isanFcRpresentinseveralisoformsonawidevarietyofcellsincludingBcells,monocytesandneutrophils. OnBcells, theFc[gamma]RIIb isoformactstodownregulateIgGproductionindirectoppositiontotheBcellco-receptorcomplexconsistingofCD19,CD21andCD81bymeansofanintracellularITIMmotif.Arecentexaminationoftheexpressionpro-fileofCD32inbovinebloodmononuclearcellsrevealedthepresenceoftwopreviouslyuncharacterizedCD32-likesequences.SequencingofthefirstuncharacterizedtranscriptindicatedittobeaCD32mRNAsplicevariant,lackinga120bpregioncorrespondingtothetransmem-braneregioninthefull-lengthmolecule.Sequencehomologywiththe

publishedbovineFc[gamma]RIIsequencewas99%and100%fortheextracellularandintracellulardomains,respectively.ItishypothesizedthattheresultingproteinisasolubleisoformofCD32;thisphenome-nonhasbeendescribedpreviouslyonlyinmiceandhumans.Analysisofthesecondsequencerevealedaputativeextracellulardomainwith96% sequence identity to CD32; however, the predicted transmem-braneandintracellularregionsdisplayed79%sequenceidentitytotheFc[gamma]RIII(CD16)molecule.ThisisofparticularnoteasCD16,throughnon-covalentassociationwitheithertheFc[epsilon]RIgammachainor theTCRzetachain,acts tocausecellactivationbymeansof ITAMmotifspresent in theircytoplasmic tails. Wehavedetectedsoluble CD32 transcript in B cells, CD4+ and CD8+T cells, CD14+monocytesandneutrophilsisolatedfrombovineblood.TranscriptfortheCD32/CD16chimericsequencewasfoundinBcells,CD4+TcellsandCD14+monocytes. Furthermore, culturedbovineBcells (BL3)werefoundtosecreteaproteinreactivewithananti-CD32monoclonalantibody(CCG32,IAHCompton).ThisisthefirsttimeasolubleFcRhasbeenreportedinaspeciesotherthanmurineorhuman.Theexis-tenceofthesenovelreceptorsaddsanewlevelofcomplexitytocellregulatoryfunctionsinvolvingIgGimmunecomplexes.Key words:solubleFcreceptor;CD16;CD32Species:ruminants

mi209. VARIATION IN ExPRESSION OF FC GAMMA RECEPTOR IIB AND CD21 ON BOVINE LyMPHOCyTES WITH

AGEKSCHATTHA

Pathobiology,OVC,UniversityofGuelph,CanadaItisdifficulttoinduceactiveimmuneresponsesinneonates,partly

due to the limited functionalabilityofneonate’s immunesystemandpartly due to inhibition mediated by maternal antibodies. Fc gammareceptor IIB(CD32) isamajorreceptorresponsible forsuppressionofantibodyresponses,throughinteractionwithimmunecomplexesofmaternalantibodies(primarilyIgG)andantigens,byvirtueofitsintra-cellular immunoreceptor tyrosine-based inhibitorymotif (ITIM).CD21(CR2), a receptor for complement component C3d, is expressed byB lymphocytesandbinding results in loweringof the threshold forBcellactivation.Thus itplaysan important role inenhancingantibodyresponsestocomplement-associatedantigens.CD21(activatingcom-ponent),Bcellreceptor(membraneIgM)andCD32(inhibitorycompo-nent)formanintricatetriadfordeterminingthethresholdofactivationforBcells.BecausecellulardistributionofCD21andCD32ofcattlehasnotbeenwelldocumented,thisstudyaimedtodeterminethevariationin expression of CD21 andCD32 on bovine lymphocytes with age,particularlyemphasizingdifferencesbetweenneonatesandadults.

Bloodwascollectedfrom20healthyHolsteincalves,1to90daysofage,and8healthyadults.Thepercentage,absolutenumberandmean fluorescence intensity of CD21 and CD32 positive cells wasdetermined using fluorochrome labelled monoclonal antibodies andflow cytometry. The percentage and absolute number of CD21 andCD32positivelymphocytesincreasedwithagefrombirthuntilabout60daysofage,withCD21showingagreaterpercentageincreasecom-paredtoCD32.TheproportionofCD32positivelymphocytesexpress-ing CD21 also increased with age. In both calves and adults, thenumberofcellsexpressingCD32wasgreaterthanthoseexpressingCD21.MeanfluorescenceintensityforCD32wassignificantlyhigherforadults(P<0.01),indicatingagreaternumberofCD32receptorsperlymphocytecomparedtoneonates.RT-PCRusingRNAextractedfromPBMCsofneonatalcalvessuggestedproductionofasolubleversionof

8. MEDIATORS OF RECRUITMENT AND FUNCTION OF CELLS OF THE IMMUNE SySTEM; FC RECEPTORS AND IMMUNOGLOBULINS; SIGNAL TRANSDUCTION AND GENE ExPRESSION IN CELLS OF THE IMMUNE SySTEM:

POSTERS MI207-MI210

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FcgammaRIIB(sCD32),lackingtransmembraneregion,whichmightcontributetolowermembraneCD32expressioninneonates.

LowerexpressionofCD21 inneonates combinedwith reducedlevelsofC3dinserummaybea limitingfactor foractivationofneo-natalBcells.AnagerelatedincreaseinthepercentageandabsolutenumberofCD21andCD32positive lymphocytes, alongwith reduc-tion in the levelofmaternalantibodiesmighthelpexplain theabilityofoldercalvestoproduceeffectiveantibodyresponsescomparedtoneonates.Key words:Species:ruminants

mi210. ELEVEN PORCINE C GAMMA (Cγ) GENES: PHyLOGENy, ExPRESSION AND ALLOTyPIC VARIANTSJoHnEBUTLER1,nAnCyWERTZ1,nICKDESCHACHT2,3,

SERGEMUyLDERMAnS2,3,JoAnKLUnnEy4 1Dept.Microbiology,Univ.ofIowa,USA;2Cellularand

MolecularImmunology,VrijeUniversiteitBrussel,,BELGIUM;3DepartmentofMolecularandCellularinteraction,VIB,Vrije

UniversiteitBrusselBELGIUM;4ANRI,ARS,USDABeltsville,MD,USA

john-butler@uiowa.eduSubclassheterogeneityofswineIgGwasrecognized>40years

ago (1,2), allotypic variation was described in 1982 (3), differentialspecificityofanti-swineIgGantibodieshasbeenrecognized(4,5)andfiveCγgenesequenceshavebeenreported(6).The fullcomplexityof the system remains unresolved but we present here incrementaldata on eleven expressed Cγ genes which appear to comprise sixsubclasses.TheallelicvariantsofIgG1(IgG1aandIgG1b)accountforaverylargeproportionoftheIgGtranscribedinswine.Thisincludes

mostoftheIgGtranscribedduringfetallife.IgG2,alsowithtwoalleles,istranscribedatmuchlowerlevelsasareIgG4,IgG5andIgG6.IgG3isauniquelong-hingedvariantwhichaccountsfor>50%ofIgGtran-scriptsintheilealPeyerspatches(IPP)andMLNofnewbornsbutrela-tivetranscriptiondeclinesastheIPPinvolute(7).Sequenceanalysesconfirmthatspeciationprecededsubclassevolutionsothatasubclasswithacertainname,e.g. IgG1,doesnot implyhomologywith thoseofthesamenameinotherspecies.Thereforeestablishingstructure-function relationships must be done for each species individually.Porcine IgG subclass proteins cannot be biochemically purified soporcine-camelid chimericheavy chainantibodies (HCAb)havebeenconstructedandexpressed.Specificitytestsfailedtoidentifyanysub-class-specificmonoclonalorpolyclonalantibodiesandshowedmosttobebiasedtotheIgG1ballelicvariant.

1.Metzger, J.J. andM.Fougereau,1967.C.R.Hebd.SeancesAcad.Sci.SerD.Scinat. 265:724

2.Kaltreider,H.B.andJ.S.Johnson.1972.J.Immunol.109:9923. Rapacz, J and J. Hasler-Rapacz. 1982. Proc. 2nd World

CongressonGeneticsApplied to Livestock Production. VolVIII.EditorialGarsi,Madrid

4.Bokhout,B.A.,J.J.vanAsten-NoordijkandW.Stock.1986.Mol.Immunol.23:675.

5.VanZaane,D.andM.M.Hulst.1987.Vet.Immunol.Immunopath.16:23.

6.Kacskovics,I.,J.SunandJ.E.Butler.1994.J.Immunol.153:3536.

7.Butler,J.E.andn.Wertz.2006.J.Immunol.177:5480.Key Words:IgGsubclasses;phylogeny;fetus;IPPSpecies:swine

VA211. NATURAL KILLER CELLS IN LyMPH NODES OF HEALTHy CALVES ExPRESS CD16 AND SHOW BOTH

CyTOTOxIC AND CyTOKINE-PRODUCING PROPERTIESPREBEnBoySEn1*,GJERMUnDGUnnES2,DAnIELAPEnDE3,

METTEVALHEIM4,AnnEKSToRSET11Dept.ofFoodSafetyandInfectionBiologyand2Dept.ofBasicSciencesandAquatic;Medicine,NorwegianSchoolofVeterinary

Science,Oslo,Norway;3IstitutoNazionaleperlaRicercasulCancro,Genova,Italy;4Dept.forAnimalHealth,SectionforPathology,

NationalVeterinaryInstitute,Oslo,Norwaypreben.boysen@veths.no

Naturalkiller(NK)cellswererecentlyshowntoplayanimportantimmunomodulatoryroleinlymphnodes.Weherereportthepresence,phenotypeand functionofNKcells resident in lymphnodesof sev-eral anatomical sites of healthy calves.NK cells, definedbyNKp46expression,werepresentintheparacortexandthemedullaofbovinelymphnodes.Mostlymphnode-derivedNKcellsexpressedCD16andperforin,andalyticcapacitywasdemonstrated,whileawell-developedinterferon-gammaresponsetointerleukin-2andinterleukin-12stimula-tionwasalsoseen.Lymphnode-derivedNKcellsdifferedfromthoseinbloodbyahigherexpressionof theactivationmarkersCD44andCD25,aswellasCD8.Lselectin

(CD62L)was expressed by themajority of lymph node-derivedNKcells,consistentwithadependencyofthismoleculeformigrationtolymphnodes.Unlikeinblood,themajorityoflymphnodeNKcellshadlittleornoCD2expression.Comparedtoavailableliterature,calflymphnodescontainedNKcells innumbersequal toorhigher thanreported inhumans,andclearlyhigher than inmice.ThesefindingssuggestacytotoxicroleoflymphnoderesidingNKcells,beyondthepredominantlycytokine-producingrolepreviouslyinferredfromstudiesonhumanNKcells.Key words: natural killer cells, lymphoidorgans,CD16,cytotoxicity,interferon-gamma,Species:ruminants

VA212. CD14+ CELLS ARE REQUIRED FOR TLR7/8 LIGAND-INDUCED IL-12 AND IFN-γ RESPONSES IN BOVINE BLOOD

MONONUCLEAR CELLSJoRAMBUZA1,PonnBEnJAMIn1,JIAnZHUnGZHU1,ARTHUR

MKRIEG2,LoRnEABABIUK1,GEoRGEKMUTWIRI11Vaccine&InfectiousDiseaseOrganization,Universityof

Saskatchewan,Saskatoon,Saskatchewan,CanadaS7N1L1;2ColeyPharmaceuticalGroup,Wellesley,MA02481USA.

joram.buza@usask.caToll-like receptors (TLR) link innate and adaptive immune

responses. Single stranded viral RNA (ssRNA) was recently identi-fiedasthenaturalligandforTLR7andTLR8.ssRNAsequencesfromviruses,aswellastheirsynthetichomologuesstimulateinnateimmuneresponses in immune cells from humans and mice. In the presentinvestigations, we tested whether synthetic ss oligoribonucleotides(ORN) can activate immune cells from cattle. In vitro incubation ofbovineperipheralbloodmononuclearcells(PBMC)withORNinducedproductionofIL-12,IFNγandTNF-a.NosignificantinductionofIFN-awasobserved.Furthermore,depletionofCD14+cellsfromthePBMCresulted in the loss of IL-12 and IFN-γ response, confirming thatCD14+cellsare required forcytokine response followingstimulationwithORN.SinceTLR7/8arelocatedintheintracellularcompartment,andrequirephagosomalmaturationforeffectivesignalling,wetestedwhetherORN-inducedresponsescouldbeinfluencedbyinhibitorsofphagosomalmaturation.Pre-treatmentofPBMCwithbafilomycin(an

inhibitor of phagosomal acidification) prior to stimulation with ORNabolished the cytokine responses, confirming that the receptor(s)which mediate the ORN-induced responses are intracellular. Theseresultsdemonstrate for thefirst time thatORNhavestrong immunestimulatoryeffects incattle,andsuggest that further investigationonthepotentialofTLR7/8ligandstoactivateinnateandadaptiveimmuneresponsesindomesticanimalsarewarranted.Key words:Innateimmunity,CD14+,TLR7,TLR8,ssRNA,CpG,IL-12,IFN-a,IFN-γ,TNF-a,ELISA,ELISPOT,bafilomycin,cattleSpecies:ruminants

VA213. PROMISCUOUS EPITOPES OF MyCoBACTERIUM PARATUBERCULoSIS 70 KD HEATSHOCK PROTEIN

ACTIVATING BOVINE T CELLSAADHoEK*1,VICToRRUTTEn1,CHRISDAVIES3,ADKoETS2DepartmentsofInfectiousDiseasesandImmunology1andFarmAnimalHealth2,FacultyofVeterinaryMedicine,UtrechtUniversity,

TheNetherlands.VeterinaryMicrobiology&Pathology3,WashingtonStateUniversity,USAa.hoek@vet.uu.nl

Bovineparatuberculosis,anintestinalinflammatorydiseasecausedby infectionof calveswith the intracellular pathogenMycobacteriumaviumsubspeciesparatuberculosis(MAP),constitutesathreattocat-tleworldwide.InearlierworktherecombinantMAP70kDheatshockprotein(MAPHsp70)showedtobeanimmunodominantantigenandavaccinecandidateforparatuberculosis*.Inthepresentstudypeptideepitopes of MAP Hsp70 were identified by the capacity of overlap-pingMAPHsp7014-merpeptidestoelicitcell-mediatedresponsesofPBMCofaMAPHsp70immunizedhighrespondercow.SubsequentlyimmuneresponsivenesstoselectedMAPHsp70peptideswastestedinbovineMHCtypedcontrolanimals(n=5)andcowsthatwereMAPchallenged(n=28)andlateronimmunizedwithMAPHsp70.

Nineoutof124MAPHsp70peptidesinducedproliferationofPBMCand6Tcelllines/clonesweregeneratedbyregularrestimulationswith3outof the5most immunogenicHsp70peptidesandpeptidecoresweremappedbyPepscantechniques.PBMCofMAPchallengedandMAPHsp70immunizedcowsrespondedpositivetothese5peptidesin 14-82%of all 28 animals testeddepending of the usedpeptide.Untreatedanimalsshowedminor responses.MHC-typingof theani-malsbyoligonucleotidemicroarraysrevealed14differentBoLAclassIItypes,whichshowsthatthesepeptideswerepresentedbymultipleMHCIIalleles.TheapparentpromiscuousbindingofthesepeptidestodifferentMHCclassIIalleleswasalsofunctionallyconfirmedbyclassIIpresentationinco-culturesofmatchedandmismatchedPBMC.

Inconclusionwehaveidentified5immunodominantTcellepitopesofMAPHsp70.ThepresentationtoTcellsbyMHCclassIIandthebindingofthesepeptidesappearedtobeofapromiscuousnatureasTcellactivationcouldbemeasuredbyPBMCinavarietyof14differentBoLAclassIItypes.ThesedataprovidefurtherinsightintheworkingmechanismoftheMAPHsp70subunitvaccineandcontributestostud-iesoftheimmunopathogenesisofbovineparatuberculosis.

*KoetsA,HoekA,LangelaarM,OverdijkM,SantemaW,RuttenV.2006.Mycobacterial70kDheat-shockproteinisaneffectivesubunitvaccineagainstbovineparatuberculosis.Vaccine.24(14):2550-9.Key words:Paratuberculosis,MAPHsp70,peptides,vaccineSpecies:ruminants

VA214. PATTERNS OF SELECTIVE CONSTRAINT ON BOVINE TOLL-LIKE RECEPTOR GENES

9. INNATE IMMUNITy, INFLAMMATION AND ADJUVANTS; MEMORy, ACQUIRED IMMUNITy AND VACCINES: POSTERS VA211-VA250

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oJAnn1,SCHAnG2,TJCoFFEy3,DHAIG2,JHoPE3,JSALT4,CWARKUP5,DWERLInG6,DWBURT1,EJGLASS1

1DepartmentofGeneticsandGenomics,RoslinInstitute,Roslin,Midlothian,EH259PS,UK;2DivisionofVirology,MoredunResearchInstitute,PentlandsSciencePark,Penicuik,EH260PZ,UK;3Division

ofImmunology,InstituteforAnimalHealth,Compton,Berkshire,RG207NN,UK;4PfizerAnimalHealth,Sandwich,Kent,CT13

9NJ,UK;5Genesis-FaradayPartnership,RoslinBioCentre,Roslin,Midlothian,EH259PS,UK;6DepartmentofPathologyandInfectiousDiseases,RoyalVeterinaryCollege,HawsksheadLane,Hatfield,AL9

7TA,UKToll-like receptors (TLRs) are pattern-recognition molecules

expressed by cells of the innate immune system and cells at theinterface between the host and environment. They recognise thepresenceofpathogensandsignalalarmtothehostimmunesystem.Polymorphisms in these molecules may explain a proportion of thevariabilityindiseaseresistanceseeninmostspeciesfromDrosophilawhere theywerefirstdiscovered tomammalssuchas livestockandman. ThusTLRshave thepotential tobecandidates forselectablemarkersfordiseaseresistancethatcouldbeexploitedbythebreedingindustry. Ourconsortiumaimstodirectly linksinglenucleotidepoly-morphisms(SNPs)withfunctionalconsequencesintheinnateimmunecells,dendriticcells.WeareundertakingaSNPdiscoveryapproachforall10TLRsincattle inrelevantbreedsbydirectsequencing.BycombiningthiswithsequencingofTLRsinrelatedbovidspeciesinclud-ingsheep,goatsand ibexandothermoreexoticspecies,weaimtoundertakeaphylogeneticanalysistoidentifysignaturesofselectiononthenucleotidesequencesofbovidTLRs.Wewillthentesttheirfunc-tional relevanceby transfectingpolymorphicvariantsandmeasuringthedown-streameffectsofligandbinding.Wewillpresentphylogeneticevidence that theevolutionarypressuresonbovineTLR2andTLR4appeartohavedifferedandwillspeculateonthepossiblereasonsforthis,andtheconsequencesforthehost. OuroverallapproachaimstoidentifySNPswiththemostlikelypotentialasmarkersforselectinganimalswithenhanceddiseaseresistance.Key words: toll-like receptor, polymorphism, disease resistance,dendriticcellsSpecies:ruminants

VA215. THE IMMUNOGENICITy OF CoRyNEBACTERIUM PSEUDoTUBERCULoSIS PROTEIN AND DNA VACCINES IN

A MURINE MODELKEILADASILVACoELHo1,AnDERSonMIyoSHI1,VASCoAZEVEDo1,CRISTInAToSCAnoFonSECA2,FERnAnDA

CALDASCARDoSo2,MICHELEMBARSAnTE2,SERGIoCoSTAoLIVEIRA2,RoBERToMEyER3

1LaboratóriodeGenéticaCelulareMoleculardaUniversidadeFederaldeMinasGerais,DepartamentodeGenética;2LaboratóriodeImunologiadeDoençasInfecciosasdaUniversidadeFederaldeMinasGerais,DepartamentodeImunologiaeBioquímica;3Instituto

deCiênciasdaSaúdedaUniversidadeFederaldaBahiaCorynebacterium pseudotuberculosis is the etiological agent

of caseous lymphadenitis (LC), a chronic disease that affects smallruminants.Thispathologie ischaracterizedby theabscessdevelop-ment which leaves the production and discart of animal products.Lymphadenitis (LC)hasaworldwidedistributionand ithas receivedintenselaboratoryresearch;however,thereisnotanefficientavailablevaccineagainstthisbacteria.Onevaccinalstrategiethathasbeenpro-posedandappliedonitssolutionisthesearchforcandidateantigens,which isbasedonstudieswithphylogeneticclosedmicroorganisms.TherearesomevaccinaltestswiththeconservedHsp65(Hsps;HeatShock Proteins) Mycobacterium leprae antigen. The immunizationwith Hsp65 induced a potent immune response protecting againstMycobacteriumtuberculosischallenge.Consideringthatthepathogen-esisandtheimmunologyofMycobacteriumtuberculosisisverysimilarto C. pseudotuberculosis, the objective of thisworkwas to developproteinandDNAvaccinesbasedontheC.pseudotuberculosishsp60gene.Toconstructthevaccines,thecodingsequenceofhsp60genewasclonedinprokaryoticandmammalianexpressionvectors.PurifiedproteinassociatedwithFreund’sadjuvantandhsp60basedDNAvac-cineswereusedtoimmunizeBALB/cmice.Bothrecombinantprotein-

basedvaccineandDNAvaccineinducedsignificantproductionofIgG,IgG1 and IgG2a levels. The protein vaccine only induced the IL-10production,characterizingaregulatoryresponse.Alltheanimalsdiedafterthechallengewith106UFCofavirulentC.psedotuberculosisPus6strain.BothproteinandDNAvaccinesfailedinprotectingtheanimalsafterthechallengepossiblyduetotheirinabilitytoelicitanefficientandtherequiredcellularimmuneresponsenecessarytoprotectagainstC.pseudotuberculosis.Key words:vaccine,Hsp60,CorynebacteriumpseudotuberculosisSpecies:ruminants;other

VA216. DEVELOPMENT OF A BRV VP6 SUBUNIT VACCINE AND ITS EVALUATION IN THE CALF COLOSTRUM-

DEPRIVED MODELDIEGoDGonZALEZ,MARInAVMoZGoVoJ,DEMIAnBELLIDo,

AnDRéSWIGDoRoVITZ,VIVIAnAPARREño,MARÍAJDUSSAnToS

InstitutodeVirología,CICVyA,INTA,Castelar,BuenosAires,Argentina

dgonzalez@cnia.inta.gov.arGroupArotavirusesarethemaincauseofacuteviralgastroenteri-

tisinhumansandanimalsthroughouttheworld.Inourcountrybovinerotavirusisoneofthemaincausesofneonatal(<3weeks)calfdiarrheaandsignificanteconomiclosses.Duetotheearlysusceptibilitytotheinfectionpreventionstrategiesarebasedontheimprovementofpas-siveimmunity levelsthroughthevaccinationofpregnantcowsinthelastthirdpartofgestation.Themajorcapsidantigen(VP6)ofbovinerotavirus is themost immunogenicviralprotein.WehavepreviouslyreportedthatVP6{groupAP[1]G6}BRVC-486strainexpressedinthebaculovirussystemwasabletoinducehighlevelsofpassiveprotectioninasucklingmicemodel.TheobjectiveofthisworkwastoevaluatetheVP6immunogenicpropertiesincalves,byusingacalfmodelofpassiveprotection.

Recombinant VP6 using the baculovirus system was used toimmunizecowsinthelastthirdofpregnancy.Firstandsecondcolos-trumoftheseanimalswerecollected,pooled,andtheantibodytiterswere determined in order to generate an hyperimmune colostrumthatwasusedtofeedcolostrum-deprivedcalveswithinthefirst6hoflife.Colostrum-deprivedcalvesreceivingonedoseofpooledimmunecolostrum,orcalvesreceivingcontrolcolostrum,wereorallyinoculatedwith2*106FFUof IND(P[5]G6)BRVat2daysofage.Bothgroupsweremonitoredfordiarrhea,virussheddingandanti-BRVantibodiestitersboth in fecesandserum.The resultsdemonstrated that itwaspossibletoreducebothdiarrheaandvirusshedding(intermsofviraltiter and excretion period) in the group receiving immune colostrumagainstVP6incomparisonwiththegroupfedwithcontrolcolostrums.Key words:bovinerotavirus,colostrum,maternalantibodiesSpecies:ruminants

VA217. ExPRESSION OF A SCFV-E2T FUSION PROTEIN IN CHO-K1 CELLS AND ALFALFA TRANSGENIC PLANTS FOR THE SELECTIVE DIRECTIONING TO ANTIGEN PRESENTING

CELLSAGUSTÍnIoSTACHUK1,SEBASTIánMCHIAVEnnA1,CRISTInAGóMEZ2,AnDREAPECoRA1,MARIAnoDPéREZ-FILGUEIRA1,

JoSéMESCRIBAno3,FERnAnDoARDILA2,MARÍAJDUSSAnToS1,AnDRéSWIGDoRoVITZ1

1InstitutodeVirología,CICVyA,INTA-Castelar(BuenosAires,Argentina);2InstitutodeGenética,CICVyA,INTA-Castelar(BuenosAires,Argentina);3DepartamentodeBiotecnología,INIA(Madrid,

España)aostachuk@cnia.inta.gov.ar

Bovineviraldiarrheavirus(BVDV),apestivirusoftheFlaviviridaefamily, is an important cause of mortality, morbidity and economicallossesofcattlewithaworldwidedistribution.Subunitvaccinesprovidetheopportunitytodevelopsafevaccines.However,thechallengeistogenerateaprotectiveimmuneresponsetoacostaffordableforveteri-naryapplications.Onealternativetosolvethisproblemistoincreasethe vaccine immunogenicity. A central event in the development ofanadaptive immune response is thepresentationof theantigens to

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CD4+Tcellsfromantigenpresentingcells(APCs).Inconsequence,awaytoobtainamoreintensespecificimmuneresponseistoincreasethenumberofMHC-peptidecomplexesonthesurfaceofAPCs.Thiswould be possible by directioning antigens toAPCs, fusing them tospecific antibodies againstAPCs´ surfacemarkers. In thiswork,wereportthedevelopmentofafusionproteinbetweenasinglechainanti-bodyfragment(ScFv),recognizingaconservedregionofMHCclassIImolecules,anda truncated formof theE2glicoprotein (E2T) fromBVDV, without its transmembrane domain. The expression systemschosen to express these proteins weremammalian cells (CHO-K1)andplants(Medicagosativa,alfalfa).E2TandScFv-E2TfusionproteinwereexpressedtransientlyandstablyinCHO-K1cells,asevidencedbyWesternblotandELISAusingamonoclonalantibodyagainstE2.TheywerepurifiedbyIMAC(IonMetalAffinityChromatography)fromcell culture supernatants, resulting in an average final yield of 0.2mg/L.Alfalfatransgenicplantsweregeneratedbyinfectionofpetioleswith recombinant Agrobacterium tumefaciens, which contained theappropriate gene cloned in a pCAR vector downstream of CsVMVpromoter, fromCassavaveinmosaicvirus.Plantswereanalyzedfortheexpressionof theproteinsbyELISA,andmanyof themresultedpositive.Bindingof fusionproteinstothesurfaceofperipheralbloodmononuclearcells(PBMCs)fromcattle,pig,horse,guineapig,sheepandgoatwasstudiedbyflowcytometry.OurresultsdemonstratethatScFv-E2Texpressed inCHO-K1cellswascapableofbinding to thesurfaceofPBMCsmoreintensivelythanE2T,thussupportingtheuseofthisstrategyfordeliveringvaccineantigenstoAPCs.Studiescom-paring the immune responseselicitedbybothproteinsare currentlybeingconductedusingaguineapigexperimentalmodel.Key words: Medicago sativa; alfalfa; plant expression system;mammalianCHO-K1cells;recombinantprotein;singlechainantibodyfragment;E2glicoprotein;BovineviraldiarrheavirusSpecies:ruminants,other

VA218. PRODUCTION AND CHARACTERIZATION OF RECOMBINANT BOVINE HERPESVIRUS TyPE 5

GLyCOPROTEIN D ExPRESSED IN PICHIA PASToRISLUAnAADUMMER1,LEAnDRoqnIZoLI1,AnDRéASRRoCHA1,CARInAMMoRAES1,FABRICIoRConCEIçÃo1,2,CARLoS

GIL-TURnES1,2,TELMoVIDoR2,FáBIoPLLEITE1,31CentrodeBiotecnologia,UniversidadeFederaldePelotas(UFPel);

2FaculdadedeVeterinária,UFPel;3InstitutodeBiologia,UFPeldummer@gmail.com

Bovine herpesvirus type 5 (BoHV-5) is a pathogen of cattleresponsibleforsporadicoutbreakoffatalmeningoencephalitisinSouthAmerica.GlycoproteinD(gD),thatactinpenetrationintohostcells,isacandidatetoarecombinantvaccinesinceitinducesastrongandper-sistentcellularimmunityresponses.ProductionofgDinheterologoussystems should be able to keep the protein solubility and authenticimmugenicity.TheyeastPichiapastoris isanexpressionsystemthatsupports thisrequirement.WeusedtheexpressionvectorpPICZaB,whichisintegrative,induciblewithmethanolandallowsthesecretionofrecombinantproteintomedium.ThegDgenewasamplifiedthroughPCR using 2% of DMSO and cloned into pPICZaB to generate thepPICZaB/gD.ThisplasmidwaslinearizedandthentransformedintoP.pastorisstrainKM71H(MutS)byelectroporation.RecombinantstrainswereselectedbyZeocin resistance inYPDSplatesand theexpres-sionwasdetectedbyColonyDotBlotinBMMYplates.Theinductionwasperformedbyaddictionof 1%ofmethanol every24hduring3daysand thedetectionof the recombinant gD (rgD)wasperformedwithmonoclonalantibody(MAb)anti-histag.Onepositivecolonywasselected,inoculatedinBMGYandincubated24hat28°Cinshakingflask.Thecellswereharvestedandresuspendedin1/5volumeBMMYtoinducetheexpression.Methanolwasaddedto1%every24hdur-ing2days.Thecellswereharvestedand thesupernatantsaturatedovernightat4°Cwith80%ofammoniumsulfate.Theprecipitatedwasobtained by centrifugation, dialyzed overnight into PBS pH 7.4 andthergDcharacterizedbySDS-PAGEandWesternBlotwithserumofmiceandbovinevaccinatedwithinactivatedBoHV-5.TheSDS-PAGEshowedabandofapproximately50kDa,whichsuggestthatthergDwasglycosilated.SerumofmiceandbovinevaccinatedwithinactivatedBoHV-5recognizedtheglycosilatedrgD(~50kDa)andunglycosilated(~38kDa),demonstrating itsantigenicity.These resultssuggest that

thergDexpressedinP.pastorisisantigenic,thusitmightbeusedasanefficientlyvaccineantigen.ImmunogenicityofrgDexpressedinP.pastoriswillbeevaluatedinmiceandbovine.Key words:BovineHerpesvirustype5,Pichiapastoris,RecombinantVaccineSpecies:ruminants

VA219. INTERACTION OF NATURAL KILLER CELLS, MONOCyTES AND DENDRITIC CELL POPULATIONS IN

CATTLEREGInALDoGBASToS1,2,CARLJoHnSon1,WEnDyC

BRoWn2,WILLLGoFF11AnimalDiseaseResearchUnit,USDA-ARS,WashingtonStateUniversity,Pullman,Washington,USA;2DepartmentofVeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,

Washington,USA.Interactionsbetweeninnateimmunecellpopulations,suchasnat-

uralkiller(NK)cellswithmonocytes/macrophagesanddendriticcells(DC)havebeenofrecentinterest.Weareinvestigatinginteractionsofthesecellsfromcattleandourhypothesisisthatmicrobial-exposedDCormonocyteinteractionwithNKcellsresultintheproductionofIFN-γthuscontributing to the innate response to initial disease.ANKp46+populationwasobtainedbyincubatingtotalspleenorperipheralbloodmononuclearcellswithrecombinanthumanIL-15for2weeksfollowedbydepletionofB-lymphocytes,T-lymphocytes,γd-T-lymphocytesandmonocytes. The NKp46+ population was identified as CD3-, TcR1-CD2+/-andCD8+andwasabletoproduceIFN-γ inresponsetoexog-enousIL-12/IL-18.CD13+DCandCD172a+monocyteswereobtainedbypositiveselectionfromtotalspleenorperipheralbloodmononuclearcellsfordeterminingtheroleofsolublefactorsandcell-to-cellcontactin the interaction with NK cells. CD13+ DC or CD172a+ monocyteswere incubated for 24hrwithheat-killedMycobacteriumbovisBCG(HK-BCG)either in thepresenceorabsenceofexogenous IL-18orrecombinantbovineCD40L.NKp46+cellswerethenaddedandco-cul-turedfor24hrintrans-wellandcell-to-cellformats.ThesupernatantswerethencheckedbyELISAforthepresenceofIFN-γ.CD172a+cellsexposedtoHK-BCG-inducedIFN-γproductionfromNKp46+cells.TheinteractionwasCD40LindependentbutthisT-cellsignalsignificantlyenhancedtheinteraction(P<0.05).Moreover,theproductionofIFN-γwascompletelysuppressedbythepresenceofthep38MAPKinhibitorSB203580,demonstratingtheinvolvementofinnateNF-kBpathwayintheinteraction.Incontrast,BCG-exposedCD13+DCobtainedfromthespleenfailedtoinduceIFN-γproductionfromNKp46+cellsregardlessofcell-to-cellcontactoradditionofCD40LorinthepresenceofIL-18.WeandothershaverecentlydemonstratedthatCD13+DCrepresentan immature phenotype. Therefore, we incubated CD13+ DC for 72hoursinthepresenceofIL-4,GM-CSFandFlt3ligandpriortointerac-tionwithBCGandNKcells.Despitematuration,splenicDCmaintaineduniquecharacteristicscomparedtomonocytes.Inconclusion,wepro-videevidenceofinteractionofinnateimmunecellpopulationsthatmayhaveimportanceearlyduringaninfectionandindrivingtheacquiredimmuneresponseincattle.Key words:DendriticCells,NKCells,BovineSpecies:ruminants

VA220. DEVELOPMENT OF COMPLEMENTARy DIAGNOSTIC REAGENTS FOR BHV-1 MARKER VACCINES

FORMULATED WITH RECOMBINANT STRAINS.DAnIELMPéREZFILGUEIRA1,2,MARIAAPALACIoS1,

CARLoSPALACIoS1,ALEJAnDRARoMERA1,2,JoSéMESCRIBAno3,4,AnAMSADIR1,2

1InstitutodeVirología,CICVyA,INTA-Castelar,BuenosAires,Argentina;2ConsejoNacionaldeInvestigacionesCientíficasy

Técnicas,(CONICET),Argentina;3DepartamentodeBiotecnología,INIA-Madrid,España;4AlternativeGeneExpressionS.A.(Algenex),

Madrid,España.aromera@cnia.inta.gov.ar

Marker vaccines against Bovine Herpes Virus type 1 (BHV-1)lacking non-essencial estructural proteins arewidely utilized aroundtheworld.OurgrouphasdevelopedarecombinantBHV-1strainlack-

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ing of glycoprotein E (gE) and including β-galactosidase gene as areplicationreporter(BHV-1δgEβgal).Currently,anadditionalstrain isbeingdevelopedfollowingthesamestrategybutincludingtheE2genefrom theBovineviral diarrheavirus (BVDV)and theβ-Gusgeneasa replication reporter instead.Here,we present the development ofdiagnosticreagentstobeusedascomplementforvaccinesformulatedwiththeseBHV-1recombinantstrains.DifferentrecombinantversionsofgEhavebeenproducedusinganinsectlarvae-baculovirusexpres-sionstrategyorabacterialsystem.AtruncatedgEgene(gEc)-withoutsignalpeptideandtransmembranedomains-wasclonedintoabacu-lovirusvector (pFastMel-1) toproduce themel-gE-BAC recombinantbaculoviruswhich allowed glycosylation of the the recombinant gEc(rgEc). Trichoplusia ni larvaewere inoculatedwithmel-gE-BAC andprocessed at 72h. post-inoculation. A recombinant protein with theexpectedelectrophoreticmobilitywasreadilydetectedbyWesternblotin totalproteinextracts from infected larvae.Crudeextractscontain-ing rgEcwereused tocoatELISAplatesandBHV-1positivebovineserumsamplesaswellaspositiveandnegativecontrols included inacommercialassaywereusedtosetupoptimalconditionsforrgEc-based ELISA. Results from a limited number of samples showedthat thergEc-basedELISAwasable todetectgE-specificantibodiesinsera fromcattle infectedwithBHV-1,showingno reactionagainstnegative samples. However, results using samples from immunizedcattle–usingcoventionalormarker vaccines-provedmorevariable,inasimilarwaytothoseobtainedwithacommercialELISA.Inordertoobtainaconfirmatoryassay,animmunoblottingtesthasalsobeenassayedusingotherversionsofgEproducedinaprocarioticsystem.PreviouspublicationshadidentifiedregionswithinthegEwhichweretoxicforbacteriaandhinderedexpressionoffulllenghtversionsofthegene.Basedonthis,2fragmentsof571and779bplongwereselectedandexpressedinE.Coli.Solublefractionsofinducedbacterialcultureswere resolved by 12% SDS-PAGE and transferred to nitrocellulosestrips.PreliminaryresultsshowedthatgEc-specificmAbsrecognizedasinglebandforbothconstructs,whileshowingnoreactionwithsero-negative samples or control antigen from non-transformed bacteria.AlltogethertheseresultsshowedthefeasibilityofdevelopingeffectiveandinexpensivediagnosticassaysasessentialcomplementsforBHV-1markervaccines.Key words:BHV-1markervaccines,diagnosis,recombinantproteins,insectexpressionsystemSpecies:other;ruminants

VA221. LIPOARABINOMANNANS FROM MyCoBACTERIUM AVIUM SUBSP. AVIUM AFFECT BOVINE IMMUNE

RESPONSES TO DIFFERENT ANTIGENSSILVIACoLAVECCHIA,AnAJoLLy,AnASTEMPLER,ELoy

FERnánDEZ,SILVIAMUnDoAreaInmunología.FacultaddeCienciasVeterinarias.Universidadde

BuenosAires;Chorroarín289.BuenosAires.Argentina.scolave@fvet.uba.ar

Lipoarabinomannans(Lams)are thestructurallymore importantoutercellwallcomponentofallmycobacteria.LamsareubiquitousofMycobacteriaspp.andappearas themostpotentnon-peptidicmol-eculestomodulatethehostimmuneresponse.

OurobjectivewastodeterminethemodulatingeffectofLamsontheimmuneresponsetosolubleandparticulatesantigensincattle.

Lams were obtained fromMycobacterium avium subsp. avium(Maa), purified and characterized by ELISA and Western blot asdescribedbyHamasurycol.,(1999).

Lams(1mg)wereinjectedthreetimestocalvessubcutaneously,inamixturewith thefollowingantigens:1mgOvalbumin(OVA),108

cellsofCanineErythrocytes(CE)or2mgofMaawholebacteria.OVA,MaaandPBS(ascontrol)wereemulsifiedwithFreund´s incompleteadjuvant (AFI) andCEwas injected inPBS.Groupsof calveswereinjectedwiththeantigenswithoutLamsascontrols.

Immuneresponsesofcalvesweretestedbeforeand21daysafterthethird immunization. Innate immuneresponseswereevaluatedbyserumelectrophoresis (EP)and respiratoryburst (RB)onperipheralcells.Specificimmuneresponseswereevaluatedbylymphoprolifera-tion (LP)assay,serumELISAandHemaglutination (HA) test invitro

anddelayed-typehypersensitivity(DTH)invivo.DifferencesbetweengroupswereanalizedbyKruskalWallisorStudentunpairedt-test.

Our results show that Lams decrease significantly the RB ofperipheralcells.ThisresultagreewithLepraeandTuberculosisLamseffectdescribedinhumanmacrophagesinvitrobyChanetal.(1991)whoconsideritasasurvivalmechanismtoevadephagocytosis.

Inaddition,wedetectedincreaseincellularimmuneresponseinLaminoculatedanimals.Ontheotherhand,specificantibodiestitersweredecreasedbysimultaneousinoculationwithLams.

OurresultsconfirmthatsimultaneousapplicationofsolubleandparticulatedantigenswithLamsmodifiestheimmuneresponseagainsttheseantigensincattle.

References:Chan,Jetal1991Infect.Immun.59:1755-1761.Hamasur, B .et al. 1999. FEMS. Immunol. Med. Microbiol. 24:

11-17.Key words:Mycobacterium,Lipoarabinomannan,immunomodulation,bovineSpecies:ruminants

VA222. SAFETy OF BRUCELLA ABORTUS RB51 VACCINATION OF ADULT COWS: BACTERIOLOGICAL

EVALUATION OF MILK KARInALEITEMIRAnDA

UniversidadeFederaldeMinasGerais,BeloHorizonte,MG.30240-060,Brazil

karinalmi@gmail.comBrucellosis caused by Brucella abortus is a chronic disease of

cattleofworldwidepublichealthandeconomic importance, resultinginabortionandinfertility.B.abortusRB51isa lipopolysaccharideO-antigen-deficientmutantofthevirulentstrainB.abortus2308usedasanalternativevaccine to theS19,which inducesantibodies thataredetectedbytheroutineserologicaltests.InBrazil,RB51isapprovedforusinginadultcowsandthereisonlyscantydataontheeliminationofRB51inmilk.Theaimofthisstudywastoevaluatebyculturetheelimination of B. abortus in themilk of cows vaccinatedwithRB51.Ten brucellosis-free cows, vaccinatedwith S19 as calf, between 30and60daysafter parturitionwere selected from theherdofEscolade Veterinária –UFMG, at Pedro Leopoldo –MG, Brazil. The cowsweregivensubcutaneousinjectionsof1.0mLofPBScontaining1.3x1010CFUofB.abortusstrainRB51,intheday0ofexperiment.Milksampleswerecollectedfromcowstwiceaday,insterilepolypropylenetubesondays1,2,3,4,7,14,21,28,35,42,49,56and63aftervac-cination.Thefirstmilkstreamsofeachteatwerediscarded,then50mLofmilksamplesweremanuallycollectedfromallquartersandstoredat-20°C.Sampleswerethawedandcentrifugedat2500xG,for15minutes,theintermediatedphasewasdiscardedandthesupernatantwasmixedwiththepellet.Themixtureswereimmediatelyinoculatedinduplicateplatesoftryptoseagarwithantibiotics(Farrel’ssupplement).Moreover,1.0mLofeachmixturewasinoculatedin9.0mLofenrich-mentmedia (tryptose brothwith Farrel’s sellective supplement) andincubated into 5% CO2 at 37 °C for 5 days, and then inoculated intryptoseagarwithantibiotics.Allplateswereincubatedinto5%CO2at37°Cfor9days.NoBrucellaabortuswasisolatedfromanysampletested.Inordertoconfirmtheseresults,allfrozenmilksampleswillbetestedbyamoresensitivePCRtechnique.Key words:Brucellaabortus;vaccine;lipopolysaccharideO-antigen-deficient;S19antigenSpecies:ruminantts

VA223. CONSTRUCTION OF IMMUNODIAGNOSTIC AND THERAPEUTIC BOVINE ANTIBODIES AS SINGLE CHAIN FV

(SCFV)MADHURIKoTI1,EVAnAGy2,AZADKKAUSHIK1

1DepartmentofMolecularandCellularBiology,UniversityofGuelph,Canada;2DepartmentofPathobiology,UniversityofGuelph,Canada

mkoti@uoguelph.ca

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Ourlabhasbeenengagedindefiningthestructuralandfunc-tionalaspectsofbovineantibodiesthatdemonstratedlimitedgermlineantibody diversity both at the heavy (VH, DH and JH gene elements)andlightchain(VLandJLgeneelements)locusincattle.Thesestud-iesalsodemonstratedthatsomeofthecattleantibodiesareamongstthe largest known because of an exceptionally long variable regioninfluencedbyanatypicalCDR3H.Sinceantibodiesare important inimmunodiagnostics,clinicaldiagnosisand immunotherapy,engineer-ing of antibodies as minimal antigen binding fragments e.g. scFvprovidesanimportantstrategicapproach.ForthesereasonswehaveexpressedbovineIgGantibodyasscFvwithdifferentlinkersizes.TheexpressedbovineIgG,asscFvswithdifferentlinkersizes,areindeedfunctionalasrevealedbyvariousimmunologicalassays.Theseexperi-mentshaveledtoconcludethatbovineantibodiescanbeexpressedasscFvsthatarefunctionalandcanbeproducedinlargeamountsinanappropriatesystem,whichareofdefinitiveimmunodiagnosticandtherapeuticpotential.

(SupportedbyNSERCresearchgrant,Canada)Key words:scFv,Immunodiagnostic,BovineantibodySpecies:ruminants

VA224. CHARACTERIZATION OF SPECIFIC IMMUNE RESPONSES IN CATTLE IMMUNIZED WITH PLASMID DNA

CONSTRUCT AND RECOMBINANT SEMLIKI FOREST VIRUS PARTICLES ENCODING CU, ZN SUPEROxIDE DISMUTASE

(SOD) OF BRUCELLA ABoRTUS

ANGELA.OÑATE,DARWINR.SÁEZ,INGRIDI.GUZMÁN,EDILIAM.ANDREWSANDALEXC.CABRERA

MolecularImmunologyLaboratory,DepartmentofMicrobiology,FacultyofBiologicalSciences,UniversidaddeConcepción,

Concepción.Chile.aonate@udec.cl

TheimmuneresponsesofcattleimmunizedwithaplasmidDNAconstruct and a recombinant Semliki Forest virus (SFV) particlesexpressingtheCu,Znsuperoxidedismutase(SOD)ofBrucellaabortuswasstudied incattle.Wehavepreviouslydemonstrated inmicethatplasmidDNAcarryingtheSODgene(pcDNA-SOD)andtheSFVpar-ticlescarryingrecombinantRNAencodingSOD(SFV-SOD)wereableto induce a protective immune response that correlated with induc-tion of antigen-specific lymphoprolipheration, INF-γ production andcytotoxicactivity.Two independient trialswereconducted incattle todeterminetheabilityofthesetwogeneticvaccinestoelicitantigen-spe-cific immune responses.Cattle injected intramuscularlywithpcDNA-SOD,butnot thecontrolplasmidpcDNA3,developedasignificant,butweak,SOD-specificIgGantibodiesofpredominantlyIgG1isotype.Inaddition,theDNAvaccineelicitedaSOD-specificT-cellproliferativeresponse.Animals vaccinatedwithSFV-SODdid not developSOD-specificantibodies,atleastuntilweek10afterimmunization(theendoftheexperiment),andinvitrostimulationoftheirperipheralbloodmono-nuclear cells (PBMC)exhibiteda significatT cell immune response.Inbothexperimentssecretionofgammainterferon,IL-4orTNF-abythelymphocyteswasnotdetecteduponinvitrostimulationwithSOD.TheseresultssuggestthepotentialofusingpcDNA-SODorSFV-SODtoinducecellularimmuneresponseincattleagainstaprotectiveanti-genofBrucella.ThisisthefirstreportedvaccinationstudyinatragethostspecieswithDNAorRNAvaccinesagainstBrucella.

This work was supported by grant 1010851 and 1050054from the Fondo Nacional de Investigación Científica y Tecnologíca(FONDECYT),Santiago,Chile.Key words: Genetic vaccine, Brucella abortus, DNA vaccine, RNAvaccineSpecies:ruminants

VA225. SEMLIKI FOREST VIRUS THAT ENCODES A BRUCELLA ABoRTUS IF3 PROTEIN PROTECTS BALB/C

MICE AGAINST INFECTIONALExC.CABRERA,DARWInR.SáEZ,EDILIAM.

AnDREWS,AnGELA.oñATE

MolecularImmunologyLaboratory,DepartmentofMicrobiology,FacultyofBiologicalSciences.UniversidaddeConcepcion,Chile.

acabrera@udec.clBrucella abortus, is a causal agent of the bovinebrucelosis. In

Chile, the disease is controlled by the vaccination withB. abortusstrainRB51however somedisadvantageshavebeendescribed, asthecapacitytoreverttothevirulentform.WithinthenewtechnologiesofvaccinationappearvectorsbasedontheSemlikiForestvirus(VSF).Theseconsistofsuicidalviralparticles,whitanautoreplicablegenomethatonlycodifiesforaninterestprotein.Inthisstudyweevaluatedtheimmune response inducedby viral particles ofVSFable to expresstheproteinIF3ofB.abortus(VSF-IF3).Forit,4groupsof10femalesBALB/c mice was immunized with PBS, VSF-lac-Z, VSF-IF3 or B.abortus strain RB51 evaluating the cellular immune response witha limphoproliferation assay and citoquine production INF-g and IL-4by ELISA sandwich. The humoral immune response was evaluatedbyELISAtestingIgM,IgG,IgG1andIgG2a.Inaddition,achallengewithpathogenicstrainB.abortus2308wasperformed,evaluatingtheconferredprotection.InthegroupSFV-IF3,lymphoproliferativeactivitywasobservedforIF3andcrudeproteinsofBrucellaandtheproductionofINF-g.Inthesamegroup,humoralimmuneresponseshowingtheproductionofIgMandIgG.TheprotectionconferredbySFV-IF3,wassimilartoobservedinthemiceimmunizedwithB.abortusRB51.TheprotectionresultsarestimulatingforthelateruseSemlikiForestvec-torsincattle,andspecialVSF-IF3inthecombatofbovinebrucellosis.Key words:Vaccine,Brucellaabortus,IntracellularpathogensSpecies:other

VA226. TOPICAL COPAíBA OIL TREATMENT OF SURGICAL WOUND ALTERS THE REPAIR AND THE CELL MIGRATION IN

THE PRESENCE OF A FOREIGN BODy IN BALB/C MICE. RAFAELCoSTAVIEIRA,ETHIAnEBoMBARDIERE,JULIERME

JoSéDEoLIVEIRA,RUyDESoUZALInoJúnIoR,LUIZAUGUSToBATISTABRITo,AnAPAULAJUnqUEIRA-KIPnISLaboratóriodeImunopatologiadasdoençasinfecciosas,Instituto

dePatologiaTropicaleSaúdePública;EscoladeVeterinária.UniversidadeFederaldeGoiás.

Copaifera langsdorffiiisaBraziliannativeleguminosaethatpro-duceresin-oil,popularlyknownascopaíbaoil.Thisoil isusedinthetreatmentofskinwoundforitsrecognizedantiinflammatoryandwoundhealingeffects.Despite,itspopularuse,therearefewpublisheddataabout the therapeutic effect of this medicinal plant. The aim of thestudywastoevaluatethetopictreatmenteffectoftheCopaíbaoilontheprocessofskinrepairandinflammationinducedbyaforeignbodysubcutanouslyimplanted.120BALB/cmiceweresubmittedtoa1cmlinear incision and one 12mm circle coverslip was subcutaneouslyimplanted.After implantation the surgical incisionwas sutured.Fourtreatmentsgroupswereestablished:control,sterilesaline(C);vehiclecontrol, sterile mineral oil, (VC); treatment 1 (T1), mineral oil pluscopaibaoil (V/V), and treatment2 (T2) copaibaoil.Theevaluationswereperformedatpre-determinedtimepoints(1,3,5,7and14days).Itwaspossible tofindfibroblasts,epithelialcellsproliferation,re-epi-thelizationandnewlyformedbloodvesselsinallgroups,however,alloiltreatedgroups(T1andT2)didnotpresentre-epithelizationatthreedayspostsurgical incision.Ondayfifth,ahigher intensityofedemaandhyperemiaonthegroupsT1andT2wasobserved,besidesthat,theT1andT2groupspresentedaserouscellularscabonthewoundsthatwasabsentontheCandVCgroups.TheinflammatoryreactionsamongthegroupsCandVCshowedmoremononuclearcellsthantheT1andT2groupsthatpresentedamixedcellpattercomposedfrombothmonoandpolymorphonuclearcells.Althoughthesurgicalwoundswerere-epithelizaded,inthegroupsT1andT2,theywerecoveredbyaserouscellularcrustandthedermistissuestillpresentedanintensemononuclearcellinflammatoryfocus.Fourteendaysafterofthesurgi-calincision,thegrossaspectsongroupsCandVCweresimilarandon groups T1 and T2, despite wound to be completely closed andwithout crusts, the skin those animals was thickened. Furthermore,thedermisongroupT2presentedmoderatefibrosis,while theothergroupspresentedslightlyones.Thecellmigrationstotheforeignbodywaschangedonlyon thefirstday,while thepredominantcellswereneutrophilsandraremonocyteormacrophageontheCgroup,theVC,

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T1andT2groupsshowedahigherpercentageofmacrophagesinthecoverslips(C=7,21,VC=18,57,T1=19,17,T2=23,76,p<0,05).Theoiltreatmentdidnotchangethecellmigrationtotheforeignbodyonthenexttimepoints.TheresultsdemonstratedthattopicaltreatmentwithC.langsdorffiioildebilitatedthenormalprocessofawoundrepairinthepresenceofaforeignbody.Key words:Copaiferalangsdorffii;copaíbaoil;woundrepairSpecies:other

VA227. ACTION OF ARToCARPUS INTEGRIFoLIA SEED ExTRACT ON THE ACTIVATION OF PHAGOCyTIC CELLS IN

INFECTION By CANDIDA ALBICANS.SFETo,MHALCALDE,LCARAnDInA,RSALVADoR,LoyoLA,WFFALM/UniversidadeEstadualNortedoParaná,Londrina,Paraná,

86062-280,Brazilwloybr@yahoo.com.br

Candidiasis is a common infection among immunosuppressedindividuals.Macrophageandneutrophilactivationisanimportantfac-torfortheprotectiveimmuneresponseagainstthefungus.Thisactiva-tionhappensmainly throughTh1groupcytokinesproducedbyCD4lymphocytes.Lectinstudieshaveshownthattheseproteinsalsohavean immuno-estimulatingaction. In thisstudy,weevaluate theactionofjacalin,alectinextractedfromtheseedofthejackfruit(Artocarpusintegrifolia),inmiceinfectedwithaC.albicans(CR15)isolatefromanHIV+patient.TheanimalswerepreviouslytreatedwithJCAorPBSinintervalsof24,48,72and96hours.Peritonealexudatewasobtainedthroughperitonealwashandwasaddedfor1hour.Afterthisperiod,C.albicanswasaddedintherateof1:5.Macrophageandneutrophilpopulations,thepercentageofphagocytisingcellsandthemeannum-berofphagocytisedparticlesinthepresenceofsera,serainactivatedby heat, as well as by the absence of opsonization. MacrophagesstimulatedbyJCApresentedanincreaseinthecandicidactivityinthe72hourinterval,eliminating76%ofinoculate,whilethecontrolanimalseliminated only 40%. The percentage of phagocyting cells and thenumberofphagocytedparticleswereatamaximumwheninpresenceofserainthe72hourinterval.TheseresultssuggestthatJCAhasastimulatingactiononphagocyticcells,increasingtheirphagocyticandcandicidability.

Support:CPG/FFALM.Key words:Macrophages,Candidaalbicans,neutrophils,jacalinSpecies:other(mice)

VA228. NEUTRALIZING ANTIBODIES IN MUSTELA VISoN AFTER VACCINATION AGAINST CANINE DISTEMPER UNDER

FIELD CONDITIONS IN ARGENTINA.RAMAyoLILIAnAG,JARAnAM,MUnDoSILVIA

L.AreadeInmunología,FacultaddeCienciasVeterinarias,UniversidaddeBuenosAires.RepublicaArgentina

Minkdistemper isa frequently lethaldiseaseofminks (Mustelavison)causedbyCanineDistemperVirus(CDV,Morbillivirusgenus).Minksareextremelysusceptibletothisvirus,andclinicalsignsofthediseasemayincluderespiratory,gastrointestinal,neurologicandcuta-neoussymptoms.Vaccinationisrecommendedtocontrolthedisease,as it elicits neutralizing antibodies that are considered to correlatedirectlywithprotectioninvivo.

InArgentina,therearenostudiesoftheserologicresponsetovac-cinationagainstCDVinotheranimalsthandogs.Inthepresentstudy,weevaluated the levelsofneutralizingantibodies induced in farmedminksby vaccination under field conditionswith a commercial com-binedvaccineforminks.(DistoxPlus®,Schering-Plough,composedbyattenuatedCDV,inactivatedminkenteritisvirus,C.BotulinumtypeCbacterin-toxoidandinactivatedPseudomonaaeruginosa).

Inexperiment1,sixteenhealthy,non-vaccinatedandwithouthis-toryofCDVinfection,fivemonthsoldminkswererandomlyassignedtovaccinated(n=8)ornon-vaccinated(n=8)groups.Serumsampleswereobtainedfourmonthsaftervaccination.CDV-specificneutralizingantibodieslevelsweredeterminedbyaneutralizationtestinVerocells.Vaccinatedminksdevelopedneutralizingantibodies (mean log10 titer

+/-SD=2.708+/-0.557)whilenon-vaccinatedanimalsdidnotshowdetectableCDVneutralizingantibodies.

Inexperiment2,neutralizingantibodieslevelswereevaluatedinfivehealthybreedingfemalesoneyearaftervaccination.Alloftheseanimalsshowedalong-lastingresponse,withtiters(log10)rangingfrom1.26tomorethan1.80(3outof5).

ThisreportshowsthatCDVvaccinationinducesaspecificneutral-izingantibody response in farmedminksofArgentina.The levelsofantibodies found in thisstudyare inaccordancewith thosedetectedbyothergroupsworldwideunderdifferentfieldconditions.Takingintoaccountthatatiterofneutralizingantibodies(log10)of1.5isconsideredasprotectingindogs,wecouldsuggestthat:(a)alltheanimalsevalu-ated4monthsafterreceivingasingledoseofCDVvaccinereachedtheprotectingstatus,and (b) thosebreeding femalesevaluatedoneyear after vaccination showed titers that were around the thresholdofprotection.Key Words: Neutralizing antibodies; - Distempe;r – Mink; Mustelavison;-VaccinationSpecies:other

VA229. STANDARDIZATION OF PROTOCOLS OF VACCINATION WITH SALMoNELLA STRAIN ExPRESSING

VAPA AGAINST RHoDoCoCCUS EqUI INFECTIONSAnDRoGSoARES,ALInEFoLIVEIRA,PATRÍCIAE

VEnDRUSCoLo,MARIA-CRISTInARoqUE-BARREIRADepartamentodeBiologiaCelulareMoleculareBioagentes

Patogênicos,FaculdadedeMedicinadeRibeirãoPreto,UniversidadedeSãoPaulo,Av.Bandeirantes,3900,14049-900,RibeirãoPreto,

SãoPaulo,BrazilRhodococcusequiisanimportantpathogenofyoungfoalsaged

upto3-5monthsold.VirulentR.equihavebeenshowntocontainapathogenicityislandthatencodes,amongothergenes,sevengenesofthevirulence-associatedprotein(vap)family.Recently,ourgrouphavedevelopedanovel oral vaccineagainstRhodococcusequi based inexpressionofvapAantigenbyS.entericaTyphimuriumstrainutilizingAsd-balanced-lethalvector-hostsystem(Oliveiraetal.,MicobesInfect.9(3):382, 2007). This vaccine has been used succesfully in murinemodel challenged with virulentR. equi strain. Also, promising datahavebeenprovidedbyimmunizationoffoals.Here,wetesteddiffer-entprotocolsofimmunizationusingS.entericaTyphimuriumvapA+inmurinemodel:(1)twooraldoses;(2)twointranasaldoses;(3)singleintranasaldose;(4)singleoraldoseandboosters.c.withAPTX(semi-purifiedvapApreparation)emulsifiedinIFA;(5)singleintranasaldoseandboosters.c.withAPTXemulsified in IFA;and(6) two intranasaldosesusingS.entericaTyphimurium vapA- (vaccine control strain).An interval of 15 days elapsed from the first to the booster antigeninoculation.Micewere challenged 36 days after the first immuniza-tion. Vaccination efficacy was evaluated trough bacterial clearance,productionofcytokinesandserumantibodieslevels,determined120hafterchallenge.CFUanalysiswasperformedinboth,liverandspleenhomogenates. Cytokines were measured in spleen homogenates.Serum levelsofantigen-specific IgG1and IgG2awerequantifiedbyELISA.ImmunizationwithSalmonellavaccine,associatedornotwithadministration of APTX, induced a significant decrease of CFU inspleenandliverofchallengedmiceincomparisonwithcontrolgroup.OralimmunizationwasonlyslightlyefficientindecreasingCFU.Serumlevelsofboth,IgG1andIgG2a,weresignificantlyhigherinmicethatreceivedaboosterdoseofAPTX.However,onlymiceimmunizedwithtwoorasingleintranasaldoseofSalmonellavaccinestraindisplayeda high quotient between IgG2a and IgG1 levels. Concentrations ofIL-12p40 were increased in all vaccinated groups, while IL-4 wasslightlyincreasedinanimalsimmunizedwithasingleintranasaldoseofSalmonellavaccinestrainandboostedwithAPTX.Thesedatasug-gestedthatprotectioninducedbyliveattenuatedSalmonellavaccinestrain expressing the vapA protein was efficient using the differentprotocols.TheintranasaladministrationofasingledoseofattenuatedSalmonellastrainexpressingthevapAprovidedgoodefficacyandpos-siblyisthemostpromisingprotocolofvaccination.Key words:Rhodococcusequi,Vaccines,Salmonellaenterica,VapAproteinSpecies:equine

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VA230. IMMUNIZATION OF PREGNANT MARES WITH RHoDoCoCCUS EqUI ANTIGENS: FIELD STUDy OF

TRANSFERRED PROTECTION IN 68 FOALSJULIEnCAUCHARD1,CoRInnESEVIn1,DAVIDCoURToIS1,MARIE-FRAnCEBREUIL1,FABIEnDUqUESnE1,JEAn-MARC

BETSCH2,SAnDRInEPETRy1,CLAIRELAUGIER1,JEAnJACqUESBALLET3

1AFSSA,Laboratoired’EtudesetdeRechercheenPathologieEquine,IPC,Goustranville,14430Dozulé,France;2Clinique

vétérinairedeMéheudin,61150Ecouche;3Laboratoired’ImmunologieetImmunopathologie,UPRES-EA2128,CHUde

Caen,14033Caen,Francej.cauchard@dozule.afssa.fr

Theaimof this studywas to evaluate foals antibody- and cell-mediated protection from pregnant mares immunized with a proteinpreparation containing VapA, from a Rhodococcus equi (R. equi)virulentstrain,associatedwithananoparticularwaterbasedadjuvant(IMS3012,SEPPIC).InfourendemicalfarmsandonefarmwithnoR.equihistory,thirtyninemareswereimmunized9and3weeksbeforetheexpecteddeliveryterm,andthirtyfivecontrolmaresreceivedonlytheadjuvant.TheirtotalIgG,IgM,IgGT,IgGaandIgGbantibodiesweremeasured inELISAs prior to and 7, 21, 45, 365 days after the firstimmunization(day0)andcolostrumIgGantibodiesdeterminedatbirth.For23mares(12immunized-11control),IFN-gamma,IL-12,IL-2,IL-4,IL-5andIL-10mRNAcontentsofperipheralbloodlymphocytesweredetermined1,2and4weeksafterday0.Concerning the foals, thepreviousantibodiesweredeterminedatbirthand24hrsand7,15daysafterbirtheverytwoweeksuntilthreemonthsofage.Clinicalfollow-upwasrecordedforthemaresandforthefoalsuntiltheageof6months.Nosideeffectwasrecordedforthemaresaftertheinjection.Comparedwithcontrolmares,anti-R.equiVapAproteinantibodiesofallisotypesincreasedsteadilyathigh levelsuntilday45post-immunization,andsignificanthigher levels (except for IgM)couldstill beobserved365daysafterthefirstinjection.IgGantibodiesweredetectedincolostra.LymphocytemRNAlevelssustainedly increasedtill4weeks for INF-gamma,IL-12andIL-2whilenodifferencewith thebasic levelswasmaintainedforIL-4,IL-5andIL-10mRNAs,thusconsistentwithapre-dominantTH1cytokineresponseprofile.Aftertakingcolostra,thefoalsexhibitedantibodylevelsclosetothatoftheirmares,whichprovedanefficientantibodytransfer.Duringthefollowing-upperiod,2outof34foalswereexcludedfromthestudyforirrelevantreasons.Concerningthe36remainingfoalsfromimmunizedmares,rhodococcosisoccuredin1followingsurgeryforcorrectionofangularlimbdeformities.Incon-trast,outofthe32controlfoals,4sufferedrespiratoryrhodococcosis.Thepresentdataconfirmtheprotectiveeffectforfoalsoftheantibod-iestransferredbycolostrums,inducedinthemaresbynanoparticularadjuvant-associatedR.equiproteinantigensresultinginapredominantTH1cytokineresponse.Key words:rhodococcusequi,mare,adjuvant,TH1Species:equine

VA231. CELLULAR AND BIOCHEMICAL ANALySES OF DIGITAL LAMELLAE FROM HORSES WITH BLACK WALNUT

ExTRACT (BWE)-INDUCED, STARCH GRUEL (CHO)-INDUCED AND CLINICAL LAMINITIS.

JLoFTUS1*,MCoynE1,DALFAnDARI1,JBELKnAP2,PJoHnSon3,SJBLACK1

1UniversityofMassachusetts,DepartmentofVeterinaryandAnimalSciences,Amherst,MA;2OhioStateUniversity,SchoolofVeterinaryMedicine,Columbus,OH;3UniversityofMissouriSchoolofVeterinary

Medicine,Columbia,MO.*PresentingAuthorJustification – BWE-induced laminitis (crippling lameness) is

muchlessdebilitatingthanCHO-inducedlaminitisandseverenaturaldisease. A comparison of inflammatory leukocytes and degradativeenzymes in lamellae from these animals may indicate those patho-physiologicalprocessesuniquelyassociatedwithfailureofthelamel-laeleadingtorotationandsinkingofthecoffinbone.

Hypothesis–Theseverity of equine laminitiswill correlatewithlamellartissuecontentofinflammatoryleukocytes,collagen-degrading

gelatinasesie.,MMP9andMMP2,andproteoglycandegradingaggre-canasesi.e.,ADAMTS-4andADAMTS-5,orcombinationsofthese.

Procedures–Archivedsampleswereusedandwerefromatleast6individualsperconditiontested.MMP9andMMP2weredeterminedinextractsoflamellaebySDS-renaturingandnon-denaturinggelatinzymography.Neutrophilcontentoflamellaewasdeterminedbyimmu-nohistochemistry using CD13 expression and nuclear morphologyforprofiling.MyeloperoxidasecontentofextractswasdeterminedbyELISA. Expression of genes encodingADAMTS-4 andADAMTS-5wasdeterminedbyRT-qPCRusingvalidatedprimersandcDNAspre-paredfromtotallamellarRNA.

Results -MMP9 (92 kDa zymogen) andMMP2 (72 kDa zymo-genand62kDacleavedform)wereelevatedinlamellarextractsfromCHO-treatedhorses,whereasMMP9zymogenonlywaselevated inlamellarextractsfromBWE-treatedhorses.Myeloperoxidasecontentwaselevated in lamellar extracts frombothBWE-andCHO-treatedhorsesandcorrelatedpositivelywithMMP9zymogenandtissueneu-trophil content. LamellarADAMTS-4geneexpressionwaselevatedin both BWE- and CHO-treated horses whereas ADAMTS-5 geneexpressionwaselevatedinclinicalandCHO-inducedlaminitisbutnotBWE-treatedhorses. Biochemicalandcellularparametersof lamel-laefromhorseswithnaturally-inducedclinicallaminitisweresimilartothoseofhorseswithCHO-inducedlaminitis.

Conclusions –ADAMTS-5 andMMP2may play critical roles indegradingtheextracellularmatrixofthedigitallamellaeinhorseswithsevereexperimentally-inducedandnaturallyacquiredlaminitis.

Keywords:Inflammation,Metalloproteinase,LaminitisSpecies:equine

VA232. AN EQUINE α-DEFENSIN: GENE TRANSCRIPTION, RECOMBINANT ExPRESSION AND CHARACTERIZATION OF

THE STRUCTURE AND FUNCTION oLIVERBRUHn1,PETRAREGEnHARD1,MATTHIASMICHALEK2,SVEnPAUL1,CHRISToPHGELHAUS3,SASCHAJUnG2,GEoRGTHALLER1,RAInERPoDSCHUn4,MATTHIASLEIPPE3,JoACHIM

GRöTZInGER2,ERnSTKALM11InstituteofAnimalBreedingandHusbandry,UniversityofKiel;2InstituteofBiochemistry,UniversityofKiel;3Departmentof

Zoophysiology,ZoologicalInstitute,UniversityofKiel;4InstituteforInfectionMedicine,UniversityofKiel

obruhn@tierzucht.uni-kiel.deDefensinsareapredominantclassofantimicrobialpeptides,which

actasendogenousantibiotics(1).Defensinsareclassifiedintothreedistinctsub-families,θ-,β-,andα-defensins.Anα-defensinsynthesisis confirmed only in primates and glires to date and is presumablyuniqueforafewtissuesincludingneutrophilsandPanethcellsofthesmall intestine.Antimicrobialactivitiesof thesepeptideswereshownagainstawidevarietyofmicrobes includingbacteria, fungi, viruses,and eukaryotic pathogens (2). Here, we report the characterizationoftheequineα-defensinDEFA1.Transcriptionanalysisrevealedthatthetranscriptof thepeptide ispresent in thesmall intestineonly.Analignmentwithknownα-defensinsfromprimatesandgliresdisplayedahomologytoPanethcellspecificα-defensins.DEFA1wasrecombi-nantlyexpressedinE.coliandsubsequentlyanalyzedstructurallybycirculardichroismspectroscopyandmolecularmodelling.Toexaminetheantibacterialandantifungalpropertiesaradialdiffusionassay(3)was performedwith 12 differentmicroorganisms and the LD90 andMBCvalueswereexamined.DEFA1showedanantimicrobialactivityagainstdifferentgrampositiveandgramnegativebacteriaandagainsttheyeastCandidaalbicans.Usingviablebacteriaincombinationwithamembrane-impermeablefluorescentdyeaswellasdepolarizationofliposomesasaminimalisticsystem(4),itbecameevidentthatmem-brane permeabilization is at least an essential part of the peptide´smodeofaction.

REFERENCES1.Zasloff,M.(2002),Nature415,389-3952.Selsted,M.E.,Szklarek,D.,andLehrer,R.I.(1984)Infect.

Immun.46,150-154

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3. Lehrer, R. I., Rosenman, M., Harwig,S.S., Jackson, R., and Eisenhauer, P. (1991) J.Immunol.Methods137,167–17

4. Leippe, M., Ebel, S., Schoenberger, O.L., Horstmann, R. D., and Müller-Eberhard (1991)Proc.Natl.Acad.Sci.USA.88,7659-7663Key words: HORSE, INNATE IMMUNITY, ANTIMICROBIALPEPTIDES,DEFENSINSSpecies:equine

VA233. IN VITRO STIMULATION OF EQUINE LEUKOCyTES WITH CPG-OLIGODEOxyRIBONUCLEOTIDES – CyTOKINE

PRODUCTION AND PROLIFERATIONEVAWATTRAnG1,AnnA-KARInPALM1,BETTInAWAGnER2

1DepartmentofParasitology(SWEPAR),NationalVeterinaryInstituteandSwedishUniversityofAgriculturalSciences,Uppsala,Sweden;

2DepartmentofPopulationMedicineandDiagnosticSciences,CornellUniversity,Ithaca,USA

Synthetic oligodeoxyribonucleotides (ODN) containing CpG-motivshavebeenidentifiedaspotentactiviatorsofimmunefunctionswithpotentialusee.g.,asvaccineadjuvants.Basedonresponsesofhumancells,threedifferenttypesofCpG-ODNhavebeenproposed,predominantlyinducinghighIFN-αβresponses(A-class)orstrongpro-liferativeresponses(B-class)orbothtypesofresponses(C-class).InthisstudyweevaluatedfourCpG-ODN,twoA-classandoneeachofB-,andC-class,respectively,forinductionofcytokineproductionandproliferationinequineleukocytes.

Cytokineconcentrationsweremeasured;IFN-αβ(bioassay)andIFN-γ,TNF-αandTGF-β(ELISA),incellculturesupernatantsafter24hofinductionandproliferationwasdeterminedafter72hofinduction.EachODNwastestedwithleukocytesfromatleast5individualhorseswithandwithouttheadditionoflipofectintocellcultures.

IFN-αβ production was induced by both B- and C-class ODNandoneoftheA-classODN.WhiletheB-andC-classODNinducedhighlevelsofIFN-αβbothinthepresenceandabsenceoflipofectin,theA-classODNonlyinducedhighlevelsofthiscytokineinthepres-ence of lipofectin.High levels of IFN-γ andTNF-αwere induced bythesameODNthatinducedIFN-αβproduction.Howeverinthiscase,allthreetypesofODNinducedsimilarlevelsofIFN-γandTNF-αandthe levelsofcytokineproducedwerenotmarkedly influencedbythepresenceoflipofectin.Similarly,proliferationwasinducedbythesameODNthatinducedcytokineproductionandtheproliferativeresponseswerenotinfluencedbyadditionoflipofectin.NoneoftheODNtestedinducedTGF-βproduction.Moreover,theactivityseemeddependentonCpG-motifssinceGpCcontrolODNwereeitherinactiveorinducedlowresponsesdependingontheparameteranalysed.However,pres-enceofCpG-motifsdidnotnecessarilyconferactivitysinceoneoftheA-classODNwasinactivedespiteofitsCpG-motifs.

Thus,withequinecellsdistinctionsininductionofdifferentimmunefunctionsbetweenA-,B-,andC-classODNwerelessobviousthanwhathasbeenobservedforhumancells.TheseobservationsfurtherstressthepresenceofspeciesdifferencesinODN-inducedresponses.Key words:CpG-ODN,cytokines,proliferation,typeIIFNSpecies:equine

VA234. INACTIVATED PARAPOxVIRUS OVIS AS AN IMMUNOMODULATOR IN FOALS

CoRMACCBREATHnACH,DAVIDWHoRoHoVDepartmentofVeterinaryScience,GluckEquineResearchCenter,

UniversityofKentucky,Lexington,KY40546-0099cormac@uky.edu

Reduced IFNγ production is a characteristic of neonatal foalimmunodeficiency.ItindicatesdecreasedcellularimmunefunctionandexplainstheincreasedsusceptibilityoffoalstointracellularpathogenssuchasRhodococcusequi,whoseclearanceisheavilydependentonIFNγ-mediatedeffects.Therefore,safeaugmentationoffoalIFNγpro-duction iscritical toprotect foals from infectiousdisease. Inactivatedparapoxvirusovis(iPPVO)isaknownimmunomodulatormarketedforuse in horses.This study tested the ability of commercial iPPVO to

elevatefoalIFNγproductioninvitroandinvivo.PBMCwerepreparedatregularintervalsfrom6ponyfoalsandculturedinthepresenceofmediumalone,iPPVOorConAfor72hr.AllsampleswerethenpulsedwithPMAandionomycininthepresenceofbrefeldinAfor4hrtopro-moteintracellularaccumulationofIFNγandTNFαfortheirsubsequentdetectionbyflowcytometry.Also,PBMCculturedwithiPPVOorConAfor24hrweretestedforalteredcytokinegeneexpressionbyreal-timePCR. Notably, foal PBMC cultured with iPPVO, but not with ConA,demonstratedincreasedIFNγproductionfollowingPMAandionomycinpulse.This indicates that iPPVO increases the IFNγ-producing abil-ityoffoalPBMC.ToinvestigatewhethertreatmentwithiPPVOhasasimilareffectinvivo,20ponyfoalsweredividedinto3groups.Group1foals(n=6)receivedsalinei.m.(0.9%NaCl)ondays0,2and9.Group2foals(n=7)receivediPPVOi.m.ondays0,2and9.Group3foals(n=7)receivediPPVOi.m.onday0only.All foalswerelessthan10daysofageonday0ofthestudy.PBMCsamplespreparedfromeachfoalatregularintervalsthrough6monthsofagewerestimulatedwithPMAand ionomycin tomeasure intracellular IFNγproduction.SerialwholebloodRNAsampleswerealsopreparedtomeasuretheeffectsofiPPVOtreatmentonexvivocytokinegeneexpression.TreatmentofyoungfoalswithiPPVOfollowinglabelrecommendationsdidnotsig-nificantlyaltertheirabilitytoproduceIFNγ.However,theaboveinvitrodataconfirmthatiPPVOdoesincreasetheIFNγproducingcapacityoffoalPBMC.VaryingdosageandrouteofadministrationofiPPVOwillbepursuedtoreproducethiseffectinvivo.Key words: horse, immunomodulator, immunostimulant, interferongammaSpecies:equine

VA235. IL-18 RECEPTOR INDUCTION By IL-12 IN THE CHICKEN: IL-12 AND IL-18 SyNERGISTICALLy STIMULATE

IFN-γ PRODUCTIONJESSEDTHoMAS1,2,3,DALEIGoDFREy2,JoHnW

LoWEnTHAL1,AnDREWGDBEAn11CSIROLivestockIndustries,AAHL,Geelong,Australia;2University

ofMelbourne,Melbourne,Australia;3AustralianPoultryCRCjesse.thomas@csiro.au

Thecontrol of viral infections isof critical importance topoultryindustriesworldwideandthisishighlightedbycostlyinfectionoutbreakssuch as Avian Influenza. Currently, increasingly hyper-virulent viralstrainsarebecomingmoreapparentand thereforenewvaccineandanti-viralstrategiesarerequiredtoamelioratetheimpactoftheseinfec-tions.Todeveloptheseimprovedstrategiesagreaterunderstandingoftheanti-viralresponseiscritical.Inthechicken,evidenceisstillbeinggatheredontheexistenceofadefinedTh1andTh2immuneresponse,however,supportforthetheoryofthisdichotomousresponseisgrow-ingrapidly.Followingthereleaseofthefullchickengenomesequencein 2004,many immune genes identified were similar to those seenin themammaliansystem.Nowaclearerpicture isavailableofhowadichotomous immune response in thechickencouldhaveevolvedsimilarly in function and in structure to its mammalian counterpart.Thisinformationprovidesnewavenuesfortargetingtherapiestowardsincreasinglyhyper-virulentinfectiousagents.Predominantly,anti-viralstrategiestargetandmodulatevariousaspectsoftheTh1armoftheimmuneresponse.Inthemammaliansystem,theinnatephaseoftheTh1responseischaracterisedbycytokinesIL-12andIL-18,actinginsynergy, toup-regulate theexpressionof IL-18and IL-12 receptors,respectively.Itisthisimportantinteractionduringaviralresponsethatinducestheproductionofthekeyanti-viralproteinIFN-γ.Thisresponseprocessisacharacteristicsequenceofinteractionsandeventsidenti-fied incellmediated immunity inmammals. In thisstudy,we investi-gated the status of theChIL-18R andChIFN-γ following stimulationwithE.coliexpressedrecombinantcytokinesChIL-12andChIL-18.Itwas found that following 48-72 h incubation of chicken splenocyteswithChIL-12therewassignificantup-regulationofChIL-18RmRNA.Using this information,we investigated if thisChIL-12 inducedChIL-18Rexpressionshoweddownstreamfunctionality in thepresenceofChIL-18.Wefoundthatpre-treatingchickensplenocyteswithChIL-12for48-72hfollowedbya24-48hincubationwithChIL-18inducedup-regulationofChIFN-γproductioninadosedependantmannerrelativetosingleandmultiplecytokinecontrols.ThisprovidesfurtherevidencefortheconservationofaTh1-likesysteminanon-mammalian(avian)

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species.Thesefindingscouldprovidepotentialnew targetareas forstrategiestowardstheproductionoftherapeuticsandanti-viralagentsagainst increasinglypathogenic infections important topoultry indus-triesworldwide.Key words:Interleukin(IL)–12,IL-18,Interferon(IFN)gamma,IL-18ReceptorSpecies:avian

VA236. IMMUNOPOTENTIATING EFFECT OF IMMUPLUS-A POLy HERBAL PREPARATION

RAVInDRAPV,RSCHAUHAn1DivisionofVeterinaryBiotechnologyIndianVeterinaryResearch

InstituteIzatnagar-243122,Bareilly,UP,India;1JointDirector,CentreforAnimalDiseaseResearchandDiagnosis,IndianVeterinary

ResearchInstituteIzatnagar-243122,Bareilly,UP,Indiaraviravindra1@rediffmail.com

Thedevelopmentofantibioticresistancetovariousantibioticspro-pelledtheuseofimmunomodulationtherapyasanalternativestrategytocombatvariousdiseases. Immunomodulation therapyalsohasanadditional advantage of amplifying the specific immune response tovaccines. In the present study, Immuplus, a poly herbal preparationcontainingWithaniasomnifera,ocimumsantctum,tinosporacordiflia,andEmblicaofficinalis was tested for its immunopotentiating effectsonthemacrophagefunctionsinchicken.Atotalof80,day-oldbroilerchicks were procured and randomly divided in two groups.Group Iservedascontrol,whilegroupIIweregivenImmuplus@1gm/100mlinwaterforaperiodoftwomonths.Macrophagefunctiontestwascarriedoutat fortnightly intervalbyNitroblue tetrazoliumreductionassay.Attheendoftheexperiment,thecountofNBTpositivecellswasupby22percentinthegroupIIincomparisontocontrols.ItwasconcludedthatImmuplussignificantlyenhancedtheengulfmentpowerofmacro-phagesandneutrophilsandthusup-regulatingtheimmunepowerofbirds.Key words: Immuplus, up-regulation, macrophage function,immunopotentiationSpecie:avian

VA237. PROTECTION OF CHICKENS AGAINST HETEROGENEOUS IBV STRAINS WITH RECOMBINANT

FOWLPOx VIRUS ExPRESSING IBV S1 GENE AND CHIFNγ GENE

yUn-FEnGWAnG,xInG-MInGSHI,GUAnG-ZHITonG*DivisionofAvianInfectiousDiseases,NationalKeyLaboratoryof

VeterinaryBiotechnology,HarbinVeterinaryResearchInstitute,TheChineseAcademyofAgriculturalScience

gztong@hvri.ac.cn.Introduction: Infectiousbronchitis isanacute,highlycontagious

viral respiratorydiseaseofchickens,whichcauseseconomic lossesinpoultryindustryworldwide.IBVposesaconstantlychangingthreattopoultryindustryasnewserotypesandvariantsemergingworldwide[1-2].Thus,developingnewvaccinesisnecessaryforthecontrolofthisdisease.Inordertosolvethequestion,arecombinantvirusexpressingchickentypeIIInterferongeneandIBVS1genewasconstructed,andimmune potency for heterogeneous strains was tested. Four-week-old SPF chickens were randomly divided into 10 groups, 5 groupswerevaccinatedwith1000PFUrecombinantfowlpoxvirusexpressingIBV S1 gene and ChIFNγ gene (rFPV-ChIFNγS1) under wing web,and others were unvaccinated control. Parental IBV LX4 strain andheterogeneousfieldisolatesLHB,LHLJ04XI,LSC99I,LTJwerechal-lengedintranasallythreeweekspost-vaccination.Theresultsindicatedall vaccinated groups produced IBV specific antibody quickly aftervaccination,while protection is significantly different amongdifferentstrains.LX4,LHBandLHLJ04XIstrains,belongedtoGenotypesIandIIandhadphylogeneticalanalogyaccordingtoS1protein,werewellprotected,whileLSC99IandLTJstrainswereincompletelyprotected.ThepercentageofCD4+andCD8+Tlymphocytesinvaccinatedgroupswas significantly different from control groups after challenged withLX4,LHBandLHLJ04XI,whileLSC99IandLTJarenotremarkable.PathologicalchangesofvaccinatedgroupsafterchallengedwithLX4,LHBandLHLJ04XIissignificantlydifferentwhencomparedwithcontrol

groups.Inaddition,LX4,LHBandLHLJ04XIstrainsshowshortenedvirusreleasetimeandquantity,whileLSC99IandLTJstrainsarenotremarkable.Weightanalysisshowedthatvaccinatedgroupshavenoremarkablechangescomparedwithcontrolgroups.Takingallresultstogether,vaccinationofrFPV-S1/ChIFNγcanprotectchickensagainstIBVstrainsbelongtoGenotypesIandII,whilenototherstrains.

Refence:[1]FarsangA,RosC,RenstromL,etal.Molecularepizootiology

ofinfectiousbronchitisvirusinSwedenindicatingtheinvolvementofavaccinestrain.AvianPathol,2002,31(3):229-36.

[2]Wu, ZQ,Yang,QW,Fu,C, et al.Antigenic and immunogeniccharacterizationof infectiousbronchitisvirusstrainsisolatedinchinabetween1986and1995.AvianPathol,1998,27:578-85.Key words:infectiousbronchitis,recombinantfowlpoxvirus,chickentypeIIInterferon,S1Species:avian

VA238. MOLECULAR IMMUNOPHENOTyPING OF LUNGS IN NAïVE AND VACCINATED CHICKENS EARLy AFTER

PULMONARy AVIAN INFLUENZA A (H9N2) VIRUS INFECTIONWInFRIEDGJDEGEn1,JACqUELInESMITH2,BARTJAn

SIMMELInK1,ELIZABETHJGLASS2,DAVEWBURT2,VIRGILEJCSCHIJnS1

1DepartmentofVaccineTechnology&ImmunologyR&D,IntervetInternationalB.V.,P.O.Box31,5830AABoxmeer,TheNetherlands,

2DivisionofGeneticsandGenomics,RoslinInstitute,Roslin(Edinburgh),MidlothianEH259PS,UK

winfried.degen@Intervet.comIn a respiratory infection model we study immune reactions in

chickens infected with the low pathogenic avian influenza A H9N2virus strain. Formolecular immune response profiling we employeda recentlydevelopedchicken immuno-microarraycontainingapprox.5000cDNAelementsinduplicate(Smithetal.2006).Inafirstexperi-ment,broiler-typechickenswereeithermock-immunized(referredtoasnon-immune),vaccinatedwithinactivatedviralantigenonly(immune),orwithviralantigenindistinctTh1orTh2polarizingimmunopotentia-tors(immunepotentiated).ThreeweeksaftervaccinationallanimalsweregivenarespiratoryinfectionwithH9N2viatheoculo-nasal-intra-trachealroute.Fromtheseanimalslungsandspleenswereremoved,RNAwas isolatedandsubsequentlyusedformicroarrayanalysis. Ingeneral,wenotedlesshostgeneexpressionin immunepotentiated,i.e.adjuvanted,birdswhencomparedtonon-immune,infectedchick-ens. Immune potentiated birds showed reduced innate responses,mainlyrestrictedtoheatshockproteins,which,likely,aresufficienttocontrol thepulmonary infection togetherwith inducedantibodiesandTcells.Evaluationof themicroarraydatasuggestedgenepathwaysuniquetoorcommonamongstthedifferentlyimmunegroups(Degenetal.2006). Insubsequentexperimentswehaveanalyzed,andstillareanalyzing, the influenceofageand type(broilerversus layer)ofthechickens,challengeroute(oculo-nasal-intratrachealversusspray),andtheuseofotherimmuneresponsepolarizingimmunopotentiatorsonthehostgeneexpressionprofilesbyusingthemicroarrayandquan-titativeRT-PCR(Q-RT-PCR),aswellasclassicalimmuneparameters.Inthispresentationwewillhighlightourfindings.ThedatacollectedinthisnaturalinfluenzaAvirustargetspecies,i.e.chicken,areofinter-est for thedesignof futurehumanpandemicandseasonalvaccinescontainingimmuneresponsemodifyingimmunopotentiators.Key words: adjuvant, avian influenza H9N2, microarray,immunophenotypingSpecies:avian

VA239. INFLUENCE OF THE USE OF PREBIOTIC AND PROBIOTIC ON BROILER MACROPHAGE ACTIVITyARyAnADnUnES1,ISISMHUEZA2,LEonILAESTERR

RASPAnTInI2,RICARDoDEALBUqUERqUE11DepartmentofAnimalNutritionandProduction,SchoolofVeterinaryMedicineandAnimalSciencesofUniversityofSãoPaulo,S.P.,Brazil;2ResearchCenterforVeterinaryToxicology(CEPTOX),Schoolof

VeterinaryMedicineandAnimalSciencesofUniversityofSãoPaulo,

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S.P.,Brazilaryananunes@usp.br

Theuseofantibioticsonlivestock,mainlyonpoultryproduction,hasbeenrelatedtopromotebacterialresistanceofsomehumanpatho-genic microorganisms. This potential risk to public health promptedresearchestodevelopalternativesubstancesoradditivestoimproveavianperformance,asprebioticsandprobiotics.Studiesconductedinordertoverifytheeffectofthesesubstancesoninnateimmunesystemrevealedapossible immunostimulatoryeffectonmacrophageactivi-ties.Thus,theaimofthepresentstudywastoevaluatethesepossibleimmunomodulatoryeffectsonmacrophageactivity (i.e.phagocytosisand hydrogen peroxide production (H2O2)) of broilers treated withmananooligosaccharide (prebiotic) or a bacterial pool (probiotic) oravilamicin (antibiotic) during 28 days. Forty one-day-old commercialbroilerchickswereweighedandrandomlyassignedtofourgroupsof10each,includingacontrolandthreeexperimentalgroupstreatedfor28dayswithbasaldiet(BS)formulatedtomeetbroilerrequirementsin the beginning stage (1-21 days old) without antimicrobial growthpromoters. Each experimental group received, respectively, BSwithprebiotic;BSwithprobiotic;andBSwithavilamicin (10ppm).Forty-eighthourbeforesacrifice,thechicksreceivedanintraperitoneal(i.p.)injectionofaSephadexsuspension(3%) inorder topromoteperito-nealmacrophageactivation.Themacrophageactivitywasevaluatedusing theprotocolsdescribedas follow:Phagocytosis:Briefly, itwascountedatotalof200cellsbyslideofeachbirdandthemacrophagephagocytosis index (PI),werecalculatedas follows:PI=numberofphagocyticactivityx100÷200adherentcellscounted,i.e.,asPI=%ofmacrophageswithphagocytizedzymosanparticles.H2O2 release:Spontaneous and phorbol myristate-acetate solution (PMA)-inducedH2O2releasebymacrophageswascalculatedfromabsorbancemea-surements. Spontaneous andPMA-inducedH2O2 production experi-mentswererepeatedfour timesforeachbird ineachgroupandthemeanvalueof the fourcountswasused todetermineH2O2concen-tration.Nostatisticaldifferences(P>0.05)betweenthegroupswereobservedinbothanalysisphagocytosisandtoH2O2production.Eventhoughnochangesonmacrophageactivitywereobserved,itisnopos-sibletoruleoutanimmunomodulatoryeffectonothercompartmentsoftheimmunesystem.Thusmoreexperimentsarebeenconductedinourlabtoverifythepossibleimmunomodulatoryeffectofthesesubstancesonhumoralandcellularimmuneresponsesofbroilerchicks.

(FAPESP06/53354-0;FAPESP06/53349-7).Key words:Broiler,MacrophageActivity,Prebiotic,ProbioticSpecies:avian

VA240. IMMUNO-MODULATORy EFFECTS OF MUCOSAL VACCINATION WITH LACTIC ACID BACTERIA THAT CARRy

THE RECEPTOR BINDING DOMAIN OF F18 FIMBRIAEAGnETALInDHoLM

DepartmentofBasicVeterinarySciencesUniversityofHelsinkiP.O.Box66(Agnessjöberginkatu2)00014Finland

agneta.lindholm@helsinki.fiE. coli with F18 fimbriae adhere to intestinal epithelial cells in

pigsthroughtheFedFproteinandcausespost-weaningdiarrhoeaoroedemadisease that result inmajorpig losseson farmsworldwide.BothpassiveandactiveoralimmunizationstudiesagainstwholeF18fimbriaehavebeenperformedbuttodatetherearenoeffectivevaccinesagainstpost-weaninginfection.Therefore,theonlytreatmentavailable,whenpreventivemethodsfail,isantibiotictherapy,whichincludesrisksofbacterialdrugresistanceandresiduesinmeatproducts.

During the last decade, the roleof lacticacidbacteria (LAB) invariousbiologicalfunctionsofthehosthasbeenextensivelyreported.Especially probiotic properties of several LAB are of interest. Someofthedocumentedbenefitsofprobioticsareduetobalancingthegutmicrobiota,improvingcolonizationresistanceagainstintestinalpatho-gens andbeneficial immunemodulation.Theseprobiotic properties,as well as the GRAS (generally regarded as safe) and food gradestatuses of many LAB, have made them interesting candidates forantigendeliveryinmucosalvaccination.OtheradvantagesofmucosalvaccinationwithLABare,comparedtoconventionalparenteralvacci-nation,stimulationofbothlocalandsystemicimmuneresponses,easyadministration,andlowdeliverycosts.

ThegoalofthisstudyistodevelopaLABvaccinevectoragainstporcinepostweaningdiarrhoeaandoedemadiseasecausedbyF18fimbriatedE.coli.TwomodelLABhavebeenemployed.LactococcuslactisNZ9000δhtrAandLactobacillusbrevisATCC8287. InL. lactis,thereceptorbindingdomainoftheF18fimbrialadhesinFedFissur-face displayed as a fusion protein with PrtP and theAcmA cell wallbindingdomain.InL.brevisthesamereceptorbindingdomainofFedFisproducedaspartofthesurfacelayer(S-layer)proteinSlpA.Inordertotestmucosalimmunogenicityoftheconstructs,theFedFproducingstrainswereadministeredeithernasallyororallytogroupsofmiceandtheproductionofFedF -specific immunoglobulines in serum, faecesandintestinallavagehasbeenevaluated.Key words: F18 fimbriae, lactic acid bacteria, FedF, mucosalvaccinationSpecies:swine

VA241. THE ANTIVIRAL STATE INDUCED By IFNβ DIFFERS IN MARC-145 CELLS AND PAMS AS DEMONSTRATED By

INFECTION OUTCOMES WITH DIFFERENT PRRSV ISOLATESCHRISToPHERCoVEREnD1,JoSEPHAMBRoGIo1

DonGSHEnGHE2,MARVInJGRUBMAn3,AnTonIoEGARMEnDIA1

1DepartmentofPathobiologyandVeterinaryScience,UniversityofConnecticut,61N.EaglevilleRd,CT06269,USA;2Collegeof

VeterinaryMedicine,SouthChinaAgriculturalUniversity,Guangzhou,510642,China;3PlumIslandAnimalDiseaseCenter,USDA,ARS,

Greenport,NewYork,11944Porcinereproductiveandrespiratorysyndromeischallengingin

termsofbothpathogenesisandhostresponse.Relativelylittleisunder-stoodregardingtheeffectsofPRRSVonthehostimmuneresponse.Existingdatapointtotheinnateimmuneresponsesasbeingsub-opti-malandthatthisdeficitprovokesadelayedandsub-optimaladaptiveresponse.Althoughthereisahighvariabilityinbothinductionofandsensitivity to type I IFN inPRRSV, it appears thatblocking the IFN-mediatedinnateresponseisapowerfulmechanismutilizedbyPRRSVtoinfectthehostsuccessfully.MostworkontypeIIFNinthecontextofPRRShasfocusedonIFNa,whereasthere is lessunderstandingoftheactualroleofIFNβ.ThisstudyinvestigatedtheeffectsofIFNβontheinfectionofbothMARC-145cellsandporcinealveolarmacro-phages(PAM)withPRRSV.PhenotypicdifferencesinIFNβsensitivitywere identified among different PRRSV isolates. Two PRRSV fieldstrains were fully sensitive to IFNβ (MO25544, MO8981), two wereintermediatelysensitive (NVSL1257,andavaccinestrain),andonewasresistant(NVSL9301)whentestedinMARC145cells.TitrationofvirusfromIFNβ-treatedandcontrolcellsonMARC-145cells,andrealtimeRT-PCRwereusedtomeasurethesensitivity.Interestingly,whensensitivitytoIFNβwastestedinPAMs,underidenticalconditions,allstrainsappearedtobesensitiveover theconcentrationrangetested(1000-20UIFNβ).Treatmentwith2-aminopurine(2-AP),toblockthedouble-stranded RNA-dependent protein kinase (PKR), resulted invarious levels of recovery of replication for all strains inMARC-145cells that had been previously primed with IFNβ. In contrast, virusreplicationwasnot rescuedbyan identical2-AP treatmentofPAMspreviouslyprimedwithIFNβ.Thesefindingsdemonstratenotonlydif-ferentialsensitivityofseveralPRRSVstrainstoIFNβ,butalsohighlightdifferencesinthehostcellsystemsemployed.TheresultssuggestthatPKRis involved in theantiviralstate inducedby IFNβ,specifically inMARC-145cells, thoughit isapparent that thereareother importantfactors contributing to host innate immunity to PRRSV infection, asexemplifiedbythedifferingresultsobtainedwhenusingPAMs.Key words:swine,interferon,PRRSV,innateimmunitySpecies:swine

VA242. CONSTITUTIVE ExPRESSION OF INTERFERON-ALPHA By SWINE LEUKOCyTES

MASSIMoAMADoRIDepartmentofAnimalWelfareandImmunoprophylaxis,Istituto

ZooprofilatticoSperimentale,Brescia,ItalyInterferon-a (IFN-a) plays an important role in the homeostatic

regulationof the inflammatory/stress response,whichdoesnot con-

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flictwith itsmain functionsin the innate immunesystem.Constitutiveexpression in healthy subjects is in line with this tenet and it wasthereforeinvestigatedinswineleukocytes.Thesamelowprevalenceof IFN-a positive cellswasshownbyflowcytometry in control andvirus-stimulatedperipheralbloodmononuclearcells(PBMC);thelatteronlywerealsopositiveforcytokinesecretion.Resultswereconfirmedwith thesamemAbbywesternblottingonNP-40 lysatesof PBMC,spleen, lymphnodecellsandalveolarmacrophages (AM)of controlandH1N1influenzavirus-infectedpigs.PBMCandAMofcontrolandvirus-infectedpigsexpressedinfactastrong32kDa+minorbands,asopposedtospleenandlymphnodes.Interestingly,thesignalinPBMCdisappeared inwesternblottingbutnot inflowcytometryafterover-nightcultureinvitro.Resultswereconfirmedbyimmunoprecipitationof35S-methionine/cysteine,metabolicallylabelledPBMCof4healthy,SPFpiglets.Intriguingly,3outof4lysateswerealsoIFN-γpositive(40kDaband)withaseeminglyinversecorrelationtoIFN-αexpression.

The possible paracrine role of IFN-α was investigated invitro. Minute amounts of IFN-a (≥0.5 U/ml) significantly increasedtheinvitrosurvivalofCD4+TcellsafterbothmitogenandFoot-and-MouthDiseaseVirus (FMDV) recallantigenstimulation. Instead, theprevalenceofdouble-positiveCD4+/CD25high+,allegedlyTregcellswasnot increased. In thesamemodelof recallTh2 response toFMDVOManisa,as littleas5U/ml IFN-acouldsignificantlyboost the invitro serum-neutralizing (SN) antibody response; no antibody wassecretedincontrolcultureswithoutpurified146SFMDVantigen;theheightofthisresponsewasinverselyrelatedtothenumberofAntibodyProducingCellsatday12(exhaustionphase).3H-thymidineincorpo-rationwas inhibited after exposure of thesePBMC to purified 146SFMDVantigen;under thesameconditions, theprevalenceof IFN-γpositive cells (ELISPOTand flow cytometry assays)was decreasedbeneaththecontrolvaluein3animalsoutof4;thiswasreversedinthepresenceof5U/mlofIFN-a.Inconclusion,intracellularIFN-aiscon-stitutivelyexpressedinsomepopulationsofswinePBMCandAManditseeminglydiffersfromitsmature,extracellularforms.Finally,minuteamountsofofIFN-acouldsignicantlyaffectsomeThcellfunctions.Key words:pig-innateimmunity-interferonalphaSpecies:swine

VA243. TARGETING TO SIALOADHESIN RECEPTOR IMPROVES ANTIGEN PRESENTATION TO T CELLSTERESAPoDERoSo,ConCEPCIönREVILLA,BELEn

ALVAREZ,SonIACHAMoRRo,PALoMAMARTÍnEZDELARIVA,FERnAnDoALonSo,AnGELEZqUERRA,JAVIERDoMÍnGUEZ

DptodeBiotecnología,INIA,Madrid,Spainjuncal@inia.es

Themaingoalofvaccinebiotechnologyistogenerateaprotec-tiveandlong-lastingimmuneresponseagainstpathogens.Oneofthestrategiesthatarebeinginvestigatedtoenhanceresponsestoweakvaccine antigens is targeting them toAPC receptors by conjugationtospecificantibodies.Sialoadhesinisamemberofthefamilyofsialicacidbindingimmunoglobulin-likelectins(Siglecs),whoseexpressionisrestrictedtosubsetsoftissuemacrophages,inflammatorymonocytesandsomedendriticcells.Here,wehaveanalysed theexpressionofsialoadhesin in porcine tissues, its regulation by cytokines and itspotentialasanantigentargetingreceptor.Porcinesialoadhesinwasexpressedinmacrophagesofthemarginalzoneofspleenandinden-dritic cells derived frommonocytes culturedwithGM-CSF plus IL-4(MoDC).Bloodmonocytes (Mo)werenegative for thismoleculebutitsexpressioncouldbeinducedaftertreatmentwithIFN-a.ToanalyseiftargetingantigentosialoadhesinleadstoanenhancementofTcellresponseswecomparedtheproliferativeresponseelicitedbyamAbtosialoadhesin(1F1)withthatofanisotypematchedcontrolmAb(1D9).Tcells frompigs immunizedwithapoolofmouse IgswereusedasrespondercellsandMoDCorMoculturedwithIFN-aasAPCs.MAbtosialoadhesininducedTcellproliferationatconcentrations100-foldlowerthancontrolmAb,suggestinganefficientroleofsialoadhesininantigenuptakeand/orprocessinginAPCs.ConsistentwiththesedatatheAlexa488labeled1F1mAbbutnottheAlexa488-labeledcontrol1D9mAbwassignificantlyinternalizedinmacrophagesandMoDCcul-turedat37°Cfor30min.Experimentstoevaluateinvivothepotentialofsialoadhesinasavaccinetargetingreceptorareinprogress.

Key words: sialoadhesin, antigen-presenting cell, monoclonalantibody,vaccineandswineSpecies:other

VA244. ADJUVANT EFFECT OF PORCINE CHEMOKINES ON DNA VACCINATION OF PIGS

BELénALVAREZ1,CARMEnCAnTó1,JoAnPUJoLS2,IVánDÍAZ2,EnRICMATEU2,M.PILARRoDRÍGUEZ-CARREño1,ConCEPCIónREVILLA1,AnGELEZqUERRA1,FERnAnDo

ALonSo1,JAVIERDoMÍnGUEZ11DptodeBiotecnología,INIA,Madrid,Spain;2CRESA,Barcelona,

Spainfalonso@inia.es

Chemokinesplaykeyrolesintheelicitationofimmuneresponsesbyrecruitingantigen-presentingcellstothesiteofantigeninoculationandregulatingthemigrationofdifferentleukocytesubsetstolymphoidorgans. This study evaluated the effect of the co-administration ofplasmids encoding porcine MIP-3a (CCL20) and SDF-1 (CXCL12)on the immune responses induced by a DNA construct encodingtheNproteinofporcinereproductiveandrespiratorysyndromevirus(PRRSV-ORF7).Thebiologicalactivityofthesechemokineconstructswaspreviouslydemonstratedinmigrationassaysofmonocyte-derivedDCswithculturesupernatantsfromtransientlytransfectedCHOcells.Groupsoffivepigswere immunizedintramuscularly threetimeswithdifferent combinations of these constructs or with empty plasmidDNA.ThehumoralandcellularimmuneresponseswereanalyzedbyaPRRSVN protein -specific ELISA, and by an in vitro proliferativeresponseofPBMCtowholePRRSvirus,respectively.Theco-admin-istration of either SDF-1 or MIP-3a plasmid increased the antibodyresponseinducedbyDNAimmunizationwithPRRSV-ORF7construct.WhereasSDF-1significantlyenhancedanti-NproteinIgG1andIgG2antibodyresponses,MIP-3afavouredthedevelopmentofIgG1anti-bodies.NodifferenceswereobservedintheproliferativeresponsetothevirusbetweengroupsvaccinatedwithorwithoutchemokinesKey words:Adjuvant,DNA-vaccination,ChemokinesSpecies:swine

VA245. OPTIMAL COMBINATIONS OF ACUTE PHASE PROTEINS FOR MONITORING DISEASE IN PIGS.

PETERMHHEEGAARD1*,AnDERSSToCKMARR1,MATILDEPIñEIRo2,FERMInLAMPREAVE2,FIonACAMPBELL3,PDAVID

ECKERSALL3,MATHILDAJ.M.ToUSSAInT4,ERICGRUyS4,nAnnASKALLSØREnSEn1

1NationalVeterinaryInstitute,TechnicalUniversityofDenmark,Bülowsvej27,DK-1790CopenhagenV,Denmark;2Universityof

Zaragoza,Zaragoza,Spain;3UniversityofGlasgow,Glasgow,UnitedKingdom;4UniversityofUtrecht,Utrecht,TheNetherlands

pmhh@vet.dtu.dkTheacutephaseprotein(APP)responseisarobustinnatereac-

tion towards tissue injury observed as one of the earliest systemicsignsofdisease.Anydiseasecausing tissuedisturbances, includingmostinfectionsandinflammatorystateswillinduceanAPPresponse.ThisallowsusingAPPsinserumformonitoringhealthofhumansandanimals.Here,we studied the response of several porcineAPPs inserumtoanumberofexperimental infectionsandanaseptic inflam-mation model. The serum panel employed consisted of more than400samples,takenatdifferenttimepointsduringfivedifferentexperi-mentalinfections(threebacterial,oneviral,andoneparasitic)aswellas during an experimental aseptic inflammation. Immunochemicalanalyses of seven different acute phase proteins, four positive andthreenegativewereperformed inallof thesamples.ThiswasdoneusingthebestavailableassaysinfourdifferentEuropeanlaboratories.Advancedstatisticaltreatmentofthedatawasperformedusingatwo-stepprocedurefirstdefiningcut-offvalues for the individualproteinsandthenderivingdetectionprobabilitiesforcombinationsofAPPsbymultivariateanalysis.ThenameasureofoverallsensitivityforallofthediseasestatesinvolvedwasconstructedbasedonthesummedareaunderthecurveforalloftheinfectionstogetherinordertocomparethevariousAPPcombinations(thismeasuregivestheaverageprobabilityofdetecting,usingtheAPPncombinationinquestion,aninfectionwith

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all5infectionsequallyprobable).Theseoperationsallowtheconstruc-tionofameasureofinfectiousdiseasethatisabletodetectdiseasemoresensitivelythananyindividualAPPoverawidediseaseperiodandwithawidevarietyof infections.Thebest2-proteincombinationwashaptoglobinandapolipoproteinA1(apoA1)closelyfollowedbyC-reactiveprotein(CRP)+haptoglobin,whilethebest3-proteincombi-nation,givingevenbettersensitivitywasCRP+apoA1+majoracutephaseprotein(pigMAP),closelyfollowedbyhaptoglobin+apoA1+pigMAP.Suggestionsandlimitationsofitsusefordifferentpurposes,includingthemonitoringofherdhealthwillbediscussed.Key words: pig acute phase response, experimental infections,diagnosticassaySpecies:swine

VA246. DENDRIMERS AS SCAFFOLDS FOR IMMUNE-STIMULATORy MOTIFS - A NEW CLASS OF WELL-DEFINED

MOLECULAR ADJUVANTS nAnnASSoREnSEn,ULRIKBoAS,KERSTInSKoVGAARD,

GREGERSJUnGERSEn,PETERM.H.HEEGAARD.NationalVeterinaryInstitute,TechnicalUniversityofDenmark,

Bülowsvej27,DK-1790CopenhagenV,Denmark.nss@vet.dtu.dk.

ProtectionagainstmanypathogensrequiresthedevelopmentofTh1 immuneresponses,and there isastrongneed for thedevelop-ment of efficient and safe Th1-biasing adjuvants. Toll-like receptors(TLRs) are a family of pattern recognition receptors that recognizeevolutionarily conserved pathogen-associated molecular patterns(PAMPs).Binding ofPAMPsbyTLRson dendritic cells induces thecellstoexpresscostimulatorymoleculesandinflammatorycytokines,leading to a Th1-biasing antigen-presentation. Dendrimers are arelativelynewclassofhighlymonodisperse,hyperbranchedsyntheticpolymerscharacterizedbyhavingasurfacewithanextraordinaryhighnumberoffunctionalgroups.Hereitwasinvestigatedifpolypropyleneimine(PPI)dendrimerscouldbeusedforthesynthesisofanewclassof adjuvants and vaccine-vehicles incorporating dendritic cell target-ingandmaturationsignals inonemolecular constructby theuseofsimplePAMP-motifs inamultivalentdisplay, thuscreatingamolecu-larly defined, all synthetic “artificialmicrobe” to which antigensmaybelinked.ArangeofPAMP-structuresweretestedonporcinePBMCsforinductionofinflammatorycytokines(ELISAandqRT-PCR)andcellsurfacemoleculeexpression(flowcytometry).Thecytokineprofilesofvarious PAMP-structures differed, with CpG oligo-deoxynucleotidesinducing IL-12p40production,single-strandRNApoly-uridine induc-ingIL-6production,andpeptidoglycan(PGN),Pam3Cys-SK4andLPSinducing mixed responses. Preliminary data show that MDP-conju-gateddendrimers,similarlytoPGN,areabletoinducetheproductionofIL-12p40andIL-6inporcinePBMCs.Inaddition,surfaceexpres-sionofB7onSWC3-positivecellswasseento increaseafterstimu-lation withMDP-dendrimers, as was seen with PGN. Unfortunately,MDP-dendrimerswerealmostascytotoxicasuncoupleddendrimers.Theseandadditional resultsonPAMP-motif-carryingdendrimerswillbepresentedandthepossibleuseofdendrimersinthepreparationofmolecularlywell-definedadjuvantsandvaccine-deliverysystemswithtailoredcytokine-inducingpropertieswillbediscussed.Key words: adjuvants, dendrimers, pathogen-associated molecularpatterns,Toll-likereceptorsSpecies:swine

VA247. TESTING IMMUNOGENICITy OF MyCOPLASMA HyOSyNOVIAE VACCINE CANDIDATES; INDUCTION OF

ANTIBODIES AND IFN-γ RESPONSEK.TØLBØLLLAURITSEn,U.RIBER,J.nIELSEn,J.ToFT

JAKoBSEn,G.JUnGERSEnNationalVeterinaryInstitute,TechnicalUniversityofDenmark,27,

Bülowsvej,DK-1790CopenhagenV,Denmarkktl@vet.dtu.dk

Mycoplasma hyosynoviae (Mhs) is a significant cause of acutearthritis inswineabove10weeksofage.Noeffectiveprophylaxis isavailable.Thereforeattemptsaremadetodevelopavaccinecandidatewithpotential toprotectagainstarthritiscausedbyMhs.Firststepis

screening of the immunogenic effect of Mhs antigen in combinationwithvariousadjuvants.Earlierstudiesoftheinfectionhaveshownthatantibodiescanprovideacertaindegreeofprotectionagainsttheinfec-tion,butaTh1likeresponsehasalsobeenshowntoplayasignificantroleinprotection.

The four vaccine candidates tested, were based on the sameformalin fixedMhs, but combined with various EMULSIGEN® oil-in-wateradjuvantformulations(MVPLaboratories).Onedosecontained300µgantigeninaqueoussolutionandadjuvantwasaddedto20%of the totalvolume.Elevenpigsat12-weeksofage (one litter, fromMycoplasmafreeherd)wereadministeredtheprimaryvaccinationi.m.(EMULSIGEN®-D:3pigs,EMULSIGEN®:2pigs,EMULSIGEN®-BCL:3 pigs andEMULSIGEN®-P: 3 pigs).Revaccinationwas carried outwithidenticalboostersafterthreeweeks.Anotherthreeweekslaterthepigswerekilled.

When measuring Mhs-specific IgG (ELISA) in serum, all pigsseroconvertedfromday24to28afterfirstvaccination(i.e.day3and7afterboost).Plateauwasreached7to14daysafterboost,ODval-ues ranging from0.5 to1.75.Measurementof IFNγ insupernatantsfrom whole blood cultures stimulated with Mhs antigen showed thatallgroupshadpigsthatreactedtovaccinationwithanelevatedIFNγresponse.Thelevelsinthetestreachedbeyond500pg/mlproducedIFNγinallpigs,insomepigsbeyond4000pg/ml.ThePBSstimulatedsamples (negative controls) didnot exceed102pg/ml.However, nogroupofpigshadconsistentlyhigherIFNγlevelthantheothers.PigsintheEMULSIGEN-D,EMULSIGEN-BCLandEMULSIGEN-PgroupsshowedaslightproliferativeT-cellresponseinantigenspecificprolif-erationmeasuredbyflowcytometry.

EarlierattemptstodevelopavaccineagainstMhshavefocusedon the serological response. Our experiment, however, focuses ontheuseofadjuvantsthatarereportedtofavourcellmediatedimmuneresponse,whilestillgivingrisetoanantibodyresponse.Theinvestiga-tionisapartofalargerinitialscreeningforvaccinecandidatesagainstMhs.Thusmorecandidatesaretobetested,andfinallyapromisingcandidate, with ability to induce both serological end cell-mediatedimmuneresponse,willbetestedinchallengeexperiments.Key words:vaccine,Mycoplasmahyosynoviae,swine,arthritisSpecies:swine

VA248. ROLE OF TOLL-LIKE RECEPTORS IN THE PATHOGENESIS OF HERPETIC STROMAL KERATITIS IN

CATSAVöGTLIn,MFRAnCHInI,UBÜCHLER,MACKERMAnnInstituteofVirology,VetSuisseFaculty,UniversityofZurich,

SwitzerlandDetectionofpathogensby the innate immunesystem iscarried

outbyaclassofsensormoleculestermedpatternrecognitionrecep-tors,orPRRs.TheToll like receptors (TLRs)belong to this classofmoleculesandareabletodetectabroadrangeofpathogens.Althoughoriginallydescribedas receptors forsensingbacteriaand fungi, it isbecomingincreasinglyapparentthatvirusesarealsosensedbyTLRs.Herpetic stromal keratitis (HSK) is an ocular disease that occurs incatsandhumans.DespitethebroaddistributionofHSKincats,littleisknownabout itspathogenesis.Experiments inmiceusingHerpessimplexvirustype1(HSV-1)indicateanimmunopathologicalbasisforHSK.SinceHSV-1wasshowntoinducecytokineresponsesviaTLR,wehypothesizeapotentialroleofthesereceptorsinthepathogenesisoffelineHSK.Ourstudyhasthepurposetoinvestigatetheinfluenceoffelineherpesvirus-1(FeHV-1)infectionontheexpressionandactivityoffelineToll-likereceptors.

OurpreliminarydataindicatethatfelinecornealepithelialcelllinesexpressmostTLRs,andinfectionwithFeHV-1influencestheexpres-sionofTLR3andTLR9.

Anotherpartof theproject includes theconstructionofdifferentexpression vectors containing feline TLRs to overexpress the mol-eculesinfelinecells.Presently,wehaveproducedclonesforTLR3,4and9.Inaddition,werevealedthecompleteTLR2codingsequenceandcloneditaswell.CloningofTLR7isinprogress.

SinceHSV-1isknowntoinduceseveralcytokineresponses,ourgoal is to examine the potential of FeHV-1 to activate inflammatory

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responses.Therefore, real-timePCRassays for the detection of IL-6,IL-1andTNFawereestablished.FirstexperimentsusingCrandellReeseFelineKidney (CRFK)cellscouldnotclearlydemonstrateaninfluence of FeHV-1 infection on production of these cytokines inculturedCRFKcells. Insummary,weestablishedthetools tofurtherinvestigatetheroleofTolllikereceptorsintheinfectionwithFeHV-1.Key words: Herpes simplex virus; feline herpesvirus-1; cornealepithelialcells;Tolllikereceptors;HerpeticstromalkeratitisSpecies:feline

VA249. SUCCESFULL TREATMENT OF FELINE VACCINE-ASSOCIATED SARCOMA IN A 10-yEAR-OLD CAT USING

ExTENSIVE INITIAL SURGERyALDACILEnES.SILVA,REnATASCAVonE

InstitutodeCiênciasBiomédicas/UniversidadedeSãoPauloDepartamentodeImunologia

Feline vaccine-associated sarcoma (FVS) is amalignant tumorlocalized at previous vaccination sites in cats. Although almost 20yearshavepassedsincethefirstdescriptionofthisdisease,FVStreat-mentstillhasalowsuccessrate.A10-year-oldspayedfemalefelinewaspresented tous inJuly2005,witha3.0cm-diameteradherent,swollenandpoorpainfultumoronitsback.Theanimalhadbeenyearlyvaccinatedfornineyearswithcommerciallyavailablevaccines,thelasttimesevenmonthspriorthepresentation.Attheclinicalexam,thecatdidnotpresentanyothersignal,exceptbythelesiondescribedabove.ComplementarybloodexamsdidnotrevealedanyalterationsandbothFIVandFeLV testswere negative. Ultrasonographic and radiologi-calaspectswerenormal,without indicationofmetastasis.Aspirativecytology,however,suggestedamalignancy.Thesurgicalexcisionofthelesionwasperformedwithlargesafetymargins.Noadjunctchemo-therapytreatmentwasappliedduetotherefusaloftheownertodoso.Histopathological examinationof theexcised tissuedocumented thepresenceofamalignantcellpopulationwithparticlesofsomekindofdeposit(aluminumadjuvantwassuggested)embeddedinmacrophagecytoplasms; tumorwasdiagnosedasagiantcellsfibrohistiocytoma.After surgery, the cat had beenevaluated once every threemonthsduring thefirstyearand theneverysixmonths.Noalterations in itsclinicalconditionorincomplimentaryexamsresultswereobservedinthisperiod.FVS isabigchallenge forveterinarymedicine.Althoughtheestimated riskofdevelopingFVS ismuch lower than the riskofnon-vaccinatedcatsacquiringinfectiousdiseases,traditionalvaccina-tionprotocolsarebeingreevaluatedinordertoreducethenumberofvaccineadministrationstotheminimumnecessary.Sincerecentstud-iesshowedthatcatswithextensiveinitialsurgeryhadamediantimeofrecurrenceof325daysandourpatientishealthyfor,atleast,636days,prognosiscannowbeconsideredverygood.Key words: vaccine, feline sarcoma, histopathology, giant cellsfibrohistiocytomaSpecies:feline

VA250. POTENTIAL VACCINE CANDIDATE AGAINST CANINE LEISHMANIASIS USING SAND FLy SALIVARy

GLAND ExTRACT AND LEISHMANIA ANTIGENSRoDoLFoC.GIUnCHETTI1,MARTADELAnA1,AnDRéIA

TEIxEIRA-CARVALHo2,oLInDoA.MARTInS-FILHo2,BRUnoM.RoATT1,RoDRIGoAGUIAR-SoARES1,JULIAnAVIToRIAno-SoUZA1,náDIAD.MoREIRA1,RAqUELT.ABREU1,WEnDELC.VITAL1,RoDRIGoCoRRêA-oLIVEIRA2,ALExAnDREB.REIS1

1UniversidadeFederaldeOuroPreto/NUPEB/LaboratóriodeImunopatologia;2FIOCRUZ/CentrodePesquisasRenéRachou/

LaboratóriodeImunologiaCelulareMoleculargiunchetti@nupeb.ufop.br

TheNewWorldsandfly,Lutzomyia longipalpis, isan importantvectorofLeishmaniachagasi,causalagentofthevisceralleishmani-asis(VL). In thesalivaofLutzomyia longipalpis,apyrase,anticoagu-lant,vasodilatoryandpotenttimmunomodulatoryactivitieshavebeenreportedevadinghostinflammatoryandimmuneresponses.Actually,salivary proteins represent good vaccine candidates to controlLeishmania infection. Furthermore, besides the current strategy forvaccinationagainstleishmaniasisisbasedontheuseofrecombinantantigens,wholeparasitevaccinesarestillattractive.Thus,consideringthedogsasthemostimportantdomesticreservoirsofL.chagasi,theunderstandingthecanineimmuneresponsetosandflysalivaryproteinsandLeishmaniaantigensmayhelpustodefinealternativevaccinationstrategiestocontrolcanineandhumanVL.Hereinweevaluatedtheimmunogenicityofsandflysalivaryglandextract(SGE)andSGEpluskilledvaccine(KVac;L.braziliensispromastigotes)indogs.Westudiedthechangesinthecellularprofileoftheperipheralbloodmononuclearcells,productionofclassandsubclassesofimmunoglobulinsandlev-elsofsericnitricoxide.OurresultsshowedthatimmunizationwithSGEplusKVacelicitedanincreaseincirculatingT-lymphocytes,particularlythesubpopulationCD8+thatwouldberelatedtothecontroloftissueparasitism,whencomparedtoplaceboandSGEgroups.Additionally,while the impairment in the levels for total IgGanti-LeishmaniawasobservedinSGEandKVacplusSGE,increaseintheIgG1/IgG2ratiowasrelatedinSGEplusKVacgroup.Furthermore,markedincreaseinsericnitricoxideproductionisthehallmarkofSGEplusKVacgroup,suggesting a Th1 immune response. Our data point out promisingresultsachievedwhenassociatedSGEplusKVac.Furtherinvestiga-tionsfocusingchallengewithLeishmaniachagasiarebeingperformedinordertoevaluatetheefficacyoftheseexperimentalgroups.

Supportedby:FAPEMIG,PAPES IVB,UFOP,FIOCRUZ,CNPqandCAPESKey words: dog, killed vaccine, sand fly salivary gland extract,LeishmaniaantigensSpecies:canine

iP251. ANTIVIRAL EFFECTS OF BOVINE IFN-τ AND HUMAN IFN-a ON BOVINE VIRAL DIARRHEA VIRUS

JUnKoKoHARA1*,yUMIKonISHIKURA2,MoToSHITAJIMA3,MISAoonUMA2,yUICHIyoKoMIZo3

1HokkaidoAnimalResearchCenter,Shintoku,Hokkaido081-0038,Japan;2GraduateSchoolofVeterinaryMedicine,HokkaidoUniversity,Sapporo060-0818,Japan;3NationalInstituteofAnimal

Health,3-1-5Kannnondai,Tsukuba,Ibaraki305-0856,JapanBovinediarrheavirus(BVDV)isdistributedworldwideandcauses

avarietyofdiseasesyndromeincattle.However,thereislittleinforma-tionaboutantiviraldrugthatisuseforthetreatmentofBVDVinfection.Inthisstudy,theantiviraleffectsofbovineinterferon-τ(boIFN-τ)andhumanIFN-a(huIFN-a)onBVDVwereexaminedin-vitroandin-vivo.In the in-vitro experiments, bovine fetal muscular (BFM) cells weretreatedwith recombinant boIFN-τ or recombinant huIFN-a for 24 hrandthenchallengedseparatelywithBVDVgenotype1or2ofeithercytopathicornon-cytopathicstrains.After4daysincubation,theviraltitersofall theBVDVstrains inBFM-IFNtreatedcellswerereducedcompared to theuntreatedcells.The replicationofBVDVwascom-pletelysuppressedataconcentrationhigherthan102U/mlforboIFN-τandhigherthan10U/mlforhuIFN-a.Inordertoexaminethein-vivoeffectsofboIFN-τandhuIFN-a,sixHolsteincowsthatwerepersistentlyinfectedwithnon-cytopathicgenotype1BVDVweretreatedwitheitherrecombinantboIFN-τnaturalhuIFN-aorrecombinanthuIFN-a.Inthefirst experiment, three cows were subcutaneously injected with 105U/kgbodyweightofboIFN-τ for tworoundsof5day-daily injectionswithin2weeks,whiletheotherthreecowswereinjectedwith106U/kgbodyweightofboIFN-τinthesamefrequency.ThemeanBVDVtitersintheserumofthepersistentlyinfectedcowstransientlydecreasedintheboIFN-τadministrationperiodcomparedtothepre-boIFN-τadmin-istrationperiodThesecondexperimentwasperformed3monthsafterthelastadministrationofboIFN-τinthefirstexperiment.Fourofthesixcowsweregivenorallywith1U/kgofnaturalhuIFN-adailyfor5days,whiletheother2cowswereinjectedintramuscularlywith100U/kgofrecombinanthuIFN-aInthisexperiment,theadministrationofhuIFN-adidnotreducethemeanBVDVtitersintheserumofthepersistentlyinfected cows. Neither physical abnormalities nor depression weredetected inall thecattleduring theconductof the twoexperiments.TheseresultssuggestthatboIFN-τsuppressestheBVDVtitersin-vitroandin-vivo,andthattheboIFN-τpossiblyreducesthedegreeofBVDVviremiaincattlewithoutanysideeffects.Key words: Bovine viral diarrhea virus; Cattle; Interferon-τ,Interferon-aSpecie:ruminants

iP252. HEMATOLOGICAL AND BLOOD SERUM PROTEINS EVALUATION OF CONFINED AND GRAZING

BOVINE’S AT THE TIME OF SLAUGHTERING. EDIAnEBSILVA*,MARIACLoRInDASFIoRAVAnTI,LILIAnABMEnEZES,MARInAPMIGUEL,AnAPAULAJKIPnIS,DIRSon

VIEIRAUniversidadeFederaldeGoiásedineveterinaria@hotmail.com

Cattlemanagedinfeedlotaregenerallyexposedtodifferentsitu-ations,suchas increasedconcentrationofanimals,dust,accumula-tion of dejections and excess manipulation.Therefore, it is possiblethatthosefactorsgeneratesuitableconditionsfortheonsetofseveralsanitaryproblems,evensubclinical,thatcouldreflectinthewhitebloodcell countandserumparametersof theanimalorganism.Thiswork

aimedtoevaluatethechangesinthewhitebloodcellcountandsomeserumproteinsofconfinedorgrazingcattle.Ofthe120bloodsamplescollected,60wereobtainedfromconfinedNeloremalebovinesand60fromanimalswiththesamecharacteristicsbutmanagedextensively.Thesampleswereobtainedatthemomentofslaughter.Theparam-etersstudiedwerethewhitebloodcellcount,serumalbumin/globulinratioandconcentrationofplasmafibrinogen.Intheanalysisofthedatadescriptivestatisticswasused,andtheaverages,standarddeviationandcoefficientofvariationcalculatedforallparametersevaluated.Thecomparisons between averagesweremade by non-parametric test.Thegrazingcattleshowedhigherlevelsofglobulinandfibrinogenwhencomparedtotheconfinedones(globulin:GC=3,299g/dL±0,76;CC=2,99g/dL±0,60andFibrinogen:GC=872mg/dL±610;CC=633mg/dL±319).Thetotalnumberofwhitebloodcells/mLwas7,640±2,150inconfinedbovinesand7,725±1,843ingrazingcattle.Therewasnosignificantdifferencebetweenthisvariableanddifferentialwhitebloodcellcountaswellasthetotalserumprotein(g/dL)fromgrazingcattle(6,10±0,53)andconfinedcattle (5,96±0,49).The levelofalbuminfromconfinedbovines(3,01g/dL±0,43)andtheA/Gratio(1,07±8,91)weregreaterwhencomparedtocomparedtothegrazingbovines(2,82g/dL±0,45)and(0,95±0,38)respectively.Thehigherserumlevelsofalbuminfoundinconfinedherdsuggestthattheyweresubjectedtoamoreadequatenutritionaldiet.TheconstantimmunologicalchallengesufferedbytheGCcouldberesponsiblefortheelevatedserumlevelsofglobulinandfibrinogen.Theseresultsshowedthatalthoughfeedlotspresentastressfulenvironmenttheydidnotshowanybloodalterationscorrelatedtothisfact.Key words:Bovine,Leukocytes,Nelore,PlasmaproteinsSpecies:ruminants

iP253. PLANT ExTRACTS INTERFERE WITH PERIPHERAL BLOOD MONONUCLEAR CELLS PROLIFERATION IN DAIRy

COWSnICoLALACETERA1,GIoRGInAKUZMInSKy1,HUAWU2,

ALESSAnDRonARDonE11DipartimentodiProduzioniAnimali,UniversitàdellaTuscia,Viterbo,

Italy;2DepartmentofAnimalHusbandry,AnimalHusbandryandVeterinaryMedicineCollege,Xining,China

nicgio@unitus.itValidationofbioactivecompoundsofplantoriginandtheirutiliza-

tioncancontributetoimproveanimalhealthandfoodsafety.Aseriesofinvitrostudieswascarriedouttoascertainwhetherfourdifferentplantextracts (PE) tested previously as immunomodulators in humans orlaboratoryanimalscanaffectspontaneousormitogendrivenprolifera-tioninbovineperipheralbloodmononuclearcells(PBMC).ThefourPEwereagiftfromIndenas.p.a.(Settala,Milan,Italy).ThreeoutoffourPEtestedarenotpatentedyet,andwillbeidentifiedasPE1,PE2andPE3.ThefourthPEwillbeidentifiedasPE4andwasrepresentedbyapurifieddryextractfromEchinaceaangustifolia(PolinaceaTM,Indenas.p.a.).PBMCwereobtained from6earlypregnantHolsteinheifers.EachPEwasaddedtoculturemediaatconcentrationsof180,60,20or6.3µg/mlwithorwithoutconcanavalinAorpokeweedmitogen.PBMCproliferationwasassessedbymeasuringthe5-bromo-2'-deoxyuridineincorporatedduringDNAsynthesis.PE1,2and3didnotaffectPBMCproliferationintheabsenceofmitogens.Conversely,allconcentrationsofPE4testedexertedasignificantmitogeniceffect.Sucheffectwasdosedependent,andwith respect toPBMCcultured in theabsenceofPE4,thehighestandthelowest increaseinproliferativeresponsewereobservedwhenPE4wasaddedtoculturesplatesat180(10foldincrease)or6.3(2foldincrease)µg/ml,respectively.Bothintermsofopticaldensity(OD)andstimulationindex(SI;ratioofODforstimulated

10. CLINICAL IMMUNOLOGy AND IMMUNOPATHOLOGy: POSTERS IP251-IP281

cellstotheODforunstimulatedcells)thefirst2concentrationsofPE1,thefirst3concentrationsofPE2,andonlythefirstconcentrationofPE3exertedstrongnegativeeffectsonproliferationofmitogen-stimulatedPBMC. When PE4 was added to culture plates in combination withmitogens,resultsofproliferativeresponseofPBMCdiffereddependingonwhetherdatawereexpressedasSI(inhibitionornoeffect)orOD(stimulationor noeffect).Results reportedherein indicate that plantextractscanactaspotentmodulatorsof lymphocytesbiologyalsoinbovinespecies,andsuggestthatfurtherresearchshouldbecarriedouttodefinedosagestobeadministeredandclinicalconditionsthatmaybenefitfromtheirutilization.Key words:Plantextracts,Bovine,Lymphocytes,ProliferationSpecies:ruminants

iP254. COMPARISON OF TWO CHROMOGENS IN THE DEVELOPMENT OF AN IMMUNOENZIMATIC ASSAy (ELISA)

FOR DETECTION OF IMMUNOGLOBULINS SPECIFIC AGAINST CORyNEBACTERIUM PSEUDOTUBERCULOSIS.

DAnIELLEDLIMA,MIRIAMFREBoUçAS,RICARDoWPoRTELA,VERAVALE,AnDRéAPACHECo,LILIAMoURA-CoSTA,AnAPGoMES,MARCoSCoSTA-SILVA,MIGUELBARRETo,RoBSon

BAHIA,REnAToCARMInATI,RoBERTSCHAER,IVAnAnASCIMEnTo,MARILIAMARqUES,SonGELIMVFREIRE,

RoBERToMEyERLaboratóriodeImunologiadoIntitutodeCiênciasdaSaúde(ICS)da

UniversidadeFederaldaBahiaCorynebacterium pseudotuberculosis is the etiologic agent of

CaseousLymphadenitis,adisease thataffectsgoatandsheep,andcausessignificativeeconomiclosses,duetotheanimalsmortalityandreductionofweightgainandmilkproduction.Thisstudywasconductedwith the objective to develop an immunoenzimatic assay, with highspecificity and sensibility, to detect specific immunoglobulins in theseraof infectedanimals,andmorespecifically,tostabilishthebetterchromogentobeusedinourELISA.Weemployedseraof39animalsthatpresentedconfirmedclinicaldiagnosticofcaseouslymphadenitis,and39samplesfromanimalswithnoclinicalsymptomsandbreedinnon-endemicareas.TheantigenemployedintheassaywasobtainedfromthesupernatantcultureofC.pseudotuberculosis.Serawasdiluted1:100andincubatedforonehour,andaanti-goattotalIgwasusedassecondaryantibody.Thetwochromogenstestedinassayswereortho-phenyl-diamine(OPD)andtetramethyl-benzidine(TMB).IntheassaywhereOPDwasusedas chromogen,weobserved, using theROCcurveasastatisticaltesttodeterminethecut-offopticaldensitypoint,that we had 93% of sensibility and 100% of specificity.WhenTMBwasaddedtoreactwithsecondaryantibodyhorseradishperoxidase,weobtainedalevelofsensibilityandspecificityof82,1%and100%,respectively,employingthesamestatisticaltest. ItcanbeconcludedthattheELISAwhereOPDwasusedaschromogenpresentedbetterresults,sinceithadahighersensibility,thereforeitisabletodistinguishpositiveandnegativeanimalswithmoresafety.Key words: Corynebacterium pseudotuberculosis, caseouslymphadenitis,ELISASpecies:ruminants

iP255. DEVELOPMENT OF AN IMMUNOENZIMATIC ASSAy FOR DETECTION OF IMMUNOGLOBULINS SPECIFIC

AGAINST CORyNEBACTERIUM PSEUDOTUBERCULOSIS - DETERMINATION OF A CUT-OFF POINT EMPLOyING

DIFFERENT STATISTICAL METHODOLOGIES.MIRIAMFREBoUçAS,DAnIELLEDLIMA,RICARDoWPoRTELA,

RoBSonBAHIA,REnAToCARMInATI,LILIAMoURA-CoSTA,VERAVALE,RoBERTSCHAER,SonGELIMVFREIRE,RoBERTo

MEyERCaseouslymphadenitisisaninfectiousdiseasethataffectsgoat

and sheep, caused by Corynebacterium pseudotuberculosis, beencharacterized by granulomes in superficial lymph nodes and in thelung.ItisanendemicdiseaseinthenortheastofBrazil,beingrespon-sibleforsignificativeeconomiclosses,duetothemortalityofanimalsandreductionofweightgainandmilkproduction.Sincenow,itisnotwellestablishedaimmunodiagnostictesttoidentifytheseropositivity

statusofanimals.Inthiswork,wedevelopedanenzymaticimmunoas-saytest(ELISA),employingC.pseudotuberculosisculturesupernatantasantigen.Thirty-nineserafromgoatswithpositiveclinicaldiagnosticforcaseouslymphadenitisandthirty-nineserafromnegativeanimals,provenient from non-endemic areas and with no clinical symptoms,were tested. The optical density (OD) mean obtained at 450nm ofabsorbancewas0,647 forpositiveanimals,and0,181 for thenega-tivesones.WhenutilizingtheROCcurvetoestablishthecut-offpoint,weobtainedacut-offvalueof0,36,with82,1%ofsensibilityand100ofspecificity,andwiththeFreymethodologywehadacut-offvalueof0,351,showing98,7ofspecificityand92,8%ofsensibility.Consideringthese data, it can be affirmed that theELISA test developed in thisprojectwasabletodistinguishinfectedandnon-infectedanimals,andthatthemethodologyofFreywasthebestonetodeterminethecut-offpointofthetest.Key words: Corynebacterium pseudotuberculosis; enzymaticimmunoassaytest;FreymethodologySpecies:ruminants

iP256. SEROPREVALENCE AND RISK FACTORS FOR BOVINE LEUKEMIA VIRUS INFECTION IN CATTLE OF GOIáS,

BRAZIL.JGAMoRIL1,SISAMARA2,HDTARABLA3,MFCAMARGoS4,

CCVFURTADo51MAPA/LANAGRO/GO,Brazil;2FCAV/UNESP/JABOTICABAL,Brazil;3INTA&UniversidadNacionaldelLitoral,Argentine;4,5

MAPA/LANAGRO/MG,Braziljosegabriel@agricultura.gov.br

Cattleinfectionbybovineleukemiavirus(BLV)isoneoftheclini-calpatternsofenzooticbovineleukosis.Itisworldwidedistributedandleads to commercial restrictions for BLV seropositive animals. FromMay to September, 2004, a cross sectional study was performed todeterminetheprevalenceandtoexploreforriskfactorsforBLVinfec-tionintheStateofGoiás,centralareaofBrazil.Thestudywascarriedoutusingasimple randomsampleof1,229bovines from42herds.Allbovineswereslaughteredat localabattoirs,whereasinglebloodsample was taken from each animal. These samples were furtherscreennedforBLVantibodies(BLVAbs.)byanimmunodiffusiongelagar(IDGA)testdevelopedbyTECPAR(Paraná,Brazil).Theunitofstudywastheindividualanimal.VariablesunderstudyincludedIDGAresults,herdlocation,sex,age,andbreed.Allvariableswerecategori-calandwerecomparedbyχ2andoddsratios(OR),95%confidenceinterval (CI). Logistic regression (LR) was performed using IDGApositivityas thedependentvariable,andanimalcharacteristicswereofferedasindependentvariables.Referencegroupsforeachriskfactorwere:centralpartofGoiás(herdlocation),male(sex),3-4years(age),Nelore(breed).BLVAbs.weredetectedin94(7.6%)animals.Allinde-pendentvariableswerestatisticallyassociatedamongthem(p<0.05).LRresultsshowedthattheriskofBLVinfectionwashigherin:(i)ani-malscomingfromherdslocatedattheSouthofGoiás,closetoMinasGeraisborderOR=9.76(CI2.41;39.56),(ii)bovines2-3yearsoldOR=3.00(CI1.51;5.95),and(iii)HolsteinOR=4.4(CI1.02;18.92).Mestiçocattle(inourstudy:mixedbreedholdingBosindicusphenotype)wasfoundtohavealowerrisk,whilesexwasnotsignificantlyassociatedtoIDGAresults.Asinanycrosssectionalstudy,nocausalrelationshipcanbeimplied,andothermanagementfactorsnottakenintoaccountinthisstudymaybeinvolvedeitherasriskfactorsorasconfoundingvariables.HoweverastrongassociationwasfoundbetweenBLVinfec-tion,region,ageandbreed.Key words:BLV,IDGA,prevalence,riskSpecie:ruminants

iP257. VACCINATION WITH TyPE III SECRETION PROTEINS REDUCES E. CoLI O157:H7 SHEDDING AND

CONTAMINATION IN CATTLERoGAnDR1,SMITHDR2,MoxLEyRA3,PoTTERAA4,STRAUSS

CE11BionicheLifeSciencesInc.,Belleville,Ontario,CanadaK8N1E2;2DepartmentofVeterinaryandBiomedicalSciences,UniversityofNebraska–Lincoln,Lincoln,Nebraska,68538-0905USA;

138

3DepartmentofAnimalScience,UniversityofNebraska–Lincoln,Lincoln,Nebraska,68538-0905USA;4VaccineandInfectious

DiseaseOrganization,UniversityofSaskatchewan,Saskatoon,SK,CanadaS7N5E3

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is animportant human pathogen, causing severe diarrhea and hemolyticuremicsyndrome(HUS).InNorthAmerica,EHECserotypeO157:H7ismostprevalentandaccountsfor85-95%ofHUScases.CattlearetheprimaryreservoirofE.coliO157:H7andassuch,serveasamajorsourceofinfectioninhumans,eitherthroughdirectcontactorthecon-sumptionofcontaminatedmeat.

E. coli O157:H7 infection of cattle requires type III secretedproteins (TTSP) which enable the bacteria to colonize the intestinalmucosa. These EHEC TTSPs include proteins such as Tir, EspA,EspB,Intimin,inadditiontootherproteinsthatareencodedbygeneswithin the locus of enterocyte effacement (LEE) operon as well asthose that are not encoded by LEE, including NleB. Both LEE andnon-LEEencodedTTSPproteinsarerequiredfor intestinalcoloniza-tionofcattle,makingthemprimecandidatesforvaccinedevelopment.AnefficaciousvaccinecouldpreventE.coliO157:H7colonizationofcattleandfecalsheddingintotheenvironment,andultimatelyreducecarcasscontaminationwithinslaughterfacilities.

TheefficacyofBionicheAnimalHealth’sE.coliO157:H7TTSPvaccinewasevaluatedincontrolledchallengesettingsaswellasnatu-ralexposurefieldstudies.Controlledchallengestudiesdemonstratedthatvaccinationresultedina36.5%reductioninthenumberofdaysthebacteriumwasshedinthefeces(P<0.05),and2.28log10reduc-tion in thenumberofbacteriabeingshed in thefeces;resulting inavaccineefficacy(preventedfraction)of63.9%.Fieldevaluationundernaturalexposuredemonstratedthatvaccinatedcattlewere98.3%lesslikelytobecolonizedbyE.coliO157:H7attheterminalrectummucosa(TRM)(oddsratio=0.014,p<0.0001).Asecondnaturalexposurefieldevaluationconductedwith20,556headdemonstrated thatonapenbasis,vaccinatedcattlehada75%lowerprobabilityofbeingcolonizedbyE.coliO157:H7(OR=0.2,p=0.03)andthatpenswerelesslikelytotestROPES-positive(OR=0.59,p=0.0004)indicatingthatvaccina-tiondecreasesenvironmentalpen-levelprevalenceofE.coliO157:H7.It isbelievedthatE.coliO157:H7contaminationofcarcasseswithinslaughter facilities comes predominantly from hides as opposed tointestinalwaste.Cattlevaccinatedthroughoutaregionofthefeedyardwere58%lesslikelytobehaveE.coliO157:H7contaminatedhidesthananimalsinaregiontreatedwithplacebo(p=0.01).

TheseresultsindicatethatvaccinationwithE.coliO157:H7TTSPhasthepotentialtobeaneffectivepre-slaughterinterventionmethodforthereductionofE.coliO157:H7contaminationoffecesandhidesofcattle.Key words:EHEC,O157:H7,E.colivaccine,ImmunitySpecies:ruminants

iP258. LACTOGENIC IMMUNITy: MODULATION OF IMMUNE RESPONSE AND PROTECTION AGAINST RV DIARRHEA IN A

CALF MODELGISELAMARCoPPIDo1,LoREnAGARAICoECHEA1,

DAnIELARoDRIGUEZ1,MARInAMoSGoVoJ1,FERnAnDoFERnAnDEZ1,MARÍAMVEnA2,VIVIAnAPARREño1

1InstitutodeVirología-CICVyA-INTACastelar,2Biogenesis-Bagó.gmarcoppido@cnia.inta.gov.ar

Bovinerotavirus(BRV) isamajorcauseofdiarrhea inneonatalcalvesworldwide.Itispostulatedthatcontinuoslevelsofpassiveandactiveantibodies(Abs)intheintestinallumenprovideefficientprotec-tionagainstinfectionandillness.PassiveprotectionagainstRVandthemodulationoftheimmuneresponseincalvesfedsupplemented-milkwithanti-BRVAbsderivedfromimmunecolostrumwasevaluated.

Newborncalves(n=31)weredividedin4experimentalgroups(G):G1=colostrum-deprived;G2=receivingcolostrumandfedmilkwithoutAbs;G3andG4=receivingcolostrumandfedsupplementedmilkwith2concentrationsofcolostrumAbs(IgG1=1024and4096).Milksupplementationwasextendedfor14days.

The calves were orally inoculated with IND (P[5]G6) BRV at 2daysofage(0post-inoculationday(dpi)andre-challengedat21dpi.

Allcalvesofcontrolgroups(G1andG2)and50%oftheanimalsfromG3andG4wereeuthanizedat21dpi.Therestof theanimalswereeuthanized at 35 dpi.Anti-BRVAbs titers in serum were evaluatedweeklyand fecalAbsweredailyanalyzedby isotype-specificELISAandvirusneutralization.

The number of BRV-specific antibody secreting cells (ASC) inintestinalandsystemiclymphoidtissuewasevaluatedbyELISPOT.

After BRV inoculation, all control calves became infected andsufferedseverediarrhea.InG3,allthecalveswereinfected,butonly62,5%(5/8)developeddiarrhea.InG4,40%(4/10)ofthecalveswerefully protected, while asymptomatic infection was observed in other4 animals and 20% (2/10) of the calves developed diarrhea.Thesecalvesshowedasignificantdelay in theonsetofdiarrheaandvirusshedding,bothofshorterduration,comparedtocontrolgroups.

Fullprotectionagainstchallenge(21dpi)wasobserved inallofthecalvesfromG3(4/4)andin4animals(4/5)fromG4.

Adecreasingmagnitudeof the IgMand IgAmediatedanti-BRVimmuneresponseinserumandfecesinmilk-supplementedgroupswasobserved,astheamountofcolostrumaddedtothemilkincreased.

The number ofAnti-BRVASCwas lower in systemic lymphoidtissueand tended todissappear inG4.However, in themilk-supple-mented groups, a large number of IgA and IgG1 anti-BRVAS wasdetectedalongthemucosallymphoidtissue,mainlyinDuodenumandYeyunum.CalffeedingwithBRV-immunemilkduringthefirsttwoweeksoflifeisanefficientstrategytoreduceBRVinfectionwithoutinterferinginthedevelopmentoftheneonatalmucosalimmuneresponse.Key words:Colostrum,CalfDiarrhea,CalfModel,RotavirusSpecies:ruminants

iP259. DEVELOPMENT AND EVALUATION OF A MONOCLONAL ANTIBODy AGAINST MyCOBACTERIUM

AVIUM SUBSP. PARATUBERCULOSISAnASTEMPLER,AnAJoLLy,SILVIACoLAVECCHIA,

MARInASoBA,ADRIAnA,FonTAnALS,SILVIAMUnDoAreadeInmunología.FacultaddeCienciasVeterinarias,

UniversidadedeBuenosAires.ArgentinaParatuberculosis is characterized by chronic granulomatous

enteritisof ruminantsworldwide.Mycobacteriumaviumsubsp.para-tuberculosis (Map) is the etiological agent of this disease. Differentindirectdiagnosticproceduresdetectingimmunoglobulintitersorcel-lular immune responses of infected cattle are accessible, but directcharacterizationofMapinbiopsymaterialsorinfectedmanureispres-entlyunavailable.

Theaimofthepresentworkwastodevelopmonoclonalantibod-iesforthedetectionofMapininfectedmaterialtofacilitatediagnosisandthestudyoftherelationshipbetweenMapandtargetcells.

BALB/cmicewere immunized intraperitoneallywith threedosesof100µgofMapwholebacteriainFreund`sincompleteadjuvant,fol-lowedbyaboosterinjectionofproteinp34-cx(arecombinantpolypep-tidecorresponding toaspecificmembraneproteinofMapcloned inprSET-AandexpressedinE.coliBL21pLys).SpleencellswerefusedwithmyelomacelllineSP2/0.HybridomasupernatantswereassayedagainstMap,p34-cxandE.colibyELISAtests.Positivehybridomasreacting specifically with Map and p34-cx but not with E. coli wereselectedandisotypedbyacommercialmouseimmunoglobulinELISAkit(BDPharmingen).OnehybridomareactingsolelywithMapandp34-cx was isolated, subcloned and named as 1A6E10.This hybridomaproducedanti-MapIgG3kappa.Then,hybridomacellswereusedtoinoculatemiceintraperitoneally.AsciticfluidwaspurifiedbyammoniumsulfateprecipitationandevaluatedbyELISAagainstMapandothermycobacteriastrains.Resultsareshownintable1.

Mycobacteria Optical DensityM.aviumparatuberculosis 1.384

M.phlei 0.225M.bovis 0.176

M.aviumavium 0.096FlowCytometryandImmunofluorescence(IF)wereusedtoevalu-

atetheattachmentofthemonoclonalantibodytothebacterialsurface,

139

whileIFwasalsousedtodetecttheinfectionofbovinemacrophagecell lineswithMap,usingaFICTlabeledantimouseIgs(H+L)anti-body. By Flow Cytometry, we could show that 1A6E10 recognizedMap(58,89%versus18,14%obtainedwithan irrelevantasciticfluidin10.000events/sample).TheseresultswereconfirmedbyIFIusingMapwholebacteria.Wecouldalsodetectdifferencesinabovinemac-rophagecell linebetweenMap infectedandnon-infectedcellsusing1A6E10.

Insummary,theresultsshowedinthisworksupportthecontinuityoftheevaluationofmonoclonalantibody1A6E10infieldsamplesfrominfectedcattleandinthestudyoftheinteractionofMapwiththetargetcell.Key words:Paratuberculosis,monoclonalantibody,diagnosisSpecies:ruminants

iP260. DEVELOPMENT OF AN ELISA TO MEASURE SOLUBLE CD163 IN PORCINE BIOLOGICAL FLUIDS

CARLoSPéREZ,PALoMAMARTÍnEZDELARIVA,MERCEDESGARCÍA-BRIonES,nURIAGóMEZ,BELénALVAREZ,AnGELEZqUERRA,ConCEPCIónREVILLA,JAVIERDoMÍnGUEZ,

FERnAnDoALonSoDptodeBiotecnología,INIA,Madrid,Spain.

falonso@inia.esCD163isamemberofthefamilyofreceptorswithscavengercys-

teine–richdomains,whoseexpressionisrestrictedtomonocytesandmacrophages.Itfunctionsasascavengerreceptorforhemoglobin/hap-toglobincomplexesandithasalsobeenimplicatedintheregulationofinflammatoryprocesses.Stimulationofmonocytes/macrophageswithphorbol esters, LPSor cross-linkingofFcgamma receptors causessheddingofCD163fromcellsurfacebyaprotease-dependentmecha-nism.The level of solubleCD163 (sCD163) hasbeen consideredausefulmarkerofmacrophageactivation.WehavecharacterizedfournewmAbsagainst porcineCD163.The specificity of theseantibod-ieshasbeenconfirmedbytheirreactivitywithCHOcellstransfectedwithpCD163.UsingtwoofthesemAbs,thatbindtodifferentepitopesonCD163molecule,wehavedevelopedasandwichenzyme-linkedimmunoassay (ELISA) tomeasure levelsofsCD163 inporcineseraandbiologicalfluids.TheassaywascalibratedusinglysatesofCD163transfectants.Itshowedasensitivityof105cells/ml,allowingtodetectsCD163 inseraand inculturesupernatantsofactivatedmonocytes.Thisassaycanbeausefulmethodofmonitoringthedegreeofactiva-tionofmacrophages in a variety of inflammatoryand infectiousdis-easesofswine.Key words: CD163, Monocytes/macrophages, Immunoassay,InflammationSpecies:swine

iP261. PROTECTIVE EFFICACy OF SOWS WITH A RECOMBINANT PSEUDORABIES VIRUS ExPRESSING THE

GP5 OF PORCINE REPRODUCTIVE AND RESPIRATORy SyNDROME VIRUS

GUAnG-ZHITonG,ZHI-JUnTIAn,yAn-JUnZHoU,HUA-JIqIUDivisionofSwineInfectiousDiseases,NationalKeyLaboratoryof

VeterinaryBiotechnology,HarbinVeterinaryResearchInstitute,CAASNo.427MaduanStreet,Harbin150001,P.R.China

Abstract: The purpose of this study was to assess the safetyand protective efficacy of a recombinant pseudorabies virus(PRV)expressingtheGP5ofporcinereproductiveandrespiratorysyndrome(PRRS)virus(rPRV-GP5)againstPRRSinareproductivepigmodel.Six PRV- and PRRS virus-free multiparous sows were randomlydividedintothreegroups,eachsowwasinoculatedwith2×108.0PFUofrPRV-GP5byintramuscularlyroutesforsafetytestatearly,middleand lategestation, respectively.ThirteenPRV-andPRRSvirus-freemultiparoussowswere randomlydivided into twogroups,eachsowofgroupA(n=9)was immunizedwith2×107.0PFUof rPRV-GP5byintramuscularlyroutesbeforegestation;groupB(n=4)wasnon-vac-cinatedasnegativecontrols.SowsofgroupsAandBwerechallengedbyintranasalinoculationwith106.0TCID50ofPRRSvirusstrainCh-1aat80-95daysofpregnancy.The results indicated that rPRV-GP5 issafeinpregnantsows,thedetectionrateofchallengevirusfromserum

ofpigletsingroupBwas56.1%whichissignificantlyhighercomparedto24.1%ingroupA.Inspiteofnodetectableanti-PRRSvirusantibodybeforechallengeingroupA,thevaccinatedsowsshowedsignificantlyimprovedhealthyfarrowingrate(82.1versus49.1%).ItwasconcludedthattherPRV-GP5issafeandeffectiveincontrollingthereproductivefailureininducedbythevirulentvirusofPRRSchallenge.Key words:recombinantPRV,PRRSV,GP5Specie:swine

iP262. MONOSPECIFIC POLyCLONAL ANTIBODIES FOR IMMUNODETECTION OF MyCOPLASMA HyOPNEUMONIAE

IN SWINE PNEUMONIC LUNGS.LUIZAAMARALDECASTRo1,2*,DESIREéSCHUCK1,nELSon

MoRES3,ARnALDoZAHA1,HEnRIqUEBUnSELMEyERFERREIRA1,DAVIDDRIEMEIER2

1CentrodeBiotecnologia,UFRGS,PortoAlegre,RS;2SetordePatologiaVeterinária,FaculdadedeVeterinária,UFRGS,PortoAlegre,RS;3EmbrapaSuínoseAves/CNPSA,Concórdia,SC.

luiza@cbiot.ufrgs.brMycoplasma hyopneumoniae is the etiological agent of swine

mycoplasmalpneumoniain(SMP),beingthemainpathogenfoundintheswinerespiratorytractandcausingimportanteconomiclosses.Thecurrently used immunodiagnostic methodsbased in seraagainstM.hyopneumoniaewholeproteinextractspresent important limitations,both in sensitivity and specificity. The cloning and expression ofM.hyopneumoniaegenesallowedtheproductionofrecombinantproteins,among which those corresponding to lactate dehydrogenase (p36),ribonucleotidereductaseR2subunit(NrdF),andthechaperoneHsp70,allofthemwithwellestablishedantigeniccharacterand/orrelevanceforvaccination.Inthiswork,wehaveproducedpolyclonalantibodiesin rabbits against these three recombinant proteins. Each producedserawasinitiallyevaluatedbyimmunoblotandELISAagainstthecog-naterecombinantprotein,aswellasagainstproteinextractsofothersporcineMycoplasmaspeciesandofM.hyopneumoniae,forspecific-ity assessment and confirmationof expressionof the correspondingnativeantigensinvivo,respectively.Theanti-p36andanti-Hsp70serapresentedstrongandspecificreactionsagainstreferenceM.hyoneu-moniaestrains(7448,7422,JandPMS).Theanti-NrdFserum,ontheotherhand,presentedcross-reactivitywithotherporcineMycoplasmaspecies,includingMycoplasmaflocculare,MycoplasmahyorhinisandMycoplasmahyosynoviae.Theanti-p36andanti-NrdFserawereusedin thestandardizationonan immunohistochemicalassayforM.hyo-pneumoniaedetectioninlungs,usingsectionsfromswineswithSMPorSPFanimalswasperformedonlyforanti-p36andanti-NrdFserum.Thestainingobtainedwithbothseraconfirmedtheexpressionofp36andNrdFantigensbyM.hyopneumoniaeininfectionconditions.Theobserved immunoreactionwas localized closely to epithelial cells ofbronchiolesandbronchi,aswellasindebrisofepithelialcells intheexudatepresentinthelumenofbronchoalveolarducts.Theobservedstainingpatternswereessentiallysimilartothatobtainedwiththestan-dardpolyclonalantiserumagainstM.hyopneumoniaeextract,currentlyusedforimmunodiagnosis.Theanti-p36serum,however,presented,asanadvantage,alowerbackgroundincomparisontothoseobtainedwith theanti-NrdFand thepositivecontrolsera.Our resultssuggestthat immunohistochemistry methods using monospecific polyclonalantibodiesmayimprovetheefficiencyofthecurrentlyusedimmunohis-tochemistryassaysforM.hyopneumoniaedetection, increasingbothsensitivityandspecificity.Key words: swine mycoplasmal pneumonia, immunohistochemicalassay, Mycoplasma hyopneumoniae, Monospecific polyclonalantibodiesSpecies:swine

iP263. THE IMMUNE RESPONSES OF THE TASMANIAN DEVIL (SARCoPHILUS HARRISII) AND THE DEVIL FACIAL

TUMOUR DISEASEALExAnDREKREISS,GREGoRyMWooDS

MenziesInstitute,DepartmentofPathology,UniversityofTasmaniaTheTasmaniandevil(Sarcophilusharrisii)issufferingfromanewly

describeddisease,knownasDevilFacialTumourDisease(DFTD).The

140

diseaseisunusualastumourcellsaretransferreddirectlybetweenani-mals[1].Althoughtheinfectiouscanceristransmittedasan“allograft”,thereisnosignofrejectionofthetumour[2],whichraisesquestionsabout the devil’s immune system.To determinewhetherTasmaniandevils had a competent immune system,we performed in vitro andinvivo immunefunctionalassays.Wealsoanalysedthestructureofthe lymphoid tissuesof healthyandDFTD-bearingdevils. Lymphoidtissues (lymph nodes, spleen and bone marrow) from healthy andDFTDbearingdevilswereformalinfixedandhaematoxylinandeosinstained. IntactTcellareaswere identified in the lymphoid tissuesofallanimalsexamined.HyperactiveBcellareaswereseeninalldevils(healthyandDFTDbearingdevils)andareasofplasmacellprolifera-tionwerecommon,althoughmorepronouncedinDFTDbearingdevils.Lymphocyteproliferationassayswereperformedwiththreemitogens(concanavalinA,phytohaemagglutininandpokeweedmitogen).SerialdilutionsofmitogenswereincubatedwithfreshlyisolatedlymphocytesfromTasmaniandevilsfor96hours.Radioactivethymidineincorpora-tionwasusedasameasureofproliferationandresultsexpressedasstimulationindices.Mostanimalsrespondedtoallmitogens,althoughlarge variation in the responses was identified. To evaluate in vivoresponses,devilswereimmunisedeithersubcutaneouslyorintraperi-toneallywithhorseredbloodcells(HRBC).ThesubcutaneousrouteprovidedamuchstrongerresponsethantheintraperitonealrouteandbothprovidedevidenceforstrongIgMandIgGlikeactivity.Wecon-cludethatTasmaniandevilshaveafunctionalinvitroTcellresponseandinvitroandinvivoBcellresponse.Therefore,thelackofimmuneresponseagainstDFTDisunlikelytobecausedbygeneralisedimmu-nosuppressionintheTasmaniandevilpopulation.

References:[1] Pearse,A.M., Swift, K., 2006. Transmission of devil facial-

tumourdisease.Nature439,549.[2]Loh,R.etal.2006.Thepathologyofdevil facial tumourdis-

ease (DFTD) in Tasmanian devils (Sarcophilus harrisii). Vet Pathol.43:896-903.Key words:Tasmaniandevil,tumour,transmissible,mitogenSpecie:other(Tasmaniandevil(Sarcophilusharrisii))

iP264. THE POSSIBLE TOxIC EFFECTS OF MONOCROTALINE ON RAT HUMORAL IMMUNE RESPONSE

ISISMHUEZA1,LEonILAERRASPAnTInI1,PAULoCESARFRASPAnTInI1,MITSUEHARAGUCHI2,JúLIACBEnASSI1,

SILVAnALGóRnIAK11ResearchCenterofVeterinaryToxicology(CEPTOX),Schoolof

VeterinaryMedicineandAnimalSciencesofUniversityofSãoPaulo,S.P.,Brazil;2BiologicalInstituteofSãoPaulo,S.P.,Brazil.

hueza@usp.brThe phytotoxin monocrotaline (MCT) is a pyrrolizidine alkaloid

found in a variety of plant species throughout theworld, and it is atoxicological concern for livestock and man. Previous studies con-ductedinmicetreatedwithhighdosesofMCT(75–100mg/kgfor14days)showedimmunosuppressiveeffectsonbothhumoralandcellularimmunity.However,itiswellestablishedthattheratisthebestanimalmodeltoevaluateimmunotoxicity.Moreover,ratsaremoresusceptibletoMCTtoxicitythanaremice.Noreportswerefoundintheliteratureshowingtheeffectsof lowdosesofMCTontherat immunesystem.Thus,theaimofthepresentstudywastoevaluatesomeparametersofhumoralimmuneresponseinratsexposedtodosesofMCTthatdonotproducehistopathologicallesions.Fortymaleratsweredividedinto4groupswhichreceived0.0,0.3,1.0and3.0mg/kgofMCT,orally,for14days.Onday14theanimalsweresensitizedbyintravenousinjectionof2.0x109sheepredbloodcells(SRBC).Sevendayslaterallanimalswereanesthetizedforbloodcollection.Theseparatedserumwasfro-zenuntilusedforanti-SRBCtiters.Theanimalsweretheneuthanatizedand lymphoidorgans (thymusandspleen)wereharvested toevalu-atetheirrelativeweight.Singlecellsuspensionsofsplenocyteswereprepared for theplaque-formingcell (PFC)assay.Briefly, theproperamountofsplenocytesuspensions,SRBC,andguineapigcomplementwereaddedintoamicrotube(1.5mL)toachieveafinalconcentrationof 2.0 x 106 splenocytes/mL/7%SRBC, and 10%complement. FiftyμLofeachsuspensionwasplacedinasealedCunninghamchamberandincubatedinahumidatmosphereat37°Cfor3h.Thenumbers

oflysedplaquesproducedby1x105splenocyteswerecountedunderalightmicroscope.Noalterationsinrelativebodyweightoflymphoidorganswereobserved(P>0.05).Nodifferences(P>0.05)betweencontrolandexperimentalgroupsweredetected forhumoral immuneresponsesevaluatedby theanti-SRBCtiteror thePFCassay.Eventhough no changes in rat humoral immune responses were foundin this study,MCTmay have undetected immunotoxic effects.Thusexperimentsarebeingconductedinourlaboratorytoevaluatethepos-sibletoxiceffectsoflowdosesofMCToninnateandcellularimmuneresponsesinrats(FAPESP06/57174-7).Key words:Humoral immuneresponse,Poisonousplant,Crotalariaspp.,MonocrotalineSpecies:other(Rat)

iP265. MULTI-SPECIES TB TEST BASED ON THE INNOVATIVE DPPTM TECHNOLOGy

KonSTAnTInLyA1ChembioDiagnosticSystems,Inc.,Medford,NY,USA

klyashchenko@chembio.comTuberculosis(TB)remainsaseriousdiseaseinlivestockandwildlife

animals,asnumeroushostspeciesaresusceptibletoMycobacteriumbovisand/orMycobacteriumtuberculosis.Inrecentyears,TBhasbeenincreasinglyrecognizedasanemergingdiseaseofmultiplezoospe-cies.Formanyofthose,thetraditionalTBtestingtechnologies,suchasthetuberculinskintest,havenotbeenfullyvalidated.Blood-basedTBassaysareappealingforuseinTBsurveillanceofcaptivewildlife(e.g.,zoos,gamefarms)andnon-traditionallivestockastheyrequireasinglehandlingevent,therebyminimizingcapture-associatedinjuries.Serologicalassaysrepresentausefulapproach,asthesemethodsaresimple, rapid, inexpensive, andmay bemore sensitive and specificthanthecurrenttests.ChembioDiagnosticSystems,Inc.hasrecentlydevelopedDualPathPlatform(DPPTM)technologyforantibodyoranti-gendetection.Withanadditionalmembranestripforsampledelivery,the DPPTM assay has several advantages over conventional lateral-flowtests.Thisinnovativeapproachwasusedtodeveloparapidsero-diagnostictestforTBinmultiplehostspeciesincludingcattle,cervids,elephants, and otherwildlife and captive animals.The test can useserum,plasma,orwholebloodsamples.Semi-quantitativeresultscanbeobtainedwithin15minuteseithervisuallyor,ifnecessary,withanopticalreader.EvaluationoftheDPPTMteston23elephantswithTBconfirmed by positive culture and 131 negative controls has shown100% diagnostic accuracy. Studies with limited numbers of serumsamplesfromconfirmedTBcasesinblackrhinoceros,Malayantapirs,jaguars,andsealionshavefurtherdemonstratedthefeasibilityofuni-versaluseofthisTBtestingtechnology.TheproposedDPPTMimmu-noassayshouldprovidearapidandhighlyaccuratediagnostictoolfortheimprovedcontrolofTBinlivestock,wildlife,andzoosettingsthatinvolvemultiplehostspecies.Key words:Tuberculosis,Diagnosis,Immunoassay,AntibodySpecies:other

iP266. ANTIGEN CAPTURE SANDWICH ELISA FOR THE DIAGNOSIS OF LEPTOSPIROSIS

FLáVIAALEIxoVASConCELLoS1,MARIAnALonERCoUTInHo1,GABRIELAHäDRICH2,LEonARDoGARCIA

MonTE3,núBIASEyFFERT1,CLáUDIAPInHoHARTLEBEnFERnAnDES1,JoSéAnTonIoGUIMARÃESALEIxo1

1CentrodeBiotecnologia-UniversidadeFederaldePelotas;2InstitutodeBiologia-UniversidadeFederaldePelotas;3InstitutodeBiologia

-UniversidadeFederaldePelotasLeptospirosisisanimportantzoonosisthatoccursinendemicform

around theworld.The infection is caused by pathogenic Leptospiraspecies whose natural reservoirs are wild and domestic animals.Humans are accidental hosts that get infected through the contactwithcontaminatedwater,urineoranimal tissues.Leptospirosisclini-calsignsinhumans,especially intheacutephase,canbeassumedassymptomsofseveralfeverishdiseasessuchascommoninfluenza,malaria,dengue,asepticmeningitisandbrucellosis.Thus,itisdifficultto establish a clinical diagnosis and the confirmation of the diseasegenerally reliesonserological tests thatdemonstrateserumconver-

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sion.Themicroscopicagglutination test (MAT) is theserological testofchoicefordiagnosisofleptospirosis,butithaslowsensitivityintheinitialphaseoftheillnessandit islaborintensive,sincerequiresthemaintenanceofalargenumberofleptospiralserovarsinthelaboratory.ThelowsensitivityofMATisduetothefactthattheimmunesystemoftheinfectedindividualstakes8-10daystoproducedetectable levelsofantibodiesagainsttheinfectiveagent.Inthiswork,wereportonthestandardizationofanantigencaptureELISAthatusesamonoclonalantibodyagainstLipL32,anoutermembraneproteinexpressedonlyinpathogenicleptospira,ascaptureantibody,andapolyclonalchickenIgYagainstwhole Leptospira interrogans serovar copenhageni cellsasdetectionantibody.Thiscombinationofantibodieshasshownlowcross-reactivity with human sera experimentally infected with lepto-spiresandadetectionlimitof105cells/mL.TheseresultssuggestthatthesandwichELISAhaspotentialforuseinthedetectionofLeptospiraintheinitialphaseofthedisease.Key words:Leptospirosis,Leptospira,Antigen-capture,DiagnosisSpecies:other

iP267. SEROLOGICAL DIAGNOSIS OF MyCOBACTERIUM BOVIS INFECTION IN LLAMAS

DEAnGS1,TWoMEyDF2,FARRAnTL3,DELARUA-DoMEnECHR4,JAHAnSK5,LyASHCHEnKoK6,VoRDERMEIERHM11VeterinaryLaboratoriesAgency,Weybridge,UK;2VeterinaryLaboratoriesAgencyStarcross,UK;3MinistryofAgriculture,

FisheriesandFood,StateVeterinaryService,UK;4DepartmentforEnvironment,FoodandRuralAffairs,UK;5VeterinaryLaboratoryAgency,WoodhamLane,NewHaw,Addlestone,UK;6Chembio

DiagnosticSystems,USAAccurate ante mortem diagnosis of Mycobacterium bovis infec-

tioninllamasisextremelydifficultusingthetestscurrentlyavailable.Intradermal tuberculin testing in the axillary site (using the regimeapproved for camelids) has been shown not to be accurate for thediagnosis ofM.bovis infection in llamas. In this small study, a llamaherd with recurrent clinical breakdowns was investigated using twoserologicaltechniques,(anon-validatedinvitrolateralflowassay,theVetTBSTAT-PAKandamulti-antigenprintimmunoassay(MAPIA))inconjunctionwithaprogrammeof90-dayintervaltuberculinskintesting,inanattempttodetectinfectedanimalsnegativebyskintest.LlamaspositivebySTAT-PAKwereeuthanizedand tissuessent forM.boviscultureandspoligotyping.AnimalsforwhichcultureandspoligotypingdataarecurrentlyavailablewereallseentobepositiveforM.bovisspo-ligotypeSB0274(VLAtype11),thespoligotypemostcommonlyisolatedinthearea.However,occasionalclinicalcasesoftuberculosiswerestilloccurringinSTAT-PAKnegativeanimalsintheintervalsbetweenskintests.FurtherworkmakinguseoftheanamnesticresponsetoSTAT-PAKfollowingskintestiscurrentlyinprogressandtheresultsofthesestudieswillbediscussed.Key words:M.bovis,serology,tuberculosis,camelidsSpecies:other(Llama–southamericancamelid)

iP268. EVALUATION OF CLINICAL, HEMATOLOGICAL AND IMMUNOLOGICAL PARAMETERS OF DOGS WITH

ACUTE MONOCyTIC EHRLICHIOSIS BEFORE AND AFTER TREATMENT WITH TETRACyCLINE

SIMonEMAGELAMoREIRA1,2,oLInDoDEASSISMARTInSFILHo3,MúCIoFLáVIoBARBoSARIBEIRo4,CAMILADE

VALGASBASToS1,4,LyGIAMFRICHEPASSoS11DepartamentodeMedicinaVeterináriaPreventiva,Escolade

Veterinária-UniversidadeFederaldeMinasGerais–C.P.567BeloHorizonte,M.G.Brazil;2FaculdadedeEstudosAdministrativosdeMinasGerais–FEAD,Brazil;3FundaçãoOswaldoCruz,Fiocruz,

Brazil;4DepartamentodeParasitologia,ICB-UFMG,BrazilOneofthemaingoalsforresearchersworkingoncaninemonocytic

ehrlichiosishasbeentoclarifytheclinicalandhematologicalalterations,aswellastheimmunologicalmechanismsinvolvedonthepathogenesisof the disease. Usually, treatments instituted during the acute phasepromote fast improvement of the animal’s clinical conditions. In thepresentstudy,ninedogswereexperimentallyinfectedwithaBrazilianstrainofEhrlichiacanisandweretreatedwithtetracyclinehydrochloride

(22mg/Kg/perday)for21days,beginningatthethirtiethdayafterinfec-tion.Someclinical,hematologicalandimmunologicalparameterswereevaluatedduring theacutephaseof thediseaseandafter the treat-ment.Inaddition,theirimmuneresponseswereevaluatedbeforeandafter thetreatmentbycytometricanalysisandcellphenotype(FACS)usingmonoclonal antibodies to cell surfacemarkers forCD5,Thy-1,CD4,CD14,MHCII,CD45R,andIgM.Theacutephasewasclinicallycharacterizedby fever,palemucousmembranes,progressiveweightlossandmoderate lymphadenopathy.Thebiochemical-hematologicalanalysis revealeddecreaseof creatinineand totalproteinconcentra-tionsalongwithanemia,thrombocytopenia,monocytosisandincreasedALTenzymeconcentrationintheinfectedanimals.TheexvivoimmuneresponseevaluationofperipheralbloodleucocytesshowedapercentilereductionofThy1+ lymphocytesduringashortperiodof timeat thebeginningoftheacutephase,accompaniedbyaprogressivepercentilereductionofTCD4+ lymphocytes,whichwassignificantlyreducedattheendoftheacutephase.Atthistime,adecreasedpercentileofbloodMHC-II+andCD45R+lymphocyteswasobserved.Ontheotherhand,the percentage of B (IgM+) lymphocytes and CD14+ cells increasedduringtheacutephase,corroboratingwithseroconversion,observedbytheindirectfluorescentantibodytest(IFAT),andmonocytosisobservedin blood examinations.A higher percentile of CD4+ neutrophils wasobserved in thegranulocyticseriesafterexperimental infections.Theinitial response to the treatment was characterized by fast clinicalrecoverywithweightgainand increasedphysicalactivity.Therewasan evident tendency to reestablish biochemical and hematologicalparameters,withmonocytes responding immediately to theantibiotictreatment.Mostparametersofimmunofenotypingthatwerealtereddur-ing theacutephase returned tonormal,demonstrating the favorableresponseaftertreatment.TheseresultsindicatethattheEhrlichiacanisinfectionpromotes importantalterations,whichseemtomodulate theimmuneresponse,allowingpersistenceoftheagentandtheestablish-mentof theacutephase.The21-day treatment regime,on theotherhand,resultsinrestorationofimmunecondition.Key words: Canine monocytic ehrlichiosis, hematology,immunofenotyping,Ehrlichiacanis,dogSpecies:canine

iP269. SEASONALITy OF SIDE EFFECTS DURING CHEMOTHERAPy OF CANINE TRANSMISSIBLE VENEREAL

TUMOR WITH VINCRISTINE.KARIMECSCARPELLI1,MARIALUIZACRVALLADÃo1,EDSonM

SCARPELLI1,KonRADInMETZE11DepartmentofPathology-UNICAMP(StateUniversityofCampinas)

SP–BRAZILKarime@vivax.com.br

Introduction: Transmissible venereal tumor (TVT) is one of themostfrequentneoplasmsindogs.Thetumorisfrequentlyencounteredintropicalorsubtropicalclimates.Theaimofthestudywastoinvesti-gateapossiblerelationshipbetweenthefrequencyofsideeffectsdur-ingtreatmentandtheseason.Methods:Westudiedprospectively100dogswithTVToriginatingfromthezoonosescontrolcenterofTaubatécity, stateofSãoPaulo,Brazil.AtdiagnosisanimalswereclassifiedaccordingtoanadaptedKarnofskyScale(KS)Thedogsweretreatedwith0,025mg/kgofvincristinesulphate intravenousyandmonitoreduntilclinicalremission.Inordertoinvestigatewhichvariableswouldbepredicitivefortheoccurrenceofsideeffectweanalyzedthefollowingcharacteristics:age,sex,weight,statusofowneddog,tumorvolume,KSandseasonoftheyearinuni-andmultivariateCoxmodels.Results:Weobservedthatcomplicationsduringtherapyweresignificantlymorefrequent during the hot months. In themultivariate Coxmodel onlythevariablesKS (B= -0,0260;p=0,05)andseasonof theyear (B=2,0642;p=0,04)remainedas independentprognostic factors forthe occurrence of complications during chemotherapy. Discussion:Sinceenvironmentaltemperaturehasshowntomodulatetheimmunesysteminvariousspecieswesuspectthattheincreaseofsideeffectsinthehotmonthsmightbecausedbyalterationsoftheimmunologicstatusofthedogs.Key words:caninetransmissiblevenerealtumor;seasonality,tropicalcountries.Species:canine

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iP270. CHARACTERIZATION OF HEMATOLOGICAL AND IMMUNOLOGICAL PARAMETERS DURING SUB CLINICAL

PHASE OF EHRLICHIA CANIS INFECTION IN DOGSFERnAnDAFIDELISGonSALESGUIMARÃES1,JULIAnASAyURI

KURIBAyASHI1,CInTIARAqUELBoMBARDIERI1,MARISTELAMARTInSDECAMARGo1,MITIKAKURIBAyASHIHAGIWARA21InstitutodeCiênciasBiomédicas-departamentodeImunologiadaUniversidadedeSãoPaulo;2FaculdadedeMedicinaVeterináriae

ZootecniadaUSP-departamentodeclínicamédicadaUniversidadedeSãoPaulo

ThecourseofinfectionbyE.caniscanbedividedinto3phases,acute,subclinicalandchronic,basedonsymptomsandclinical-patho-logicalterations.Theacutephase,rangingfrom3to5weeksofdura-tion,ischaracterizedbythedevelopmentofmulti-systemicsymptomsduetotheintenseinflammatoryprocesstriggeredbyinfection.Uponresolutionoftheacutephase,thosedogsunabletoeliminatetheinfec-tiousagentbecomecarriers.Thesedogsareclinicallyhealthy,althoughsomepresentvariabledegreesofthrombocytopeniaandleucopenia.Withprogressionof infection, thesedogsdeveloppancytopeniawithreservedprognostic.PreviouslyitwasshownthathighlevelsofIgG,increaseinCD8+numberandinversionofCD4+:CD8+ratiodevelopduringtheacutephaseofE.canis infection.Hereinweanalyzedthehumoralresponseandlymphocytesubpopulationsin5asymptomaticdogspresentingdiscreteleucopeniaandthrombocytopenia.Fourdogshavebeen infected for fouryearsandonehasbeen infected foranundetermined period of time.Antibodies were quantified by IFI andCD4+andCD8+ lymphocyteswerequantifiedbyflowcytometry.Ascontrols,weanalyzed8clinicallyhealthydogswithnomorbidhistorysuggestive of canine erlichiosis and no hematological alterations.Theseanimalswerealsonegativeforspecificanti-ehrlichiaantibodiesandforpresenceofE.canisDNA(byPCR).Allanimalsintheinfectedgroupwere positive in thePCRanalysis forE. canis DNA and pre-sented anti-E. canis titles ranging from 40.960 and 81.920. Healthyindividualspresented45.7±6.6%ofCD4+cells,22.6± 6.1%ofCD8+cellsandaratioof2.25forCD4+:CD8+.E.canis-infecteddogspre-sented33.7±10.6%ofCD4+cells,51.6± 21.3%ofCD8+cellsandaratioof0.85forCD4+:CD8+.ThehightitleofspecificantibodiesandtheinversionofTCD4+:CD8+ratioarecompatiblewithintenseactivityoftheimmunesystemagainsttheinfectiousagent.Thehematologicalalterationsobserved in terminal canineerhliquiosismightbeassoci-atedwiththeinflammatoryandimmune-mediatedreactionstriggeredbythepersistenceoftheinfection.Key words:Ehrlichiacanis,inversionofCD4+:CD8+,dogsSpecies:canine

iP271. RNA-LOADED CD40-ACTIVATED B CELLS STIMULATE ANTIGEN-SPECIFIC T CELL RESPONSES IN

DOGS WITH SPONTANEOUS LyMPHOMA nICoLAJMASon1,2,3,CHRISTInAMCoUGHLIn1,4,JARISHnCoHEn1,THERESAACoLLIGon1,CRAIGACLIFFoRD6,AnDREASZURBRIGGEn7,KARInUSoREnMo3,RoBERTH

VonDERHEIDE1,51AbramsonFamilyCancerResearchInstitute,Universityof

PennsylvaniaSchoolofMedicine;2DepartmentofPathobiology;3DepartmentofClinicalStudies,UniversityofPennsylvaniaSchoolofVeterinaryMedicine;4DivisionofOncology,DepartmentofPediatrics,

Children’sHospitalofPhiladelphia,UniversityofPennsylvaniaSchoolofMedicine;5Hematology-OncologyDivision,Department

ofMedicine,UniversityofPennsylvaniaSchoolofMedicine;Philadelphia,PA;6OncologyService,RedBankVeterinaryHospital,RedBank,NewJersey;7InstituteofAnimalNeurology,Universityof

Bern,Bern,Switzerland.Cell-basedvaccinationstrategiestoinducetumor-specificTcells

andgenerateanti-tumorimmunityhavelargelyfocusedontheuseofautologousdendriticcells to triggerantigen-specificTcell responsesincancerpatients.CD40activatedBcells(CD40-B)loadedwithtumorantigenareahighlyefficientalternativeantigen-presentingcellcapableofprimingnaïveTcellsagainstneoantigens,boostingmemoryTcellresponsesandbreakingtolerancetotumor-associatedantigens.TheuseoftumorRNAastheantigenicpayloadforCD40-Bappearsespe-ciallypromisingandallowsforgenetransferwithouttheuseofviruses

orvectorsandpermitsanMHC-independent,multiple-antigentargetingapproach important incancerswhere fewtumor-associatedantigenshavebeendescribed.However,theinvivoefficacyofRNA-transfectedCD40-B cells to stimulate tumor-specific T cell immunity remainsimmunologicallyandclinicallyuntested.Developmentofalargeanimalmodel of spontaneous cancer to evaluate the safetyandefficacyofRNA-loadedCD40-BcellsiswarrantedpriortouseofthesepromisingalternativeAPCs inclinical vaccinationofhumancancerpatients. Inthisstudy,wecharacterizeanovelculturesystemconsistingofCD40LtransfectedK562cells(KtCD40L)togeneratefunctionalCD40-BcellsfrombothhumanandcaninePBMCs.Furthermore,wedemonstratethatRNA-loadedCD40-Bcellscaninducefunctionalantigen-specificCTLsfromhumans,healthycaninedonors,andprivatelyowneddogswith spontaneously occurring lymphoma. Our data demonstrate thatRNA-loaded autologous CD40-B can stimulate antigen-specific Tcellresponsesinvitroindogs,pavingthewaytowardthefirstclinicalimmunotherapytrialusingtumorRNA-loadedCD40-Bcellsinvivotostimulate anti-tumor immunity in a large animalmodel of spontane-ouslyoccurringneoplasia.Key words:CD40,Bcell,cancerimmunotherapy,canineSpecies:canine

iP272. HARVEST AND CHARACTERIZATION OF MESENCHyMAL CANINE STEM CELLS FROM ADIPOSE

TISSUE AND BONE MARROW ACCSILVEIRA1,RSLIMA2,EMPEnHA1,SGMACAMBIRA2,MBPSoARES2,RRIBEIRo-DoS-SAnToS2,SMBARRoUIn-

MELo1,PHPAGUIAR11EscolaMedicinaVeterináriadaUFBA,Dep.PatologiaeClínicas;

2CentrodePesquisasGonçaloMoniz–CPqGM/FIOCRUZStem cells differ from other kinds of cells in the body.All stem

cells,regardlessoftheirsource,havethreegeneralproperties:(i)theyarecapableofdividingandrenewingthemselvesforlongperiods;(ii)theyareunspecialized;and(iii) theycangiverisetospecializedcelltypes.The increasingapproach for theuseofstemcellswith thera-peuticpurposeshasbeenoccurringduetotheircapacityofhemato-poietic reconstructionandmainly to theirplasticity,whatallows theirdifferentiationindiversetissues,suchasliver,centralnervoussystem,skeletalmuscleandothers.Stemcellsarepresentinearlyembryonicdevelopmentandarefoundinmatureadults.Potentialtherapiesbasedon adult stem cells are appealing because their obtaining does notrequiredestructionofthehostandisthereforelessethicalcontrover-sial. Therefore, adult stem cells from the bone marrow stroma andadiposetissuehavebeenproposedasanalternativesource.Adiposetissueandbonemarrowarederivedfromthemesenchymeandcon-tainasupportivestromawhich iseasily isolated.Thesecellscanbeobtainedinlargenumbersathighfrequencyfromatissuesourcethatcanbeextractedinlargequantitieswithminimalmorbidity.Theaimofthisstudywastostandardizeandcharacterizeamethodforobtainingmesenchymalstemcellsfromdog’sadiposetissueandofbonemar-row. Material and Methods: Eight healthy mongrel dogs were used.Thebonemarrowaspirativepuncturewasperformedintheposterioriliaccrest.TheadiposetissuewascollectedbyliposuctionwithmanualcannulainthedorsalareaandtreatedwithCollagenaseSigmaTypeIA.StemcellswereharvestedfrombonemarrowandadiposetissuewithFicollHistopaque1119and1077andcounted.ThecellswerekeptunderculturewithDMEMmediumandanalysedbyFACSwithmono-clonalantibodiesspecificforCD34,CD105,c-Kit,GFAPandAFProtein.Results:Eachbonemarrowaspirativepunctureyieldedanaverageof7x107cells/mLwhileeachlipo-aspirationallowedanaverageof1,5x106cells/mL.ThecellswerepositiveforthemarkersCD34,CD105,c-Kit, GFAP andAFProtein. Conclusions: These preliminary resultsshow that thecellswhichwereharvested fromcaninebonemarrowandadiposetissuecouldbecharacterizedascaninestemcells.Theproceduresweresafeandallowitsuseasstemcellmodelforthera-peuticpurposeandscientificstudiesthusgivingimportantinformationinhumanpreclinicaltrialswithstemcell.Key words:MesenchymalStemCells,AdiposeTissue,BoneMarrowSpecies:canine

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iP273. IMMUNOLOGICAL CHANGES IN DOGS WITH CHRONIC RENAL FAILURE

SKRALoVA1,LLEVA2,MToMAn1,21UniversityofVeterinaryandPharmaceuticalSciencesBrno;

2VeterinaryResearchInstitute;Brno,CzechRepublictoman@vri.cz

Chronic renal failure (CRF) is the condition of decreased renalfunction,inwhichkidneysfailtomaintainstabilityofinternalenviron-menteveninbasalconditions.Thediseaseisalwaysaccompaniedbyazotemiacharacterisedbyelevatedlevelsofcreatinineandureaintheblood.Clinicalsignsareofvariousintensities.AnadverseeffectofCRFontheimmunesystemandimmunosuppressionhasbeendescribedinhumans; however, knowledge ofCRFeffect on the immune systeminanimals isonlypartial. Inourstudyon40dogs–clinicalpatientswithvariousdegreesofCRF–weinvestigatedasetofbiochemical,haematological and immunological parameters. The patients wereallocated into three groups according to two criteria. First criterionwasmanifestationofclinicalsymptoms–group1waswithoutclinicalsymptomsandwithdiagnosedazotemia,group2waswithmoderateclinicalsymptomsandgroup3wasinthefinalstageofchronicrenalfailure.Secondcriterionwasplasmacreatinine level.Ourstudycon-firmedthatdependingondevelopmentofclinicalsymptoms,anaemiaand lymphopoenia appeared and creatinine, urea and phosphorusplasma levels increased.Besidesdevelopment of clinical symptomsandchangingbiochemicalparameters,lymphopoeniawereaccompa-niedbyadecreasingactivityoflymphocytesinthetestoflymphocytetransformationbothinrestingandmitogenstimulatedcells.Wedidnotfindchangesoflymphocytesubsetsratioinaffectedanimals.Wealsotestedtheeffectofserumfromdiseasedanimalsoninvitroactivitiesoflymphocytesandneutrophils.Therelationshipbetweenrenalfailureandrespectiveparametersof immuneresponses isdiscussed in thepresentation.SupportedbygrantMZE0002716201.Key words: immunopathology, chronic renal failure, lamphocyte,immunosuppressionSpecies:canine

iP274. RT-PCR: DIAGNOSIS VALUE IN DOGS WITH SPONTANEOUS ACUTE-, SUBACUTE-, AND CHRONIC-

DEMyELINATING DISTEMPER ENCEPHALITIS.EDSonMSCARPELLI1,KARIMECSCARPELLI1,ALExAnDREM

AMUDE2,AMAURIAALFIERI2,MARIALCInTRA11DepartmentofPathology-UNICAMP(StateUniversityofCampinas)SP–BRAZIL;2LaboratoryofAnimalVirology-UEL(StateUniversity

ofLondrina)PR-BRAZILedsonms@fcm.unicamp.br

Introduction:Distemperisanendemicdisease,thatisnotrestrictedto carnivorousor toasingle specie.Encephalitisdue to caninedis-tempervirus(CDV)hasbeenassociatedwithmultiplesclerosis(MS)because of extensive demyelization in both and the postulated viralorigin in MS.A domestic dog barely spends its life without contactwithdistempervirus (DV),mainly indevelopingcountries;also,con-tactmayoccur throughvaccinationprograms.Methods:Withinthreeyears,45dogswithsignsofcaninedistemper(CD)wereattendedinZoonosisControlCenterofTaubaté,SãoPaulo,Brazil.Clinicaldatawererecordedaccordingtoaprotocol.Theanimalswereeuthanized,necropsies, photos were taken, and a brain sample was randomlyselectedforRT-PCRtoCDV.In7healthydogs(controlgroup),sub-mitted to euthanasia for different reasons, the same procedure wasadopted. This study followed guidelines prescribed by the BrazilianMedicalResearchCentreand receivedapproval from theResearchEthicsCommitteeoftheStateUniversityofCampinas.Results:in31out45dogs,apositiveRT-PCRreactionwasfoundinbrainsamples(groupA); in the 14 remained dogs, negative resultswere obtained(group B); in all the 7 healthy dogs samples, positive PCR resultswere found (group C). On histological view, chronic-demyelinatingencephalitis, typicalofCD,was identified ingroupAanimalsand in11/14dogsingroupB;3/14dogsingroupBdisplayednohistologicalabnormalities.Focal lymphoidinfiltratewasfoundin5/7animalsandnoabnormalitiesin2/7dogsingroupC.Discussion:FortheendemicnatureofCDandvaccinationprograms,positiveresultsofRT-PCRdo

notallowthedistinctionofCDVfromotherdiseasesthatsharewithitthesameclinicalmanifestation.Bytheotherside,false-negativeRT-PCRcouldbetheresultofinadequatebrainsampleselectionorbeduetotechnicalreasons.Inthisway,ourfindingsstresstheimportanceofclinicalevaluationforproperCDdiagnosis.Theresultsmaybeusefulforclinical researchonCDVandsanitaryprogramsofcontrolof thedisease.Key words: Canine Distemper, Canine Distemper Virus, Pathology,RT-PCR.Species:canine

iP275. THE ROLE OF T CELLS IN AVIAN INFLUENZA H5N1 INFECTED CATS

MATTHIASGIESE,TIMMCHARDER,JEnSPTEIFKE,THoMASCMETTEnLEITER,THoMASWVAHLEnKAMP

Friedrich-Loeffler-Institute,Greifswald-InselRiems,Germanythomas.vahlenkamp@fli.bund.de

Duringtheoutbreakofhighlypathogenicavian influenza(HPAI)H5N1virusinfectionsamongwildbirdsandpoultryinAsia,EuropeandAfricatransmissionstocatshavebeendocumentedinatleastsevencountries.AlsoduringtheoutbreakinGermanythreecatscontractedthe infection. Infections of mammals with the zoonotic HPAI virusreceivedgreatpublicattentionascatsfrequentlyliveinclosecontacttohumans.Ascatsmightdiewithinoneweekpostinfection(p.i.)stud-iesontheinteractionwiththe(innate)immunesystemarecrucialforabetterunderstandingofdiseasepathogenesis.

WeperformedexperimentalinfectionsincatsusingtheinfluenzaH5N1virusisolatedfromoneofthenaturallyinfectedcatsinGermany.Cats were inoculated via the naso-pharyngeal route with 106 EID50HPAIH5N1virus.Theinoculatedcatsdevelopedfeverandrespiratorydistresswithin 24-48hp.i.Theanimals showeda systemic infectionincluding lymphopenia, viraemiaandpresenceof virus in all organs(differentpartsoftherespiratoryandgastrointestinaltract,CNS,liver,kidney,tonsils) investigated.TheCD4andCD8positiveTcellswereenrichedfromtheperipheralbloodduringtheacutestageoftheinfec-tionbybiomagneticbeadseparationandanalyzedforthepresenceofinfluenzaviralRNA.BothTcellpopulationswerefoundtobeRT-PCRpositive. In individual animals the amount of virus detected in theTcellsnegativelycorrelatedwiththeextendoflymphopenia.LowTcellnumberscorrelatedwithhighcellassociatedviralloads.Flowcytomet-ricanalysisoftheperipheralbloodmononuclearcellsrevealedthattheCD4/CD8Tcellratiowasnotaffectedintheanimals.

Insummary,catsprovedtobesusceptibletoHPAIH5N1virusiso-latedfromanaturallyinfectedcatsinGermany.OurresultssuggestthatlymphopeniaobservedintheinfectedcatsisassociatedwithinfluenzavirusinfectionofCD4andCD8positiveTcells.InvitroexperimentsarecurrentlyperformedtoinvestigatewhethervirusinfectioncontributestotheTcelldepletionandwhichmechanismsareinvolved.Key words:Tcells,influenzavirus,viraemiaSpecies:feline

iP276. PROSPECTIVE AND MOLECULAR STUDIES OF FELL PONy SyNDROME

RLTALLMADGE1,TSToKoL2,MJBFFLAMInIo11Dept.ofClinicalSciences;2Dept.ofPopulationMedicineandDiagnosticSciences,CollegeofVeterinaryMedicine,Cornell

University,Ithaca,NYUSAFell PonySyndrome is a fatal immunodeficiency that occurs in

youngFellPony foals.Affected foalspresentwithsevereanemia,Bcelllymphopenia,opportunisticinfections,andlethargicbehavior.Thesyndrome appears to be inherited as an autosomal recessive trait.OurobjectivewastoconductaprospectivestudyofFellPonyfoalstoestablishimmunologicparametersfornormalandpotentiallyaffectedFellPonies.Weperformedlymphocytephenotyping,measuredserumimmunoglobulin concentrations, and determined complete bloodcountsfor39normal foals,oneaffectedfoal,andfiveadults.This isthefirstreportofdatafromanaffectedfoalpriortoclinicalpresenta-tion.Atbirth,theaffectedfoalappearedclinicallynormal,withneitheranemia nor lymphopenia in peripheral blood. However, hematocritand percentage of B cells steadily declined over four to six weeks,

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until clinical signs were apparent. Immunohistochemical staining ofpost-mortem lymphoid tissues for equineB andT cellmarkerswasperformedonsamples fromnormal andaffected foals.B cellsweredepletedfromlymphoidtissues,althoughTcellswerereadilydetected.MolecularanalyseswereundertakentoidentifyadefectleadingtolossofBlymphocytes.ApanelofprimersetsforBcelldevelopment,signal-ing,andmaturationwasused todeterminemRNAexpression.MostBcell developmental and signalingmarkerswereunchangedat themRNA level.Differential expressionwas found between normal andaffectedfoalsforseveraltranscriptionfactors,oneknowntobespecificforBcellsandsomeofwidespreadactivity.

These findingsadvanceour understandingof the causeofFellPony Syndrome. Ultimately, these results will be used to elucidatediagnostic indicators for affected foals, and will hopefully assist inestablishinggeneticmarkersforthedisease.Key words:immunodeficiency,BcelllymphopeniaSpecies:equine

iP277. THE EFFECT OF GLUCOCORTICOID THERAPy IN THE IMMUNE SySTEM OF THE HORSE

MJULIABFFLAMInIo1,REBECCALTALLMADGE1,ERICASECoR1,DEREKJCAVAToRTA1,MARyBMATyCHAK1,MARyB

GoRDon2,RAnDELHRAUB21CornellUniversity,CollegeofVeterinaryMedicine,Ithaca,NYUSA

and2LandOLakes-PurinaMillsFeed,SaintLouis,MOGlucocorticoids are known to bind to a cytoplasmic receptor in

immune and non-immune cells, alter the function of transcriptionalfactors, and subsequently the secretion and expression of proteins.Theobjectiveof thisstudywasto identifydose-dependenteffectsofglucocorticoidtherapyinthecellsoftheimmunesystem.Twelveadulthealthyhorseswereacclimatizedtostallsfor1weekbeforetreatment.Thehorseswere randomlyassigned todifferent treatmentgroups inadouble-blindstudydesignincludingplacebo,anddifferentdosesofintravenous glucocorticoid treatment: 0.025mg/kg, 0.05mg/kg, and0.1mg/kg.Peripheralbloodsampleswerecollectedfromthehorsesat the following timepoints:dayminus7,dayminus6, immediatelybefore treatment,4,8,12,24,and48hrsafter treatment.The testsincludedserumACTHandcortisol levels,completebloodcellcount,phagocytosis and oxidative burst activity, lymphocyte subpopulationdistribution,integrinCD11a/CD18andmajorhistocompatibilitycomplex(MHC)classIIexpression,lymphocyteproliferation,IFNγexpression,andtetanustoxoid-specificantibodyproductionusingflowcytometricassays,real-timeRT-PCRandELISA.Alphawassetatp=0.05.Noneoftheparameterswereaffectedbymanagementchangefrompasturetostall.Theplacebogrouphadnochangesintheparameterstestedduring the study period. Importantly, all the effects of glucocorticoidtherapywereindependentofthedose.AdecreaseinserumACTHandcortisol levels at 4hrs after treatment (p = 0.03) supported the sys-temic effects of exogenous glucocorticoid administration.There wasan increase inperipheralbloodneutrophilcountswithaconcomitantdecreaseinlymphocytecountsat4and12hrs,respectively(p=0.03),after treatment in all glucocorticoid groups. Glucocorticoid inducedadecrease inCD4+T lymphocytesubpopulationdistribution,withaconcomitant increaseintheCD8+Tcellpopulation,andconsequentdecreaseintheCD4/CD8ratioat4hrs(p=0.01)aftertreatment.ThemeanfluorescenceintensityoftheintegrinmoleculeCD11a/CD18andMHCclassIIweregreater in the lymphocytesofhorsestreatedwiththe higher dose of glucocorticoid at 12hrs (p = 0.02). Phagocytosisandoxidativeburstactivity,Bcellsubpopulationdistribution,lympho-cyte proliferation, IFNγ cytokinemRNA and protein expression, andhumoralresponsetotetanustoxoidvaccinationwerenotaffectedbytreatment.Insummary,theeffectsofglucocorticoidwereindependentof thedose,andrevealedan increase inneutrophilanddecrease inlymphocytecounts,decreaseinCD4/CD8ratio,andanincreaseintheCD11a/CD18expressiononlymphocytes.Key words: equine, immunologic testing, glucocorticoid,immunosupressionSpecies:equine

iP278. CHANGES IN INFLAMMATORy CyTOKINE RESPONSE IN OLDER MARES AFTER PREDNISONE

TREATMENTKATHARInAKoHLER1,KAREnEWoLFSDoRF2,AMAnDAA

ADAMS1,DAVIDWHoRoHoV11MaxwellH.GluckEquineResearchCenter,Departmentof

VeterinaryScience,UniversityofKentucky,Lexington,KY,USA;2HagyardEquineMedicalInstitute,Lexington,Kentucky,USA

kkohl0@uky.eduImmunosenescence in theaged individual ischaracterizedbya

decline in lymphoid cell numbersand function.Somewhat paradoxi-cally,advancedageisalsoassociatedwiththeincreasedproductionofpro-inflammatorycytokinesandotherinflammatorymediators.Thisphenomenonhasbeen termed“inflamm-aging”. Inolderhorses, thisprocess may contribute to chronic inflammatory conditions such asendometritis and recurrent airway obstruction (RAO). Modulation ofthisinflammatoryresponsecouldimprovethehealthandwell-beingofaffectedhorses.Prednisone,asyntheticglucocorticosteroid,hasbeenusedinhorsestotreatavarietyofinflammatoryconditions,includingRAOandendometritis.Wehypothesizethatprednisoneadministeredorally toagroupofoldermareswill reduce inflammatoryresponses,asmeasuredby reducedproduction invitroof twopro-inflammatorycytokines:interferon-gamma(IFN-γ)andTumorNecrosisFactor-alpha(TNF-a). Five young (7-9 yrs) and four old mares (≥ 19 yrs) wereplaced into the treatment group and five additionalmares (four >19andone8yearold)wereusedascontrols.Themaresinthetreatmentgroupweregivenprednisone(200mgperos)onadailybasis for9weeks. Then the dosages were reduced to 100 and 50 mg for twoweekseach.Thecontrolmaresreceivednotreatment.Bloodsampleswere collected weekly into heparinized tubes from which peripheralblood mononuclear cells (PBMC) were isolated. The PBMC werestimulatedwithphorbol12-myristate13-acetate(PMA)andionomycinandthenstainedforIFN-γ andTNF-a.Sampleswereanalyzedusingaflowcytometertodeterminethepercentageofpositivelystainedcellsandcorrespondingmeanfluorescentintensity.Beforetreatment,oldermareshaveasignificantlyincreasedproductionofbothIFN-γandTNF-a when compared to younger mares.Treatment of the older mareswith 200mgof prednisoneover time causeda significant decreasein IFN-γ production, but not TNF-a. No significant treatment effectwasobservedintheyoungermares.Wecanthereforeconcludethattreatingagedmareswithoralprednisoneover timecan reducepro-inflammatorycytokineproductioninvitro,possiblyreflectingareducedpro-inflammatorystate.Key words:Inflammation,Aging,Prednisone,CytokinesSpecies:equine

iP279. WNV CANARyPOx VECTOR HORSE VACCINE: ACTIVE SPECIFIC IMMUNITy IN THE ABSENCE OF

INHIBITING ANTI-VECTOR RESPONSESHELGARCH1,JMMInKE1,JREHDER2,SRICHARD1,C

ToULEMonDE3,SDInIC3,CAnDREonI1,JCAUDonnET1,RnoRDGREn4,VJUILLARD1*

1MerialSAS,DiscoveryResearch,254rueMarcelMérieux,69342Cedex07Lyon,France;2MerialLimited,115TranstechDrive,Athens,

GA,USA;3MerialSAS,ClinicalOperationsSaintVulbas,France;4MerialLimited,3239SatelliteBlvd.,Duluth,GA,USA

SuccessfulvaccinationagainstWestNileVirus(WNV)isthoughttorequire inductionofbothneutralizingantibodiesandcell-mediatedimmune responses. In this study,wehaveassessed theability of arecombinant ALVAC®-WNV vaccine (RECOMBITEK® WNV) to elicitneutralizing antibodies and virus specific T cell-mediated immuneresponsesinhorses.Inaddition,weexaminedwhetherpriorexposuretoALVAC®-WNVvaccinewould inhibitBandTcell specific immuneresponses against the transgene product upon subsequent boosterimmunizationswiththesamevaccine.Theresultsdemonstratedthatthe recombinantALVAC-WNV vaccine induced neutralising antibod-iesandprM/E insert-specific IFNγ+Tcell responsesagainstWNV invaccinated horses. Prior exposure toALVAC®-WNV vaccine did notimpair the ability of horses to respond to two subsequent boosterinjectionswith thesamevaccine,althoughanti-vector-specificTandBcell responseswere induced invaccinatedhorses.Weshowed in

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thisstudythatALVACvectorallowsforastrongimmunogenicityoftherecombinantantigenintheabsenceofaninhibitoryanti-vectorimmuneresponseevenaftermultipleinjections.Inthisregard,ALVACvectorssuccessfullycombinethesafetyofaninactivatedvaccinewiththeeffi-cacyofamodifiedlivevaccine.Key words: Canarypox vaccines, West Nile Virus, horses, cellularimmuneresponseSpecies:equine

iP280. INSECT BITE HyPERSENSITIVITy IN ICELANDIC HORSES: IL-10 AND TGF-β1 REGULATION OF IL-4

PRODUCTION.EMAnHAMZA1,BETTInAWAGnER3,THoMASWJUnGI1,

JELEnAMIRKoVITCH1,ELIAnEMARTI21InstituteofVeterinaryVirologyand2DepartmentofClinical

VeterinaryMedicine,VetsuisseFaculty,UniversityofBern,Bern,Switzerland,and2J.A.BakerInstituteofAnimalHealth,Collegeof

VeterinaryMedicine,CornellUniversity,Ithaca,NY,USAeman.hamza@ivv.unibe.ch

Insectbitehypersensitivity(IBH)isarecurrentallergicdermatitisofhorsesoccurringinsummerandcausedbyIgE-mediatedreactionsto bites of midges of the genus Culicoides. IBH does not occur inIcelandduetotheabsenceofCulicoides.However,IcelandichorsesexportedtocontinentalEuropeasadults(1stgeneration)havea>50%incidenceofdevelopingIBH.Incontrast, theirprogenyborn inconti-nental Europe (2nd generation) has a <10% incidence of contractingIBH.Recently,weshowedthatthediseaseincidenceisassociatedwiththelevelofIL-4productioninculturesofPBMCstimulatedpolyclonally,orwiththeIBHallergen.ThisraisedthequestionwhetheralowlevelofIL-4productionin2ndgenerationhealthyIcelandichorsesisduetosup-pressionofIL-4production,orduetoabsenceofresponseinculturesofPBMCstimulatedeitherpolyclonally,orwiththespecificallergen.Inthepresentstudy,weexaminedwhether there isdown-regulationofIL-4productioninhealthy2ndgenerationIcelandichorses.Tothisend,PBMCfrom1stgenerationhorsesand2ndgenerationIBHhorseswereculturedwithCulicoidesallergenorConcanavalinA(ConA)togetherwithpooledsupernatantsfromhealthy2ndgenerationIcelandichorses.These experiments showed that the addition of supernatants fromPBMCculturesof2ndgenerationhealthyhorsesinducedasuppressionofIL-4productionbyPBMCfrom1stgenerationIBHhorses,asmea-suredbyflowcytometryorELISA.ApotentialroleofIL-10andTGF-β1wasexaminedintwoways.AdditionofrecombinantIL-10andrecombi-nantTGF-β1combined,butnotofsinglecytokines,ledtoasignificantreductionofIL-4levelsin4dayculturesofPBMCfrom1stgenerationhorsesand2ndgenerationIBHhorses.Conversely,additionofanti-IL-10andanti-TGF-β1toculturesofPBMCfrom2ndgenerationhealthyhorsespromptedanincreaseinIL-4levelsandIL-4-producingcellsin4dayPBMCcultureswhichwerestimulatedbyCulicoidesallergenorbyConA.Thissuggests(i)thatthelowlevelofIL-4productioninPBMCfrom2ndgenerationhealthyhorsesisduetosuppressionratherthanabsenceofresponse,(ii)thatIL-10andTGF-β1combinedcontributetoIL-4suppression,and(iii)thatIL-10andTGF-β1combinedpresentinsupernatantsfromPBMCof2ndgenerationhealthyhorsesregulateIL-4levels.Thefindingssupport thenotionthat inthisnaturallyoccurring

IgE-mediatedallergicdisease,thegenerationofallergen-specificTh2cellsisregulatedbyIL-10andTGF-β1.Key words: IBH, Icelnadic horses, IL-4 regulation, IL-10 and TGF-beta1;InsectbitehypersensitivitySpecies:equine

iP281. INSECT BITE HyPERSENSITIVITy (IBH) IN HORSES: SENSITIZATION, PREVALENCE, AND PERSISTENCE.

JRoHWER,CHKoBELT,WLEIBoLDInstituteofImmunology,UniversityofVeterinaryMedicine,BischofsholerDamm15,30173HANNOVER,Germany

Clinical signs of IBH (or summer eczema) in horses are basedonan immediatehypersensitivityreaction(typeIallergy) toantigensprovided by various insects such as Culicoides spp. and others.ResponsiblefortypeIallergicsymptomsareproinflammatorymedia-torsreleasedbytypeIeffectorcells(mastcells,basophils)uponcrosslinkingof theiractivatingFc-receptors.Suitableequine(eq) immuno-globulinisotypessuchaseqIgEorsomeeqIgGareabletobindtheseFc-receptors,thus,sensitizingthemforepitopestheyrecognize(spe-cificsensitization).CrosslinkingofactivatingFc-receptorsviabridgingtheirsensitizingantibodiesresultsinanactivationincludingdegranula-tion(releaseofstoredmediators)andnewgenerationofinflammatorymediators.SufficientsensitizationoftypeIeffectorcellsisanessentialprerequisiteforthedevelopmentofatypeIallergysuchasIBH.Itcansensitively beassessedbymeansof a functional in vitro test (FIT),developedinourlab:Washedhorsebloodcellsareinvitroexposedtodifferentconcentrationsofsuspectedallergens inorder todeterminequalitatively and semi quantitatively the specific sensitization of anindividual.Inordertomonitorexclusivelythereactivityofbasophilswequantifytheirhistaminereleaseasreadout.

ComparingclinicalsymptomsandbasophilsensitizationofhorsesagainstCulicoidesnubeculosus(Cn)weaskedthreequestions:

1:Howearlyinlifeshowjuvenilehorses(0to3years)afunctionalsensitization?

2:Howcomparessensitizationand IBH inadulthorses inaoneyearfollowupstudy?

3:How persistent is a functional sensitization under abstentionfromallergenforoneupto15years?

Foalsandyounghorseshardlydisplayhypersensitivityreactionsbutcandisplayspecificsensitization forCnasearlyas fourmonthspostdeliveryanddefinitelyduringtheirfirstyearoflife,longbeforeanyclinicalsymptoms.

Therearethreegroupsofadulthorses:ThosewhichhavenoIBHandareFIT-negative(doublenegatives).ThosebeingdoublepositiveforIBHaswellasfortheFIT.ThesestayFITpositivealsoduringtimeswhenall IBHsymptomsaregone (e.g. inwintertime)and thehorselooks–temporarily–healthy.AndthosewhichareFIT-positivebuthavenot(yet?)shownanysignofIBH.

HorsesonceIBHaffectedcanstaysensitizedforatleast15yearsunderallergenabstention.Key words:allergy,atopicdermatitis,insectbitehypersensitivitySpecies:equine

Author index

Family Name, First Name ............................... pages

AAASTED, BENT ......................................................77ABATEPAULO, ANTONIO RR ...............14,43,85,105ABEL, LCJ .....................................................107,108ABREU, RAQUEL TRÓPIA DE .......................14,135ACKERMANN, M ..................................................134ADAMS, AMANDA A ........................................82,144ADANIA, CRISTINA H .......................................... 117AERTS, J ................................................................54AGGER, ELSE-MARIE ...........................................77AGUIAR, CS ..........................................................111AGUIAR, PAULO HENRIQUE PALIS ...........14,16,97AGUIAR, RODRIGO DIAN DE OLIVEIRA ............ 114AGUIAR-SOARES, RODRIGO ............................135AGUIRRE, N ...........................................................72AITKEN, ROB ....................................................... 119ALBUQUERQUE, RICARDO DE .........................131AL-BUSAIDY, RASHILD M .....................................79ALCALA-CANTO, YAZMIN ...................................109ALCALDE, MH ......................................................128ALCANTARA, AC DE ............................................111ALEIXO, JOSÉ ANTONIO GUIMARÃES .............140ALESSI, ANTONIO CARLOS .................................91ALFANDARI, D .....................................................129ALFIERI, AMAURI A .............................................143AL-HABSI, KHALID ................................................79ALIBERTI, JCS .......................................................98ALLEGRETTI, SILMARA M .................................. 117ALMEIDA, DENISE E DE .......................................67ALMEIDA, JULIANA FRANCO ..........................59,60ALONSO, FERNANDO ............................95,133,139ALONSO, GABRIELA RODRIGUEZ ......................82ALVAREZ, BELÉN ....................................95,133,139ALVES, TELMA MARIA ..........................................69ALVES, TM .............................................................68AMADORI, MASSIMO ..........................................132AMI, YASUSHI ........................................................77AMORIL, JG .........................................................137AMUDE, ALEXANDRE M .....................................143ANANIAS, RZ .......................................................108ANATRIELLO, E .....................................................61ANDERSEN, PETER ..............................................77

ANDERSON, IAN ...................................................77ANDERSON, JM ....................................................60ANDERSSON, LEIF ...............................................58ANDERSSON-EKLUND, LENA ..............................53ANDO, ASAKO .......................................................58ANDO, JUNICHI .....................................................79ANDRADE, CAROLINE P DE .........................16,102ANDRADE, ELVA....................................................86ANDRADE, JOSIANA G DE .................................101ANDRÉ, MARCOS R ............................................ 117ANDRÉO, ROGÉRIO ...........................................107ANDREONI, C .......................................71,90,91,144ANDREWS, EDILIA M ..........................................127ANGUITA, JUAN ....................................................42APPLEGATE, TODD J ............................................76APPOLINÁRIO, CM ................................................76ARAGÃO, LS DE .................................................. 117ARAÚJO, BÁRBARA SILVEIRA ........................16,69ARAÚJO, CC ..........................................................80ARDILA, FERNANDO ..........................................124ARESTEGUI, MB ...................................................72ARTIS, DAVID ........................................................40ASCARATEIL, S .....................................................78AUCOUTURIER, J .................................................78AUDONNET, JC ..............................................90,144AWATA, TAKASHI .............................................58,59AZEVEDO, VASCO ..............................................124

BBABIUK, LORNE A ...............................................123BACIGALUPE, DIANA ..........................................109BAHIA, MARCUS V ..............................................108BAHIA, MARIA TEREZINHA ................................ 112BAHIA, ROBSON .................................................137BAKER, MICHELLE L.............................................57BALAZUC, ANNE-MARIE .......................................94BALDWIN, CYNTHIA ....................................15,17,40BALLET, JEAN JACQUES ...................................129BALLINGALL, KEITH ........................................14,53BALMELLI, CAROLE ..............................................31BALU, SUCHARITHA .............................................16BANNERMANN, DOUG .........................................42BAQUEIRO, T ...................................................... 117BARBEITO, CLAUDIO G ......................................120BARNES, A .............................................................50

BARRETO, MIGUEL .............................................137BARROSO, CE ..................................................... 118BARROUIN-MELO, STELLA MARIA .................22,70BARROW, PAUL .....................................................22BARSANTE, MICHELE M ....................................124BARTLEY, PAUL M ............................................... 116BASIRICÒ, LOREDANA .........................................79BASON, MELINA .................................................. 113BASSO, WALTER .................................................109BASTOS, CAMILA DE VALGAS ...........................141BASTOS, REGINALDO G ....................................125BAUHOFER, OLIVER ............................................31BAXTER, REBECCA ..............................................95BEAL, RICHARD ....................................................22BEAN, ANDREW GD ............................................130BEARSON, SMD ....................................................49BECHARA, GERVÁSIO HENRIQUE 23,55,85,94,102BEGIN, K ................................................................86BEHLING-KELLY, ERICA .......................................65BELKAID, YASMIN .................................................23BELKNAP, J ..........................................................129BELLIDO, DEMIAN .........................................67,124BELLINZONI, RODOLFO .......................................69BEMQUERER, MARCELO .....................................60BENASSI, JÚLIA C ...............................................140BENDER, BALÁZS .................................................43BENGTEN, EVA ................................................17,31BENJAMIN, PONN ...............................................123BERG, MIKAEL .................................................31,89BERGMAN, INGRID-MARIA ..................................58BERNABUCCI, UMBERTO ....................................79BESSO, R ...............................................................72BETSCH, JEAN-MARC ........................................129BEVILACQUA, CLAUDIA .......................................94BIASI, C ................................................................ 118BITTENCOURT, DVV ............................................111BJØRKMAN, C .....................................................109BLACK, SJ .......................................................15,129BLIXENKRONE-MØLLER, MERETE .....................77BLUM, S .................................................................88BOAS, ULRIK .......................................................134BODROGI, LILLA ...................................................43BOHACH, GREGORY A ....................................31,62BOMBARDIERE, ETHIANE .................................127BOMBARDIERI, CINTIA RAQUEL .......................142

147

BOND, JP ...............................................................55BONNEAU, MICHEL ..............................................94BORHACH, GREGORY .........................................31BOSCH, RV ..................................................... 80,118BOURDOISSEAU, GILLES ....................................49BOUTRUP, TORSTEN S ........................................71BOVINE, JAMES WOMACK ..................................21BOYD, PATRICIA....................................................15BOYSEN, PREBEN .........................................32,123BRAECKMANS, D ..................................................32BRAGA, SAMUELL .............................................. 114BRANDÃO, LG ...........................................60,61,102BRCIC, MARIJA .....................................................64BREATHNACH, CORMAC C ..........................82,130BREUIL, MARIE-FRANCE ...................................129BRÍGIDO, MARCELO DE MACEDO ......................60BRITO, LUIZ AUGUSTO BATISTA .......................127BROERE, FEMKE ..................................................63BROWN, WENDY C ..........................................23,33BROWNLIE, J .........................................................94BRUHN, OLIVER ..................................................129BSZE, ZSUZSANNA ...............................................43BÜCHLER, U ........................................................134BUDDLE, BRYCE M ...............................................65BURRELLS, A .......................................................106BURT, DAVE W ..........................................40,75,131BUSEK, SOLANGECU ......................................... 114BUTLER, JOHN E .............................................33,40BUXTON, DAVID .................................................. 116BUZA, JORAM .....................................................123

CCABRERA, ALEX C ..............................................127CAETANO, AG .................................................75, 75CAETANO, ALEXANDRE R ...................................54CAMARGO, MARISTELA MARTINS DE ..............142CAMARGO, MM .....................................................97CAMARGOS, MF .................................................137CAMBULI, C ...........................................................55CAMPBELL, FIONA ..............................................133CAMPOS, PATRÍCIA COTA ....................................69CAMPOS, VINICIUS E B ......................................104CAÑAVATE, CARMEN ..........................................111CÂNDIDO, TERESINHA CRISTINA ..................... 113CANTÓ, CARMEN ...............................................133CAPOFERRI, R ......................................................55CAPPARELLI, FAUSTO EMÍLLIO .....................59,60CAPRERA, A ..........................................................55CARANDINA, L.....................................................128CARDOSO, FERNANDA CALDAS ......................124CARDOSO, RONE ............................................59,60CARIOLET, ROLAND .............................................97CARMINATI, RENATO .........................................137CARNEIRO, CLÁUDIA MARTINS ..................112,114CARRILLO, EUGENIA ...........................................111CARVALHO JR, ARIOLDO .....................................62CARVALHO, GABRIEL DOMINGOS ....................104CARVALHO, L PONTES DE ................................ 117CARVALHO, MARIA DAS GRAÇAS .................... 114CARVALHO, WANESSA A ..................43,85,105,122CASE, RUTH ..........................................................16CASSALI, GIOVANI .............................................. 116CASSATARO, JULIANA .........................................64CASTRO, LUIZA AMARAL DE .............................139CASTRO, MÁRCIO BOTELHO DE ...................... 110CATCHPOLE, B .....................................................50CAUCHARD, JULIEN ...........................................129CAVA, ANTONIO LA ...............................................23CAVASSANI, KA .....................................................98CAVATORTA, DEREK J ........................................144CERVENAK, JUDIT ................................................43CGORNI, CGORNI .................................................55CHAMBERS, TOM .................................................82

CHAMLEY, LAWRENCE W .................................. 119CHAMORRO, SONIA ...........................................133CHANG, S .......................................................53,124CHAPAT, L ....................................................71,90,91CHAPPELL, LUCY .................................................22CHARDON, PATRICK ............................................58CHARLESTON, BRYAN ..................................41,121CHARLEY, BERNARD ...........................................94CHARREYRE, C ....................................................90CHATTHA, KULDEEP S .......................................121CHAUHAN, RS .....................................................131CHAVATTE-PALMER, PASCALE ...........................23CHEEVER, ALLEN .................................................42CHEN, S ........................................................107,108CHIANINI, FRANCESCA ...................................... 116CHIAVENNA, SEBASTIÁN M ..........................66,124CHICHARRO, CARMEN .......................................111CHILMONCZYK, STEFAN .....................................94CHINCHAR, G ........................................................15CHINCHILLA, LEONARDO A ...............................120CHRISTOPHER-HENNINGS, JANE ......................59CINTRA, MARIA L ................................................143CISSELL, ROBIN L.................................................68CLAIRE, ROGEL-GAILLARD .................................58CLIFFORD, CRAIG A ...........................................142CLIFFORD, DJ .......................................................66COAD, M ................................................................66COELHO, KEILA DA SILVA ..................................124COFFEY, TJ ....................................................53,124COHEN, JARISH N ..............................................142COLAVECCHIA, SILVIA ....................................50,69COLLIGON, THERESA A .....................................142COLVIN, CHRIS .....................................................63COLVIN, CHRISTOPHER J....................................67CONCEIÇÃO, FABRICIO R ............................74,125CONNELLEY, T ..........................................54,95,106CONTI, LUÍS HENRIQUE A ..............................53,86CONTRERAS, VANESSA ......................................94CORL, CM ..............................................................86CORREA, JP ........................................................ 116CORRÊA-OLIVEIRA, RODRIGO ............112,114,135COSTA, GM ..................................................... 80,118COSTA, MIRELA TINUCCI .....................................91COSTA, RUI GIL DA ...............................................86COSTA-SILVA, MARCOS .....................................137COTTORELLO, APC ..............................................68COUGHLIN, CHRISTINA M .................................142COURBET, ALEXANDRE .......................................94COURTOIS, DAVID ..............................................129COUSSENS, P .......................................................55COUSSENS, PAUL M .......................................63,67COUTINHO, LUIZ L ..............................................107COUTINHO, MARIANA LONER ...........................140COUTURE, OP .......................................................49COWAN, PHILIP E ............................................... 119COX, ERIC ........................................................40,53COYNE, M ............................................................129CRABB, BRENDAN S ............................................74CRAIG, PATRICIO O ..............................................67CRAWFORD, ALLAN M ....................................21,54CRUZ, APR ..........................................................103CTOULEMONDE, CTOULEMONDE ......................90CUELLO, MARIA F .................................................81CYRINO, LARISSA TAVARES ..............................104CZUPRYNSKI, CHARLES J ...................................65

DDALIN, AM ..............................................................88DASSANAYAKE, ROHANA P ............................67,68DAVID, JORGE M ................................................108DAVID, JUCENI P.................................................108DAVIDSON, LJ .......................................................50DAVIES, CHRIS ...................................................123

DAVIES, CJ ............................................................80DAVILA, ALBERTO .................................................21DAVIS, WILLIAM C ......................................31,62, 80DAWSON, HARRY D ................................. 23,55,110DAY, MICHAEL J .................................................. 115DEAN, GS ............................................................141DEANE, DAVID .................................................14,77DEBENHAM, SALLY ..............................................16DEEG, CORNELIA .................................................33DEGEN, WINFRIED GJ .......................................131DEKKERS, JC ........................................................49DELGADO, G .........................................................72DELGADO, MONICA ..............................................40DENAPOLI, P .......................................................107DENIS, MICHEL .....................................................65DENKERS, ERIC ....................................................41DERETIC, VOJO ....................................................40DESCHACHT, NICK .............................................122DEVÉNS, BRUNA ALVES ....................................104DEVILLE, S ............................................................78DÍAZ, IVÁN ...........................................................133DIEZ-TASCON, CRISTINA .....................................21DINIC, S ...............................................................144DINIS, MÁRCIA ......................................................86DINY, NICOLA ........................................................96DOMENECH, NIEVES ...........................................95DOMI, MAYUMI ......................................................79DOMÍNGUEZ, JAVIER .............................95,133,139DONNELL, ROBERT L ...........................................68DOS-SANTOS, WLC .....................................111, 117DOWNING, ALISON ...............................................75DOYLE, SARAH .....................................................50DRIEMEIER, DAVID .............................................139DUMMER, LUANA A ........................................74,125DUPUIS, L ..............................................................78DUQUESNE, FABIEN ..........................................129

eECHAIDE, S TORIONI DE .....................................72ECKERSALL, P DAVID ........................................133EDEN, WILLEM VAN .........................................41,63EDFORS-LILJA, INGER .........................................58EGGEN, ANDRÉ ....................................................43EGNUNI, TEKLU ....................................................66EGUCHI-OGAWA, TOMOKO .................................59EICHER, SUSAN D ................................................76EKMAN, ANNA .......................................................93ELLIS, SHIRLEY A ............................................58,93ELNEKAVE, ELDAD ...............................................42ELSWAIFI, SHAADI F ............................................65ENTRICAN, GARY .......................................14,50,80EPARDAUD, MATHIEU ..........................................94ESCRIBANO, JOSÉ M ..................................124,125ESPINO-SOLIS, GP ...............................................98ESTEIN, SILVIA M ..................................................64ESTES, DON M ......................................................22ETO, SF ................................................................128EVERTS, ROBIN E ...........................................23,55EZQUERRA, ANGEL ................................95,133,139

fFADAEI, M ..............................................................83FARIA, GERMANO K .............................................70FARIAS, NARA R ................................................. 115FARIAS, SANDRA E .............................................100FARRANT, L .........................................................141FATTORI, KARINA REINALDO .................. 59,60,116FAVARON, P ......................................................... 118FEITOSA, MM ...................................................... 115FEITOSA, RRS ..................................................... 117FELIPPE, PAULO AN ........................................... 117FERNANDES, CAMILA C .......................................75

148

FERNANDES, CLÁUDIA PINHO HARTLEBEN ...140FERNANDEZ, ANA .................................................53FERNANDEZ, E .....................................................69FERNÁNDEZ, ELOY ............................................126FERNANDEZ, FERNANDO ............................69,138FERREIRA, ANNA PAULA B RIBEIRO ................104FERREIRA, BR ....................60,61,85,86,98,102,105FERREIRA, CAS ..................................................103FERREIRA, HENRIQUE BUNSELMEYER ..........139FERREIRA, PAULA ................................................86FERRONATO, BO ................................................ 118FGAUNA, ADRIANA B VIVAS HECTOR ................83FILGUEIRA, DANIEL M PÉREZ ...........................125FILIPPI, JORGE .....................................................69FIORAVANTI, MARIA CLORINDA S.....................136FIORENTINO, MARÍA A .........................................64FIORETTO, ET .......................................................80FIRMINO, ALEXANDRE .......................................103FIRTH, MATT A .....................................................121FISCHER, G ...........................................................77FITZGERALD, BARRY ...........................................82FITZPATRICK, JULIE .............................................87FLAMINIO, M JULIA BF ....................................60,82FLORES-MENDOZA, L ..........................................97FLYNN, JOANN ......................................................31FONSECA, CRISTINA TOSCANO .......................124FONTANA, PAULA A ............................................120FONTANALS, ADRIANA .......................................138FOREST, L ...................................................71,90,91FOREYT, WILLIAM J ..............................................67FOSSATI, CARLOS A .............................................64FOSSUM, CAROLINE ......................58,73,81, 88, 89FOX, LAWRENCE K...............................................62FRANÇA, ANGELA .................................................86FRANCHINI, M .....................................................134FRANCO, ANA C ....................................................65FRANÇOIS, LEFEVRE ...........................................58FRANKE, CR .........................................................111FRANZIN, ALESSANDRA M ......................63,86,103FREIRE, SONGELI M V ...............................63,67,70FRESCHI, ANA PAULA PEREZ .......................59, 60FRETWELL, N ........................................................50FURTADO, CCV ...................................................137FUXLER, LISBETH ...........................................73,81

gGAÉTAN, LEMONNIER ..........................................58GAMA, MARILIA NOGUEIRA DA .........................104GAMA, ROG ..........................................................111GANDY, JC .............................................................86GANNE, V ..............................................................78GARAICOECHEA, LORENA ................................138GARCH, H EL ........................................71,90,91,144GARCIA, CÉSAR A ................................................70GARCIA, CF .........................................................107GARCIA, GR ......................................60,85,102, 103GARCIA, M ...........................................................108GARCÍA-BRIONES, MERCEDES ........................139GARMENDIA, ANTONIO E ..................................132GAZZINELLI, RICARDO TOSTES .........................33GELHAUS, CHRISTOPH .....................................129GENOY-PUERTO, A ............................................. 118GENTILE, N ............................................................72GERARDI, DANIEL ................................................91GERDTS, VOLKER ................................................50GERNER, WILHELM ..............................................96GERZOSCHKWITZ, TATIANA DE OLIVEIRA ...... 113GIAMBARTOLOMEI, GUILLERMO ........................64GIBERTONI, ALIANDRA M ....................................75GIESE, MATTHIAS ...............................................143GILKERSON, JAMES R .........................................74GIL-TURNES, CARLOS ....................................65,77GIMENO, EDUARDO J ........................................120

GIOTIS, EFSTATHIOS S ........................................75GIRÃO, FLÁVIA ARAÚJO ......................................60GIUNCHETTI, RODOLFO CORDEIRO ... 66,114,135GLASS, EJ ....................53,56,75,87,95,105,124,131GLEESON, DIANNE M ......................................... 119GNUDI, M ...............................................................90GODDEERIS, B ......................................................90GODFREY, DALE I ...............................................130GOFF, WILL L ....................................................42,66GOLDAMMER, TOM ............................................ 119GOLDBAUM, FERNANDO A .............................66,67GOMES NETO, CMB ............................................111GOMES, ANA P ....................................................137GOMES, JOSIANA ...............................................102GÓMEZ, CRISTINA ..............................................124GÓMEZ, NURIA ...................................................139GONÇALVES, MARIANA C M ................................75GONZALEZ, DIEGO D ....................................67,124GONZÁLEZ, FERNANDO .....................................111GORDON, MARY B ..............................................144GORIN, STÉPHANE ..............................................97GÓRNIAK, SILVANA L .....................................99,140GORYO, MASANOBU ............................................93GOTO, H .............................................................. 115GOUBIER, A .................................................71,90,91GOULART, LUIZ RICARDO .......................60,67,124GRAHAM, S .........................................................106GRAHAM, SIMON P ...............................................93GRECO, CECILIA R .....................................81,82,83GREEN, CHERYL A ...............................................23GRIEBEL, P ............................................................94GROSSO, MARIA C ...............................................81GRÖTZINGER, JOACHIM ...................................129GRUYS, ERIC ......................................................133GUALTIERI, CAS ...................................................72GUEDES, MARIA TEREZA B ...................67,108,109GUERRA, LUANDA L ........................................... 114GUIDRY, CATHERINE A .........................................15GUIMARÃES, ANDREA RSC ............................... 114GUIMARÃES, FERNANDA F GONSALES ..........142GUIMARÃES, MARCO PEZZI ............................. 116GUNNES, GJERMUND ........................................123GURROLA-BRIONES, GA .....................................98GUZMAN, EFRAIN ..........................................41,121GUZYLACK-PIRIOU, LAURENCE .........................31

hHAARLEM, DAPHNE VAN .....................................16HÄDRICH, GABRIELA .........................................140HAERTLE, STEFAN ............................................. 119HAGIWARA, KATSURO .........................................79HAGIWARA, MITIKA KURIBAYASHI ....................142HAIG, D ...........................................................53,124HAIG, DAVID M ......................................................77HAMIR, AN .............................................................32HAMMARSTRÖM, LENNART ................................43HAMMER, SABINE ................................................96HAMMER-MUNTZ, O .............................................88HAMZA, EMAN .....................................................145HANDKE, KRISTIN A .............................................83HANNANT, DUNCAN ........................................14,16HANSEN, J .............................................................15HANSEN, JOHN D .................................................17HARAGUCHI, MITSUE ...................................99,140HARDEGGER, ROLAND .......................................66HARDER, TIMM C ................................................143HARO, SERGIO S DE ............................................40HAROLD, WITOLA WILLIAM ............................... 117HARRIS, JAMES ....................................................40HARRIS, JIM ..........................................................40HARRIS, REUBEN .................................................32HÄRTLE, STEFAN .................................................57HARTLEY, CAROL A ..............................................74

HASTINGS, NICOLA ....................................53,87,95HATHAWAY, JENNIFER .........................................57HE, JOSEPH AMBROGIO DONGSHENG ...........132HEEGAARD, PETER MH ...........................70,71,134HELLER, D .............................................................88HENDRIKS, JUDITH ..............................................16HENEINE, LUIZ GUILHERME DIAS ......................69HERMOGENES, MÁRCIA SILVA ...........................69HERNÁNDEZ, J ................................................81,97HERNDON, CAROLINE N .....................................67HEWINSON, R GLYN ........................................66,70HICKSTEIN, DENNIS .............................................33HIKONO, HIROKAZU ...........................................120HILLEGAS, JULIA M ..............................................17HODGINS, DOUGLAS C ......................................121HOEK, AAD .....................................................94,123HOGE, AYA .............................................................97HOLLAND, OLIVIA J ............................................ 119HOLMES, KATHRYN V ........................................ 119HOLTMEIER, WOLFGANG ....................................96HONDA, YOSHIKAZU ............................................93HONG, YEONG ......................................................16HOPE, J ................................................................124HOPE, JAYNE ....................................14,16,40,80,94HOPE, JC ...............................................................53HOROHOV, D .........................................................15HOROHOV, DAVID W ....................14,82,83,130,144HUDGENS, EDWARD ............................................17HUE, ISABELLE .....................................................23HUEZA, ISIS M ..............................................131,140HUGNET, CHRISTOPHE .......................................49HULST, MARCEL ...................................................89

iIBARRA-VELARDE, FROYLAN ...........................109IBELLI, ADRIANA MG ..........................................107ICOM, RICHARD ....................................................17IIVANAINEN, ANTTI ...............................................93IKEDA, FA ............................................................. 115IKEDA, MANABU ..............................................63,93IMAMURA, SAIKI ............................ 100,101,102,117IMBUGA, MABEL ...................................................56INNES, ELISABETH A .......................................... 116INUMARU, SHIGEKI .......................................87,120INZANA, THOMAS J ..............................................65IQBAL, MUHAMMAD .............................................16IRAQI, FUAD A .......................................................56ISABEL, KF ..........................................................103ISHIGURO, NAOTAKA ...........................................93ITO, YUKO ...........................................................100IUNES, RS ....................................................... 80,118IVO, MA ................................................................ 118IZUEL, MERCEDES ...............................................69

JJAHANS, K ...........................................................141JAKOBSEN, JEANNE TOFT ........................71,73,81JAMIN, AGNÈS ......................................................97JANAGAMA, H .......................................................55JANN, O ..........................................................53,124JAR, ANA M ..........................................................128JENNE, CRAIG N ...................................................87JENSEN, K ......................................................56,105JENSEN, TIM K ......................................................71JENSEN, TRINE H .................................................77JEPSON, CATHERINE ...........................................14JGRUBMAN, MARVIN .........................................132JIWAKANON, J.......................................................88JOENSUU, J ...........................................................90JOF, PAULA ............................................................86JOHANSSON, AMELIE ..........................................58JOHNSON, CARL.................................................125

149

JOHNSON, EUGENE H .........................................79JOHNSON, P ........................................................129JOHNSON, RODGER ............................................59JOHNSTON, CLAIRE E .........................................74JOISEL, F ...............................................................90JOLLY, ANA ..............................................69,126,138JONES, C ...............................................................50JONES, ETHIOPIA BESHAHYOLANDA .............. 110JR, ALCEU GS .......................................................74JUILLARD, V .........................................71,90,91,144JUNG, SASCHA ...................................................129JUNGERSEN, GREGERS ..............................71,134JUNGI, THOMAS W ........................................64,145JUNQUEIRA JR, DANILO G ..................................70JUNQUEIRA-KIPNIS, ANA PAULA ...................62,75JUTILA, MARK A ..........................................64,75,86

kKABARA, E .............................................................55KACSKOVICS, IMRE .............................................43KAI, CHIEKO ..........................................................77KAISER, PETE ........................................14,16,22,75KALKS, KARLOS HENRIQUE M ...........................60KALM, ERNST ......................................................129KAMMERER, ROBERT ................................... 57,119KANIA, STEPHEN A ...............................................68KÄSER, TOBIAS ....................................................96KASHINO, SS .........................................................85KATAEVA, GALINA .................................................53KATEPALLI, MADHU ..............................................82KATZER, FRANK .................................................106KAUFMAN, JIM ............................................14,16,33KAUSHIK, AZAD K ..........................................53,126KEANE, JOSEPH ...................................................40KEANE, ORLA M ....................................................21KEID, LARA ............................................................70KEMP, JM .............................................................106KENNEDY, LAURIE J .............................................87KENNEDY, LJ .........................................................50KER, HENRIQUE GAMA ...................................... 112KERR, DE ...............................................................55KERSTENS, HINRI ................................................89KFOURY JR, JR .............................................. 80,118KHUMTHONG, R ...................................................42KIELING, K .............................................................80KIPNIS, ANA PAULA J ..........................................136KIPNIS, ANDRÉ .....................................................62KISHIMA, MASATO ..............................................120KLASING, KIRT C ..................................................81KLEVAR, S ...........................................................109KLOSS, C ...............................................................55KOBELT, CH .........................................................145KOBUNE, FUMIO ...................................................77KODAMA, MICHI .................................................. 117KOETS, AD .................................................41,63,123KOETS, AND ..........................................................94KOHARA, JUNKO ...........................................63,136KOHLER, KATHARINA ....................................82,144KONNAI, SATORU ...................... 63,93,100, 102,117KOOCYK, YVETTE VAN ........................................41KOOL, JOLANDA ...................................................94KOTI, MADHURI .............................................53,126KRALOVA, S ........................................................143KREISS, ALEXANDRE .........................................139KRIEG, ARTHUR M ..............................................123KRIFUCKS, O .........................................................88KUBOTA, TAKAYUKI .......................................87,120KUCKLEBURG, CHRISTOPHER J ........................65KUHAR, D .........................................................49,59KULBERG, S ........................................................109KUNTZ-SIMON, GAËLLE .......................................97KURIBAYASHI, JULIANA SAYURI .......................142KUROIWA, Y ..........................................................32

KUZMINSKY, GIORGINA ................................79,136KWONG, LAI SHAN ...............................................16KYBURZ, ANNIKA ..................................................66KYDD, JULIA H .................................................16,79

lLABRESH, JOANNA .........................................15,17LACETERA, NICOLA ......................................79,136LAGE, AP ..........................................................68,69LAGER, KELLY D ...................................................72LAMPREAVE, FERMIN ........................................133LANA, MARTA DE ......................................... 112,135LARANGEIRA, DANIELA FARIAS ........................111LARSEN, ALEJANDRA ........................................109LATORRE, ANDREIA O..........................................99LATSHAW, M ..........................................................55LAUGIER, CLAIRE ...............................................129LAURENCE, FLORI ...............................................58LAURITSEN, K TØLBØLL ....................................134LAVAL, A .................................................................78LAVOIE, JEAN-PIERRE .........................................33LAW, A ....................................................................54LAW, Y ....................................................................95LAZZERI, B ............................................................55LEAL, ALEXANDRE T ..........................................100LEE, SANG UN ......................................................62LEIBOLD, W ....................................................98,145LEIPPE, MATTHIAS .............................................129LEITE, FÁBIO PL...............................................77,81LEITNER, G ............................................................88LEMKE, CAITLIN D ................................................72LESMERE, JEAN-LOUP ........................................49LEVA, L .................................................................143LEVY, M SUSANA ..................................................66LEWIN, HARRIS A ........................................23,55,82LI, FAN ....................................................................74LIAUDAT, ANA ........................................................83LICEA-NAVARRO, AF ............................................98LILLEHOJ, HYUN .........................................16,17,82LIMA, AR ......................................................... 80,118LIMA, DANIELLE D ..............................................137LIMA, FERNANDA W DE MENDONÇA ........108,109LIMA, FLÁVIA L .................................................... 110LIMA, LIZIANE MARIA DE .....................................60LIMA, RS .................................................111,117,142LIMA, VALÉRIA MARÇAL FELIX DE ..............113,116LIMA, VMF .................................................76,115,116LIN, TSANG L .........................................................76LINDHOLM, AGNETA ...........................................132LINO JR, RUY DE SOUZA ...................................127LISA, PRM DE ......................................................103LISBOA, CS .......................................................... 118LIU, WEIGUO ....................................................67,72LOFTUS, J ............................................................129LOGULLO, CARLOS ..............................100,101,102LONGO-MAUGERI, I .....................................107,108LOPEZ, APRIL ........................................................57LÓPEZ, JORGE A ................................................108LÓPEZ, VIRGINIA ..................................................69LOUKAS, ALEX ......................................................40LÖVGREN, TANJA .................................................73LOWENTHAL, JOHN W .......................................130LOYOLA, W ..........................................................128LUND, MOGENS S ................................................53LUNNEY, JOAN K ......................................49,85,105LUVIZOTTO, MARIA CECÍLIA RUI ...................... 113LYA, KONSTANTIN ..............................................140LYASHCHENKO, K...............................................141

mM, REZENDIZ .........................................................81MACAMBIRA, SG .................................................142

MACHADO, GF ......................................................76MACHADO, MM .....................................................68MACHADO, RZ ............... 86,91,103,110,113,114,115MACHUGH, N .................................................95,106MACHUGH, NIALL D..............................................93MACKINNON, KATHRYN M .................................106MACPHERSON, GORDON ....................................50MADDEN, KATHLEEN ......................................... 110MAGE, ROSE G .....................................................56MAGNUSSON, ULF ...............................................89MAIA, ANTONIO AM ..............................................86MAKINS, GD ........................................................105MARANGUNICH, LAURA .......................................69MARANHÃO, ANDRÉA QUEIROZ .........................60MARCOPPIDO, GISELA ......................................138MARIA, AB ..............................................................42MARINHO, M ..........................................................76MARQUES, FERNANDA K ....................................70MARQUES, MARILIA ...........................................137MARTEL, CYRIL J ..................................................77MARTI, ELIANE ....................................................145MARTINEZ, RAMIROA ...........................................81MARTINS FILHO, OLINDO DE ASSIS ....... 49,87,112MARTINS, HELEN RODRIGES ........................... 112MARTINS, MF ......................................................108MARUYAMA, SRC ..........................................60,102MASON, NICOLA J ..............................................142MASSOCO, C .........................................................97MASTER, SHARON S ............................................40MASUDA, A ..........................................................103MASUDA, AOI ...............................................100,101MATEU, ENRIC ....................................................133MATTOS, RT ........................................................103MATUSHIMA, ER ................................................. 118MATYCHAK, MARY B .....................................82,144MAYER, BALÁZS ...................................................43MAYER, NORA ..................................................82,83MAZINI, AM ............................................................76MCCULLOUGH, KENNETH C ..........................88,89MCEWAN, JOHN C ................................................21MCINNES, COLIN ..................................................14MCKEEVER, DECLAN J .........................14,40,53,88MCLAUGHLIN, KRISTIN E ....................................82MCLIMA, ANNA ......................................................70MEDEIROS, CARLA LEITE .............................60,104MEDEIROS, MARIA LÚCIA S ..............................100MEEUSEN, ELS .....................................................25MEEUSEN, ENT ...................................................106MEGID, J ................................................................76MELEIRO, MZ ........................................................97MENEZES, LILIANA B ..........................................136MENTINK-KANE, MARGARET M ..........................42MÉRANT, CATHERINE ..........................................14MERLOT, ELODIE ..................................................81METTENLEITER, THOMAS C .............................143METZE, KONRADIN ............................................141MEULEN, JAN VAN DER .......................................89MEYER, J DE .........................................................90MEYER, ROBERTO .................................70,124,137MIAH, S ..................................................................94MICHALEK, MATTHIAS .......................................129MICHELY, MICHELY ............................................. 110MIGUEL, MARINA P .............................................136MILLER, N ..............................................................15MILLER, NORMAN W ............................................17MILLER, ROBERT D ..............................................57MILLER, ROBERT ..................................................32MILLING, SIMON ...................................................50MILTIADOU, DESPOINA ........................................53MINEO, JOSÉ R .....................................................88MINEO, TIAGO WILSON PATRIARCA ....113,114,115MINKE, JM ......................................................90,144MIRANDA, KARINA LEITE ...................................126

150

MIRET, JORGE .....................................................111MIRKOVITCH, JELENA ........................................145MIYOSHI, ANDERSON ........................................124MOLDAL, T ...........................................................109MOLINA, RAMON ..................................................59MONTAÑO, JAVIER ANTÔNIO BENEVIDES ......104MONTASSIER, HÉLIO J ...................................75,93MONTASSIER, MARIA DE FATIMA S ...............75,93MONTE, LEONARDO GARCIA ............................140MONTEIRO, GER ......................................85,94,102MONTEIRO, JM .............................................. 80,118MORAES, CARINA M ......................................74,125MORAES, GILSON P .............................................70MORAES, JORGE L DA C ...................................101MORAR, DARSHANA ............................................16MORÉ, DANIELA D ....................................85,93,105MORÉ, GASTÓN ..................................................109MOREIRA, NÁDIA DAS DORES .....................93,135MOREIRA, SIMONE MAGELA .............................141MORELLI, JOÃO ....................................................85MORENO, JAVIER ...........................................49,111MORERA, PATRIZIA ..............................................79MORES, NELSON ................................................139MORO, JULIANA ....................................................91MOROZUMI, TAKEYA ............................................59MORRISON, W IVAN .............................................93MORRISON, WI .........................................54,95,106MORTENSEN, SHILA ............................................71MOSGOVOJ, MARINA .........................................138MOURA, MARIA I ...................................................62MOURA-COSTA, LILIA .........................................137MOXLEY, RA ........................................................137MOZGOVOJ, MARINA V .................................67,124MUELLE, Y ........................................................71,91MUILU, M ...............................................................90MULENGA, ALBERT ..............................................42MULLARKY, ISIS K ..............................................106MUNARI, DP ........................................................ 115MUNDO, S .........................................69,126,128,138MURTAUGH, MICHAEL .........................................40MUSSALEM, JS ............................................107,108MUTWIRI, GEORGE K .........................................123MUYLDERMANS, SERGE ..............................43,122MWANGI, DUNCAN M ...........................................93MWANGI, WAITHAKA .......................................14,43

nNAGY, EVA ...........................................................126NAKAGHI, ANDRÉA CH ....................................... 117NAKAJIMA, CHIE .......................................... 100,117NAKATA, LILIANE C .............................................107NARDELLI, CJ ........................................................85NARDONE, ALESSANDRO ............................79,136NASCIMENTO, IVANA .........................................137NEGRÃO-CORRÊA, DÉBORAH .......................... 116NEGREA, AUREL ...................................................16NETTLETON, D ......................................................49NEVES, MARIA FRANCISCA ............................... 110NEW, DD ................................................................80NGANGA, JOSEPH ................................................56NGUGI, D .............................................................106NGUGI, DANIEL ...................................................106NGUYEN, LIEN THI ...............................................71NIELSEN, J ..........................................................134NIELSEN, LARS P .................................................77NIETO, JAVIER .....................................................111NIEWOLD, THEO ...................................................89NIKLANDER-TEERI, V ...........................................90NISHIKADO, HIDETO .......................................... 117NISHIKURA, YUMIKO ..........................................136NIZOLI, LEANDRO Q ...........................................125NOGUEIRA, CARLOS EW .....................................74NORDGREN, R ...............................................90,144

NORIMINE, JUNZO ................................................14NORON, ANTONIO CARLOS F DE ..................... 110NOTTER, DAVID R ..............................................106NUNES, ARYANA D .............................................131

oOESCH, BRUNO ....................................................66OHASH, KAZUHIKO ............................................102OHASHI, KAZUHIKO ............................... 63,100,117OHOTA, MASATO ................................................120OHTA, MASATO .....................................................87OKADA, KOSUKE .............................................63,93OKUMURA, NAOHIKO ...........................................59OLIVEIRA, ALINE F .........................................73,128OLIVEIRA, AP ........................................................75OLIVEIRA, AS ...................................................... 118OLIVEIRA, CJF ......................................................98OLIVEIRA, DIOGO COELHO DE PADUA ............104OLIVEIRA, GERALDO GILENO DE SÁ ................111OLIVEIRA, JULIERME JOSÉ DE .........................127OLIVEIRA, LJ .........................................................80OLIVEIRA, MÁRCIA CS .......................................107OLIVEIRA, MARIA ZD ............................................70OLIVEIRA, PAULO R .............................................70OLIVEIRA, PS ...................................................... 117OLIVEIRA, ROSANE .........................................23,55OLIVEIRA, SERGIO COSTA ................................124OLIVEIRA, T M F DE S ................... 100,101,102,113OLLIER, WER ........................................................50OLLIER, WILLIAM ..................................................50OLSEN, I ..............................................................109OMWANDHO, CHARLES OA...............................120OÑATE, ANGEL A .................................................127ONUMA, MISAO ........ 63,70,93,100,101,102,117,136ONUMA, SAIKI IMAMURA MISAO .......................100OSORIO, FERNANDO A ........................................72OSTACHUK, AGUSTÍN I .................................66,102OSWALD, ISABELLE .............................................50OVEREND, CHRISTOPHER C ............................132

pPACHECO, ANDRÉA ...........................................137PAJUABA, ANA CAM .............................................88PALACIOS, CARLOS ...........................................125PALACIOS, MARIA A ............................................125PALIS, PAULO ........................................................70PALM, ANNA-KARIN ............................................130PALMER, GUY H ....................................................14PALMER, MITCHELL .............................................66PANIAGO, JULIANA DEL GIÚDICE .....................104PANZITTA, SVIOLINI F ..........................................55PAOLICCHI, FERNANDO A ...................................64PAPIEROK, GÉRARD MARIE .........................49,103PARASAR, P ..........................................................80PAREEK, R .............................................................55PARIZI, LUÍS FERNANDO ...................................100PARK, JOO YOUN .................................................62PARK, YONG HO ..............................................31,62PARRA, ZULY E .....................................................57PARREÑO, VIVIANA ................................69,124,138PASCALE, FLORENTINA .......................................94PASCUAL, DAVID W ..............................................64PASSOS, LYGIA M FRICHE.................................141PATARROY O, JOAQUÍN HERNAN ................60,104PATHANIA, UDAY ..................................................16PATRICK, CHARDON ............................................58PATTERSON, JOHN A ...........................................76PATTNAIK, ASIT .....................................................72PAUL, SVEN .........................................................129PAULA, JOF ...........................................................85PAULA, RENATA CRISTINA DE ........................... 116PAULAN, SILVANA C ........................................... 110

PECONICK, ANA PAULA .......................................60PECORA, ANDREA .........................................66,124PEDROZA, KELY C ..............................................109PEIRO, JR ..............................................................76PELLÉ, ROGER .....................................................93PENDE, DANIELA ................................................123PENHA, EM ..........................................................142PERALTA, L ............................................................72PÉREZ, CARLOS ............................................95,139PÉREZ-FILGUEIRA, MARIANO D .......................124PERRI, SHV ...........................................................76PERSSON, E ..........................................................88PESCE, JOHN T.....................................................42PETRY, DEREK ......................................................59PETRY, SANDRINE..............................................129PIEDRAFITA, DM .................................................106PIÑEIRO, MATILDE .............................................133PINELLI-AAVEDRA, A ............................................81PINTARIČ, MAŠA ...................................................96PIRAS, F .................................................................90PODEROSO, TERESA .........................................133PODSCHUN, RAINER .........................................129POHL, PAULA C ...................................................101PONTES-DE-CARVALHO, LAIN ...........................111POPP, TANJA .................................................. 57,119PORTELA, RICARDO W ......................................137PORTIANSKY, ENRIQUE L ..................................120POSSANI, LD .........................................................98POTIER, MARIE-FRÉDÉRIQUE LE .......................97POTTER, AA .........................................................137POULET, H ........................................................71,91POULSEN, KARIN TARP .......................................71PROFES, MARCOS S ............................................65PRUDENCIO, CARLOS ROBERTO..................59,60PUDRITH, CHARLES B .........................................63PUJOLS, JOAN ....................................................133

qQIU, HUA-JI ..........................................................139QU, L ......................................................................49QUEIROZ, NINA MARI GP DE ............................. 110

rR, ALEXANDRE B ................................................135RAEBER, ALEX ......................................................66RAMALINGAM, THIRUMALAI R ............................42RAMAYO, LILIANA G ...........................................128RASPANTINI, LEONILA ESTER R ................108,140RASPANTINI, PAULO CESAR F ..........................140RAUB, RANDEL H................................................144RAVINDRA, PV ....................................................131RAZZAK, ANTHONY ..............................................23REBOUÇAS, MIRIAM F .......................................137RECH, HERBERT ................................................100REECE, JOSHUA J ..............................................120REEDY, STEPHANIE .............................................82REETH, K VAN .......................................................32REGENHARD, PETRA .........................................129REGITANO, LUCIANA CA ....................................107REGO NETO, OB ...................................................85REHDER, J ......................................................90,144REICHHART, JEAN-MARC ....................................33REINER, STEVE ....................................................50REIS, ALEXANDRE BARBOSA .........49,109,112,114RENARD, JEAN-PAUL ...........................................23RESÉNDIZ-SANDOVA, M ......................................97RESTREPO, MARTA ............................................ 110REVILLA, CONCEPCIÓN ........................95,133,139REYNOLDS, JOHN D ............................................87RHIJN, ILDIKO VAN ..........................................41,94RHODES, SG .........................................................66RIBEIRO, DÂMASO P ............................................88

151

RIBEIRO, JM ..........................................................61RIBEIRO, JMC ................................................60,102RIBEIRO, MÚCIO FLÁVIO BARBOSA .................141RIBEIRO-DOS-SANTOS, R .................................142RIBER, U .........................................................71,134RICCA, LG ............................................................108RICHARD, S ..........................................71,90,91,144RICHT, JA ...............................................................32RICHT, JUERGEN ..................................................32RIJSEWIJK, FRANS AM ........................................65RITEAU, BÉATRICE ...............................................94RIVA, PALOMA MARTÍNEZ DE LA ..........95,133,139ROATT, BRUNO MENDES ......................110,114,135ROBERTS, ESTEBAN ...........................................40ROBINSON, CARL .................................................16ROBINSON, N ......................................................106ROBL, J ..................................................................32ROCCHI, MARA ................................................53,80ROCHA, ANDRÉA S R ....................................74,125ROCHA, HUGO GUIEIRO RIBEIRO ...............60,104RODRIGUES, ALINE APARECIDA REZENDE ......59RODRIGUES, MARINA QUADRIO RAPOUSO BRANCO ................................................................60RODRIGUEZ, DANIELA ..................................69,138RODRIGUEZ, NANCY ......................................82,83RODRÍGUEZ-CARREÑO, M PILAR ....................133RODRIGUEZ-ZAS, SANDRA L ..............................23ROEHE, PAULO M .................................................65ROGAN, DR .........................................................137ROHWER, J ....................................................98,145ROMBOUT, JAN .....................................................32ROMERA, ALEJANDRA .......................................125ROOS, TALITA BANDEIRA ....................................77ROQUE-BARREIRA, MARIA-CRISTINA .........73,128ROSSETTI, BEATRIZ F DE MIRANDA SANTOS 103ROSSI, CN ........................................................... 115ROSSI, S .............................................................. 118ROTHWELL, LISA .............................................16,75ROWAN, LA ..........................................................105ROWLAND, RRR ...................................................59RUA-DOMENECH, R DE LA ................................141RUGGLI, NICOLAS ................................................31RUSSELL, GEORGE .............................................77RUTTEN, VICTOR PMG ................ 41,63,94,112,123

sSA, RENATA GUERRA ......................................... 114SAAL MÜLLER, ARMIN..........................................96SADIR, ANA M ......................................................125SÁEZ, DARWIN R ................................................127SAHANA, GOUTAM ...............................................53SAKAI, MONICA ................................................80,99SALGADO, BRENO SOUZA ..................................60SALT, J ............................................................53,124SALVADOR, R ......................................................128SAMARA, SI .........................................................137SANJUAN, CRISTINA DE JUAN ............................41SANTOS, AG ..........................................................77SANTOS, ISABEL KF DE MIRANDA .94,105,113,116SANTOS, MARÍA J DUS .................................67,124SANTOS, MARINA J DUS ......................................66SANTOS, R RIBEIRO DOS .................................. 117SANTOS, RL ..........................................................68SANTOS, ROBERTO RB DOS .....................108,109SANTOS, SILVIA CAROLINE O .................... 109,114SANTOS, WASHINGTON LUIS DOS ....................49SARANDÓN, AURELIA ..........................................83SÁ-ROCHA, LC .................................................... 118SÁ-ROCHA, VM ................................................... 118SATO, HIROKI ........................................................77SAUNDERS, KELLY ...............................................16SAURER, LESLIE ..................................................89SAUTER, KAY-S .....................................................64

SCARPELLI, EDSON M ................................141,143SCARPELLI, KARIME C ...............................141,143SCAVONE, RENATA ............................................135SCHAER, ROBERT ..............................................137SCHARFSTEIN, JULIO ..........................................17SCHAROVSKY, OG ...............................................72SCHIJNS, VIRGIL EJC .........................................131SCHILLER, IRENE .................................................66SCHNEIDER, ZITA .................................................43SCHNITZLEIN, WILLIAM .......................................15SCHUCK, DESIREÉ .............................................139SCHULMAN, NINA .................................................53SCHW, ISABELLE ..................................................94SCOTT, ALISTAIR NJ .............................................75SDINIC, SDINIC .....................................................90SECOMBES, CHRISTOPHER J ............................33SECOR, ERICA ....................................................144SELA, S ..................................................................88SEO, KEUN SEOK ............................................31,62SEVIN, CORINNE ................................................129SEYFFERT, NÚBIA ..............................................140SHANTHALINGAM, SUDARVILI ............................72SHARP, ALANA ......................................................57SHAW, RJ .............................................................107SHEA-DONOHUE, TEREZ .................................. 110SHEIBANI, MOHAMMADT ................................... 118SHEWEN, PAT .......................................................50SHEWEN, PATRICIA E.........................................121SHI, XING-MING ..................................................131SHINKAI, HIROKI ...................................................59SHORT, AD .............................................................50SIFFRIN, EC ...........................................................98SILVA JR, E RAMIRO DA .......................................85SILVA, ALDACILENE S ........................................135SILVA, C .................................................................76SILVA, CARLOS HENRYQUE SOUZA E ........60,104SILVA, DEISE AO ...................................................88SILVA, EDIANE B ............................................62,136SILVA, FERNANDA EK.........................................101SILVA, JS DA ........................................................105SILVA, JS ..........................................61,85,86,98,105SILVA, KETTY F ................................................... 117SILVA, LUCIANA MARIA ........................................69SILVA, MARCO A M................................................62SILVA, SS .............................................................103SILVA, VMG .......................................................... 117SILVA-CAMPA, E ....................................................97SILVEIRA, ACC ..............................................111,142SILVEIRA, ANA CAROLINA TROMPIERI...............91SIMMELINK, BARTJAN .......................................131SINGER, BERNHARD B ................................. 57,119ŠINKORA, MAREK .......................................72,95,96ŠINKOROVÁ, JANA ..........................................95,96SKOVGAARD, KERSTIN ................................71,134SKYBERG, JEROD A .............................................64SMITH, ADRIAN ...........................................21,22,25SMITH, DR ...........................................................137SMITH, JACQUELINE ..........................................131SMITH, L ..............................................................105SMITH, SADIE L .....................................................23SMITS, MARI ..........................................................89SNEKVIK, KEVIN ...................................................72SOARES, MBP .............................................. 117,142SOARES, RODRIGO DOA ................................... 114SOARES, SANDRO G ....................................73,128SOBA, MARINA ....................................................138SOLANO-AGUILAR, GLORIA .............................. 110SOMMER, SANDRA ...............................................63SOMMERS, ALLISON ............................................55SOPP, PAUL ......................................................14,16SORDILLO, LM ......................................................86SORDILLO, LORRAINE .........................................24SORENMO, KARIN U ..........................................142

SØRENSEN, NANNA SKALL ........................120,134SORGINE, MARCOS ...........................................101SOSSAI, SIDIMAR ..........................................60,104SOTO, C .................................................................32SOTO, E .................................................................81SOUZA, ADRIANO LUIS SOARES ...................... 116SOUZA, DANIEL M .............................................. 114SOUZA, GUILHERME ROCHA LINO DE ........60,121SOUZA, JULIANA VITORIANO DE ...................... 112SQUAIELLA, CC ...........................................107,108SREEVATSAN, S....................................................55SRIKUMARAN, S .........................................67,68,72STALKER, A ...........................................................94STANCIOLI, EFB ....................................................68STARKE-BUZETTI, WILMA A ............................... 110STEINBACH, FALKO .............................................33STEMPLER, A .........................................69, 126,138STILZ, JP ................................................................82STOCKMARR, ANDERS ......................................133STOKOL, T ...........................................................143STORSET, A ....................................................95,109STORSET, ANNE K .........................................31,123STRAUSS, CE ......................................................137STUDDERT, MICHAEL J ........................................74STURGILL, DVM TRACY L ....................................83STURGILL, TRACY ................................................82SUAREZ, C ............................................................80SUEIRO, FELIPE AUGUSTO RUIZ .......................91SUMMERFIELD, ARTHUR ....................31,89,94,124SUZUKI, KOHEI .....................................................59SWA, SANDI...........................................................77SWILSON, MARK ...................................................42SZABÓ, MATHIAS P ..............................................85SZABÓ, MATIAS PABLO JUAN ........................... 110

tTAFURI, WASHINGTON LUIZ ............................. 112TAJIMA, MOTOSHI ..............................................136TAJIMA, TOMOKO .................................................70TAKAHASHI, HIDEYUKI ........................................87TALBOT, R ..............................................................56TALBOT, RICHARD T .............................................75TALLMADGE, REBECCA L ......................82,125,144TANAKA, MAIKO ...............................................58,59TARABLA, HD ......................................................137TARACHA, E ........................................................106TARACHA, EVANS L N ..........................................93TASCA, KAREN I ................................................. 110TAVARES, DELFINA ...............................................86TAYLOR, GERALDINE .........................................121TEIFKE, JENS P ..................................................143TEIXEIRA, MAURO MARTINS ............................. 116TEIXEIRA-CARVALHO, ANDRÉA .................126,135TENORIO, S ......................................................... 110THALLER, GEORG ..............................................129THOMAS, JESSE D .............................................130THOMPSON, ROBERT W......................................42THOMSEN, BO ......................................................53TIAN, X CINDY .......................................................23TIAN, ZHI-JUN .....................................................139TIJHAAR, EDWIN J ................................................16TIMMUSK, SIRJE ..............................................73,81TODD, DANIEL .......................................................75TOMAN, M ............................................................143TOMPKINS, DANNIELLE .......................................17TONG, GUANG-ZHI ......................................131,139TONIAZZI, FE .......................................................107TOULEMONDE, C ................................................144TOUSSAINT, MATHILDA JM ................................133TRIGO, GABRIELA ................................................86TRINDADE, CHARLENE NS ................................ 115TRUJILLO, JONATHAN .........................................57TUGGLE, CK ..........................................................49

152

TWOMEY, DF .......................................................141

uUENISHI, HIROHIDE ........................................58,59UNZAGA, JUAN M ...............................................109URBAN JR, JOSEPH F ......................15, 24,120,129USUI, TATSUFUMI .................................................63UTHE, JJ ................................................................49

vVA, MARLENE ISABEL ........................................104VAHLENKAMP, THOMAS W ................................143VALE, VERA .........................................................137VALENTE, FABRÍCIO LUCIANI ...........................104VALENTINA, MARIANI ...........................................58VALENZUELA, JG .............................................60,61VALHEIM, METTE ................................................123VALLADÃO, MARIA LUIZA CR ............................141VANGIMALLA, VANDANA ......................................15VARGAS, AGUEDA PC ..........................................74VASCONCELLOS, FLÁVIA ALEIXO ....................140VASCONCELOS, RO ........................................... 115VAZ JR, ITABAJARA DA SILVA .....................103,129VELOSO, VANJA MARIA ..................................... 112VENA, MARÍA M ..............................................69,138VENDRUSCOLO, PATRÍCIA E.............................128VENTURINI, LUCILA ............................................109VENTURINI, MARÍA C .........................................109VERDONCK, F .......................................................90VERMA, SUBHASH ............................................. 119VERONEZ, VIVIANE APARECIDA ....................... 110VICK, MANDI M ......................................................82VIDOR, T ................................................................77VIDOR, TELMO ....................................................125VIEIRA, DIRSON ..................................................136VIEIRA, RAFAEL COSTA .....................................127VIINALASS, HALDJA .............................................53VILKKI, JOHANNA .................................................53VILLIERS, ETIENNE P DE .....................................93VILORIA, MARLENE ISABEL VARGAS ...............104VINCENT, AMY .......................................................72

VINCENT, ISABELLE E ..........................................31VITAL, WENDEL COURA .......................131,135,140VITORIANO-SOUZA, JULIANA ............................135VIUFF, BIRGITTE ...................................................77VIVAS, ADRIANA B .........................................82,132VIVEIROS, RITA DE CASSIA S ........................... 110VLEESCHAUWER, A DE .......................................32VÖGTLIN, A ..........................................................134VONDERHEIDE, ROBERT H ...............................142VORDERMEIER, H MARTIN ...................66,132,141VORDERMEIER, MARTIN .....................................66

wWADA, MAKOTO ...................................................70WADDINGTON, D ..................................................56WAGNER, BETTINA............................15,17,130,145WAHAB, SHAHIRA ABDEL ....................................68WALKER, ALAN R ................................................106WANG, YF ..............................................................49WANG, YUN-FENG ..............................................131WARKUP, C .....................................................53,124WARR, GREGORY ................................................32WATANABE, SATOKO ....................................87,120WATERS, RAY........................................................66WATSON, DONNA .................................................14WATTEGEDERA, SEAN ...................................14,80WATTRANG, EVA ............................................73,130WEBER, PATRICK .................................................72WEDLOCK, D NEIL ................................................65WEI, X-Q ................................................................77WEISBELITH, L ......................................................88WERLING, D ..............................................53,94,124WERTZ, NANCY ..................................................122WHELAN, AO .........................................................66WIDDISON, STEPHANIE .......................................40WIGDOROVITZ, ANDRÉS ..............................67,134WIGLEY, PAUL ..................................................22,74WIKSTRÖM, FRIDA HASSLUNG .....................73,81WILLIAMS, JOHN L .........................................87,135WILSON, M .................................................15, 42,55WIT, AGNES DE .....................................................89

WITOLA, WILLIAN ...............................................102WOLFSDORF, KAREN E .....................................144WOODS, GREGORY M .......................................139WRIGHT, NICOLA ..................................................16WRIGHT, STEPHEN E ......................................... 116WU, CHING C ........................................................76WU, HUA ..............................................................136WU, ZHIGUANG .....................................................16WYNN, THOMAS ...................................................42

yYAMADA, SHINJI .......................................... 100,117YAMAGE, MAT .......................................................93YANG, XIANGZHONG ...........................................23YASUDA, MASAHIRO ............................................87YOKAYAMA, MAKIKO ..........................................120YOKOMIZO, YUICHI ............................................136YONEDA, MISAKO ................................................77YOUNG, FIONA ......................................................87YOUNG, JOHN .......................................................16YRLID, ULF ............................................................50

zZACHARIAS, FAROUK .................................108,109ZAHA, ARNALDO .................................................139ZAKERI, A ...............................................................83ZAKERI, S ..............................................................83ZANUZZI, CAROLINA N .......................................120ZARLENGA, DANTE ..............................................15ZHAO, SH ...............................................................49ZHAO, YAOFENG ..................................................43ZHENG, J ...............................................................55ZHOU, YAN-JUN ..................................................139ZHU, JIANZHUNG ................................................123ZHU, YAOHONG ....................................................89ZHUANG, YAN .......................................................14ZIMMERMAN, JEFFREY .......................................59ZIMMERMANN, WOLFGANG ......................... 57,119ZUCKERMANN, FEDERICO ..................................15ZURBRIGGEN, ANDREAS ..................................142ZYLBERMAN, VANESA .........................................64

Key words index

key word ......................................................... pages

0-92x to 7x coverage ...................................................57

Aautoimmunity ........................................................ 117A. hebraeum .........................................................102ab CD4+ T cells ....................................................121abomasum ............................................................107abortion ...................................................................80activity ...................................................................120adaptive traits .........................................................88Adipose Tissue .....................................................142adjuvant .................................77,78,108,129,131,133adjuvants ..............................................................134aged........................................................................83aging ................................................................82,144alfalfa ....................................................................125all trans retinoic acid ...............................................89allergy ...................................................................145amblyomma cajennense .............................85,95,102Anaplasma marginale .............................................69anatomy ................................................................ 118anthelminthic ........................................................107antibodies ......................................16,17,115,117,109antibody ...............................................74,91,104,140Antibody Diversity ...................................................53Antibody kinetics ................................................... 115Antibody libraries ....................................................60antigen ..................................................................109Antigen-capture ....................................................141Antigenicity ...........................................................106antigen-presenting cell .........................................133antigens ................................................................ 114ANTIMICROBIAL PEPTIDES ...............................130anti-tick vaccine ....................................................100anti-venom ..............................................................99APCs. .....................................................................95arthritis ..................................................................134Ascaris suum ........................................................ 110atopic dermatitis ...................................................145autoimmunity ..........................................................77

avian infectious bronchitis virus ..............................75avian influenza H9N2 ...........................................131Avidity-Immunoblot .................................................88

BB cell .....................................................................142B cell lymphopenia ...............................................144B cells ................................................................93,96B. microplus .....................................................60,101B220 .......................................................................82Babesia bovis .......................................................104Babesia canis ....................................................... 117bafilomycin ............................................................123Balb/c mice .............................................................77Basophil ..................................................................85B-cell repertorie ......................................................87Beluga .................................................................. 118BHV-1 marker vaccines ........................................126BHV-5 .....................................................................77bighorn ....................................................................68Biological parameters ...........................................105blood .......................................................................41BLS (Brucella Lumazine Synthase) ........................67BLV ..................................................................63,137body condition scores .............................................82BoLA .......................................................................95bone marrow .................................................. 114,142boophilus microplus ..................................101,103,60Boophilus Yolk Pro Cathepsin ..............................101Bovigam® ...............................................................66Bovine .............. 66,79,85,86,95,119,125,126,136,137Bovine antibody ....................................................127Bovine gene ............................................................54bovine genital .........................................................69Bovine herpesvirus 5 ..............................................65Bovine Herpesvirus type 5 ....................................125Bovine Leukemia Virus ...........................................56Bovine Rotavirus ........................................67,70,124Bovine viral diarrhea virus .............................125,136bovines ...................................................................86Broiler ...................................................................132broiler chickens .............................................83Brucella ...................................................................65Brucella abortus ........................................88,126,127

Brucella canis .........................................................70Brucella suis ...........................................................72BTA5 ..................................................................... 119BVDV ......................................................................67

CC. fetus subsp. Venerealis .................................68,69C18 .........................................................................68Calf Diarrhea ........................................................138Calf Model ............................................................138calreticulin .............................................................100camelids ...............................................................141Canarypox vaccines .............................................145cancer immunotherapy .........................................142Candida albicans ..................................................128canine ...................................................................142Canine Distemper .................................................143Canine Distemper Virus ........................................143canine leishmaniasis ............................................ 112Canine Leishmaniasis .......................................... 113Canine monocytic ehrlichiosis ..............................141canine transmissible veneral tumor .................92,141canine visceral leishmaniasis .........................111,114caprine .................................................................. 110carcinoembryonic antigen ................................ 57,119caseous lymphadenitis .........................................137cattle ...................................66,67,69,88,105,123,136Cattle immune response .........................................70CCL3 .................................................................... 117CD molecule ...........................................................15CD molecules .........................................................17CD1 ........................................................................59CD134 ...............................................................71,91CD134+ B cells .......................................................91CD14 ......................................................................16CD14+ ..................................................................123CD16 .............................................................121,123CD163 ..................................................................139CD20 ......................................................................82CD21 .................................................................82,93CD25+Foxp3+ T regulatory cells ............................97CD32 ....................................................................121CD4+ ................................................................. 115,8CD4+ T cells ...........................................................41

154

CD4+CD134+ T cells ..............................................91CD40 ...............................................................82,142CD40 signaling .......................................................63CD79A ....................................................................82CD79a ...............................................................93,96CD79B ....................................................................82CD8+ T cell .............................................................79CD8+ T cells ............................................. 41,115,121cDNA libraries .........................................................97CDR3H ...................................................................53cell apoptosis ..........................................................93cell proliferation ............................................... 63,117cell response ........................................................ 112cell surface molecules .......................................17,57cell-surface antigens ...............................................17Cellular function Evaluation .................................. 119cellular immune response ...........................77,90,145cellular immunity ................................................... 116cement-like proteins ...............................................60CFSE ......................................................................80Chagas disease .................................................... 112chain repertoire .......................................................41chemokine ..............................................................55chemokine rcceptors ..............................................17chemokines ............................. 17,62,98,105,117,133chemokines expression ..........................................68chicken .................................................................120chicken anaemia immunosuppression ...................75chicken type II Interferon ......................................131chimeric ..................................................................64Chlamydophila abortus ...........................................80Chlamydophila psittaci ............................................92chronic infection ......................................................71chronic renal failure ..............................................143circoviruses ............................................................73Class II haplotypes .................................................50Classical MHC Class I ............................................81Classical swine fever virus .....................................97cloning ........................................................68,75,101clotting time ..........................................................103cobalt ......................................................................79Coconut meal .........................................................76colostrum ......................................79,90,124,138,109commercial antibodies ............................................17Comparative Analysis .............................................61comparative immunology ........................................57copaíba oil ............................................................128Copaifera langsdorffii ............................................128copy number variation ............................................58corneal epithelial cells ..........................................135Corynebacterium pseudotuberculosis ...........124,137costimulation ...........................................................57CpG .................................................................90,123CpG oligonucleotide ...............................................94CpG-ODN .............................................................130Cratylia mollis e Caesalpinia piramidalis .............108Crohn’s disease ......................................................55cross-species reactivity ..........................................14Crotalaria spp. ......................................................140CTL antigenic peptides ...........................................93CTL response .......................................................106CTLA-4 ...................................................................50culture .....................................................................95culture filtrate protein 10 .........................................66cutaneous basophilia ..............................................85cutaneous reactions ...............................................86CXCR4 ...................................................................91cynomolgus monkey ...............................................77cytidine deaminase ............................................... 119cytokine ...............................16,68,77,87,89,106,107,Cytokine mRNA expression .................................. 115cytokine receptors ..................................................17cytokine regulation. .................................................59

cytokines ........................ 15,17,74,77,86,110,130,144cytotoxicity ............................................................123

Dd T cells ..................................................................96daignosis ................................................................66defensins .............................................................. 110DEFENSINS .........................................................130Delayed seroconversion ....................................... 115dendrimers ............................................................134dendritic cells ...................59,62,89,97,98,99,124,125development ...........................................................96DH genes ................................................................53diabetes ..................................................................50diagnosis ..................... 111,113,116,126,139,140,141diagnostic assay ...................................................134differentiation ..........................................................96disease ................................................................. 117disease resistance ......................................56,95,124Distempe; .............................................................128DLA ....................................................................50,97DNA cytosine deamination .....................................33DNA vaccine .........................................................127DNA-vaccination ...................................................133Dog ............................................... 70,91,110,135,141Dog Tick..................................................................61Dogs ................................................. 111,113,115,142

eE. coli ......................................................................89E. coli vaccine .......................................................138E2 glicoprotein ......................................................125EHEC ....................................................................138Ehrlichia canis ................................... 71,117,141,142eimerosis .................. 70,74,110,111,113,116,123,137ELISPOT ..............................................................123embryogenesis .....................................................101endothelial ..............................................................65Endothelial cell .......................................................87enterotoxigenic E. coli ............................................90enzootic bovine leukosis .........................................93Enzootic Calcinosis ..............................................120enzymatic immunoassay test ...............................137Eosinophil ...............................................................85epitope profile .........................................................60epitope spreading ...................................................90epitopes ................................................................ 113EqStim® .................................................................83equine .........................................................74,83,144equine MHC class I B2 ...........................................16erythropoiesis ....................................................... 114Escharichia coli .............................................76,75,88experimental infection ............................................111experimental infections .........................................134expression ..............................................................75Expression Variation ...............................................56

fF18 fimbriae ..........................................................132F4 fimbriae ..............................................................90Fasciola hepatica .................................................. 117FedF .....................................................................132feline herpesvirus-1 ..............................................135feline sarcoma ......................................................135ferret .......................................................................77Fetal immunoglobulins ............................................82fetus ......................................................................122FIV ..........................................................................71FIV Gut-associated lymphoid tissue .......................91Flow Cytometric .................................................... 119flow cytometry .........................................................82Fluorescent Body Test ............................................76

Foot ......................................................................121Fowlpox virus ..........................................................92Foxp3 .................................................................62,94Frey methodology .................................................137

gGALT ......................................................................71gamma interferon .................................................109gastrointestinal function ........................................ 110gd T-cells ..............................................................109Gene .......................................................................58gene expression ..............................................86,107Genetic resistance ..................................................53Genetic vaccine ....................................................127Genome ..................................................................54genome assemblies ................................................57genomics ................................................................58giant cells fibrohistiocytoma ..................................135gilt ...........................................................................89globule leukocytes. ...............................................106glucocorticoid ........................................................144glycogen synthase kinase ....................................101GM-CSF .................................................................87goats ....................................................79,95,102,108GP5 ......................................................................139guinea pig ............................................................. 110Guinea pig immune response .................................70

hHaemaphysalis longicornis ...................................100Haemonchus .................................................108,109Haemonchus contortus ...........................106,107,108Haemonchus spp ..................................................107haplotypes ..............................................................53HCDR3 ...................................................................72heat shock ..............................................................79heat shock protein ..................................................64HeLa cells ...............................................................68helminthiasis .........................................................108helminths ..............................................................107hematology ...........................................................141hemoglobinolytic ...................................................108hepatic lipidosis ......................................................79Herpes simplex virus ............................................135Herpesvirus ............................................................77Herpetic stromal keratitis ......................................135Histochemical ........................................................111histology ............................................................... 118histopathology ......................................................135Homology ...............................................................56horse .....................................................................130Horse TickTranscriptomes ......................................61horses ...................................................................145host immune response .........................................103HSP ...................................................................... 118Hsp60 ...................................................................124human ...................................................................120Humoral immune response .............................104,14humoral immunity ...................................................83humoral response ................................................. 112hypervitaminosis D ...............................................120

iIBH ........................................................................145IBVD .......................................................................76Icelnadic horses ....................................................145IDGA .....................................................................137IFN ...........................................................16,82,90,94IFN- ...............................................................79,94,96IFN- Bronchoalveolar lavage cells ..........................83IFN-a ...........................................................73,97,123IFN-g ................................................................80,123

155

IgD ..........................................................................96IgE positive monocytes ...........................................98IgG antibodies ...................................................... 113IgG Isotypes ...........................................................72IgG subclasses .....................................................122IgM ..........................................................................96IgM deficiency .........................................................98IL-10 ........................................................50,81,94,97IL-10 .....................................................................145IL-10homologue ......................................................68IL-12 ...........................................................96,97,123IL-15 .......................................................................16IL-18 ................................................................96,131IL-18 Receptor ......................................................131IL-2 .........................................................................96IL-4 .........................................................................50IL-4 regulation .......................................................145IL6...........................................................................16ileal Peyer’s patch ..................................................87Immune dysregulation ............................................72immune evasion .....................................................65immune gene expression .......................................59immune modulator ..................................................83immune parameters ................................................84immune response ................................................. 110immune system ....................................................120immune tolerance ..................................................80Immunity .............................................. 65,72,110,138Immunoassay ..................................................139,14immunoblot ...........................................................104Immunoblotting .....................................................102Immunocontraception ...........................................120immunodeficiency .................................................144Immunodiagnostic ................................................127Immunodiagnostics .................................................70Immunodominance ...............................................106immunofenotyping ................................................141immunoglobulin allotypes .......................................57immunoglobulin G .................................................103immunohistochemical assay .................................139immunohistochemistry .............................86,91,92,95immunologic testing ..............................................144Immunomodulation ................................77,90,99,126immunomodulator .................................................130immunopathology .................................................143immunophenotyping .............................................131immunopotentiation ..............................................131immunossupressed .............................................. 112immunostimulant ..................................................130Immunosuppression ...................................74,77,143immunosuppressor ............................................... 117immunosupression ...............................................144immunotherapy e chemotherapy. ......................... 112Immuplus ..............................................................131inactivated vaccine .................................................90indoleamine 2,3 dioxygenase .................................80indoleamine 2,3-dioxygenase (IDO) .......................80infection. .................................................................55infectious bronchitis ..............................................131Infectious Bronchitis Virus ......................................76Infectious Bronchitis Vírus (IBV) .............................75INF-g ......................................................................81infiltrating T lymphocytes ........................................92Inflammation ................................. 86,87,117,144,139inflammatory ...........................................................68influenza ............................................................77,83influenza virus .......................................................143Innate immune response ........................................53innate immunity .......................33,55,58,123,130,132innate immunology .................................................71iNOS .......................................................................76Insect bite hypersensitivity ....................................145insect expression system ....................................126

intereukin4 ............................................................120interferon ..............................................................132Interferon (IFN) gamma ........................................131interferon gamma ..............................................71,13Interferona ............................................................136interferon-g ...........................................................121interferon-gamma .................................................123interferon-Species: ruminants .................................41Interferon-t ............................................................136Interleukin (IL) – 12 ...............................................131Interleukin-10 ..........................................................66interleukin-15 ..........................................................77Interleukins .............................................................56intestinal genomics .................................................89intestinal immunity ..................................................90Intracellular pathogens .........................................127inversion of CD4+:CD8+ .......................................142IPP ........................................................................122

Jjacalin ...................................................................128jejunal Peyer’s patch ..............................................87Johne´s disease ................................................55,63

kK-39 ...................................................................... 116killed vaccine ........................................................135Kinetics ................................................................. 113

llactic acid bacteria ................................................132lactose fermentation ...............................................88LAM ........................................................................69lamphocyte ...........................................................143large intestine .........................................................41larval establishment ..............................................106Lawsonia ................................................................72lectin .....................................................................104LEISHIMMUNE VACCINE .................................... 116Leishmania ..............................................113,114,116LEISHMANIA ........................................................ 116Leishmania (Leishmania) chagasi ........................ 114Leishmania antigens .............................................135Leishmania braziliensis ....................................... 114Leishmaniose Visceral Canina ..............................111leptin .......................................................................79Leptospira .............................................................141Leptospirosis ........................................................141leptospirosis ...........................................................77lesions .................................................................. 110leukocyte antigens ..................................................14Leukocyte kinetic ..................................................104leukocyte receptor complex .................................. 119Leukocytes .................................................... 119,136Leukotoxin ..............................................................68Lipoarabinomannan ..............................................126lipopolysaccharide O-antigen-deficient .................126liver ....................................................................... 118LPS. .......................................................................88lymph node ......................................................41,107lymph nodes ...........................................................95Lymphocyte ............................................................80lymphocyte proliferation ..........................................79lymphocytes ..................................82,86,105,108,137lymphocytic proliferation .........................................79lymphoid organs ...................................................123

mM protein .................................................................74M. bovis ................................................................141macrophage .....................................................63,106

Macrophage Activity .............................................132macrophage function ............................................131macrophages ....................................56,62,76,92,128macrophages migration ........................................ 117Malignant Catarrhal Fever .................................68,77mammalian CHO-K1 cells ....................................125Mammalian system .................................................67mammary gland ......................................................89Mammary neoplasias .............................................91Mannheimia haemolytica ........................................68MAP Hsp70 ..........................................................123Maproumea ..........................................................108mare .....................................................................129marsupial ...........................................................57,12mass spectrometry .................................................69mast cells ..............................................................106Mastitis ..........................................54,55,86,87,88,89maternal antibodies ..............................................124maturation ...............................................................96MD-2 .......................................................................64measles virus ..........................................................77Medicago sativa ....................................................125Mesenchymal Stem Cells .....................................142MHC ......................................................53,58,59,120MHC class I chain-related molecules .....................41MHC class II ......................................................... 115MHC class I-restricted CD8+ epitopes .................121Micobacterium bovis ...............................................62microarray ........................................................88,131microarray analysis .................................................55microarrays .............................................................71mitogen .................................................................140mixed leucocyte reactions ......................................98model .....................................................................77monoclonal antibodies ...............14,15,69,95,133,139Monoclonal scFv antibody ......................................76Monocrotaline .......................................................140Monocytes/macrophages .....................................139Monospecific polyclonal antibodies ......................139Montanide ...............................................................78MOS .......................................................................83mouse ...................................................................120mouth disease virus ..............................................121mucosal imprinting ..................................................89mucosal vaccination .............................................132Mustela vison ........................................................128mycobacteria ..........................................................63Mycobacterium .....................................................126Mycobacterium avium paratuberculosis .................67mycobacterium avium ssp. Paratuberculosi ...........64Mycoplasma hyopneumoniae ...............................139Mycoplasma hyosynoviae ....................................134myelopoiesis ......................................................... 114myocarditis .......................................................... 117

nn-alkil ferulates. ....................................................108natural interferon ....................................................94Natural Killer cells ............................................96,123Nelore ...................................................................136Neospora caninum ...............................................109NEOSPORA CANINUM ........................................109Neospora caninum ............................................... 115Neutralizing antibodies .........................................128neutrophils .......................................................79,128nitric oxide ..............................................................62NK ...........................................................................95NK cells .........................................................109,125Non-classical MHC Class I .....................................81Norepinephrine .......................................................82nymphs ...................................................................85

156

oO157:H7 ...............................................................138oestrogen ................................................................80ontogeny ............................................................93,96Ontogeny of immune system ................................ 115Opossum ................................................................57oral immunisation ...................................................90outer membranes ...................................................69oviduct ....................................................................89ovine brucellosis .....................................................64Ovine herpesvirus-2 ...............................................68

pp24..........................................................................71Paratuberculosis ..................................67,69,123,139passive immunity ..................................................109pathogen receptor ................................................ 119pathogen-associated molecular patterns ..............134pathogen-host coevolution ................................... 119Pathogen-host interaction .......................................55Pathology ..............................................................143Pattern recognition receptors .................................53PBMCs ...................................................................83PCR ....................................................16,111,116,120PCV2 ......................................................................90Pedigreed NIAID Rabbits .......................................57Peptide libraries ......................................................60peptides ....................................................61,105,123perforin ...................................................................96Phage display .................................................. 60,113phagocytosis ...........................................................79Phrynops geoffroanus .......................................... 119phylogeny .............................................................122Piaractus mesopotamicus .................................... 118Pichia pastoris ......................................................125pig ...........................................................................90pig - innate immunity - interferon alpha ................133pig acute phase response ....................................134pig host response ...................................................71piglets .....................................................................89pigs .........................................................................72Placenta ..................................................................80plant expression system .......................................125Plant extracts ........................................................137Plasma proteins ....................................................136plasmacytoid dendritic cells ...............................94,97platelet ....................................................................65Platelet activating factor .........................................87Poisonous plant ....................................................140Polymorphism ..................................................58,124Polymorphisms .......................................................55polymorphonuclear leukocytes ............................. 114Porcine Reproductive ........................................59,97postnatal .................................................................87Prebiotic ................................................................132Prednisone ...........................................................144pregnancy ...............................................................81prenatal ...................................................................87prenatal stress ........................................................84prevalence ............................................................137probiotic ......................................................... 110,132Probiotics ................................................................77progesterone ..........................................................80PROINFLAMMATORY CYTOKINES ....................105proliferation ....................................................130,137Propionibacterium acnes .................................76,107Proteases .............................................................108protection ..........................................................72,111protein ...................................................................120protozoan .........................................................56,106PRRSV ...................................................................73PRRSV ..........................................................132,139

PrV ..........................................................................59pseudoafferent lymph duct cannulation ..................41

qQTL .........................................................................54Qtl ...........................................................................56Quantitative trait loci ...............................................56

rr – Mink .................................................................128rabbits ...................................................................120rabies ......................................................................76RAG-1 ................................................................82,93RAG-2 ................................................................82,93rams ........................................................................64RANTES .................................................................16rats .........................................................................84reagents ..................................................................15real time PCR .......................................................107Receptor ............................................................68,73receptors. ................................................................17Recombinant antibody ............................................76recombinant antigen ...............................................62recombinant fowlpox virus ....................................131recombinant nucleoprotein .....................................75recombinant protein .........................................74,125Recombinant proteins ......................................17,126recombinant PRV .................................................139recombinant vaccine ........................................92,125Recombination ......................................................106Regulatiory T cell ....................................................62Regulatory T cells ...................................................67Reproductive performance .....................................72Reproductive tract ..................................................74reservoir ..................................................................71Resistance ..............................................................54Resistance ..............................................................54resistance ...............................................................85resistance ...............................................................88resistance/susceptibility ..........................................59Resistence ............................................................105respiratory disease. ................................................92Respiratory Syn ......................................................59respiratory syndrome ..............................................97respiratory virus ......................................................74review ...................................................................120Rhipicephalus (Boophilus) microplus. ....................61Rhipicephalus appendiculatus ..............................100Rhipicephalus microplus ...........................60,103,104Rhipicephalus sanguineus ................................. 98,11Rhodococcus equi ....................................74,128,129Riphicephalus (Boophilus) microplus ...................104Riphicephalus (Boophilus) microplus ...................100risk ........................................................................137RNA interference ....................................................56RNA vaccine .........................................................127rotavirus ..................................................................89Rotavirus ..............................................................138RT-PCR. ...............................................................143

sS1 .........................................................................131S1 Glycoprotein ......................................................75S19 antigen .........................................................126Saccharomyces cerevisiae .....................................75saliva ....................................................................104salivary gland ........................................................ 117salivary glands ............................................60,61,103Salmonella ..............................................................74Salmonella enterica .........................................74,128sand fly salivary gland extract ..............................135scFv ......................................................................127

Scorpion toxin .........................................................99seasonality ............................................................141seminal plasma .......................................................89serology ................................................................141Sheep .......................................................53,107,108Sialoadhesin ....................................................73,133single chain antibody fragment .............................125single nucleotide polymorphism .............................56skin ...............................................................41,85,86Skin-test ..................................................................66SLA .........................................................................58small intestine .........................................................41SNP ...................................................................54,55Solanum glaucophyllum .......................................120soluble Fc receptor ...............................................121Sows .......................................................................72Speothos venaticus .............................................. 116spleen .............................................................. 41,118spleen cells ...........................................................109ssRNA ...................................................................123stability ....................................................................87stable transduction .................................................64Strangles ................................................................74Streptococcus agalactiae ......................................86Streptococcus equi .................................................74stress .................................................................... 118subcellular vaccines ...............................................64subclass ................................................................ 113subunit vaccine .......................................................69sukcling mice model ...............................................67Superantigen .....................................................62,72swine ............................................90,96,132,133,134swine mycoplasmal pneumonia ............................139synthetic immunogens ..........................................104

tT Cell .....................................................41,57,65,143T lymphocytes ...................................................69,82T regulatory cells ....................................................94Tasmanian devil ....................................................140Taurine ..................................................................103Tax gene .................................................................56T-cell receptor chains .............................................17T-cells ...................................................................106TCR ..............................................................17,54,57Tdt ..........................................................................82Terrestrial animals ................................................ 118tetramers ................................................................16TGF beta ................................................................89TGF- b2 ..................................................................81TGF-b .....................................................................94TGF-beta1 ............................................................145TH1 .......................................................................129Th1 cytokines .........................................................82Th2 cytokines ....................................................81,82thap.......................................................................101Theileria ................................................................106Theileria parva .....................................................93,1therapeutics ............................................................87thrombus .................................................................65thymus. ................................................................. 118Tick ............................................... 54,86,101,105,117tick antigens .....................................................86,103Tick infestation ......................................................105Ticks ..................................................................85,98tissue parasite load ............................................... 114TLR ....................................................................53,58TLR2 .......................................................................64TLR4 .......................................................................64TLR7 ................................................................76,123TLR8 .....................................................................123TLR9 .......................................................................90TNF ....................................................................16,82

157

TNF- .......................................................................97TNF-a ..............................................................63,123TNF-receptors ........................................................63Toll Like Receptors ..........................................56,135toll-like receptor ............................95,98,106,124,134tool kit .....................................................................16toxic plants ..............................................................99transcriptome ....................................56,59,60,75,103transfected mouse P815 cells ...............................81transferrins ..............................................................92transmissible .........................................................140Trasfectant .............................................................73triosephosphate isomerase ..................................101Triton X-114 ............................................................88tropical countries. .................................................141Trout ..................................................................... 118Trypanosoma cruzi .........................................111,115Trypanosomosis .....................................................56tuberculin early secretory antigenic target 6 ...........66Tuberculosis .............................................66,140,141

Tumor Necrosis Factor Receptor I..........................93Tumor Necrosis Factor Receptor II.........................93tumour ..................................................................140type I IFN ...........................................................94,13

uup-regulation .........................................................131uterine mononuclear cells .......................................81

vVaccination ...........................................................128vaccination ..............................................................64vaccine .......................................................................60,61,66,67,77,78,100,101,104,105,116,123,126,127,133,134,135,vaccine DNA .........................................................101vaccines ......................................................70,74,128VapA protein ....................................................74,128vasculitis .................................................................65

viraemia ................................................................143viral IL-10 ................................................................68virginamycine ..........................................................83virion host shutoff ...................................................65virulence ................................................................111virus ........................................................................72virus restriction .......................................................33Visceral Leishmaniasis ...................................113,116Vitamin E ................................................................81VP8 .........................................................................67

wWest Nile Virus .....................................................145wild felids .............................................................. 117wound repair .........................................................128

zZebuine .................................................................103

All .................................................................................................................................................................................. 14 All species ................................................................................................................................................................ 16,78Avian ............................................................................................................................... 17,74,75,76,83,92,120,131,132Bovines ......................................................................................................................................................................... 62Canine ...........................................................................................................50,57,70,71,91,92,97,111-116,135,141-143Dog, guinea pig ............................................................................................................................................................110Domestic horse (Equus caballus) ................................................................................................................................. 14Equine ............................................................................................................... 16,17,74,82,83,98,128,129,130,144,145Feline ...................................................................................................................................................71,91,117,135,143Fish .........................................................................................................................................................................17,118Human and ferret .......................................................................................................................................................... 77Llama (south american camelid) ................................................................................................................................. 141Marsupial ..................................................................................................................................................................... 120Mice ................................................................................................................................................................... 56,77,128Monkeys ........................................................................................................................................................................ 78Other ..................................................................... 15,57,60,61,76,77,98,99,116,117,119,120,126,127,128,133,140,141Rabbit ................................................................................................................................................................ 57,77,120Rat .......................................................................................................................................................................... 84,140Reptile (turtle) ...............................................................................................................................................................119Ruminant ....................17,33,41,53-56,62-70,79,80,81,85-88,93,94,95,100-110,121-127,136-139,108,109,119,136,137Swine ....................................................................15-17,58,59,71-73,81,82,89,90,95-97,110,122,132,133,134,139,139Tasmanian devil (Sarcophilus harrisii) ......................................................................................................................... 140

Species index

List of participants

familyname,firstname.................Department,institute-Country.................................................................................................................................................................email

AAbel, Lucia C.J. ................................. Immunology, Universidade Paulista - Brazil ........................................................................................................................ luciaabel@uol.com.brAdams, Amanda ................................Department of Veterinary, Maxwell H. Gluck Equine Research Center - USA .............................................................. amanda.adams@uky.eduAguiar, Paulo .....................................Departamento de Patologia e Clínicas, Escola de Medicina Veterinária - UFBA - Brazil................................................................ phpa@ufba.brAlcala-Canto, Yazmin ........................Parasitologia, Univ. Nacional Autonoma de Mexico - Mexico .....................................................................................yazmin@servidor.unam.mxAlmeida, Denise ................................Animal Science, Michigan State University - USA.................................................................................................................. almeidad@msu.eduAlonso, Fernando ..............................Biotechnology, INIA - Spain .......................................................................................................................................................... falonso@inia.esAlves Dummer, Luana .......................Centro de Biotecnologia, Universidade Federal de Pelotas - Brazil......................................................................................dummer@gmail.comAlves, Telma Maria ............................Preventive Veterary Medicine, Escola de Veterinária - Brazil ......................................................................................... telmavet@yahoo.com.brAmadori, Massimo ............................. Italy ......................................................................................................................................................................................... mamadori@bs.izs.itAmaral De Castro, Luiza ...................UFRGS, Centro de Biotecnologia - Brazil .............................................................................................................................. luiza@cbiot.ufrgs.brAmoril, José .......................................LANAGRO/GO, MAPA - Brazil .............................................................................................................................. josegabriel@agricultura.gov.brAnatriello, Elen ..................................Basic and Applied Imunologia, College of Medicine of Ribeirão Preto - Brazil ..................................................................................elen@usp.brAndrade, Caroline ..............................Centro de Biotecnologia, UFRGS - Brazil .................................................................................................................carolandrade@cbiot.ufrgs.brAndré, Marcos Rogério ......................Veterinary Pathology, UN of Pennsylvania - USA ..............................................................................................................dartis@vet.upenn.eduAscarateil, Stephane ........................Developement, SEPPIC - France ......................................................................................................................sebastien.deville@airliquide.comAsgarali, Zinora .................................veterinary school, university of the west indies - Trinidad and Tobago .................................................................................zinoraa@yahoo.comAyoola, Emmanuel Akinniyi ...............Animal Husbandry, Osun State Development Project - Nigeria ................................................................................. akinniyiayoola@yahoo.com

BBaldwin, Cynthia ................................Veterinary and Animal Sciences, University of Massachusetts - USA........................................................................cbaldwin@vasci.umass.eduBallingall, Keith ..................................Epidemiology and population Biology, Moredun Research Institute - UK ............................................................ keith.ballingall@moredun.ac.ukBannerman, Douglas .........................ARS - Bovine Functional Genomics Lab, United States Department of Agriculture - USA ......................................dbanner@anri.barc.usda.govBarbosa Reis, Alexandre ...................Análises Clínicas, Escola de Farmácia - Brazil ................................................................................................................alexreis@nupeb.ufop.brBarçante, Joziana ..............................Parasitologia, - Brazil ...................................................................................................................................................... jozithales@hotmail.comBarreto, Maria ....................................Universidade Federal Fluminense, Instituto de Biologia - Brazil .........................................................................................mlbarreto@gmail.comBastos, Reginaldo .............................Veterinary Microbiology and Pathology, Washington State University - USA ............................................................. rgbastos@vetmed.wsu.eduBaxter, Rebecca ................................Genetics and Genomics, Roslin Institute - UK ......................................................................................................... rebecca.baxter@bbsrc.ac.ukBechara, Gervasio H. ........................Patologia Veterinaria, Fac. Cienc. Agr. Vet. - UNESP - Brazil .......................................................................................... bechara@fcav.unesp.brBelkaid, Yasmine ...............................LPD, NIAID - USA ............................................................................................................................................................. ybelkaid@niaid.nih.govBellido, Demian ................................. Instituto de Virologia INTA Castelar - Argentina ...........................................................................................................demianbellido@gmail.comBent, Aasted ......................................Department of Veterinary Pathobiology, Faculty of LIFE Sciences - Denmark .............................................................................. bas@life.ku.dkBergman, Ingrid-Maria .......................Biomedicine, School of Pure and Applied Natural Sci - Sweden ............................................................................ ingrid-maria.bergman@hik.seBertho, Nicolas ..................................Virologie, INRA - France.............................................................................................................................................. nicolas.bertho@jouy.inra.frBilej, Martin ........................................Department of Immunology, Institute of Microbiology ASCR - Czech Republic .................................................................mbilej@biomed.cas.czBlum, Eduardo (Shlomo) ...................Clinical Bacteriology, Kimron Veterinary Institute - Israel ........................................................................................................... blums@012.net.ilBochoeyer, Diego ..............................R+D, Biogenesis Bago - Argentina ..........................................................................................................diego.bochoeyer@biogenesisbago.comBohach, Gregory ...............................MMBB, University of Idaho - USA ..................................................................................................................... gbohach@uidaho.eduBoysen, Preben .................................Dept for Animal Health, National Veterinary Institute - Norway ..................................................................preben.boysen@vetinst.noBreathnach, Cormac ..........................Veterinary Science, University of Kentucky - USA ....................................................................................................cormac@uky.eduBroere, Femke ................................... Infectious Disease & Immunology, Fac. Veterinary Medicine, Uni. Utrecht - Netherlands ....................................... f.hauet@vet.uu.nlBrown, Wendy ...................................Veterinary Microbiology and Pathology, Washington State University - USA ..............................................wbrown@vetmed.wsu.eduBruhn, Oliver ......................................Agricultural and Nutritional Sciences, Institute of Animal Breeding - Germany ....................................... obruhn@tierzucht.uni-kiel.de

160

Buddle, Bryce ....................................AgResearch, Hopkirk Research Institute - New Zealand .................................................................. bryce.buddle@agresearch.co.nzButler, John ........................................Microbiology, University of Iowa - USA ............................................................................................................. john-butler@uiowa.eduButtke, Danielle .................................The Baker Institute for Animal Health, Cornell College of Veterinary Medicine - USA ...........................................db334@cornell.eduBuza, Joram ......................................Vaccine and Infectious Disease Organizat - Canada ........................................................................................ joram.buza@usask.caBuzetti, Wilma ....................................Biologia e Zootecnia, University of São Paulo State-UNESP - Brazil ........................................................... starke@bio.feis.unesp.br

CCabrera, Alex .....................................Molec. Immun. lab, Dept. Microbiology, Universidad de Concepción - Chile ...........................................................acabrera@udec.clCaetano, Alexandre ...........................NTBIO, Embrapa - CENARGEN - Brazil .......................................................................................... acaetano@cenargen.embrapa.brCamargo, Maristela ........................... Immunology, Institute of Biomedical Sciences - Brazil ............................................................................................ mmcamar@usp.brCançado, Guilherme .......................... Imunologia Celular e Molecular, Centro de Pesquisas René Rachou - Brazil ......................................guilhermegrossi@terra.com.brCaperucci, Débora .............................Brazil .......................................................................................................................................................................debio@rc.unesp.brCarvalho, Gabriel ...............................Veterinary Department, Federal University of Vicosa - Brazil ............................................................... gabrielmedvet@yahoo.com.brCarvalho, Wanessa ........................... Immunology and Biochemistry, FMRP/USP - Brazil .........................................................................................wanac30@hotmail.comCassin Duz, Ana Luiza ......................Laboratório de Imunopatologia, Universidade Federal de Ouro Preto - Brazil...............................................anacd07@yahoo.com.brCauchard, Julien ................................Microbiology Immunology, French Food Safety Agency - France ............................................................. j.cauchard@dozule.afssa.frChattha, Kuldeep S. ..........................Pathobiology, OVC, University of Guelph - Canada ......................................................................................... kchattha@uoguelph.caCissell, Robin .....................................Comparative & Experimental Medicine, University of Tennessee - USA .....................................................................rcissell@utk.eduCoad, Michael ....................................TB Research Group, Veterinary Laboratories Agency - UK ....................................................................m.coad@vla.defra.gsi.gov.ukCoffey, Tracey .................................... Immunology, Institute for Animal Health - UK .............................................................................................. tracey.coffey@bbsrc.ac.ukColavecchia, Silvia ............................ Inmunología, Fac. Ciencias Veterinarias. UBA - Argentina ............................................................................. scolave@yahoo.com.arConnelley, Timothy ............................Dept of Veterinary Clinical Science, University of Edinburgh - UK ............................................................... s0239639@sms.ed.ac.ukCottorello, Ana Claudia ......................Preventive Veterinary Medicine, Veterinary School - Brazil .......................................................................... acottorello@hotmail.comCox, Eric ............................................Laboratory of Immunology, UGent, Faculty of Veterinary Medicine - Belgium ........................................................ eric.cox@ugent.beCrawford, Allan ..................................Animal Genomics, AgResearch - New Zealand .............................................................................. allan.crawford@agresearch.co.nzCruz, Ana Paula Rottini .....................FABIO, PUCRS - Brazil ..................................................................................................................................paularottini@pop.com.brCuisinier, Anne Marie .........................R&D, Virbac Laboratories - France ...........................................................................................................................acuisini@virbac.frCunha, Silvia .....................................Departamento de Palotogia, FMVZ - Universidade de São Paulo - Brazil ..................................................silvialearth@yahoo.com.brCzuprynski, Charles ..........................Pathobiological Sciences, University of Wisconsin - USA .................................................................czuprync@svm.vetmed.wisc.edu

DDa Silva Vaz Junior, Itabajara ............Centro de Biotecnologia, Universidade Federal do Rio Grande do Su - Brazil ......................................................... ita@cbiot.ufrgs.brDalloul, Rami .....................................Animal & Poultry Sciences, Virginia Tech - USA .......................................................................................................... rdalloul@vt.eduDavies, Christopher ...........................Veterinary Microbiology and Pathology, Washington State University - USA .............................................. cdavies@vetmed.wsu.eduDávila, Alberto ...................................Oswaldo Cruz Institute, FIOCRUZ, - Brazil ..........................................................................................................davila@ioc.fiocruz.brDawson, Harry ...................................NRFL, BHNRC, ARS-USDA - USA...........................................................................................................harry.dawson@ars.usda.govDe Medeiros Costa, Gerlane .............University of São Paulo - Brazil ............................................................................................................ gerlanemcosta@yahoo.com.brDe Miranda Santos, Isabel K. F. ........Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto - Brazil................................................... imsantos@fmrp.usp.brDe Paula, Renata ..............................Parasitologia, ICB-UFMG - Brazil ...........................................................................................................renatacpaula@yahoo.com.brDeeg, Cornelia ................................... Institute of Animal Physiology - Germany .................................................................................. deeg@tiph.vetmed.uni-muenchen.deDegen, Winfried .................................Vaccine Technology and Immunology R&D, Intervet International BV - Netherlands ............................ winfried.degen@intervet.comDenapoli, Priscila ............................... Imunology, Universidade Paulista - Brazil ............................................................................................. prisciladenapoli@hotmail.comDenkers, Eric .....................................Microbiology and Immunology, Cornell University - USA ......................................................................................... eyd1@cornell.eduDevlin, Joanne ...................................The School of Veterinary Science, - Australia ................................................................................................devlinj@unimelb.edu.auDian De O. Aguiar Soares, Rodrigo ...Núcleo de Pesquisa em C.Biológicas, UFOP - Brazil ................................................................................... sapodian@yahoo.com.brDominguez, Javier .............................Biotechnology, INIA - Spain ............................................................................................................................................juncal@inia.esDoyle, Sarah ......................................School of Biochemistry and Immunology, Trinity College Dublin - Ireland .....................................................................doylesl@tcd.ie

eEdfors-Lilja, Inger ..............................School of Pure and Applied Sciences, University of Kalmar - Sweden ........................................................... inger.edfors-lilja@hik.seEguchi-Ogawa, Tomoko ....................Animal Genome Research Unit, NIAS - Japan ....................................................................................................egutomo@affrc.go.jpEicher, Susan ....................................USDA-ARS-MWA-LBRU - USA ........................................................................................................................... spruiett@purdue.eduEkman, Anna .....................................department of basic veterinary medicine, University of Helsinki - Finland ......................................................anna.ekman@helsinki.fiEldaghayes, Ibrahim ..........................Microbiology and Parasitology, Faculty of Veterinary Medicine - Libya ...................................................... ibrahim1971@hotmail.comEllis, Shirley ....................................... Immunology Division, Institute for Animal Health - UK ...................................................................................shirley.ellis@bbsrc.ac.ukEntrican, Gary ....................................Bacteriology, Moredun Research Institute - UK .................................................................................... gary.entrican@moredun.ac.ukEsfandiari, Javan ...............................R&D, Chembio Diagnostic Systems, Inc. - USA...........................................................................................jesfandiari@chembio.comEspino, Gerardo Pavel ......................Medicina Molecular y Bioprocesos, Instituto de Biotecnologia - UNAM - Mexico ............................................... espino@ibt.unam.mxEstein, Silvia Marcela ........................SAMP, F.C.V., U.N.C.P.B.A - Argentina .................................................................................................... silmares@vet.unicen.edu.arEstes, Mark ........................................Pediatrics and Microbiology and Immunol., University of Texas Medical Branch - USA .......................................dmestes@utmb.edu

fFarias, Sandra Estrazulas .................Centro de Biotecnologia, UFRGS, Biociências - Brazil ......................................................................................sandra@cbiot.ufrgs.brFattori, Karina ....................................Brazil ........................................................................................................................................................... karinafattori@hotmail.comFerreira, Beatriz .................................MISP, Escola de Enfermagem - USP - Brazil ............................................................................................................... brferrei@usp.brFerreira, Carlos A. Sanchez ..............Biologia Celular e Molecular, FaBio - Brazil ............................................................................................................ cferreira@pucrs.brFirth, Matt ..........................................Pathobiology, University of Guelph - Canada........................................................................................................mfirth@uoguelph.caFiuza, Jacqueline Araújo ...................Laboratório de Imunologia Celular e Mol, Centro de Pesquisas René Rachou - Brazil ............................ jacqueline@cpqrr.fiocruz.brFlaminio, M. Julia ...............................Clinical Sciences, Cornell University - USA..............................................................................................................mbf6@cornell.edu

161

Flynn, Joanne ....................................Molecular Genetics and Biochemistry, W1157 BSTWR - USA .................................................................................... joanne@pitt.eduFontana, Paula ..................................Preclinic Sciences, Pathology - Argentina .................................................................................................... pfontana@fcv.unlp.edu.arFossum, Caroline ..............................Veterinary Immunology, Swedish University of Agricultural Scien - Sweden ............................................ caroline.fossum@bvf.slu.seFranchini, Marco ................................ Immunology, Veterinary Virology - Switzerland ..................................................................................marco.franchini@vetvir.unizh.chFranco Mahecha, Olga Lucia ............Colombia ................................................................................................................................................olgaluciafrancom@gmail.comFranzin, Alessandra ...........................Biochemistry and immunology, School of Medicine of Ribeirão Preto-USP - Brazil ................................................alefranzin@usp.brFujiwara, Ricardo ...............................Cellular and Molecular Immunology, IRR/FIOCRUZ - Brazil ......................................................................... fujiwara@cpqrr.fiocruz.brFurlan, Luiz ........................................Animal Breeding and Nutrition, FMVZ-UNESP - Brazil ........................................................................................cedral@fca.unesp.br

gGallo, Guillermo .................................Pfizer Animal Health - USA ........................................................................................................................guillermo.gallo@pfizer.comGama Ker, Henrique ..........................Núcleo em Pesquisa em C. Biológicas, UFOP - Brazil ......................................................................................... hgker@hotmail.comGandy, Jeffery ...................................Large animal clinical science, Michigan State University - USA.................................................................................gandy@msu.eduGazzinelli, Ricardo Tostes ................. Imunology, Institute René Rachou - Brazil ........................................................................................................ritoga@cpqrr.fiocruz.brGenoy Puerto, Alexander ..................Pathology, Veterinary - Brazil .........................................................................................................................alexandergenoy@usp.brGerdts, Volker ....................................Vaccine & Infectious Disease Organizatio, - Canada .....................................................................................volker.gerdts@usask.caGerner, Wilhelm .................................Universtiy of Veterinary Medicine Vienna, Clinical Immunology - Austria .............................................wilhelm.gerner@vu-wien.ac.atGhvinjilia, Gia ....................................Risk mang. and Comunication Division, Food Saf. Vet. and Plant Prot. Nat.Serv. - Georgia ....................... dr_gia_vet@hotmail.comGibertoni, Aliandra .............................Microbiology, UNESP - Brazil ........................................................................................................................... aligiber@fcav.unesp.brGiese, Matthias ..................................Vaccine Development, Fraunhofer Institute for Cell Therapy an - Germany .....................................matthias.giese@izi.fraunhofer.deGinêz, Karen De O. Serrador ............Universidade Metodista de São Paulo - Brazil ............................................................................................. ka_biovet@yahoo.com.brGiunchetti, Rodolfo ............................NUPEB, ICEB - Brazil ..................................................................................................................................giunchetti@nupeb.ufop.brGlass, Liz ...........................................Genetics and Genomics, Roslin Institute - UK ................................................................................................... liz.glass@bbsrc.ac.ukGoff, Will ............................................Animal Disease Research Unit, USDA-ARS - USA.........................................................................................wgoff@vetmed.wsu.eduGonçalves De Souza, Fabiane ..........Programa de Mestrado Ciência Animal, Univ. Federal de Mato Grosso do Sul - Brazil ................................. fabicientista@gmail.comGonzalez, Diego ................................ Instituto de Virología, CICVyA, INTA - Argentina .......................................................................................dgonzalez@cnia.inta.gov.arGoubier, Anne ....................................R&D, MERIAL - France ...............................................................................................................................anne.goubier@merial.comGraham, Simon .................................Virology, Veterinary Laboratories Agency - UK ....................................................................................s.graham@vla.defra.gsi.gov.ukGreco, Cecilia Rosa ........................... Inmunology, Facultad de Ciencias Exactas - Argentina .................................................................................cgreco@exa.unrc.edu.arGriebel, Philip ....................................Vaccine and Infectious Disease Organizat - Canada ...................................................................................... philip.griebel@usask.caGual P. De Queiroz, Nina Marí ..........Biologia e Zootecnia, Universidade Estadual Paulista - UNESP - Brazil ....................................................... nina_gual@hotmail.comGuedes, Maria ...................................Analises, Faculdade de Farmácia. UFBa - Brazil.............................................................................................. tipameri@terra.com.brGuerra, Luanda .................................Lab. de Imunologia Celular e Mole, Centro de Pesq. René Rachou-FIOCRUZ - Brazil ................................. luanda@cpqrr.fiocruz.brGuimarães, Andréa ............................ Imunologia Celular e Molecular, Centro de Pesquisas René Rachou - Brazil ............................................. andrearizia@hotmail.comGuimarães, Fernanda Fidelis G. ....... Immunology, Institute of Biomedical Sciences - Brazil .................................................................................zezao_70@yahoo.com.brGuzman, Efrain ..................................Detp. of Immunology, Institute for Animal Health - UK ............................................................................. efrain.guzman@bbsrc.ac.ukGuzman, Ingrid ..................................Microbiology, Universidad de Concepcion - Chile .................................................................................................... iguzman@udec.cl

hHagiwara, Katsuro .............................Veterinary Medicine, Rakuno Gakuen University - Japan .................................................................................... k-hagi@rakuno.ac.jpHaig, David ........................................Division of Virology, Moredun Research Institute - UK .............................................................................. david.haig@moredun.ac.ukHamza, Eman .................................... Immunology, Institute of Veterinary Virology - Switzerland ........................................................................eman.hamza@ivv.unibe.chHarris, Jim .........................................Clinical Medicine, Trinity Centre for Health Sciences - Ireland...................................................................................... jaharris@tcd.ieHarris, Reuben ..................................Dept of Biochemistry, University of Minnesota - USA ..................................................................................................... rsh@umn.eduHastings, Nicola .................................Genetics and Genomics, Roslin Institute - UK ........................................................................................ nicola.hastings@bbsrc.ac.ukHeegaard, Peter ................................Veterinary Diagnostics and Research, National Veterinary Institute - Denmark .......................................................pmhh@vet.dtu.dkHein, Wayne ......................................Animal Health, AgResearch - New Zealand .........................................................................................wayne.hein@agresearch.co.nzHernández, Jesús ..............................Mexico .......................................................................................................................................................................... jhdez@ciad.mxHerndon, Caroline .............................Veterinary Microbiology and Pathology, Washington State University - USA ........................................... cherndon@vetmed.wsu.eduHickstein, Dennis ...............................Experimental Transplantation, National Cancer Institute - USA ........................................................................ hicksted@mail.nih.govHoek, Aad .......................................... Immunology, Veterinary Faculty, Utrecht University - Netherlands ........................................................................... a.hoek@vet.uu.nlHolland, Olivia ...................................Obst. and Gynaecology/Molecular Eco, Univ. of Auckland/Landcare Res. - New Zealand ............ hollando@landcareresearch.co.nzHope, Jayne ...................................... Immunology, Institute for Animal Health - UK ................................................................................................ jayne.hope@bbsrc.ac.ukHovde, Carolyn ..................................MMBB, University of Idaho - USA ...................................................................................................................... cbohach@uidaho.eduHueza, Isis .........................................Department of Pathology, FMVZ/USP - Brazil ...............................................................................................................hueza@usp.br

iIbelli, Adriana .....................................Federal University of São Carlos - Brazil ......................................................................................................adriana.ibelli@gmail.comIivanainen, Antti .................................Department of Basic Veterinary Sciences, University of Helsinki - Finland.................................................antti.iivanainen@helsinki.fiImamura, Saiki ................................... Infectious Diseases, Hokkaido University - Japan ................................................................................. saiki-i@vetmed.hokudai.ac.jpInnes, Elisabeth .................................Parasitology, Moredun Research Institute - UK .......................................................................................... lee.innes@moredun.ac.ukInumaru, Shigeki ................................Research Team for Advanced Biologicals, National Institute of Animal Health - Japan ........................................inumaru@affrc.go.jpIraqi, Fuad .........................................Human Microbiology, Tel-Aviv University - Israel .................................................................................................. fuadi@post.tau.ac.il

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JJaso-Friedmann, Liliana .................... Infectious Diseases, University of Georgia - USA ................................................................................................... ljaso@vet.uga.eduJesse, Thomas .................................. Innovative Disease Control, CSIRO - AAHL - Australia ....................................................................................jesse.thomas@csiro.auJiwakanon, Jatesada .........................Clinical Sciences, SLU - Sweden .......................................................................................................... jatesada.jiwakanon@kv.slu.seJohnson, Eugene H. ..........................Animal and Veterinary Sciences, Sultan Qaboos University - Oman ............................................................... ejohnson@squ.edu.omJohnston, Claire .................................Veterinary Pathology, University of Liverpool - UK ............................................................................................ c.e.johnston@liv.ac.ukJolly, Ana ...........................................Argentina ..............................................................................................................................................................anajolly@yahoo.comJuillard, Veronique .............................R&D, MERIAL - France .........................................................................................................................veronique.juillard@merial.comJungersen, Gregers ...........................Veterinary Diagnostics and Research, National Veterinary Institute - Denmark ........................................................... gju@vet.dtu.dkJungi, Thomas ................................... Institute of Veterinary Virology - Switzerland .............................................................................................. thomas.jungi@ivv.unibe.chJunqueira, Danilo ...............................Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia - Brazil ..................................dan_hp2002@yahoo.com.brJunqueira-Kipnis, Ana Paula ............. Immunology, Federal University of Goias - Brazil ............................................................................................ ana.kipnis@pq.cnpq.brJuppa, Ana .........................................Brazil ........................................................................................................................................................ana.crissiuma@terra.com.brJutila, Mark ........................................Veterinary Molecular Biology, Montana State University - USA .......................................................................... uvsmj@montana.edu

kKacskovics, Imre ...............................Department of Immunology, Institute of Biology, Faculty of Science - Hungary ..........................................................ikacsko@elte.huKai, Chieko ........................................ Institute of Medical Science, The University of Tokyo - Japan ......................................................................... ckai@ims.u-tokyo.ac.jpKammerer, Robert .............................Tumor Immunology Laboratory, LIFE Center, LMU Munich - Germany ................................... robert.kammerer@med.uni-muenchenKano, Rika .........................................Graduate School of Veterinary Medicine, Hokkaido University - Japan ................................................. rkano@vetmed.hokudai.ac.jpKatzer, Frank .....................................Moredun Research Institute, Pentland Science Park - UK ............................................................................... frank.katzer@ed.ac.ukKaufman, Jim ...................................... Immunology, Institute for Animal Health - UK ...............................................................................................jim.kaufman@bbsrc.ac.uk Kaushik, Azad ....................................Molecular and Cellular Biology, University of Guelph - Canada .......................................................................akaushik@uoguelph.caKeila, Coelho .....................................Brazil ............................................................................................................................................................... keilavet@yahoo.com.brKemp, Joanna ...................................Physiology, Monash University - Australia.................................................................................... joanna.kemp@med.monash.edu.auKerr, David .........................................Animal Science, University of Vermont - USA ......................................................................................................david.kerr@uvm.eduKfoury Jr., J. Roberto .........................Anatomy, Faculdade de Medicina Veterinária e Zoot - Brazil .............................................................................robertok@fmvz.usp.brKohara, Junko ...................................Animal Research Center of Hokkaido - Japan .................................................................................... koharajn@agri.pref.hokkaido.jpKohler, Katharina ...............................Veterinary Science, 108 Gluck Equine Research Center - USA ....................................................................... kkohl0@email.uky.eduKonnai, Satoru ...................................Graduate School of Veterinary Medicine, Hokkaido University - Japan ................................................konnai@vetmed.hokudai.ac.jpKreiss, Alexandre ...............................Pathology, Menzies Institute-University of Tasmania - Australia ......................................................................... akreiss@utas.edu.auKruger, Ernesto ..................................departamento de antígenos, instituto de tecnologia do paraná - Brazil ...................................................... errekruger@yahoo.com.brKuntz-Simon, Gaelle ..........................Swine Virology Immunology Unit, AFSSA-LERAPP - France ......................................................................... g.kuntz.simon@afssa.fr

lLa Cava, Antonio ................................Medicine, UCLA - USA ............................................................................................................................... alacava@mednet.ucla.eduLacetera, Nicola .................................Produzioni Animali, - Italy ........................................................................................................................................2_nicgio@unitus.itLacetera, Nicola .................................Produzioni Animali, - Italy ............................................................................................................................................nicgio@unitus.itLarangeira, Daniela ...........................LIVE, UFBA - Brazil .......................................................................................................................... danielalarangeira@yahoo.com.brLatorre, Andreia Oliveira ....................Pathology, Faculty of Veterinary Medicine - Brazil ...................................................................................................... alatorre@usp.brLavoie, Jean-Pierre ...........................Département de Sciences Cliniques, Faculté de Médecine Vétérinaire - Canada............................ jean-pierre.lavoie@umontreal.caLeibold, Wolfgang ..............................University of Veterinary Medicine, Immunology - Germany ...............................................................................wldoma@hotmail.comLeite, Fabio ........................................Microbiology and parasitology, Federal University of Pelotas - Brazil ........................................................... fabio_leite@ufpel.tche.brLeitner, Gabriel ..................................National Mastitus, Kimron Veterinary Institute - Israel ........................................................................................ leitnerg@moag.gov.ilLeoncio Braga, Samuel ..................... ICEB/NUPEB, Universidade Federal de Ouro Preto - Brazil................................................................. samueleoncio@yahoo.com.brLewin, Harris ...................................... Institute for Genomic Biology, University of Illinois - USA ...................................................................................h-lewin@igb.uiuc.eduLiboni, Marcio ....................................R&D, Vallée S.A. - Brazil ......................................................................................................................................liboni@vallee.com.brLima, Ana Rita De ..............................Faculdade de Medicina Veterinária, Universidade de São Paulo - Brazil ......................................................................arlima@usp.brLima, Valéria ......................................Brazil .............................................................................................................................................................. vmflima@fmva.unesp.brLogullo, Carlos ...................................LQFPP, Centro de Biociencias e Biotecnologia - Brazil ............................................................................................... logullo@uenf.brLoner Coutinho, Mariana ...................Brazil .......................................................................................................................................................marianaloner@yahoo.com.brLoukas, Alex ...................................... Infectious Diseases and Immunology, Queensland Institute of Medical Research - Australia ......................alex.loukas@qimr.edu.auLövgren, Tanja ...................................Biomedicine and Veterinary Public Health, Swedish University of Agricultural Scien - Sweden ....................tanja.lovgren@bvf.slu.seLoyola, Wagner .................................Patologia Geral, Universidade Estadual Norte do Paraná - Brazil .................................................................... wloybr@yahoo.com.brLund, Arve ......................................... Immunoprophylaxis, National Veterinary institute - Norway ................................................................................ arve.lund@vetinst.noLunney, Joan .....................................Animal Parasitic Diseases Laboratory, USDA ARS BARC - USA ............................................................ jlunney@anri.barc.usda.govLyashchenko, Konstantin ..................R&D, Chembio Diagnostic Systems, Inc. - USA........................................................................................................ kl@chembio.com

mMachado Z., Rosangela ....................Patologia Veterinaria, FCAV-UNESP - Brazil ..................................................................................................zacarias@fcav.unesp.brMackinnon, Kathryn ...........................Genetics, Bioinform, and Comp. Biology, Virginia Tech - USA ................................................................................. kmackinn@vt.eduMacpherson, Gordon .........................Sir William Dunn School of Pathology, University of Oxford - UK ................................................ gordon.macpherson@path.ox.ac.ukMage, Rose .......................................Laboratory of Immunology, National Institutes of Health, NIAID - USA ............................................................... rmage@niaid.nih.govMaisonnave, Jacqueline ....................Area de Inmunología, Fac.de Veterinaria-UDELAR - Uruguay ..................................................................jacmaiso2004@yahoo.comMalovrh, Tadej ...................................Veterinary faculty, Microbiology and parasitology - Slovenia ........................................................................tadej.malovrh@vf.uni-lj.siMansoor, Muhammad ........................Veterinary MIcrobiology, University of Agriculture - Pakistan .......................................................................mansoor03@hotmail.comMarchiori Filho, Moacir ......................P&D - Vacinas, Vallée S/A - Brazil ..................................................................................................................... moacir@vallee.com.brMartel, Cyril .......................................Veterinary Pathobiology, University of Copenhagen - Denmark ............................................................................. cmartel@life.ku.dk

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Martins-Filho, Olindo .........................Laboratório de Doença de Chagas, Instituto René Rachou - FIOCRUZ-Minas - Brazil...............................oamfilho@cpqrr.fiocruz.brMaruyama, Sandra ............................ Immunology and Biochemistry, FMRP - USP - Brazil......................................................................................... 2_sandrarcm@usp.brMason, Nicola ....................................Clinical Studies/ Pathobiology, University of Pennsylvania - USA ..................................................................nmason@vet.upenn.eduMasuda, Aoi .......................................Centro de Biotecnologia, UFRGS, Biociências - Brazil ............................................................................................aoi@cbiot.ufrgs.brMc Cullough, Kenneth ....................... Institute of Immunology and Immunoprophy - Switzerland ............................................................ kenneth.mccullough@ivi.admin.chMckeever, Declan ..............................Pathology and Infectious Diseases, Royal Veterinary College - UK ......................................................mckeever.declan@gmail.comMedeiros, Maria Lucia .......................Centro de Biotecnologia, UFRGS - Brazil .............................................................................................................malu@cbiot.ufrgs.brMeeusen, Els .....................................Physiology, Monash University - Australia.................................................................................... els.meeusen@med.monash.edu.auMendonça Lima, Fernanda ................Análises Clínicas e Toxicológicas, Faculdade de Farmácia-UFBA - Brazil .......................................................ferwlima@terra.com.brMenezes Souza, Daniel .....................Laboratório de Imunologia Celular e Mole, Instituto René Rachou/FIOCRUZ - Brazil........................... danielsouza@cpqrr.fiocruz.brMerlot, Elodie .....................................Biomedical Sciences and Veterinary Publi, Swedish University of Agricultural Scien - Sweden..............elodie.merlot@rennes.inra.frMiller, Robert .....................................Biology, University of New Mexico - USA .................................................................................................................rdmiller@unm.eduMineo, Tiago ......................................Brazil ............................................................................................................................................................... tiagomineo@gmail.comMiranda, Karina .................................Preventive Veterinary Medicine, Universidade Federal de Minas Gerais - Brazil ...............................................karinalmi@gmail.comMiriam, Rebouças ..............................Biointeraction Department, Institue of Health Sciences - Brazil .........................................................miriamreboucas@yahoo.com.brMoraes, Carina ..................................Cenbiot/UFPel, UFPel - Brazil ................................................................................................................ carina_moraes@terra.com.brMoré, Daniela ....................................Bioquemistry and Immunology, School of Medicine of Ribeirão Preto - Brazil ............................................................ ddmore@usp.brMoreira, Nádia ...................................Brazil ................................................................................................................................................... nadiabelladona@yahoo.com.brMoreno, Javier ...................................Ciencia de Proteínas. Lab. 3000, C. Investigaciones Biológicas - CSIC - Spain ........................................ javier.moreno@cib.csic.esMorrison, William Ivan .......................Division of Veterinary Clinical Sciences, Royal (Dick) School of Veterinary Studi - UK ................................. ivan.morrison@ed.ac.ukMulenga, Albert ..................................Entomology, Texas A & M University - USA ....................................................................................................... a-mulenga@tamu.eduMundo, Silvia Leonor ......................... Inmunologia, Facultad de Ciencias Veterinarias UBA - Argentina .......................................................................smundo@fvet.uba.arMunuera Monteiro, Janaína ...............de Cirurgia-FMVZ, Universidade de São Paulo - Brazil ........................................................................ janaina.mmonteiro@ig.com.brMurtaugh, Michael .............................Veterinary and Biomedical Sciences, University of Minnesota - USA .................................................................. murta001@umn.eduMuyldermans, Serge .........................Molecular and cellular interactions, VIB - Belgium ..............................................................................................svmuylde@vub.ac.beMwangi, Waithaka .............................Veterinary Pathobiology, Texas A&M University - USA ................................................................................wmwangi@cvm.tamu.edu

nNaessens, Jan ................................... ILRI, Int’l Livestock Research Institute, - KenyaNganga, Joseph ................................Biochemistry, Jomo Kenyatta University of Agric. & Tec - Kenya ............................................................................ jnganga@cgiar.orgNielsen, Morten .................................Center for biological sequence analysis, BioCentrum, DTU - Denmark................................................................... mniel@cbs.dtu.dkNiewold, Theo ....................................Nutrition and Health, Katholieke Universiteit Leuven - Belgium ...........................................................theo.niewold@biw.kuleuven.beNunes, Aryana Duckur .......................Nutrição e Produção Animal, FMVZ/USP - Brazil ...............................................................................................aryananunes@usp.br

oOkada, Kosuke ..................................Veterinary Pathology, Iwate University - Japan ..................................................................................................kosuke@iwate-u.ac.jpOliveira, Andressa Peres De ............. IMUNOVIR, UNESP - Brazil ...................................................................................................................... andressa225@terra.com.brOliveira, Carlo ....................................Bioquimica e Imunologia, Faculdade de Medicina de Ribeirão Preto - Brazil ..................................................................carlo@usp.brOliveira, Maria Zoraida ......................Pathology and clinics department, Federal University of Bahia - Brazil .......................................................... zoraida.vet@gmail.comOliveira, Rosane ................................Animal Sciences, University of Illinois - USA ............................................................................................................rosane@uiuc.eduOliveira, Sílvia .................................... imunologia, UFBA - Brazil .............................................................................................................................. silviakarol@hotmail.comOliveira, Tricia ....................................Veterinary Patology, Universidade Estadual Paulista - Brazil ...................................................................triciaoliveira@yahoo.com.brOliveira,, Aline Ferreira De ................Biologia Celular, Molecular e Bioagentes, Faculdade de Medicina de Ribeirão Preto - Brazil ..........................alinefopereira@usp.brOliver, Miguel .....................................Battelle Memorial Institute - USA ........................................................................................................................ oliverml@battelle.orgOllier, William .....................................CIGMR, The University of Manchester - UK ................................................................................................ cigmr@manchester.ac.ukOlsen, Ingrid ......................................Department of Animal Health, National Veterinary Institute - Norway ..............................................................ingrid.olsen@vetinst.noOnate, Angel ......................................Molec. Immun. lab, Dept. Microbiology, Universidad de Concepción - Chile .............................................................. aonate@udec.clOnodera, Takashi ..............................School of Agriculture, University Of Tokyo - Japan ............................................................................aonoder@mail.ecc.u-tokyo.ac.jpOnuma, Misao ...................................Disease Control, Graduate School of Veterinary Medicine - Japan .................................................. monuma@vetmed.hokudai.ac.jpOstachuk, Agustin .............................. Instituto de Virologia, CICVyA, INTA - Argentina .......................................................................................aostachuk@cnia.inta.gov.arOswald, Isabelle ................................Pharmacology-Toxicology, INRA - France ......................................................................................................ioswald@toulouse.inra.fr

pP V, Ravindra .....................................Veterinary Biotechnology, Indian Veterinary Research Institute - India .................................................. raviravindra1@rediffmail.comPaniago, Juliana Del Giúdice ............veterinary, Federal University of Viçosa - Brazil .................................................................................................. jpaniago@gmail.comPapierok, Gérard ...............................Bio Veto Test - France ....................................................................................................................................................... fbrun@bvt.frPardini, Lais Lujan .............................Laboratorio de Inmunoparasitología, Fecultad de Ciencias Veterinarias UNLP - Argentina ...................... laispardini@fcv.unlp.edu.arParizi, Luís Fernando ........................D. de Biologia Molecular e Biotecnologia, Centro de Biotecnologia - UFRGS - Brazil ..................................luisfparizi@cbiot.ufrgs.brPark, Joo Youn ..................................Veterinary Medicine, Washington State University - USA .........................................................................jooyounp@vetmed.wsu.eduParra, Zuly .........................................USA ...........................................................................................................................................................................zulyep@unm.eduPassos, Lygia ....................................Medicina Veterinaria Preventiva, Universidade Federal de Minas Gerais - Brazil ................................................... lygia@vet.ufmg.brPaulan, Silvana De Cassia ................Biology, UNESP - Brazil ........................................................................................................................scpaulan@aluno.feis.unesp.brPauletti, Rebeca ................................Brazil ............................................................................................................................................................... rebecabp@hotmail.comPeconick, Ana Paula ..........................Veterinary, Federal University of Viçosa - Brazil ................................................................................... anappeconick@yahoo.com.brPenha, Tânia .....................................Divisão de Antígenos, Instituto de Tecnologia do Paraná - TECP - Brazil .............................................................. trpenha@tecpar.brPereira De Faria Jr., Sebastião .........Livestock, Schering-Plough - Brazil ........................................................................................................ sebastiao.faria@spcorp.comPerez-Filgueira, Daniel ......................Argentina ....................................................................................................................................................... mperez@cnia.inta.gov.arPintaric, Majda ...................................Zasebna zobna ambulanta dr. Pintaric - Slovenia ................................................................................................ majda.dent@siol.netPintaric, Masa ....................................University of Veterinary Medicine Vienna, Clinical immunology - Austria ..................................................... masha_pin@hotmail.com

164

Plaxton, Ken ......................................Netherlands ..................................................................................................................................................... k.plaxton@elsevier.comPohl, Paula ........................................Centro de Biotecnologia, Biologia - Brazil ............................................................................................................paula@cbiot.ufrgs.brPortela, Ricardo ................................. Institute of Health Sciences, Federal Univesity of Bahia - Brazil..........................................................................rwportela@oi.com.brProfes, Marcos ..................................Brazil ........................................................................................................................................................ marcos_s_p@yahoo.com.brPrudêncio, Carlos .............................. INGEB - Brazil ................................................................................................................................................crprudencio@uol.com.br

rRadcliffe, Robin .................................Wildlife Health Service, Cornell University College of Vet Medici - USA ...............................................................robinr@fossilrim.orgRaquel, Trópia De Abreu ................... ICEB/NUPEB, Universidade Federal de Ouro Preto - Brazil...................................................................raquel_tropia@yahoo.com.brReichhart, Jean-Marc .........................CNRS, IBMC - France .......................................................................................................................... jm.reichhart@ibmc.u-strasbg.fr Reiner, Steven ...................................Medicine, University of Pennsylvania - USA ..........................................................................................sreiner@mail.med.upenn.eduReque Ruiz, Valeska Regina .............Brazil ................................................................................................................................................................. vamedvet@uol.com.brRibeiro, Dâmaso ................................ Immunology, Bimedical Science - Brazil ........................................................................................... damasopacheco@yahoo.com.brRibeiro, Daniela ................................. Imunology, Institute René Rachou - Brazil ....................................................................................................danibye@cpqrr.fiocruz.brRibeiro, Flávia ....................................Microimunoparasitologia, IPEC/ FIOCRUZ - Brazil .............................................................................................flavia@ipec.fiocruz.brRiber, Ulla ..........................................Veterinary Diagnostics and Research, National Veterinary Institute - Denmark ................................................................. ur@vet.dtuRice, Jennifer .....................................Dow AgroSciences - USA ............................................................................................................................................jrice3@dow.comRicht, Juergen ...................................NADC-ARS-USDA, VPDLRU - USA ......................................................................................................... juergen.richt@ars.usda.govRoatt, Bruno ......................................NUPEB, UFOP - Brazil .................................................................................................................................... bmroatt@yahoo.com.brRocha Gracia, Gustavo .....................Biochemistry and Immunology, university the São Paulo - Brazil ............................................................................ gugarg10@usp.brRocha, Andréa ...................................Cenbiot/UFPel, UFPel - Brazil ..........................................................................................................................ararocha@terra.com.brRocha, Hugo ......................................UFV - Brazil ........................................................................................................................................................ hguiero@hotmail.comR. Abatepaulo, Antonio Roberto ........Biochemistry and Immunology, Medicine school of Ribeirão Preto - Brazil ...........................................................abatepaulo@usp.brRodrigues, Aline ................................Research and Development, Vallee SA - Brazil ................................................................................................... aline@vallee.com.brRogel-Gaillard, Claire ........................ Institut National de la recherche Agrono - France ...............................................................................claire.rogel-gaillard@jouy.inra.frRombout, Jan ....................................Dept. of Animal Sciences, Wageningen University - Netherlands ......................................................................... jan.rombout@wur.nlRoos, Talita Bandeira ........................Brazil ............................................................................................................................................................... talitaroos@terra.com.brRosalinski-Moraes, Fernanda ............Patologia Básica, UFPR - Universidade Federal do Paraná - Brazil................................................................ fermoraes@uol.com.brRutten, Victor Pmg ............................ Infectious Diseases and Immunology, Fac of Veterinary Medicine, Utrecht Univ - Netherlands .............................. v.rutten@vet.uu.nl

sSaalmueller, Armin .............................Clinical Immunology - Austria ...........................................................................................................armin.saalmueller@vu-wien.ac.atSalgado, Breno ..................................Brazil ........................................................................................................................................................... brenosalgado@globo.comSantana, Vania ..................................Bacteriologia, Ministério da Agricultura - Brazil ......................................................................................vanialucia@agricultura.gov.brSantos, Filipe .....................................Veterinary Medicine, Universidade Estadual de Maringá - Brazil................................................................... felipe_fd_@hotmail.comSá-Rocha, Luiz Carlos .......................Pathology, School of Veterinary Medicine - São Paul - Brazil .................................................................................... lcsaroch@usp.brSato, Hiroki ........................................ Institute of Medical Science, The University of Tokyo - Japan ......................................................................satohi@ims.u-tokyo.ac.jpScarpelli Cury, Karime .......................Department of Pathology, State University of Campinas - UNICAMP - Brazil ................................................... karime@vivax.com.brScarpelli Martins, Edson ....................Department of Pathology, State University of Campinas - UNICAMP - Brazil ........................................... edsonms@fcm.unicamp.brScavone, Renata ...............................Brazil ............................................................................................................................................................... rescavone@yahoo.comScharfstein, Julio ............................... Immunobiology, Instituto de Biofisica C Chagas Filho - Brazil .................................................................................scharf@biof.ufrj.brScheerlinck, Jean-Pierre ...................Centre for Animal Biotechnology, The University of Melbourne - Australia ........................................... j.scheerlinck@unimelb.edu.auSchiller, Irene .....................................Prionics AG, - Switzerland ........................................................................................................................ irene.schiller@prionics.comSchwartz-Cornil, Isabelle ...................France ....................................................................................................................................................isabelle.schwartz@jouy.inra.frSebastian Matias, Chiavenna ............Instituto de Virología - Argentina ............................................................................................................. schiavenna@cnia.inta.gov.arSecombes, Chris ...............................Biological Sciences, University of Aberdeen - UK .........................................................................................c.secombes@abdn.ac.ukSeo, Keun Seok .................................MMBB, University of Idaho - USA ................................................................................................................... baram717@uidaho.eduSerafim, Tiago ...................................UFOP - Brazil ....................................................................................................................................................tiagodon@iceb.ufop.brShanthalingam, Sudarvili ...................Veterinary Microbiology and Pathology, Washington State University - USA ................................................. sudar@vetmed.wsu.eduShareck, Michael ...............................Battelle Memorial Institute - USA ..................................................................................................................... shareckm@battelle.orgSheibani, Mohammad ........................Department of Basic Sciences, Faculty of Veterinary Medicine - Iran............................................................... tsheibani@yahoo.comShewen, Patricia ................................Pathobiology, University of Guelph - Canada.................................................................................................. pshewen@uoguelph.caSilim, Amer ........................................Pathologie Et Microbiologie, Faculte De Medecine Veterinaire - Canada ...............................................................silima@yahoo.comSilva Aguiar, Cristiane ........................Pathology and Clinics, UFBA - Brazil ............................................................................................................. aguiarcs@yahoo.com.brSilva, Aldacilene ................................ Immunology, Institute of Biomedical Sciences - Brazil ......................................................................................... alda.vet@gmail.comSilva, Ediane Batista .......................... Immunology, Instituto de Patologia Tropical e Saúde - Brazil ............................................................ edianeveterinaria@hotmail.comSilva, Fernanda .................................Centro de Biotecnologia, UFRGS - Brazil ................................................................................................... fekleinsilva@cbiot.ufrgs.brSilveira, Ana Carolina ........................Pathology and Clinics, UFBA - Brazil .............................................................................................................aninhaccs@hotmail.comSimplício, Kalina ................................UFRPE - Brazil ..............................................................................................................................................kalinamms@hotmail.comSinkora, Marek ..................................Sector of Immunology and Gnotobiology, Institute of Microbiology - Czech Republic .........................marek.sinkora@worldonline.czSkyberg, Jerod ..................................Veterinary Molecular Biology, Montana State University - USA ...................................................................... jskyberg@montana.eduSmith, Adrian ..................................... Immunology, Institute for Animal Health - UK .............................................................................................. adrian.smith@bbsrc.ac.ukSommer, Sandra ................................ANS, Michigan State University - USA .................................................................................................................... sansom@msu.eduSordillo, Lorraine ...............................Large Animal Clinical Sciences, Michigan State University - USA ........................................................................... sordillo@msu.eduSorensen, Nanna Skall ......................Veterinary Diagnostics and Research, National Veterinary Institute, DTU. - Denmark ................................................ nss@vet.dtu.dkSouza, Fernando ...............................São Paulo University, - BrazilSrikumaran, Subramaniam ................Veterinary Microbiology and Pathology, Washington State University - USA .......................................ssrikumaran@vetmed.wsu.eduStalker, Amanda ................................Pathology and Infectious Diseases, Royal Veterinary College - UK ...................................................................... astalker@rvc.ac.ukStecca Iunes, Renata ........................Universty of São Paulo - Brazil ............................................................................................................................... riunes@gmail.com

165

Steinbach, Falko ................................Virology, VLA - UK ............................................................................................................................. f.steinbach@vla.defra.gsi.gov.ukStempler, Ana ....................................Facultad De Ciencias Veterinarias, U.B.A - Argentina ............................................................................... anastempler@hotmail.comStorset, Anne K ..................................Norwegian School of Veterinary Science, Food Safety and Infection Biology - Norway .................................. anne.storset@veths.noSturgill, Tracy .....................................Veterinary Science, University of Kentucky - USA ...................................................................................................... tlstur2@uky.eduSummerfield, Artur ............................. Immunology, Institute of Virology and Immunoprophylx - Switzerland ................................................artur.summerfield@ivi.admin.ch

tTaguchi, Takashi ................................Veterinary Sciences, Osaka Prefecture University - Japan ....................................................................... tt0206@vet.osakafu-u.ac.jpTajima, Tomoko ..................................Laboratory of Veterinary Microbiology, Osaka Prefecture University - Japan ............................................tajima@vet.osakafu-u.ac.jpTakayuki, Kubota ...............................National Institute of Animal Health - Japan ............................................................................................................ tkubota@affrc.go.jpTallmadge Ingram, Rebecca ..............Clinical Sciences, Cornell University - USA.................................................................................................................rlt8@cornell.eduTan, Sheila .........................................Veterinary Biologics, Canadian Food Inspection Agency - Canada ..................................................................tans@inspection.gc.caTavares Cyrino, Larissa .....................Veterinary, UFV - Brazil .......................................................................................................................... larissatavaresc@hotmail.comTijhaar, Edwin ....................................Cell Biology and Immunology, Animal Sciences Group, Wageningen Univ. - Netherlands ..................................edwin.tijhaar@wur.nlToivanen, Auli ....................................Medicine, Turku Univ - Finland ..........................................................................................................................................aultoi@utu.fiToivanen, Paavo ................................Med Microbiology, Turku Univ - Finland ...........................................................................................................................paatoi@utu.fiTølbøll Lauritsen, Klara .....................National Veterinary Institute, Technical University of Denmark - Denmark .................................................................... ktl@vet.dtu.dkToman, Miroslav ................................ Immunology, Veterinary Research Institute - Czech Republic ........................................................................................ toman@vri.czTong, Guang-Zhi ................................National Key Lab of Vet Biotechnology, Harbin Veterinary Research Institute - China ........................................... gztong@hvri.ac.cnTrompieri Silveira, Ana Carolina ........Veterinary Pathology, UNESP - Brazil ........................................................................................................actrompieri@yahoo.com.br

uUenishi, Hirohide ...............................Division of Animal Sciences, Natl. Inst. of Agrobiological Sci. - Japan ................................................................. huenishi@affrc.go.jpUrban, Jr., Joseph F. ..........................Diet, Genomics, and Immunology Laborator, Beltsville Human Nutrition Research Cent - USA ................... joe.urban@ars.usda.gov

vVahlenkamp, Thomas ........................Friedrich-Loeffler-Institute - Germany ...............................................................................................thomas.vahlenkamp@fli.bund.deVan Kooyk, Yvette .............................Molecular Cellbiology and Immunology, VUmc - Netherlands ............................................................................ y.vankooyk@vumc.nlVan Reeth, Kristien ............................Lab of Virology, Faculty of Veterinary Medicine - Belgium ........................................................................ kristien.vanreeth@ugent.beVan Rhijn, Ildiko ................................. Infectious Diseases and Immunology, Veterinary Medicine, UU - Netherlands .................................................... i.vanrhijn@vet.uu.nlVanrompay, Daisy ..............................Molecular Biotechnology, UGent, Faculty of Bioscience Engineering - Belgium ...................................... daisy.vanrompay@ugent.beVena, Maria .......................................Technical Service, R&D - Argentina ................................................................................................maria.vena@biogenesisbago.comVerardi, Paulo ....................................Vet Med: ILMB, University of California Davis - USA ...................................................................................... phverardi@ucdavis.eduVerma, Subhash ................................ Infection and Immunity, University of Glasgow - UK ............................................................................ s.verma.1@research.gla.ac.ukVeronez, Viviane Aparecida ...............patologia animal, fcav-UNESP - Brazil .........................................................................................................viviveronez@hotmail.comVieira, David .......................................Executive Secretariat, University of São Paulo - Brazil ....................................................................................... secretariat@8ivis.orgVieira, Pedro ...................................... Imunopatologia e Alergia, Incor - USP - Brazil .............................................................................................. pedrommmv@gmail.comVital, Wendel ..................................... Instituto de Ciências Biológicas, Universidade Federal de Ouro Preto - Brazil....................................... wendelcoura@yahoo.com.brVitoriano-Souza, Juliana ....................Núcleo em Pesquisa em C. Biológicas, UFOP - Brazil ................................................................................. juvsouza@yahoo.com.br

wWang, Yun-Feng ................................NKLVB, Harbin Veterinary Research Institute - China ............................................................................................yfwang@hvri.ac.cnWarr, Gregory ....................................Biochemistry, Medical University of South Carolina - USA ..................................................................................... warrgw@musc.eduWattegedera, Sean ............................Bacteriology, Moredun Research Institute - UK ............................................................................ sean.wattegedera@moredun.ac.ukWattrang, Eva ....................................Department of Parasitology (SWEPAR), National veterinary institute - Sweden ............................................gunilla.lindgren@sva.seWeinlich, Ricardo ...............................Department of Immunology, Institute of Biomedical Sciences - USP - Brazil ...................................................... rwpinas@yahoo.comWerling, Dirk ......................................Pathology and Infectious Diseases, Royal Veterinary College - UK ......................................................................dwerling@rvc.ac.ukWiddison, Stephanie .......................... Immunology, Institute for Animal Health - UK ................................................................................... stephanie.widdison@bbsrc.ac.ukWigley, Paul .......................................Veterinary pathology, University of Liverpool - UK ....................................................................................paul.wigley@liverpool.ac.ukWilkie, Bruce ......................................Pathobiology, The University Of Guelph - Canada .............................................................................................. bwilkie@uoguelph.caWilliams, John ................................... IDRA Laboratory, ILS-PTP - Italy ........................................................................................................... john.williams@tecnoparco.orgWilson, Mark ......................................LPD, NIAID, NIH - USA .................................................................................................................................wilsonmar@niaid.nih.govWomack, James ................................Veterinary Pathobiology, Texas A&M University - USA ................................................................................. jwomack@cvm.tamu.eduWood, Paul ........................................VMRD, Pfizer Animal Health - Australia ............................................................................................................paul.wood@pfizer.com

yYasuda, Masahiro ..............................Veterinary Anatomy, University of Miyazaki - Japan ..............................................................................yasudaja@cc.miyazaki-u.ac.jp

zZakeri, Afshin .....................................poultry diseases, animal department, Islamic Azad University branch Tabriz - Iran ............................................ azakerii@yahoo.comZhu, Yaohong ....................................Department of Clinical Sciences, SLU, SLU, Uppsala - Sweden .................................................................... yaohong.zhu@kv.slu.se