Eleventh Congress of the Bulgarian Microbiologists program and...2 October 5, 2006 October 6, 2006...
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1 PROGRAM AND ABSTRACTS Eleventh Congress of the Bulgarian Microbiologists with International Participation International House of Scientists Frederic Joliot-Curie St. Constantine, Varna October 5-7, 2006
Transcript of Eleventh Congress of the Bulgarian Microbiologists program and...2 October 5, 2006 October 6, 2006...
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PROGRAM AND ABSTRACTS
Eleventh Congress
of the Bulgarian Microbiologists
with International Participation
International House of ScientistsFrederic Joliot-Curie
St. Constantine, Varna
October 5-7, 2006
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Table of Contents
Congress Organizers ……………………………………………..Congress Organizing Committee……………………………………..Congress SponsorsCongress SectionsSocial ProgramFINAL PROGRAM
Opening CeremonyORAL PRESENTATIONSThursday, October 05
Hall1: Memorial Session Dr. Stamen Grigorov Tuberculosis
Hall1: Virology, Session IHall2: Medical Microbiology, Sessions I and IIHall3: Veterinary Microbiology, Sessions I and IIHall4: Infectious Immunology and Parazitology, Sessions I and II
Friday, October 06Hall1: General and Applied Microbiology, Sessions I, II, III and IVHall2: Medical Microbiology, Sessions III and IVHall2: Plant and Soil Microbiology, Session IHall3: Virology, Sessions II and III
Saturday, October 07Hall1: Plenary Session “Actual Problems of the BioScience”
POSTER PRESENTATIONSThursday, October 05
Lobby: Poster Session I:General and Applied MicrobiologyVeterinary Microbiology
Friday, October 06Lobby: Poster Session II:
Medical MicrobiologyVirologyInfectious ImmunologyParazitologyPlant and Soil Microbiology
ABSTRACTSFirst Authors’ Index
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CONGRESS ORGANIZERS
Union of Scientists in Bulgaria –
Bulgarian Society for Microbiology
with associated
Bulgarian Society of Medical MicrobiologyBulgarian Society of Medical Virology
Bulgarian Society of Plant Virology “Prof. Dr. D. Atanasoff”
and
The Stephan Angeloff Institute of Microbiology,Bulgarian Academy of Sciences
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CONGRESS ORGANIZING COMMITTEE
President: Prof. Angel S. GALABOV, DSc,Corr. Member of BAS
Secretary General: Assoc. Prof. Hristo NAJDENSKI, PhD
Members:
Assoc. Prof. Angel ANGELOV, PhDProf. Maria ANGELOVA, DScProf. Radka ARGIROVA, DScAssoc. Prof. Zheko BAICHEV, PhDAssoc. Prof. Milyana CHUCHKOVA, PhDAssoc. Prof. Svetla DANOVA, PhDAssoc. Prof. Stefan DENEV, PhDProf. Raycho DIMKOV, PhDAssoc. Prof. Lyuba DOUMANOVA, PhDAssoc. Prof. Elka EMANUILOVA, PhDAssist. Prof. Elica GOLKOCHEVA, PhDProf. Stoyan GROUDEV, DScAssoc. Prof. Veneta GROUDEVA, PhDAssoc. Prof. Irina HAYDUSHKA, PhDAssoc. Prof. Tsonka HRISTOZOVA, PhDProf. Iskra IVANOVA, DScAssoc. Prof. Todor KANTARDJIEV, PhD
Assoc. Prof. Mira KOJOUHAROVA, PhDAssoc. Prof. Rossica KOTSEVA, PhDAngel KUNCHEV, PhDAssoc. Prof. Rossitsa KURDOVA, PhDProf. Jordanka KUZMANOVA, DScProf. Ivan MITOV, DScProf. Ivan MURGOV, DScAssoc. Prof. Nedelcho NEDELCHEV, PhDProf. Plamen NENKOV, DScProf. Bogdan PETRUNOV, DSc,Corr. Member of BASAssoc. Prof. Maria SHISHINIOVA, PhDAssoc. Prof. Penka SOTIROVA, PhDAssoc. Prof. Maria SREDKOVA, PhDAssist. Prof. Antoniy STOEV, PhDTencho TENEV, MDAssoc. Prof. Pavel TEOHAROV, PhDAssoc. Prof. Rossica VATCHEVA, PhD
Local organizing committee:
Assoc. Prof. Velina YONKOVA, PhD(Coordinator)Branimir SHMATOV, MD (Secretary)
Secretariat:
Liliana HARALAMPIEVA, MS,(Coordinator)Assist. Prof. Radoslav ABRASHEV, MSAssist. Prof. Nelly SLAVOVA-AZMANOVA, MDElka GENOVA, BSIvailo GEORGIEV, MS Eng.Mihail ILIEV, MS
Assoc. Prof. Vesselin RUSSEV, PhD
Assist. Prof. Ralitca NEDKOVA, MSKrassya NENOVA, ClerkAssist. Prof. Lubomira NIKOLAEVA-GLOMB, PhDEmilia POPOVA-AYVAZOVA, BSDessislava TODOROVA, MSRalitsa VASSILEVA, MS
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ORGANIZING SECRETARIAT
The Stefan Angeloff Institute of Microbiology,Bulgarian Academy of Sciences
26, Acad. G. Bonchev Str.1113 Sofia, Bulgaria
Tel.: +359 2 8701081, fax: +359 2 8700109e-mail:[email protected]/congress11
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CONGRESS SPONSORS
GENERAL SPONSORS
БИОКОМ Tрендафилов ЕООД
IDEXX - USA
представлявана за България от фирма КРОСПОЙНТ ЕООДчрез фирма HATO Handelsgesellschaft mbH - Germany
SPONSORS
Medical Technics Engineering Ltd.GlaxoSmithKline
Roche Bulgaria Ltd.BioSystems Ltd.
AQUAlabEcopharm Ltd.
TECOM Analytical Systems Ltd.Аквахим ЕООД
Anti-sell / Selidis Bros Bulgaria Ltd.L.K.B. Bulgaria Ltd.
БУЛ БИО-НЦЗПБ ЕООДBIOLAB Ltd.
ФОТ ООД - официален представител на SIGMA-ALDRICH за БългарияSevex Pharma
ЕЛТА’ 90М ООДDr. Herbert Knauer Ltd. Berlin, Germany
5040 Сървисиз ООДThermo Electron LED GmbH Germany
Диамед ООДИНФОМЕД ЕООД (OLYMPUS)
DMG Clinic Ltd.Лаборбио ООД
CONTRIBUTORSНационална ветеринарномедицинска служба, МЗГИнститут по микробиология „Стефан Ангелов”, БАН
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CONGRESS SECTIONS
Tuberculosis (Tb)
General and Applied Microbiology (GAM)
Medical Microbiology (MM)
Veterinary Microbiology (VM)
Virology (V)
Parasitology (P)
Infectious Immunology (II)
Plant and Soil Microbiology (PSM)
Actual Problems of the BioScience (BS)
SOCIAL PROGRAM
Thursday, October 05, 19:00Welcome Party
Friday, October 06, 20:00Congress Banquet
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FINAL PROGRAM
Registration: Wednesday, October 04 16:00 – 19:00Thursday, October 05 08:00 – 18:00Friday, October 06 08:00 – 14:00
Thursday, October 05
Opening Ceremony
Hall 1
Chairpersons: Angel S. GalabovHristo Najdenski
9:00 Welcoming Addresses
President of the Organizing CommitteeMayor of the City of VarnaPresident of the Dr. Stamen Grigorov FoundationOthers
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ORAL PRESENTATIONS
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Thursday , October 05Hall 19:00 – 9:20 Opening Ceremony
Memorial Session Dr. Stamen GrigorovTuberculosisChairpersons: A. Engibarov, Sofia
H. Najdenski, SofiaM. Chouchkova, Sofia
9:20 Tb1 Vitae of Dr. Stamen GrigorovA. Galabov, Sofia
9:40 Tb2 BCG vaccinesM. Chouchkova, T. Stefanova, Sofia
10:05 Tb3 Renaissance of tuberculosis. Actual problemsZ. Yankova, Plovdiv
10:30 Break11:00 Tb4 Control of tuberculosis and introduction of DOT strategy
in BulgariaD. Stefanova, Sofia
11:25 Tb5 Tuberculosis among the children in the past and todayP. Minchev, Sofia
11:50 Tb6 Contemporary microbiological diagnostics oftuberculosis and methods for epidemiological tracingT. Kantardjiev, St. Panaiotov, E. Bachiiska, Sofia
12:15 Tb7 Molecular and epidemiologycal characteristics ofMycobacterium tuberculosis strains originating fromdifferent regions of BulgariaV. Valcheva, I. Mokrousov, O. Narvskaja, N. Markova,Sofia, St. Petersburg
12:45 Tb8 Intra-species determination of mycobacteria by PCRM. Bonovska, H. Najdenski, Sofia
13:00 Confocal microscopy and microbiology applicationsJ. Pala, Leica Microsystems, Brno, Czech Republic
13:30 Session end
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Thursday, October 05, 2006
Hall 1: Virology
Session IChairpersons: A. S. Galabov, Sofia
F. Wild, LyonL. Doumanova, Sofia
14:00 V1 Henipaviruses, a new family of paramyxoviruses,Plenary which have emerged in Asia and Australia
F. Wild, Lyon, France
14:30 V2 Structural basis for antiviral drug-resistance of aPlenary VP1 mutated enterovirus
A. S. Galabov, I. Nikolova, R. Petkova,S. Chakarov, B. Atanasov, Sofia
15:00 V3 The hierarchical QSAR technology for effectivePlenary virtual screening and molecular design of
potential antiviral agentsV. E. Kuzmin, A. G. Artemenko, E. N. Muratov,L. N. Ognichenko, A. I. Hromov, A. V. Liahovskij,P. G. Polischuk, Odessa, Ukraine
15:30 Break
16:00 V4 Christo Russeff Memorial LecturePlenary Oxoglaucine: a new highly potent antienteroviral
compoundL. Nikolaeva-Glomb, S. Filipov, A. S. Galabov,Sofia
16:20 V5 Rational treatment course schedule effectiveagainst enteroviral neuroinfection in miceR. Vassileva-Pencheva, A. S. Galabov, Sofia
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16:35 V6 Еffects of picornavirus replication inhibitors againstcalicivirus FCVJ. D. Tumbarski, A. S. Galabov, Sofia
16:50 V7 In vivo effective anti-flu combination of antiviralsL. Simeonova, G. Gegova, A. S. Galabov, Sofia
17:05 V8 Must monesine preparative be used for prophylaxis ofbird fluS. Dundarov, Sofia
17:20 V9 In vitro anti-influenza virus effect of a protease inhibitorfrom Streptomyces chromofuscus 34-1J. Serkedjieva, L. Angelova, I. Roeva, I. Ivanova, Sofia
17:35 V10 Anti-herpes activities of Pseudomonas sp. S-17rhamnolipid and its complex with alginateM. Remichkova, I. Roeva, D. Galabova, A. S. Galabov, Sofia
17:50 V11 Antioxidant effects of plant polyphenols quercetin andrutin in influenza virus infected miceM. Mileva, A. S. Galabov, Sofia
18:05 V12 A plant polyphenol extract reduces oxidative stress inthe livers of influenza virus-infected miceE. Krumova, S. Abarova, L. Tancheva, M. Angelova,J. Serkedjieva, Sofia
18:20 Session end
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Thursday , October 05
Hall 2: Medical Microbiology
Session I
Chairpersons: G. Terziiski, SofiaM. Petrovska, Skopie, R. MacedoniaM. Sredkova, Pleven
14:00 MM1 Polyphase identification analysis of clinicalPlenary isolates from Burkholderia cepacia complex
(BCC)M. Sredkova, S. Mihailova, E. Moor, Pleven,Goteborg, Sweden
14:30 MM2 Study of vaginal lactobacilli from BulgarianPlenary women
G. D. Stoyancheva, S. P. Dimitonova,P. M. Petrova, R. N. Aleksandrova, I. Tzvetkova,S. T. Danova, Sofia
14:50 MM3 Frequency of the triggering infection at patientsPlenary with reactive arthritis and unidentified
oligoarthritis and necessity for theirexaminationR. Stoilov, Sofia
15:10 Real-Time PCR in molecular microbiologyV. Kardjeva, Biosystems Ltd., Sofia
15:30 Break
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Session IIChairpersons: G. Terziiski, Sofia
M. Petrovska, Skopie, R. MacedoniaM. Sredkova, Pleven
16:00 MM4 Microbiological investigation of importedBrucella infection among Bulgarian citizensR. Nenova, T. Kantardjiev, I. Ivanov, I. Tomova,B. Popov, Vl. Novkirishki, Sofia
16:15 MM5 Characteristics of the cultivation ofBifidobacterium bifidum 1 in media withpalatinoseD. Blazheva, Z. Denkova, A. Krastanov, Sofia
16:30 PCR-DIAPOPS method for detection ofLegionella in waterE. Domínguez, ZEU-INMUNOTEC, Zaragoza, Spain
17:00 MM6 Contamination of hotels from Bulgarian BlackSea Coast with different branches of Legionella– ecological precondition for colonizationI. Tomova, U. Helbig, V. Levterova, R. Nenova,I. Ivanova, Sofia
17:15 MM7 Normal flora is found in human bloodE Kalfin, Sofia
17:30 MM8 Q-fever – an insufficiently known andunderestimated infection in Bulgaria.V. Serbezov, Sofia
17:45 MM9 Clinical aspects of lung mycotic infectionsV. Pencheva, D. Petrova, O. Georgiev,T. Kantardjiev, Tz. Mondeshki, Tz. Terzieva,D. Valchev, Sofia
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18:00 MM10 PCR-based methods for diagnosis of systemicmycoses and genotyping of pathogenic fungiP. Angelov, T. T. Kantardjiev, V. Levterova,E. Zamfirova, M. Leseva, R. Vacheva, E. Shopova,E. Bobcheva, Sofia
18:15 Session end
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Thursday, October 05
Hall 3: Veterinary Microbiology
Session I
Chairpersons: I. Ivanov, SofiaH. Najdenski, Sofia
14:00 Microbiological methods for detection ofantibiotic residues in foodE. Dominguez, ZEU-IMMUNOTEC, Zaragoza,Spain
14:20 VM1 Development and application of rabbit polyclonalPlenary monospecific affinity purified antibody against
synthetic sequence from peptide P32 to detectsheep pox virus by immunohistochemical polymerstaining technique on frozen tissue sectionsV. Bumbarov, R. Eligulashvili., H. Yadin,Beit Dagan, Izrael
14:40 VM2 Findings and serotyping of Listeriamonocytogenes in meat of chicken broilersR. Karakolev, Veliko Tarnovo
15:00 VM3 Optimization of PCR-DGGE for direct detectionPlenary and molecular typing of Campilobacter jejuni and
Campylobacter coli in bird cecal samplesH. Najdenski, Sofia
15:30 Break
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Session IIChairpersons: R. Karakolev, Veliko Tarnovo
N. Korudjiisky, Sofia
16:00 Rapid solutions for food safety – A review of methodsand industry trendsF. Martens, Hygiena International Ltd., Hertfordshire,United Kingdom
16:30 Reducing the risk of antibiotic drug-residuecontamination and ensuring milk safety and quality withthe reliable IDEXX SNAP® test kitsG. Trifonov, IDEXX-USA, Sofia
16:50 VM4 Comparative researching of pH in some musselsfrom sacrified animalsA.Kuzelov; O. Kirovska Cigulevska, Sveti Nikole,Skopje, R. Macedonia
17:10 VM5 Ecological alternatives of antibiotic prophylacticsand therapyH. Arnaudov, R. Karakolev, P. Kalcheva, Veliko Tarnova
17:35 VM6 Drug resistant bacteria from the genusMoraxella, isolated from rabbits with pneumoniaS. Ivanova, N. Korudjiisky, T. Galabinova, B. Mitov, Sofia
17:55 VM7 Disinfection of objects in the apiculture contaminatedwith Paenibacillus alvei and Ascosphaera apisK. Gurgulova, D. Ilieva, J. Hristov, S. Karadjov, E. Iliev, Sofia
18:25 VM8 Microbiological investigation on pseudotuberculosis insheeps and goatsN. Korudjiisky, S. Ivanova, B. Gurov, D. Todorov,Zv. Dikova, Sofia
18:40 Session end
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Thursday, October 05
Hall 4: Infectious Immunology
Session I
Chairpersons: I. Smirnov, Moscow, RussiaT. Vassilev, Sofia
14:00 Intro of IDEXX Lab Inc main subject WATERCLT / CLT 18S. Herterich, G. Trifonov, IDEXX EUROPE B.V.,Schiphol-Rijk, Netherland; Sofia
14:30 II1 Quality of analysis in clinical microbiologyPlenary I. Smirnov, Moskow, Russia
14:50 II2 Improved pooled IgG prevents death inPlenary experimental sepsis
T. Vassilev, J. Dimitrov and N. Ivanovska, Sofia
15:10 II3 Effect of Yersinia pseudotuberculosis YopKPlenary protein on invasion mediated endocytosis from
HELA cellsE. Ivanova, H. Najdenski, A. Vesselinova,F. Deleuil, H. Wolf-Watz, Sofia, Umea, Sweden
15:30 Break
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Session II
Hall 4: Parazitology
Chairpersons: R. Kurdova-Mintcheva, SofiaP. Petrov, Sofia
16:00 Conversation of the EUDWD into National LawM.G. Czapp, HATO Handelsgesellschaft mbH,Achim, Germany
16:45 P1 Emerging and reimurging parasitic diseases inPlenary Bulgaria
R. Kurdova-Mintcheva, D. Jordanova,T. Marinova, M. Ivanova, N. Tsvetkova,I. Marinova, R. Harizanov, Sofia
17:15 Session end
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Friday, October 06
Hall 1: General and Applied Microbiology
Session I
Chairpersons: E. Emanuilova, SofiaV. Groudeva, Sofia
9:00 GAM1 Mycological studies in ContinentalPlenary Antarctica: an overview
S. Tosi, Pavia, Italy
9:30 GAM2 Microbiological control of detoxificationbioremediation technologiesY. Topalova, R. Dimkov, C.V.Keer,C. Y.Cheng, C.Y.Cheng, M. Nunes, Sofia,Gent, Belgium; Porto, Portugal
9:45 GAM3 Diversity of Synechococcus community infreshwater fake George, NYD. Gouliamova, Sofia
10:00 GAM4 Microbial status of 7 Thracian vaults nearto Kasanlak, Bulgaria and methods forpreventationTz. Groudeva, A. Doycheva, Sofia
10:15 GAM5 Taxonomical identification of arsenicresistant and arsenic transforming sulfate- reducing bacteriaK.S. Krumova, V.I. Groudeva, Sofia
10:30 Break
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Session II
Chairpersons: S. Groudev, SofiaZ. Alexieva, Sofia
11:00 GAM6 Low-temperature biodegradation ofPlenary phenol
R. Margesin, Innsbruck, Austria
11:30 GAM7 In situ bioremediation of soil polluted withcrude oil and heavy metalsV.I.Groudeva, A.S.Doycheva andS.N.Groudev, Sofia
11:45 GAM8 Comparative characteristics of oil-degrading activity of microorganisms withdifferent taxonomic statusV. Groudeva, I. Ivanova, A. Doycheva,M. Dumitru, A-R. Vasculesku, Sofia,Bucharest, Romania
12:00 GAM9 Comparative characteristics of toxicityassessment of heavy metal polluted soilswith different microorganismsI. Ivanova, D. Dimova, A. Stojanova,V. Groudeva, Sofia
12:15 GAM10 Study for intensification of the biogasproduction from agricultural and agro-industrial wastesD. Galabova, D. Karakashev, A. Mirkov,L. Nikolov, I. Simeonov, Sofia
12:30 Lunch
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Session III
Chairpersons: R. Dimkov, SofiaI. Ivanova, Sofia
14:00 GAM11 Molecular biomarkers of oxidative stress infilamentous fungiM. Angelova, Sofia
14:15 GAM12 New mitochondrial catalase in yeastSaccharomyces cerevisiaeV. Petrova, Sofia
14:30 GAM13 Proteomics in target-specific antibacterial drugdiscovery based on UMP kinaseN. Slavova-Azmanova, C. Evrin, L. Assairi,H. Najdenski, O. Bârzu and A.- M. Gilles, Sofia,Paris, France
14:45 GAM14 Heat shock response of Streptococcusthermophilus industrial strainsP. Petrova, D. Gouliamova and G. Stoyancheva,Sofia
15:00 GAM15 Second generation of glucooligosaccharides withprebiotic properties, synthesied byglycosiltransferases from Leuconostocmesenteroides LM 286I. Iliev, T. Vassileva, Plovdiv, Sofia
15:15 GAM16 Isolation and properties of extracellular â-xylosidase produced by Aspergillus niger B03G. Dobrev, I. Pistijski, L. Koleva, Plovdiv
15:30 Break
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Session IV
Chairpersons: Ch. Chomakov, SofiaI. Abrashev, Sofia
16:00 GAM17 Polyhydroxyalkanoic acids (PHA) –Plenary interesting bacterial polymers and aspects
of their productionJ-U. Ackermann, G. Mothes, Dresden,Leipzig, Germany
16:20 GAM18 Peculiarities of biofilm systemPlenary implementation in microbial
biotechnologiesL. Nikolov, Sofia
16:45 GAM19 Evolution modeling of bacterial oxidationof ferrous ions in biofilm systemL. Nikolov, E. Petrova , V. Mamatarkova,Kl. Mladenov, St. Stoytchev, Sofia
17:00 GAM20 Precondition for producing of organic foodin MacedoniaA. Kuzelov, O. Cigulevska, Skopie,R. Macedonia
17:15 GAM21 Heterogeneity of L. plantarum isolatesfrom artisanal white brined cheeseR. Aleksandrova, S. Dimitonova,I. Ivanovaand S. Danova, Sofia
17:30 GAM22 Comparison of fermentative capacity ofLactobacillus bulgaricus cultivated onmedia with oligosaccharidesTz. Ignatova, I. Ivanova, I. Iliev, Shumen,Sofia, Plovdiv
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17:45 GAM23 Amino acids use and production - currentstatus and prospectsA. Ratkov, Sofia
18:00 GAM24 Mathematical identification of L-valinefed-batch fermentation processK. Todorov, I. Dimov, Tz. Georgiev,J. Kristeva, V. Ivanova, Al. Ratkov, Sofia
18:15 GAM25 Carcinogen-induced transposition ofSaccharomyces cerevisiae Ty1 transposondepends on mitochondrial functionT. Stoycheva, P. Venkov, Ts. Tsvetkov, Sofia
18:30 Session end
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Friday , October 06
Hall 2: Medical Microbiology
Session III
Chairpersons: I. Mitov, SofiaT. Kantardjiev, SofiaE. Keuleyan, Sofia
8:30 MM11 Etiological structure of drug resistancePlenary and consumption of antibiotics in
BulgariaT. Kantardjiev, M. Petrov, Ts. Velinov,A. Bachvarov, P. Angelov, Sofia
8:55 MM12 Investigation of the virulence factors andPlenary antibiotic resistance of Moraxella
catarrhalisI. Mitov, R. Gergova, V. Ouzunova,R. Markovska, D. Kuncheva, Sofia
9:20 Resistance of Streptococcus pneumoniaeto βββββ-lactam antibiotics. New dose form ofAmoxicillin/Clavulanate in the therapy ofrespiratory and ORL infections-AugmentinES 600B. Markova, GlaxoSmithKline, Sofia
9:50 MM13 Species affiliation and antibioticsresistance of clinical isolates fromhemoculturesD. Rukanova, E. Staikova, K. Rachkova,I. Dukova, H. Djeneva, M. Baicheva,G. Lazarova, Stara Zagora
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10:00 MM14 Study on the species affiliation andantibiotic sensitivity of strains isolatedfrom urine of patients with chronicpyelonephritisV. Grigorova, V. Todorov V. Edreva,K. Dragoev, Pleven
10:15 MM15 Antimicrobial susceptibility of urinepathogenes isolated from patients withbenign prostatic hyperplasia treated withurostim .V. Grigorova, S. Stratev, F. Shargabi,N. Kolev, P. Panayotov, R. Kostov,O. Mihaylov, Ts. Georgiev, V. Dunev,B. Atanasov, Pleven
10:30 Break
Session IV
Chairpersons: I. Mitov, SofiaT. Kantardjiev, SofiaE. Kyolean, Sofia
11:00 MM16 Susceptibility of S. pneumoniae clinical isolatesto some antibioticsK. Bojkova, T. Stoeva, V. Kaludova, V. Kamenova,V. Russev, Varna
11:15 MM17 Antibacterial and antifungal activities of apolyphenol-Rich extract from Geraniumsanguineum L.M. Gulluce, F. Sahin, A. Sokmen, A. Teodosieva,J. Serkedjieva; Erzurum, Sivas, Turkey; Sofia
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11:30 MM18 Investigation of the inhibitory effect of lactic acidbacteria on the cells of Escherichia coliNBIMCC 8739P. Nedelcheva, Z. Denkova, R. Nikolova, Plovdiv
11:45 MM19 Probiotics and probiotic foods in the protection ofhuman healthI. Murgov, Z. Denkova, Plovdiv
12:00 MM20 Sepsis associated with central venous cathetersat patients in critical situation – causative agentsand frequencyD. Terziiski, N. Petrov, N. Mladenov, Sofia
12:15 MM21 Molecular diagnostics of the causative agents ofatypic pneumoniaN. Brankova, T. Kantardjiev, V. Levterova,E. Georgieva, I. Tomova, S. Panajotov, Sofia
12:30 Lunch
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Friday, October 06
Hall 2: Plant and Soil Microbiology
Session I
Chairpersons: N. Bakardjieva, SofiaD. Sakalieva, Plovdiv
14:00 PSM1 Spread and detection of phytoplasmaPlenary diseases
D. Sakalieva, Plovdiv
14:45 PSM2 Manifestation of tolerance to sharkaPlenary (plum pox) virus of plum cultivars
imported in BulgariaA. Stoev, P. Iliev, Kostinbrod, Dryanovo
15:15 PSM3 Proposals for improvement of the varietyPlenary testing of wheat and barley accordingly
their reaction to the most important inBulgaria virusesN. Bakardjieva, A. Stoev, Kostinbrod
15:45 Break
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Friday, October 06
Hall 3: Virology
Session II
Chairpersons: S. Dundarov, SofiaR. Argirova, SofiaR. Kotseva, Sofia
8:30 V13 SARS-coronavirus interaction with ACE2Plenary receptor; implications for virus adaptation and
vaccine designA.P. Andonov, Winnipeg, Canada
9:00 V14 Evidence supporting the replicative homeostasisPlenary hypothesis concerning HIV replication in cell
cultureR. Argirova, Sofia
9:30 V15 Strain diversity and mechanisms of evolution ofrotavirusesZ. Mladenova, N. Korsun, S. Gyurova, Sofia
9:45 V16 Diagnostic of the first suspected patients inBulgaria for avian influenza virus subtypeА(H5N1).T. Hadjiolova, S. Pavlova, R. Kotseva, Sofia
10:00 V17 Diagnostic studies of etiological role ofrespiratory-syncytialvirus in hospitalized children.S. Pavlova, T. Hadjiolova, R. Kotseva, Sofia
10:15 V18 Diagnostic aspects of haemorrhagic feversD. Velcheva, Sofia
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10:30 Break
11:00 V19 Studies on CCR5, CXCR4 And CCR2 GeneticPolymorphism in HIV-Infected BulgariansK. Borisov, A. Savov, I. Kremensky, S. Raleva,L. Froloshka, R. Markova, V. Terzieva, K. Kostov,R. Argirova, Sofia
11:15 V20 An experimental study of HIV-1 epitopestructure changes under inhibition ofglycosylationR. Gavazova, S. Ivanov, D. Ivanov, P. Genova,S. Raleva, L. Froloshka, D. Dundarova,R. Argirova, Sofia
11:30 V21 Spread of the genetic forms of HIV-1 circulatingin BulgariaM. Peneva, D. Beshkov, I. Aleksiev, V. Georgieva,K. Kostov, I. Elenkov, T. Varleva, I. I. Elenkov,Sofia
11:45 V22 Study of the spread of human T-lymphotropicviruses HTLV-I and II among BulgarianpopulationI. Aleksiev, D. Beshkov, V. Georgieva, M. Peneva,S. Bakalova, M. Genova, M. Atanasova,I. Elenkov, Sofia
12:00 V23 HIV-1 variants (mutants) after long-termpassaging in presence of newly synthesixed4-hydroxicoumarins (4-Hc)S. Raleva, A. Yordanova, Y. Gradinarova,A. Savov, L. Froloshka, P. Genova, I. Manolov,D. Dundarova, R. Argirova, Sofia
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12:15 V24 Antiretroviral resistance of HIV-1 amongBulgarian seropositive patients (2002-2006)D. Beshkov, I. Aleksiev, M. Peneva, V. Georgieva,K. Kostov,I. Elenkov, T. Varleva, I. I. Elenkov, Sofia
12:30 Influenza and influenza pandemic: epidemiologyand current possibilities for prophylactic andtreatmentМ. Kamenova (Roche Bulgaria Ltd.), Sofia
13:00 Lunch
Hall 3: Virology
Session III
Chairpersons: P. Teoharov, SofiaV. Serbezov, SofiaZ. Kalvachev, Sofia
14:00 V25 Diagnostics of the viral hepatitis -Plenary achievements and problems
P. Teoharov, Sofia
14:30 V26 Molecular characteristics and pathogeneticPlenary potential of human polyomaviruses
Z. Kalvachev, Sofia
15:00 V27 Rapid identification of polyomavirus hominis-1(ВКV) in the urine of patients with kidneytransplantationS.. Slavov, I. Nenkov, A. Petrova, L. Hristova,P. Simeonov, D. Dimova, Z. Kalvachev, Sofia
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15:15 V28 Polyomavirus hominis-2 (JCV) in the urine ofpatients with kidney transplantationI. Nenkov, S. Slavov, A. Petrova, L. Hristova,P. Simeonov, Z. Kalvachev, Sofia
15:30 V29 Clinical features and serological verification ofacute EBV infectionA. Gotseva, T. Kuzmova, D. Velcheva, Sofia
15:45 V30 Bird flu – the new global threat: epidemiology,spread and possible use as biological weaponK. Mekouchinov, Sofia
16:00 Session end
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Saturday, October 07
Hall1
Plenary Session Actual Problems of the BioScience
Chairpersons: Angel S. Galabov, SofiaHristo Najdenski, Sofia
9:30 BS1 Science funding in BulgariaA. Vutsova, Ministry of Education and Science, Sofia
10:15 BS2 Bioinformatics resources in the microbiology: challengesand perspectivesD. Vassilev, AgroBio Institute, Sofia
11:00 Congress Adjourn
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POSTER PRESENTATIONS
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Thursday, October 05
Poster session I
16.00 – 18.30
General and Applied Microbiology
GAM26 Phenol hydroxylase dependance on the variouse hydroxyphenols utilized as substrates by Trichosporon cutaneumR57Z. Alexieva, M. Gerginova, B. Atanasov, Sofia
GAM27 Creating oligonucleotide primers for pcr analysis andphyA gene sequencing in Trichosporon cutaneum R57strainY. Manasiev, T. Primov, M. Gerginova, N. Peneva,Z. Alexieva, Sofia
GAM28 Dot-blot analysis by biotin labeled probe for identifyingPHYA gene in microbial strainsM. Gerginova, Y. Manasiev, P. Petrova, A. Krastanov,Z. Alexieva, Sofia, Plovdiv
GAM29 Influence of toxic phenolic compounds on the phenolhydroxilase activity in Trichosporon cutaneumM. Gerginova, Y. Manasiev, N. Shivarova, Z. Alexieva, Sofia
GAM30 Microflora in a natural wetland used fpr treatment of acidmine drainageV.I. Groudeva , S.G. Bratkova and S.N. Groudev, Sofia
GAM31 Biodegradation of mixed phenol compounds by microbialassociation of Aspergillus awamori and ThermoascusaurantiacusI. Stoilova, A. Krastanov, H. Bui, V. Stanchev, Plovdiv
GAM32 Benzonitrile and 4-cyanopiridine degradation byimmobilized cells of Bacillus sp. UG-5B in a columnbioreactorL. Kabaivanova, E. Dobreva, E. Emanuilova, B. Samuneva,G. Chernev, Sofia
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GAM33 Biological properties of biosurfactant-complex fromPseudomonas sp. PS-17A. Sotirova, D. Spasova, E. Vasileva-Tonkova, D. Galabova,Sofia
GAM34 Decolorization of the acid orange 7 by resting Alcaligenesfaecalis and Rhodococcus erythropolis cells: acomparative studyT. Avramova, L. Stefanova, B. Angelova and S. Mutafov, Sofia
GAM35 pH–Related equilibrium study on copper biosorption byPenicillium cyclopiumM. Ianis, K. Tsekova, P. Marinov and D. Todorova, Sofia
GAM36 Immobilization of Penicillium cyclopium cells in PVAhydrogels for heavy metal ions biosorption applicationsD. Christova, K. Tsekova, S. Ivanova, M. Ianis, S. Ganeva,Sofia
GAM37 Biosorption of binary mixtures of copper and cobalt byPenicillium brevicompactumK. Tsekova, M. Ianis, V. Dencheva, S. Ganeva, Sofia
GAM38 Sensitivity of Saccharomyces cerevisiae yeast toarsenateT. Todorova, S. Vuilleumier, A. Kujumdzieva, Sofia,Strasbourg, France
GAM39 Transport of arsenat in yeast cellsS. Shilev, Sofia
GAM40 Isolation, identification and selection of arsenic andcadmium resistant yeastN. Krumov, V. Gotcheva, Ts. Hristozova, C. Posten,A. Angelov, Sofia, Karlsruhe, Germany
GAM41 Characterisation and identification of bacterial communityisolated from metalworking fluidsS. Bakalova, P. Hristova, R. Dimkov, Veneta Groudeva, Sofia
GAM42 Taxonomic identification of Xenorhabdus(Enterobacteriaceae) species by Restriction analysis ofPCR-Amplified 16S rDNA genesJ.H. Georgieva, V.I. Groudeva, M.D. Shishiniova, Sofia
GAM43 Preliminary investigations of thermal sources biodiversityfrom Rupite region
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A. Terziyska, R. Mandeva, D. Lyutzkanova,M. Stoilova-Disheva, G. Radeva, M. Kambourova, Sofia
GAM44 Biodiversity of carbohydrate degrading cultivable bacteriafrom Bacillus group, isolated from bulgarian hot springsA. Derekova, C. Sjøholm, R. Mandeva, M. Kambourova,Sofia;
GAM45 Spatial pattern of microbial numbers in the free water ofthe Srebarna lakeS. Naumova, A. Petrova, Sofia
GAM46 Comparison of bacterioplankton development betweencontrol and fertilized fish pondsH. Kalcheva, D. Tersiisky, R. Kalchev, Sofia
GAM47 Adaptive stress response of Humicola lutea 103to copperexposureE. Krumova, Y. Gocheva, A. Dolashki, P. Dolashka,S. Stefanovic, W. Voelter, M. Angelova, Sofia, Tuebingen,Germany
GAM48 Physiological response of Antarctic fungi to long-termtemperature stressY. Gocheva, E. Krumova, L. Slokoska, J. Miteva,M. Angelova, Sofia
GAM49 Cellular response and antioxidants enzymes in Aspergillusniger strain against temperature stressR. Abrashev, A. Dolashki, S. Stevanovic, P. Dolashka,W. Voelter, L. Stefanova, S. Pashova, R. Hristova,M. Angelova, Sofia, Tuebingen, Germany
GAM50 Biosynthesis of proteolytic enzymes from Antarcticactinomycete strainsD. Dimitrova, P. Dorkov, B. Gocheva, Sofia
GAM51 Biosynthesis of antimicrobial substances from Antarcticactinomycete strainsD. Dimitrova, P. Dorkov, B. Gocheva, Sofia
GAM52 Biosynthesis of proteinase inhibitor from Antarcticactinomycete strainsD. Dimitrova, P. Dorkov, B. Gocheva, Sofia
GAM53 Cyclodextrin glucanotransferase production bymagnetically responsible Bacillus circulans atcc 21783 cells
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M. Safarikova, N. Atanasova, V. Ivanova, S. Engibarov,I. Safarik, A. Tonkova, Ceske Budejovice, Sofia, Plovdiv
GAM54 Molecular analysis of a thermostable gellan lyase byMECC/HPLCM. Atanassova, J.I.G. Sanchez, A. Terziiska, A. Derekova,R. Mandeva, M. Kambourova, Sofia, A Coruña, Spain
GAM55 Purification and characterisation of keratinolyticproteases produced by Streptomyces albidoflavusV. Stefanova, N. Kirilov, K. Tsiroulnikov, M. Dalgalarrondo,J-M. Chobert, I. Ivanova, T. Haertlé, Sofia, Moskow, Russia;Nantes, France
GAM56 BNMPK: a web based suite of bacterial nucleotide monophosphte kinasesAjay Bikumandla, Sai Guduru, Paulina Daalova, Alexandra Shosheva, Petya Christova, Petras Kundrotas,Emil Alexov, Sofia
GAM57 Screening of phytase producing yeastsD. Georgiev, S. Gargova, Plovdiv
GAM58 Mutagenic treatment of strain Aspergillus awamori K-1,producer of xylanaseY. Evstatieva, S. Ilieva, D. Nikolova, V. Savov, A. Atev,Sofia
GAM59 Studying of bioactive metabolites from Arctic cold-adaptedstreptomycetesD. Lyutskanova, M. Stoilova-Disheva, , M. Kolarova,K. Alexieva, V. Peltekova, V. Ivanova, Sofia
GAM60 Properties and immobilization of fungal cellulase onpolyamideG. Delcheva, I. Pistijski, G. Dobrev, Plovdiv
GAM61 Primary characterization of a newly discoveredbacteriocin-like substance duracin produced fromEnterococcus durum M-3S.G. Dimov, Sofia
GAM62 Novel bacteriocin from Enterococcus faecium 3587 –spectrum of activity and some molecularcharacteristicsV.M. Peeva, P.M. Ivanova, S.G. Dimov, Sofia
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GAM63 Lactococcus lactis subsp. Lactis HV219 – a probiotic?S.D. Todorov, M. Botes (neé Brink), S.T. Danova, L.M.T.Dicks, Sofia, Stellenbosch, South Africa
GAM64 Effect of medium components on production ofbacteriocins JW3BZ and JW6BZ by Lactobacillusplantarum isolated from bozaJ.W. von Mollendorff, S.D. Todorov, L.M.T. Dicks,Stellenbosch, South Africa, Sofia
GAM65 Factors affecting the adsorption of bacteriocin ST194BZto Lactobacillus sakei and Enterococcus faeciumS.D. Todorov, M. Meincken, LMT Dicks, Stellenbosch, SouthAfrica, Sofia
GAM66 Deformation of bacterial cells as a result of bacteriocinsproduced by lactic acid bacteriaM. Meincken, S.D. Todorov, J.W. von Mollendorff, L.M.T.Dicks, Stellenbosch, South Africa, Sofia
GAM67 Partial characterization of a bacteriocin produced byLactobacillus curvatus isolated from cervelat salamiA. van den Worm, S.D. Todorov, L.M.T. Dicks, Stellenbosch,South Africa, Sofia
GAM68 Sanionins: antiinflammatory and antibacterial agents withweak cytotoxicity from the Antarctic moss Sanioniageorgico-uncinataV. Ivanova, K-J. Dornberger, A. Haertl, U. Moellmann,H-M. Dahse, M. Kolarova, K. Aleksieva, N. Chipev, Sofia,Jena, Germany
GAM69 Malonyl,4-5-dihydroniphimycin: new polyol macrolideantibiotic, produced by Streptomyces hygroscopicusV. Ivanova, M. Kolarova, K. Aleksieva, Sofia
GAM70 Diphenylether and macrotriolides occurring in a fungalisolate from the antarctic lichen NeuropogonV. Ivanova, U. Graefe, B. Schlegel, M. Kolarova,K. Aleksieva, Sofia, Jena, Germany
GAM71 Microbiaeratin, a new natural indole alkaloid from aMicrobispora aerata strain, isolated from Livingstonisland, AntarcticaV. Ivanova, U. Gräfe, H-M. Dahse, M. Kolarova,
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K Aleksieva, H Laatsch, Sofia, Jena, Göttingen, GermanyGAM72 An investigation on the opportunities for increasing
Streptomyces ambofaciens biosynthetic activity throughinduced mutagenesisN. Hristova, V. Baloutzov, S. Karafizova, Sofia
GAM73 Permeabilization of yeast cells for ΑΑΑΑΑ-keto acid productionD.D. Kostova, A. Kujumdzieva, Sofia
GAM74 Study of effect of oxygen mass transfer on biosynthesis ofexopolysaccharide P-45A.V. Atanassova, K. Pavlova, G. Atanassova, A.I. Tonchev,V.V. Lossev, V.G. Nasarov, Pestera, Plovdiv
GAM75 Scaling-up of amino acid biosynthesis using oxygen masstransferA.V. Atanassova, A.I. Tonchev, V.V. Lossev,S.B. Petkov,V.G. Nasarov, Pestera
GAM76 Quantitative assay for gentamicin in blood plasma bymicrobiological methodP. Yankova, A. Varssanova, Pestera
GAM77 Designing a whole-cell biotransformation double-phaseoxidation system with Streptomyces roseochromogenesB. Marinov , D. Koleva, I. Kostova, Razgrad
GAM78 Biophysical characteristics of bacterial biosurfactantsE. Stoimenova, E. Vassileva-Tonkova, M. Ivanova,Ch. Petkova, A. Jordanova, A. Sotirova, D. Galabova, Z.Lalchev, Sofia
GAM79 Combination of methods for species identification ofLactobacillus delbrueckii ssp. bulgaricusT. Savova, Z. Urshev, M. Spassova, I. Petrova,D. Ishlimova, P. Alexieva, Sofia
GAM80 Selection of Streptomyces thermophilus strains forimprovement of starter cultures for direct applicationK. Pashova-Baltova, N. Ninova, Z. Urshev, M. Michailova,Sofia
GAM81 Valuation of proteolytic activity in cultures ofLactobacillus delbrueckii ssp. bulgaricusZ. Urshev, N. Fachikova, K. Pashova-Baltova, I. Petrova,Sofia
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GAM82 Antimicrobial activity of lactic acid bacteria isolated fromBulgarian rye acid doughL. Iovcheva, G. Dobreva, R. Vassileva,S. Antonova-Nikolova, Sofia
GAM83 Milk-clotting enzymes from submerge cultivatedbasidiomycetesT. Dmitrieva, I. Feist, M. Shamtsyan, St. Petersburg, Russia
GAM84 Intensification of yeast biomass accumulation and ethanolfermentation processesI. Koltyga, T. Dmitriyeva, M. Shamtsyan St. Petersburg,Russia
GAM85 Ballistic disintegration of biomass from Lactobacillusdelbrueckii subsp. bulgaricus BTCC 50D. Nikolova, V. Savov, Y. Evstatieva, S. Ilieva, P. Dalev,A. Atev, Sofia
GAM86 Hybridization of Lactobacillus plantarum 226-15 andLactobacillus casei subsp. casei CA. Slavchev, I. Murgov, Z. Denkova, Plovdiv
GAM87 Investigation of the inhibitory effect of lactic acid bacteriaon the cells of Escherichia coli NBIMCC 8739P. Nedelcheva, Z. Denkova, R. Nikolova, Plovdiv
GAM88 The effect of DNA replication on the repair of damagedplasmids in Saccharomyces cerevisiaeM. Koprinarova, A. Gospodinov, G. Russev, Sofia
GAM89 Ageing in brewing yeastT. Ginova, S. Mileva, Sofia
GAM90 Growth inhibitory properties of chalcones against variousyeast speciesK.L. Lahtchev, D.I. Batovska, St. Parushev, V. Bankova, Sofia
GAM91 Electro-optical method as a new approach of investigationand discrimination of two strains Esherichia coliA. Gurova-Chausheva, E. Velichkova, R. Aleksandrova,S. Danova, S.P. Stoylov, Sofia
GAM92 Application of co-polymer microparticles forimmobilization of trypsinT. Ivanov, M. Kamburov, V. Ivanova, J. Hristov, Sofia
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GAM93 Immobilization of trypsin on co-polymers of acrylonitrileand maleinic anhydrideT. Ivanov, M. Kamburov, V. Ivanova, J. Hristov, Sofia
Veterinary Microbiology
VM9 Comparative studies on the methods for detection ofMycobacterium bovis in animalsM. Bonovska, S. Milashki, A. Abass, T. Savova, E. Gjurova, Sofia
VM10 Influence of karbofuran on capability of Fusariummoniliforme to produce fumonisins upon maizeL. Borisova, Y. Tasheva, V. Vrabcheva, Sofia
VM11 Determination of Pseudomonas aeruginosa in bull andboar semen by reaction co-agglutinationG. Vassilev, Stara Zagora
VM12 Experiments of induction of toxigenous mutants ofClostridium perfringens type CE. Iliev, D. Ilieva, M. Petkov, N. Korudgiisky, Sofia
VM13 Changes in the pathogenic potential of Yersiniaenterocolitica during storage of contaminated pig meatM. Iliev, H. Najdenski, Sofia
VM14 Detection of pathogenic serotypes of Yersiniaenterocolitica in contaminated milkM. Iliev, H. Najdenski, Sofia
VM15 Exactingness bacteria in etiology of endometritispuerperalis cow and possibility for therapyN. Korudjiiski, Sofia
VM16 Drug resistant bacteria of the genus Alcaligenes, isolatedfrom rabbits with intestinal disordersT. Galabinova, B. Mitov, N. Korudjiiski, S. Ivanova,L. Angelov, Sofia
VM17 Neoplasms in farm-raised Russian sturgeonV. Chikova, V. Kolarova, A. Tsekov, Sofia, Varna
VM18 Occurrence of aeromonosis infection among culturedPaddlefish (Polyodon spathula)V. Chikova, T. Hubenova,V. Kolarova, R. Atanasova,A. Zaikov, Sofia, Varna
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Friday, October 06
Poster session II
16:00-18:30
Medical Microbiology
MM22 Emm Sequence typing of clinical isolates Streptococcuspyogenes recovered in BulgariaA. Decheva, V. Karjeva, D. Beshkov, I. Alexiev, Sofia
MM23 Recombinant Borrelia Burgdorferi proteins as antigensfor serologic diagnosticof lyme borreliosisI. Christova, M. Lesseva, G. Miloshev, Sofia
MM24 Detection of Borrelia burgdorferi sensu lato, Anaplasmaphagocytophilum and Francisella tularensis in wildrodents from an endemic focus of tularemia in BulgariaT. Gladniska, I. Christova, E. Taseva, R. Nenova, Sofia
MM25 New focus of tularemia established in North-East Bulgariaduring 2004 – 2005N. Gotev, K. Mladenov, Tz. Tzvetanov, E. Penkov,N. Korudjiysky, S. Ivanova, V. Doicheva, Sofia
MM26 Fenotypic characteristics of Francicella tullarensisisolated in Bulgaria during 1961 – 1965 and 1998 – 2005N. Gotev, K. Mladenov, Sofia
MM27 Treatment of experimental tularemia infectionK. Mladenov, H. Najdenski, Tz. Tzvetanov, N. Gotev, Sofia
MM28P Rapid identification and biodiversity of Lactobacillusspecies in vaginal samplesS. Dimitonova, P. Grozdanov,A. Galabov, B. Bakalov,R. Aleksandrova, G. Stoyancheva and S. Danova, Sofia
MM29 Virulence determinants of Aeromonas spp. isolated fromfood, drinking water and patients in BulgariaP. Orozova, I. Abrashev, Sofia, Bulgaria
MM30 Determination of expiry term of antimicrobial disksCefoxitin and Ceftriaxon and their introduction for
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manufacturingD. Chankova, D. Pencheva, Sofia
MM31 Laboratory methods for drug susceptibility testing ofmedically important yeast and mouldsA. Kouzmanov, T. Kantardjiev, Z. Ivanova, L. Boyanova,Sofia
MM32 Study of hypocholesterolic effect of HigherBasidiomycetesA. Popov, A. Panchenko, O. Chistova, N. Petrishchev,N. Denisova, M. Shamtsyan, St. Petersburg, Russia
MM33 Resistance and genotypic diversity of multidrug resistantAcinetobacter baumanii in university hospitaR. Vatcheva-Dobrevski, E. Savov, A. Bernards,van den Barselaar, Sofia, Ceiden, Netherlands
MM34 Immunomodulating and antitumour action of higher fungiV. Spiridonova, P. Tsvetkov, A. Panchenko,A. Korchmaryova, N. Petrischev, M. Shamtsyan,St. Petersburg, Russia
MM35 Antioxidant properties of higher mushroomsN. Dubyago, I. Shugaley, E. Tozik. M. Shamtsyan,St. Petersburg, Russia
MM36 Indirect immunofluoroscence method for detection ofHelicobacter pylori directly from stomach biopsyK. Ivanova, Tz. Ilieva, M. Mariana, I. Mitov, B. Vladimirov,J. Churchev, Sofia
Virology
V31 Effects of some antivirals against rhinovirus H-14I. Georgieva, A. S. Galabov, Sofia
V32 Study of virus RNA synthesis in cell cultures infected withbovine viral diarrhea virus (BVDV) using hypertonicNaCl solutionsYu. P. Abashev, L. Mukova, L. Wassilewa, A. S. Galabov,Sofia
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V33 Biological activity of extract from Orthosiphon stamineusBenthS. Shishkov, K. Kostova, E. Georgieva, V. Kapchina-Toteva, Zh. Iordanova, V. Chipeva, S. Trandeva, Sofia
V34 Virological surveillance of acute flaccid paralysis inBulgaria during 2001-2006 periodN. Korsun, S. Gyurova, Z. Mladenova, Sofia
V35 Effect of thiosemicarbazone on HSV-1 and HSV-2replication in vitroN. Vilhelmova, M. Gacovska, S. Shishkov, P. Souza, Sofia;Madrid, Spain
V36 Seroepidemiological studies of influenza in Varna for1990-2004 periodV. Rusev, L. Ivanova, A Shtilianova, R. Ivanova,B. Shmatov, Varna
V37 Influence of osmotic swelling on the dhpc lipid bilayerscontaining ndv glycoproteinsP. Borissova, V. Neitchev, L. Doumanova, Sofia
V38 Detection of canine parvovirus serotype 2 in fecalsamples from dogs by polymerase chain reactionC. Filipov, P. Grozdanov, Z. Raikov, H. Haralambiev,A. S. Galabov, Sofia
V39 Molecular diagnostic of the Lewandowsky-Lutz syndrome(Еpidermodysplasia Verruciformes)B. Boneva, M. Sajedg, G. Mateev, Z. Kalvachev, Sofia
V40 Varicella zoster virus (VZV) and pregnancyL. Ivanova, Varna
V41 Demonstration of rabbit myxoma virus in cell cultures bydirect immunoperoxidase method
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R. Bostandjieva, Z. Dimitrova, R. Peshev, Sofia
V42 Evaluation of the efficacy of the broad-spectrumdisinfectant on some viruses pathogenic for the fishesD. Ilieva, Sofia
V43 Comparative studies of whales for diagnostic of somevirus infections in fishesV. Chikova, D. Ilieva, Sofia
Infectious Immunology
II4 Rapid immunohistochemistry method for detection of TSEprion protein on frozen brain tissue sectionsE. Lubashevsky, H. Yadin, S. Perl, N. Adry, A. Gorochov,V. Bumbarov, Beit Dogan, Israel
II5 “Respivax” modulates the expression of CD86 ondifferent circulating APC subsetsD.Stankulova, A. Mihova, H. Taskov, Vl. Maximov,M. Nikolova, Sofia
II6 Skin test for assessment of post-vaccine BCG allergy.Quantity of Bulgarian PPD tuberculin for human useE. Sapungieva, E. Jordanova, Sofia
II7 Fifty-year immunoprophylactics of tetanus with BulgarianvaccineE. Sapungieva, I. Todorova, E. Jordanova, J. Hristova,M. Demireva, R. Malchanova, Sofia
II8 Monitoring of cytotoxic CD8 T lymphocytes in HIV-1+patients subjected to HAARTM. Muhtarova, M. Nikolova, S. Magaev, H. Taskov, Sofia
II9 Protection against whlooping cough in children between 0-6 years oldR. Alexiev, K. Hadjiisky, S. Malchanova, V. Demireva,Pl. Nenkov, Sofia
II10 Investigation on the immune status of the populationagainst diphtheria during the period 2001 – 2005
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R. Alexiev, K. Hadjiiski, S. Malchanova, V. Demireva,Pl. Nenkov, Sofia
II11 Determination of minimal sensitizing doze of BCG vaccine(substrain Sofia SL222) in guinea pigT. Stefanova, M. Chouchkova, S. Nikolaeva, Sofia
II12 Personal experience for diagnostics and differentialdiagnostics of avian flueV. Lupke, B. Yonkova, V. Lyoutzkanova, Veliko Tarnovo,Varna
II13 Chlamydia trachomatis antibodies in serum and genitalfluids in infertile couplesV. Yonkova, V. Lyoutzkanova, V. Savouleva, Y. Yonkov,Varna
II14 Immunoblot analysis of antibody response to plasmidencoded released proteins of Yersinia enterocolitica inpatients with reactive arthritisE. Golkocheva, H. Najdenski, R. Stoilov, Sofia
II15 Attenuation and preserved immunogenic potential ofYersinia pseudotuberculosis mutant strains evidenced inoral pig modelH. Najdenski, E. Golkocheva, E. Ivanova, V. Kussovski,A. Vesselinova, S. Garbom, H. Wolf-Watz, Sofia, Umea,Sweden
II16 Hemocyanins as immunostimulatorsR. Toshkova, E. Ivanova, M.-D. Nastke, L. Velkova,S. Stevanovic, R. Hristova, A. Dolashki, M. Gardeva,I. Dimitrov, W. Voelter, P. Dolashka-Angelova, Sofia,Tuebingen, Germany
Parazitology
P2 Epidemiological aspects of human trichinellosis inBulgaria (2001 – 2005)M. Ivanova, R. Kurdova, D. Jordanova, N. Tsvetcova, Sofia
P3 Study on the diagnostic value of specific IgE antibodies inhuman echinococcosis
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I. Marinova, G. Nikolov, A. Mihova, R. Kurdova,B. Petrunov, Sofia
P4 Detection of cross-reactive bands in cystic fluid bywestern blot analysisI. Marinova, I. Rainova, A. Tchernov, R. Kurdova, Sofia
P5 Application of IgG аvidity for diagnosis of acutetoxocarosisI. Rainova, Sofia
P6 Antibodies against Toxoplasma gondii in human IgpreparationsI. Rainova, J. Nacheva, A. Tchernov, Sofia
P7 Identification of free-living amoebae by PCR.N. Tsvetkova, R. Kurdova, Sofia
P8 Visceral leishmaniasis in BulgariaR. Harizanov, G. Filipov, D. Yordanova, R. Kurdova, Sofia
P9 Echinococcosis distribution among children andadolescents in Bulgaria (1990 – 2005)D. Yordanova, R. Kurdova, Sofia
P10 Clinical forms and chemotherapy of trichinosisD. Vuchev, K. Anichina, K. Eneva, V. Blagoeva, A. Russinova,M. Darakchieva, G. Stancheva, Sofia, Plovdiv, Smoljan
P11 Epidemiological features of trichinosis in central southernBulgaria (Plovdiv, Pazardjik and Smolian regions)D. Vuchev, K. Eneva, V. Blagoeva, A. Russinova,M. Darakchieva, G. Stancheva, N. Paliiska, Plovdiv,Smoljan, Pazardjik
Plant and Soil Microbiology
Chairpersons:A. Stoev, SofiaR. Donkova, SofiaN. Kaloianova, Sofia
PSM4 Morphologic and cultural variation in PhomopsisfoeniculiIR. Rodeva, J. Gabler, Sofia, Aschersleben, Germany
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PSM5 In vitro and in planta interaction between three sclerotialplant pathogensR. Rodeva, R. Pandeva, Sofia
PSM6 Biological properties and frees-drying of apple chloroticleave pop virus isolates from fruit tree species inBulgariaA. Borisova, A. Yordanova, Kjustendil, Sofia
PSM7 Effect of inoculation with Azospirillum and AM fungi onthe plant biomass of white thistle (Sylibium marianumL.)E. Jonova, N. Kaloyanova, Sofia
PSM8 Effect of inoculation with local phosphate decomposingbacterial strains on the rye-grassK. Nedjalkova, Sofia
PSM9 Taxonomical identification of arsenic resistant andarsenic transforming sulfate – reducing bacteriaK. Krumova, V. Groudeva, Sofia
PSM10 Effect of protein hydrolysate from keratin wastes on thesoil microfloraM. Nustorova, A. Gushterova, D. Braikova, E. Vasileva,Sofia
PSM11 Response of soybean genotypes to inoculation withBradyrhizobium japonicum
A. Markova, R. Altimirska, Y. Kirkova, G. Stoimenov, SofiaPSM12 Microbiological activity in soybean rhizosphere at
different soil moistureR. Altimirska, A. Markova, Hr. Stoykov, K. Chachev, Sofia
PSM13 The influence of herbicide Relay on the main properties ofBradyrhizobium japonicumR. Donkova, Sofia
PSM14 Microbiological characteristic of soils in the area of non-ferrous metals factory, town of Plovdiv, BulgariaR. Donkova, N. Dinev, Sofia
PSM15 Cadmium influence on microbiological activity ofcalcareous chernozemG. Petkova, R. Donkova, Sofia
17:30 General discussion
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ABSTRACTS
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TUBERCULOSIS
Tb1VITAE OF DR. STAMEN GRIGOROV
Angel S. GalabovThe Stephan Angeloff Institute of Microbiology, BAS
Tb2BCG VACCINES
M. Chouchkova, T. StefanovaBB-NCIPD Ltd., Sofia
The BCG vaccines celebrate the 100th anniversary of their discovery in a decade at thebeginning of 21 century since Albert Calmette and Camille Guérin had presented it beforethe Academie des Sciences in 1908. Over a period of 13 years, from 1908 to 1921, the bothresearchers produced ever less virulent subcultures by cultivating bovine tubercle bacilliover and over again. More than three billion doses of BCG vaccine have been given overthe past 80 years. We would again emphasize the immense role played by BCG immunizationin the struggle against tuberculosis in children.
The evolution of BCG strains and the diversity of strains used for production as wellas a need to explore their potential impact in efficacy and safety of BCG vaccines inhumans have been considered at the WHO meetings in the last few years. WHO hadidentified the need for better characterization of BCG substrains in the presently usedBCG vaccines. The Bulgarian BCG substrain is one of the three substrains used in theworld for the production of BCG vaccines, which have been prequalified by WHO for theUN agencies. The moderate residual virulence, the adequate postvaccinal tuberculinsensitivity and the genetic stability of the strain as well as the consistency of the productionhave been confirmed in the recent studies.
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Tb3РЕНЕСАНС НА ТУБЕРКУЛОЗАТА. АКТУАЛНИ
ПРОБЛЕМИ
Зл. ЯнковаКлиника по пулмология - МУ, гр. Пловдив
Може да се твърди, че туберкулозата е в “топлистата” на 10-тте заболявания,завършващи най-често със смърт. СЗО наблюдава туберкулозната епидемия вповечето страни на Света.
В последните години честота на туберкулозата е около 6,3 - 11,1 милионаболни ежегодно. Смив положителните са 2,8 - 4,9 милиона. Фаталният изход е23%. Заболяването поразява млади хора и за високо рисковите райони – често сесреща при родилки. В 22 страни са съсредоточени 80% от всички заболели -Индия, Китай, Пакистан, Бангладеш; Африка - южно от Сахара и др. За периодот 1 година /1997-1998/ 36500 инфектирани с НІV са починали от туберкулоза.Бедните страни имат средно 2,6 пъти по-често туберкулоза от богатите.
Няма тясна корелация между отделяните средства за борба с туберкулозата иефективност на Националните програми /СЗО/. Има 4 фактора, с които е свързанаепидемиологията:
1.Разрушаване на принципите на туберкулозния контрол и появата нарезистентност в Източна Европа.
2.Инфектиране с HIV в Африка. Появяване на асоциирани инфекции от дветезаболявания.
3.Миграция на инфектирани с ТБК от бедните - в индустриализираните страни.4.Нарушението на туберкулозния контрол в Латинска Америка и Азия.У нас Национална програма за борба с туберкулозата действува от 1950
год., когато са създадени първите диспансери. От 400 на 100 хиляди население,туберкулозата се редуцира до 28 на 100х. За съжаление след 1992 годиназаболяването зачести и сега се движи между 42 – 45 на 100 хиляди за известнататуберкулоза, но има хронично болни с недоказана туберкулоза. Един важен инерешен проблем остава доказването на Латентната туберкулозна интоксикация.Предлагаме за обсъждане диагностичен алгоритъм за ежедневната клиничнапрактика.
Tb4КОНТРОЛ НА ТУБЕРКУЛОЗАТА И ВЪВЕЖДАНЕ НА DOТ
СТРАТЕГИЯ В БЪЛГАРИЯ
Д. Стефанова
Увеличаването на заболеваемостта от туберкулоза в глобален мащаб през
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последните две десетилетия стана един от водещите проблеми, ангажиращисветовната общественост. В резултат на съчетанието на множествонеблагоприятни фактори в някои страни туберкулозата взе застрашителниразмери. Затова целите на Туберкулозния контрол са насочени към :
1. Намаляване заболеваемостта и смъртността от туберкулоза2. Ограничаване на лекарствената резистентностЕдинственно рационалната химиотерапия може да спре развитието на
туберкулозния процес. Навременното започване на комбинирана, продължителнаи непрекъсната терапия е гаранция за ефективно лечение. През 1990 год.Световната Здравна Организация изрази тревогата си от нарастване наразпространението на туберкулозата и започна въвеждането на DOTS стратегията– Директно наблюдавано лечение в съкратени срокове.DOTS се превърна вповратна точка в контрола на туберкулозата. През 1998 г. започна въвежданетона стратегията у нас, което продължи до 2003 год. Лечебните режими на DOTSстратегията се основават на най-ефективни съчетания от противотуберкулознипрепарати и синергичното им действие върху Туберкулозния микобактерий. Тъйкато лечението при всички болни не може да се провежда само с краткосрочнирежими СЗО ревизира стратегията и въведе DOT /директно наблюдавано лечение/. DOT позволява и индивидуализирани програми с вариации в комбинациите притежки прогресиращи форми на туберкулоза.
След започването на модерната химиотерапия Туберкулозният микобактерийпоказа резистентност към различните медикаменти, която компрометираНационалните програми за контрол и лечение на туберкулозата. От същественозначение е мултирезистентността, която се определя от резистентност къмосновните туберкулостатици – Тубоцин и Римицид при наличие или отсъствие нарезистентност към другите туберкулостатици. В България мултирезистентносттаварира в последните години. Най-висока е тя през 1998 г. – 7,3% , а за 2005 г. е4.1% . Намаляването на процента на мултирезистентност се дължи на включванена стратегията DOT+ с етиоамиди, макролиди, нови генерации аминоглюкозиди,респираторни хинолони и въвеждането на хирургическо лечение при болни бездисеминация на туберкулозния процес и съхранени кардио-респираторни резерви.
Традиционно резистентността се разделя на първична и придобита.Първичната резистентност се развива при болни, при които няма анамнеза запровеждано туберкулостатично лечение. Първичната резистентност се задържависока до 40%, което показва, че в страната циркулират вирулентнимултирезистентни щамове. Вторичната резистентност се развива когато сепровежда лечение с неефективни лекарствени режими или лечението е прекъснатопреди определените срокове.
За първи път у нас ще се представи и новата стратегия на СЗО / 2006- 2011 / заКонтрол на туберкулозата.
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Tb5ТУБЕРКУЛОЗАТА СРЕД ДЕЦАТА – В МИНАЛОТО И
ДНЕС
П. МинчевУниверситетска Детска Клиника по Белодробни БолестиМедицински Университет – София
Разпространението на туберкулозата в България сред детското население следвойната е изключително голямо – 90 на 100 000 души. За период от 45 годинизаболеваемостта намалява и през 1990 година е в границите на 8 на 100 000души. В структурата на заболяемостта настъпват промени, като значителнонамаляват относителните дялове на хематогенно – дисеминираните форми итуберкулозния менингит. Основният фактор за благоприятните тенденции вепидемиологията на първичната туберкулоза е провеждането на специфичнаимунопрофилактика с ваксината БЦЖ. България заедно с Хонг – Конг въвеждазадължителна БЦЖ ваксинация при децата още през 1951 година.
След 1990 година започва неблагоприятна тенденция в епидемиологията иструктурата на първичната туберкулоза. Причините за това са комплекснообусловени. През 2005 година заболяемостта от туберкулоза сред децата е вече25,8 на 100 000 души. Съществен проблем е и латентната туберкулозна инфекция,която налага контролирана химиопрофилактика.
Лечението на туберкулозата при децата се извършва с въведените у настерапевтични режими за всяка форма на първична туберкулоза. DОТS енеприемлива за деца в целия свят, поради различията между туберкулозата увъзрастни и деца. Основната обективна разлика е, че бацилоотделянето при деца ев ниски граници /6-10%/ и не е единствения критерии за поставянето на диагнозата.
Туберкулозата сред децата и възрастните е заболяване, което не може да бъдеизцяло инактивирано. Абсолютно сигурно е че е възможно да бъде поставено поднепрекъснат контрол.
Tb6СЪВРЕМЕННА МИКРОБИОЛОГИЧНА ДИАГНОСТИКА
НА ТУБЕРКУЛОЗАТА И МЕТОДИ НАЕПИДЕМИОЛОГИЧНО МАРКИРАНЕ
Т. Кантарджиев, Ст. Панайотов, Ел. Бачийска,НЦЗПБ, София
Представени са възможностите на НРЛ по туберкулоза и НРЛ по молекулярнамикробиология към Националния център по заразни и паразитни болести в
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съвременната микробиологична диагностика на туберкулозата. Посочени саосновните – класически методи, както и нови, не конвенционални методи вкултивирането на M.tuberculosis. Изтъква се голямото диагностично значение наметодите за епидемиологично маркиране на туберкулозните щамове – RFLP,AFLP, сполиготипиране.
Tb7МОЛЕКУЛЯРНО-ЕПИДЕМИОЛОГИЧНА
ХАРАКТЕРИСТИКА НА ЩАМОВЕ MYCOBACTERIUMTUBERCULOSIS ОТ РАЗЛИЧНИ РЕГИОНИ НА БЪЛГАРИЯ
Виолета Вълчева1, Игорь Мокроусов2, Ольга Нарвская2, Надя Маркова1
Институт по Микробиология, БАН ,София1;Санкт Петербургский ИнститутПастьор, Русия2
В България броят на новооткритите случаи на туберкулоза се е увеличил от41.0/100,000 през 2000г. до 42.4/100,000 през 2004г. Цел на изследването бешемолекулярно епидемиологично охарактеризиране на популационната структурана M. tuberculosis в България и анализ на мутациите, свързани с резистентносттакъм противотуберкулозни препарати. Въз основа на метода spoligotyping, 113щама бяха подразделени на 35 сполиготипа: 13 уникални профили и 15 профили,включващи от 2 до 29 щама; индексът на Хънтър-Гастон беше 0.9. Сравнениетосъс световната база дани SpolDB 4.0 показа присъствието на два глобалнисполиготипа ST53 (25.7%) и ST47 (6%). 19 (16.9%) и 7 (6%) щама принадлежаха наST125 и ST41. 8 (7%) сполигопрофили не бяха намерении в SpolDB 4.0. Щамове сгенотип Beijing не бяха открити в изследваната колекция. Типирането с методитеIS6110-RFLP, MIRU-VNTR и DRE-PCR позволи диференциране на щамовете вътре вST53, ST47, ST41 и ST125. 17 (15%) щама бяха генотипно резистентни къмрифампицин, въз основа на анализ на rpoB гена. 13 от тях имаха мутация в rpoB531TCG>TTG докато един щам беше хетерорезистентен. Мутация в katG315 сеустанови в 9 щама. 8 щама имаха мутация в embB306, а 3 от тях - мутацииедновременно в rpoB и katG. При сравнение с фенотипните тестове, генотипнатадетекция на резистентността даде 80% чувствителност за рифампицин (анализ наrpoB) и само 30% за изониазид (анализ на katG315). Въз основа на полученитерезултати, беше установено, че изследваната популация на M. tuberculosis е достахетерогенна и в нея преобладават няколко глобално разпространени и балканскисполиготипа. Трансмисията на мултирезистентни щамове в България (21.6% вколекцията) не е свързана с глобалното распространение на генотип Beijing, койтоочевидно все още не е достигнал страната ни.
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Tb8ВЪТРЕВИДОВО ОПРЕДЕЛЯНЕ НА МИКОБАКТЕРИИ
ЧРЕЗ PCR
Магдалена Боновска1, Христо Найденски2
1Централен Научноизследователски Ветеринарномедицински Институт, София,2Институт по микробиология “Стефан Ангелов” – БАН
Различни представители на род Mycobacterium са диференцирани успешно смултиплексен формат на PCR, прилагайки две и три двойки праймери в различниколичествени съотношения. Използвани са двойките праймери ТВ15/ТВ19, IS41/IS43 и PT1/PT2 и геномна ДНК от M. bovis, M. bovis BCG, M. tuberculosis, M. avium,M. kansasii, M. intracellulare, M. malmoense, M. phlei, M. chelonae и M. scrofulaceum.
При мултиплексна PCR с праймери ТВ+РТ в съотношение1:1, се наблюдаваPCR продукт с големина 600 bр при M. bovis, M. avium, M. kansasii, M. intracellulare,M. malmoense и M. chelonae. При ТВ+РТ (2:1,7) само M. bovis, M. avium и M.malmoense амплифицираха PCR продукт от 600 bр, а при ТВ+РТ (2:1,5) - само M.bovis. M. tuberculosis амплифицира два фрагмента (400 и 600 bр) при всичкиколичествени съотношения на праймерите. Комбинацията ТВ+IS (2:1)амплифицира два фрагмента от 300 и 600 bp с M. bovis, M. bovis BCG и M.tuberculosis. Подобни амплификати се наблюдаваха при M. bovis, M. bovis BCG иедин щам M. tuberculosis и при съотношение на праймерите 1,7:1. Другите 2 щамаM. tuberculosis показаха един фрагмент от 300 bp. При ТВ+IS (1,5:1) двуфрагментниамлификати показаха M. bovis и M. bovis BCG, а всички M. tuberculosis бяхаеднофрагментни. При амплификация с три двойки праймери ТВ+IS+РТ (2:1:2) сенаблюдават три фрагмента - 300, 400 и 600 bp при M. tuberculosis, два (300 и 600bp) при M. bovis и M. bovis BCG и един (600 bp) при M. avium, M. phlei и M.scrofulaceum. При ТВ+IS+РТ (2:1:1,5) амплификати се установяват само при M.bovis и M. bovis BCG (двуфрагментни) и при M. tuberculosis (трифрагментни).
Получените резултати показват, че мултиплексната PCR с 2 и 3 двойкипраймери, при подходящо съотношение между тях, може да се използва успешноза специфично и бързо вътревидово разделяне на представителите на M.tuberculosis complex от една страна, а от друга между тях и осталите микобактерии,което не винаги е възможно с нормална PCR, използваща за амплификация еднадвойка специфични праймери.
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VIROLOGY
V1HENIPAVIRUSES, A NEW FAMILY OF PARAMYXOVI-
RUSES, WHICH HAVE EMERGED IN ASIA AND AUSTRALIA
F. WildINSERM U404, Immunity and Vaccination, CERVI, IFR 128, Lyon, France.
During the past ten years in Southern Asia and the Western pacific a number ofviruses have emerged in which the natural host is the fruit bat (Pteropus). Amongst thesewere two closely related viral pathogens Hendra and Nipah, which appeared ingeographically distinct regions. The viruses were shown to belong to the Paramyxovirusfamily. In 1994 in Australia, Hendra virus crossed the species barrier infecting horses andeventually man. In 1998 in Malaysia, Nipah virus was found in pigs and subsequently manbecame infected. Since this time, it has been shown that a high proportion of the fruit batpopulation in Asia and Australia are infected. Further studies have identified epidemics inhumans in India and Bangladesh. Infection in pigs gives mainly an acute respiratorydisease with approximately 10% mortality, whereas humans develop encephalitis with upto 70% mortality. For these reasons, the virus has been classified as a P4 virus i.e. can onlybe handled in laboratories with the highest security levels.
In order to analyse the various processes during infection, we have developed thehamster as an animal model. The infected animals develop fatal encephalitis and thepathology is similar to that observed in humans. Immunisation of hamsters with either theG or F glycoproteins protected the animals from a clinical infection. Further, either polyclonalor monoclonal antibodies directed against either the G or F glycoprotein when givenpassively protected animals against infection. Thus, we have defined the main actors inboth preventive (vaccination) and treatment (passive) of the virus infection.
V2
STRUCTURAL BASIS FOR ANTIVIRAL DRUG-RESISTANCEOF A VP1-MUTATED ENTEROVIRUS
Angel S. Galabov1, Ivanka Nikolova1, Roumena Petkova2, Stoyan Chakarov2 andBoris Atanasov3
1Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria;2Scientific Biological Service, Ltd., Sofia, Bulgaria; 3Institute of Organic Chemistry,Bulgarian Academy of Sciences, Sofia, Bulgaria
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Twenty years ago was found that antiviral WIN-compounds (such as arildone,pleconaril, disoxaril and etc) inhibit virus uncoating. Based on direct crystal X-ray analysisof virus-inhibitor complexes it was shown that the primary target structure of this action isthe VP1 capsid protein: inhibitors, inserted into a twisted â-sheet formed hydrophobiccleft, increased structural rigidity of VP1 subunit and thus prevent virus “undressing”.Following Darwinian evolution all WIN-treated wild viruses are blocked and the onlyinhibitor-resistant generation survived – a phenomenon also well desribed.
Using “Darwinian selsction” approach two disoxaril-resistant mutant strains of thecoxsackievirus B1 from a wild-type disoxaril-sensitive (Connecticut 5) were obtained. Oneof them was produced in FL cells and the other one isolated from brains of newborn mice,infected with coxsackievirus B1 and treated with disoxaril. They were object of furthermolecular genetic studies.
Analysis of the RNA sequence of an RT-PCR assay which primer sets selected from aregion of the coxsackievirus B1 genome coding for the capsid protein VP1 was carried out.A parallel comparative analysis of the sequences of resulting fragments from the disoxarilmutant studied and the Gen-Bank sequence of origin of the VP1 gene of coxsackievirus B1was performed with the BLAST alignment tool. Distinct alternations in the VP1 locus ofthe disoxaril-resistant compared to the sequence of origin from the Gen-Bank (namely, adeletion of UUG at ntt. 2749-2751 and an insertion of UUU at nt. 2769) were observed. Theresistant mutant obtained in mice was found to be very similar to the strain, dependent incell cultures. A crucial important change in disoxaril-resistant strain was two point mutations– M213H and F237L – both in ligand-binding pocket. As general, amino acid sequences ina large VP1 peptide 195-255 is highly different in comparison with the correspondingpositions of the wild protein.
A putative 3D-models of coxsackievirus B1 VP1 protein – wild (Sofia variant) and itsdisoxaril-resistant mutants – was constructed using as template known X-ray structure ofcoxsckievirus B3 (pdbcov.ent file) with “Composer-4” and “MOLIDE” programmingpackets. Also palmitate at B3-VP1 virus complex was replaced to disoxaril using GROMOS-96 molecular dynamics program at very restricted degree of freedom. Generated tetramericproteins of wid and resistant mutant forms (both with myristilate as amide bond to VP4 N-terminal group) was studied in terms of their intra-/inter molecular electrostatic andhydrophobic interactions. Specific stabilizing effect of VP1 on tetramer assembling; acooperative effect of ligand binding supported by all chains and sterically forbiddenaccess to VP1-binding cavity in the resistant mutant were obtained. A mechano-chemicalhypothesis explaining vital difference between palmitate and disoxaril complexes will bediscussed.
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V3
THE HIERARCHICAL QSAR TECHNOLOGY FOR EFFEC-TIVE VIRTUAL SCREENING AND MOLECULAR DESIGN OF
POTENTIAL ANTIVIRAL AGENTS
V. E. Kuz’min, A. G. Artemenko, E. N. Muratov, L. N. Ognichenko, A. I. Hromov,A. V. Liahovskij, P. G. PolischukA. V. Bogatsky Phys.-Chem. Institute of the Ni=ational Academy of Sciences ofUkraine, 86 Lustdorfskaya doroga, Odessa 65080, Ukraine; E-mail:[email protected]
Hierarchical QSAR (Quantitative Structure Activity Relationship) technology (HT) isdestined for optimization of new effective antiviral agents creation process. HT allows tosolve the QSAR task not ab ovo, but with the use of information received from a previousstage by mean of the system of improved solutions. The unique and principle feature ofthe HT consists in multiple-aspects hierarchical strategy that related to: models of molecularstructure descripion; models of atoms description in molecular simplexes; scales of activityestimation; mathematical methods of analysis the structure-activity relationship; finalaims of QSAR research (prediction, interpretation, structure optimization, molecular design).The set of different QSAR models that are supplementing each other is the result ofapplication of HT. These models all together, in complex, solve the problems of virtualscreening, evaluation of structural factors influence on activity, modification of knownmolecular structures and design of new high-performance potential drugs. Innovativeaspect and main advantages of HT: simplex representation of molecular structure, that isproviding universality, diversity and flexibility of description of compounds related todifferent structural types; HT that depending on the concrete aims of research allows toconstruct the optimal strategy of QSAR models generation. HT does not have therestrictions of such well-known and widely used approaches as CoMFA and HASL, usageof the lasts is limited in the structurally homogeneous set of molecules and only oneconformer. The efficiency of the HT shown on the example of compounds that possessingantiviral activity. If in an initial set there were only 12% of active compounds, afterapplication of the HT 75% of new designed compounds turned out perspective antiviralagents.
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V4CHRISTO RUSSEFF MEMORIAL LECTURE:OXOGLAUCINE: A NEW HIGHLY POTENT
ANTIENTEROVIRAL COMPOUND
Lubomira Nikolaeva-Glomb1, Stephan Filipov2, Angel S. Galabov1
1Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria;2Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academyof Sciences, Sofia, Bulgaria
A series of aporphinoid alkaloids isolated From Glaucinum flavum L. or obtainedsynthetically were tested in vitro for antiviral activity against viruses belonging to picorna-,orthomyxo-, paramyxo- and herpesviruses. One of them, oxoglaucine, manifested a wellpronounced inhibitory effect on poliovirus 1 replication in FL cells measured by the semi-quantitative agar-diffusion plaque-inhibition test. In virucidal activity testing the compounddid not show direct virucidal effect on the extracellular virus. Oxoglaucine’s 50% inhibitoryconcentration for poliovirus 1 (Mahoney) was found to be 0.188 µg/ml in the CPE-inhibitiontest and 0.041 µg/ml in the classical plaque-inhibition test. Similar values were obtained forthe vaccinal poliovirus type 1 strain, LSc-2ab. The antiviral effect of oxoglaucine on thereplication of viruses belonging to another enteroviruses was tested, i.e. coxsackie andechoviruses (HEV-B group). Coxsackievirus A-9, the six coxsackie B viruses and 6echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine bythe end-point dilution method in the multi-cycle CPE inhibition set-up in FL cells.Oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. Theconcentrations that reduced virus titer by 1 lg ranged from 0.01 µg/ml to 1.0 µg/ml. Selectivityindex was greater than 100 and even greater than 1000 for some of the viruses tested.Time-of-addition study showed that the susceptible period to oxoglaucine’s effect is thelatent and lag phase of the virus replication cycle.
V5
RATIONAL TREATMENT COURSE SCHEDULE EFFECTIVEAGAINST ENTEROVIRAL NEUROINFECTION IN MICE
Ralitsa Vassileva-Pencheva and Angel S. GalabovThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Sofia, Bulgaria; E-mail: [email protected]
Enteroviral infections are a basic indication for the application of antiviral chemotherapy.The lack of an effective therapeutic registered for clinical use, in spite of the substantial
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number of enteroviral replication inhibitors found in vitro, is due mainly to the rapiddevelopment of resistance in vivo. This phenomenon is a consequence of an accumulationof resistant population of quasispecies as a result of countless number of point mutations.One of the main possible approaches for an attempt of preventing the occurrence ofresistance is the method of combined aplication of antiviral inhibitors. Because of themultiple viral replication cycles in the presence of the partners in the combination, theusual scheme of administration of antiviral drugs – all partners are administered at once inone day, give no guarantee that the problem with the resistance would be resolved. Inorder to find a solution to this relevant question of present interest we propose anotherscheme for combined administration of inhibitors - consecutive administration of thepartners, as in double combinations they are applied every other day, in triple combinations–every third day, and in quadriple combination –every fourth day. In previous study ofour team we found out that two of the triple combinations – Dis/Oxo/PTU-23 and Dis/Oxo/Guan show significant effect of protection. In order to optimize this combined course oftreatment we studied the influence of chronology of the arrangement of inhibitors. In theexperiments that we carried out, three substances were applied in a combination – disoxaril(WIN compound), oxoglaucine (a new antiviral drug, developed in our laboratory) andguanidine-hydrochloride (a classic enteroviral inhibitor), on the model of a neurotropicinfection with coxsackievirus B1 in newborn mice. As a result of this investigation, itcould be concluded that the start of the treatment course with disoxaril has certain priorities,especially when disoxaril is followed by guanidine-hydrochloride. The effect of the triplecombination starting with oxoglaucine, followed by guanidine-hydrochloride is moderate.The combination in which guanidine-hydrochloride is the first of the partners to be applied,proved to be ineffective.
V6
EFFECTS OF PICORNAVIRUS REPLICATION INHIBITORSAGAINST CALICIVIRUS FCV
Julian D. Tumbarski and Angel S. GalabovThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Sofia, Bulgaria; E-mail address: [email protected]
The search of substances suppressing replication of caliciviruses is of special interestdue to their particular role in the human infectious pathology. Calicivirises belonging toNorovirus genus are among the principal causative agents of viral gastroenteritides (inchildren predominantly, and in all age groups in the developed countries). Caliciviridae isa comparatively recently differentiated family, initially (till 1979) included as a separategenus in Picornaviridae. Caliciviruses possess a (+) RNA genome; they are close instructure, but reveal a markedly different genome strategy. Application of anti-calicivirus
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chemotherapy is indicated in contrast to the lack of systematic search for antiviralsefficient vs. caliciviruses.
The aim of present report is the testing anti-calicivirus effects of several highly efficientinhibitors of picornavirus replication. Study was carried out on the feline calicivirus (FCV),F9 strain, a surrogate norovirus, grown in the Crandell’s feline kidney cell line (CrFK),highly susceptible to FCV replication. The antiviral screening carried out included thefollowing compounds: arildone, disoxaril and S-7 (inhibitors of early stages of thepicornavirus replication cycle), guanidine hydrochloride, PTU-23 and HBB (picornavirusspecific RNA synthesis inhibitors), ribavirin (a broad-spectrum antiviral agent) andoxoglaucin (a recently described in this laboratory enterovirus replication inhibitor). Anti-norovirus activity was tested through the CPE inhibition test in the microplate monolayercell cultures vs. virus inoculation doses ranging within 1 and 10 000 CCID50. The neutralred uptake and the routine visual microscopic methodical variants were used formeasurement of both compound antiviral effect and cytotoxicity. Results obtained manifesta pronounced efficacy of HBB (IC50 of 7.0 ìM), a marked activity of PTU-23 (IC50 67.4ìM), ribavirin (IC50 6.6 ìM) and oxoglaucin (IC50 0.076 ìM). Inhibitors of earlystages in picornavirus growth cycle (arildon, disoxaril and S-7) and guanidinehydrochloride did not show an anti-norovirus effect.
V7
IN VIVO EFFECTIVE ANTI-FLU COMBINATION OFANTIVIRALS
Lora Simeonova, Galina Gegova and Angel S. GalabovThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Sofia, Bulgaria; E-mail: [email protected]
The combination effect of rimantadine hydrochloride and oseltamivir phosphate(prodrug of the active compound oseltamivir carboxylate) on experimental infection inmice with influenza A/Aichi/2/68(H3N2) virus was studied. Compounds were administeredsimultaneously in a 5-day-treatment course, starting on the day of virus inoculation, 4hours before intranasal infection of animals with 10 or 20 viral 50% mouse lethal doses(MLD50). Initially, we tested a series of compound combinations in a checkerboard order,using oseltamivir at daily doses of 0.05, 0.1 and 0.2 mg/kg/day and rimantadine – 2.5, 5.0and 7.5 mg/kg/day. Mortality rate dynamics was followed till the 14th day post infection,mean survival time (MST) and protection index (PI) being evaluated. It was established: (i)PI values of all combinations of oseltamivir with 5.0 and 7.5 mg/kg rimantadine reaching87%, while the individual effect of oseltamivir - between 0 and 10% only, and of rimantadine– 0% at 5 mg/kg and 18.7-29.6% at 7.5 mg/kg; (ii) markedly lengthened MST in combination-treated groups. The combination effect was characterized as synergistic by the three-
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dimensional method of Prichard and Shipman, modified for in vivo studies. In furtherexperiments, we studied in more details the antiviral activity of oseltamivir at a daily doseof 0.05 mg/kg (200 times lower than the compound optimal effective dose of 10 mg/kg) incombinations with rimantadine 5 mg/kg (8-16 times lower than the optimal effective doseof 40-80 mg/kg). Measurement of mouse pneumonia parameters (lung virus titer in MDCKcells, lung index and consolidation score) proved the high effectiveness of this combinationfor treating of influenza virus A(H3N2) infection. At the 48-60th hour post infection (thepeak of lung virus growth) a 2.8 log10 CCID50 lower titer was recorded in the combination-treated group, compared to that in the placebo group, in contrast to 0.1-1.0 log10 and 1.1-1.4 log10 in the groups treated with oseltamivir and rimantadine, respectively. These dataemphasize the high anti-influenza A potential of the combination. As a next step we testedthe combination selected activity following a therapeutic treatment course: onset on days1, 2 etc. post virus inoculation. Moreover, efficiency of higher doses of combinationpartners (the ratio rimantadine/oseltamivir 99:1 been preserved) was studied.
V8
ТРЯБВА ЛИ ДА СЕ ИЗПОЛЗВА ПРЕПАРАТАМОНЕНЗИН ЗА ПРОФИЛАКТИКА НА ПТИЧИЯ ГРИП
С. Дундаров
Монензинът е полиетерен йонофорен антибиотик. За него са известни следнитеданни: 1.Притежава изключително широк противоинфекциозен спектър, в койтоса включени много и различни бактерии, фунги, паразити и вируси ( американскипатент); 2. Широко се използва от над 30 години в цял свят като антикокцидиалносредство и за угояване на птици и добитък (американски патент); 3. Притежаваспособност да преминава през клетъчните мембрани като променя обмена на солии образува микрофисури. Това му позволява да улеснява вътреклетъчния достъпна препарати с противораков ефект (френски патент). 4. От над 20 год. успешносе използва като препарат за локално приложение при лечение на повърхностнизаболявания, причинени от HSV1, HSV2 ,VZV и аденовирусни конюнктивити(български патент на препарат Модувир); 5. Противохерпесният му ефект сеобуславя от комплексна инхибиция на синтезата на вирусните белтъци и коственовъздействие върху репликацията на вирусната ДНК. Продукцията наинфекциозен вирус е редуцирана в над 99%; 6. Доказан е като мощен индуктор наинтерферон (българско авторско свидетелство); 7. Според някои автори може дапредотврати събличането на грипния вирус в ендозомите като алкализира средатаим и ограничава ефекта на ензима клатрин; 8. Проведени наскоро у насизследвания показаха, че Монензин инхибира развитието на грипните вирусиА(H1 N1 ) и A(H7 N1 ) в клетъчна линия МДСК (резултатите ще бъдат докладвани
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от авторите). Тези предпоставки ни дават основание да го препоръчаме катохранителна добавка за профилактика срещу заразяване от птичи грип нарисковите групи птици.
V9
IN VITRO ANTI-INFLUENZA VIRUS EFFECT OF A PRO-TEASE INHIBITOR FROM STREPTOMYCES
CHROMOFUSCUS 34-1
Julia Serkedjieva1, Lidiya Angelova1,2, Ivana Roeva1, Iskra Ivanova2
1Institute of Microbiology “Stefan Angelov”, Bulgarian Academy of Science, 26,Acad. Georgy Bonchev St.; 2Department of Microbiology, Sofia University, 8Dragan Tzankov Blvd., Sofia, Bulgaria
Twenty three Streptomyces strains, producers of typical serine protease inhibitors,were tested for inhibitory activity on the replication of influenza virus A/Germany/34,strain Rostock (H7N1) (A/Rostock) in Madin-Darby canine kidney cells. Eleven of them(52.2%) significantly inhibited viral reproduction. The most effective was the inhibitor,produced by Streptomyces chromofuscus 34-1 (SS 34-1). Its in vitro anti-influenza viruseffect was studied in more detail. As a first approach we assessed the susceptibility ofrepresentative influenza viruses to the inhibitory action of SS 34-1; most sensitive toinhibition were A/Rostock and A/PR8/34 (H1N1). By the use of complementary virologicalassays it was demonstrated that the expression of the viral haemagglutinin on the surfaceof infected cells, the virus-induced cytopathic effect and the infectious virus yields, usedas measures of A/Rostock virus growth, were all reduced at non-toxic concentrations ofSS 34-1. In addition the preparation protected mice from mortality in the experimentalinfluenza A/Aichi virus infection. The isolated novel protease inhibitor (PISC-2002) waspurified by anion-exchange chromatography and reversed phase-HPLC analysis. It was ahydrophobic and a termostable protein, had a molecular mass of 11.2 kDa, isoelectricpoint of 7.5 and a high content of hydrophobic amino acids and proline. The N-terminalsequence demonstrated its homology to the Streptomyces subtilisin inhibitors family.
V10
ANTI-HERPESVIRUS ACTIVITIES OF PSEUDOMONAS SP.S-17 RHAMNOLIPID AND ITS COMPLEX WITH ALGINATE
Mimi Remichkova, Ivana Roeva, Danka Galabova, Angel S. Galabov
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The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad. Georgi Bonchev str. 26, 1113 Sofia, Bulgaria
Pseudomonas sp. S-17 produces rhamnolipid (biosurfactant) and alginate(polysaccharide). In the present study, we investigated the antiviral activity of rhamnolipidand its complex with alginate against herpes simplex virus (HSV) types 1 and 2. Rhamnolipidand its complex with alginate significantly inhibited the herpes virus cytopathic effectassessed in MDBK cell line. The suppressive effect of the compounds on HSV replicationwas dose-dependent and occurred at concentrations lesser than critical micelleconcentration (CMC) of the surfactant. The 50% inhibitory concentration (IC50) ofrhamnolipid was 14.5ìg/ml for HSV-1 and 13ìg/ml for HSV-2. The IC50 values of thecomplex were 435ìg/ml for HSV-1 and 428ìg/ml for HSV-2. Similar results wereobtained from the virus yield reduction assay. We observed that the antiviral propertiesof rhamnolipid in a complex with a alginate were more pronounced than rhamnolipid alone.
V11ANTIOXIDANT EFFECTS OF PLANT POLYPHENOLS
QUERCETIN AND RUTIN IN INFLUENZA VIRUS INFECTEDMICE
Milka Mileva1, Angel S. Galabov2
1Department of Medical Physics and Biophysics, Medical University, 2 Zdrave Str.,Sofia-1431, Bulgaria; 2Institute of Microbiology, Bulgarian Academy of Sciences,Akad. G. Bonchev Str. 26, Sofia-1113 Bulgaria
In this study an investigation and comparison of the effects of plant flavonoidpolyphenols quercetin ant its sugar-containing homologue (rutinoside) rutin on the“oxidative stress” in liver, lung as well as in blood plasma isolated from influenza virus A/Aichi/2/68 (H3N2) (2.0 of LD50) inoculated mice, is carried out. It was found that experimentalinfluenza virus infection is accompanied with a significant increase of lipid peroxidationproducts and a decrease of natural antioxidants vitamin E, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-N-demethylase and amidopyrine-N-demethylase) as compared to control (non-infected) animals. The preliminary (5 days)supplementation of mice with rutin, quercetin or its combination and their subsequentvirus inoculation was accompanied with accumulation of lower levels of malondialdehyde(MDA) and fluorescent lipofuscine-like products and of higher levels of endogenousvitamin E in comparison with animals, subjected to influenza virus infection only. Theprotective effect of rutin against influenza virus-induced lipid peroxidation and activitiesof CYP and liver monooxygenases was better expressed than the effect of quercetin maybe due to containing of rutinoside part or difference of its metabolism.
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V12
A PLANT POLYPHENOL EXTRACT REDUCES OXIDATIVESTRESS IN THE LIVERS OF INFLUENZA VIRUS-INFECTED
MICE
Ekaterina Krumova1, Silviya Abarova2, Lyubka Tancheva2, Maria Angelova1, JuliaSerkedjieva1
1Institute of Microbiology, 2Institute of Physiology, Bulgarian Academy of Sciences,Akad. G. Bonchev Str. 26, Sofia-1113, Bulgaria
Plant polyphenols aroused considerable interest because of their broad pharmacologicalpotential which has been ascribed to their antioxidant and free-radical scavengingcapacities. We have found that a polyphenol extract, obtained from the medicinal plantGeranium sanguneum L. (PC), was highly inhibitory to influenza virus replication in vitroand protected mice from mortality in the experimental influenza infection (EIVI, Manolovaet al., 1986). At the same time it was shown that PC significantly restored and stimulatedthe antioxidant activities in the lungs of virus-infected mice (VIM, Abarova et al., 2004). Inthe present study we followed the effect of PC on the total antioxidant activity (AOA), onthe levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) aswell on the lipid peroxidation (LPO) in the 9000x g liver supernatant of albino mice on the2, 6 and 9 days after the challenge with influenza A/Aichi/2/68 (H3N2) virus. The free–radical character of EIVI (Tancheva et al., 2001) was confirmed clearly. The highconcentrations of LPO products as markers of the oxidative stress, correlated proportionallywith decreased AOA, particularly on day 9 p.i. (180% and 60% of healthy control, HC,resp.). The preventive nasal treatment with 10 mg/kg of PC successfully normalized bothparameters. EIVI induced also a significant increase in SOD and CAT activities in thelivers of VIM on day 2 p.i.; their values reached 280% and 160% of HC resp. PC-treatmentreduced to normal the elevated enzyme levels. The virological parameters of the infectionwere followed in parallel. PC-application to VIM led to noticeable reduction of mortalityrates (index of protection = 77.8%) and marked prolongation of mean survival time (+ 5.2days). The protective effect of PC on EIVI is related to both the antioxidant activity andthe specific antiviral effect of the extract.
V13
SARS-CORONAVIRUS INTERACTION WITH ACE2 RECEP-TOR; IMPLICATIONS FOR VIRUS ADAPTATION AND
VACCINE DESIGN
Anton P. Andonov, University of Manitoba, Winnipeg, Canada
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V14
EVIDENCE SUPPORTING THE REPLICATIVE HOMEOSTA-SIS HYPOTHESIS CONCERNING HIV REPLICATION IN
CELL CULTURE
R. ArgirovaLab. for Retroviruses, Dept. of Virology, National Center of Infectious and ParasiticDiseases, Sofia, Bulgaria
The replicative homeostasis hypothesis, launched in 2005 by R. Sallie deals withviruses replicating via RNA intermediates and capable for persistent infection like hepatitisC (HCV), hepatitis B (HBV) virus and Human Immunodeficiency Virus (HIV). Their extremegenetic and antigenic diversity is paradoxically combined with their stability. Thehypothesis reviews the auto-regulation of replication of these viruses and presents amechanism mediating this auto-regulation – namely replicative homeostasis. It proposesa rational basis for all observed viral behaviors and host responses during chronicinfections caused by these viruses. Here we present in vitro data confirming the fact thatthe HIV-1 replication is autocontrolled and independent of cellular or humoral immunefunction. Studying newly derived HIV-1 mutants after serial passages of the virus in thepresence of increasing concentrations of IM-7 (designed to be HIV-1 integrase inhibitors)an over-accumulation of 2-LTR DNA over linear cDNA (normally integrated) and un-integrated DNA was observed in cell culture. This fact, similarly to emergency of viralmutants presents strong evidence in support of autoregulation of HIV-1 replication – thecenter of replicative homeostasis hypothesis.
V15ЩАМОВО РАЗНООБРАЗИЕ И МЕХАНИЗМИ НА
ЕВОЛЮЦИЯ ПРИ РОТАВИРУСИТЕ
З. Младенова, Н. Корсун, Сн. ГюроваНационална референтна лаборатория по Ентеровируси -Национален център по заразни и паразитни болести, София
Човешките ротавируси от група А са главен причинител на тежкидехидратиращи гастроентерити при децата на възраст под 5 години в целия свят.По данни на СЗО те са отговорни за 20-70% от хоспитализациите и за 20% отсмъртните случаи, дължащи се на диария, в тази възрастова група. Понастоящемединствената стратегия за контрол и превенция на тежките ротавирусни инфекциие ваксинацията, тъй като мерките, целящи подобряване на хигиената и
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водоснабдяването не са ефикасни за ограничаване на заболяемостта.Използването на съвременните антигенни методи за характеризиране на
серотипа, генотипирането чрез RT-PCR и нуклеотидното секвениране презпоследните 10 г. показа, че над 90% от циркулиращите по света ротавируснищамове са от типове G1, G2, G3 и G4, а също така позволи идентифициране нанад 42 комбинации от G/P типове. Днес проучването на щамовото многообразиепри ротавирусите е задача от първостепенно значение в световните програми занадзор над заболеваемостта от ротавирусни гастроентерити и хвърля светлинавърху потенциалните механизми на еволюция и разпространение на нови щамоверотавируси.
Разработването на ротавирусни ваксини се основава на създаване нахомотипен и хетеротипен имунен отговор срещу един или няколко от най-разпространените G и Р типове ротавируси. Лицензирането на две ротавирусниваксини в началото на 2006 г. налага мониториране на циркулацията наротавирусните щамове във всяка страна, откриване на нови щамове с глобалноили регионално разпространение, което е определящо за успеха на ротавируснитеваксинални програми.
В доклада ще бъдат представени актуални данни за типовото разнообразиесред ротавирусите, евентуалните механизми на тяхната еволюция и поява нанови типове, както и съвременните методи за диагностика на ротавируснитегастроентерити.
V16
ДИАГНОСТИКА НА ПЪРВИТЕ СУСПЕКТНИ ЗА ПТИЧИГРИПЕН ВИРУС ПОДТИП А/H5N1/ ПАЦИЕНТИ У НАС
Т. Хаджиолова, С. Павлова, Р. КоцеваОтдел Вирусология, НЦЗПБ
Направени са първите диагностични изследвания за доказване етиологичнатароля на птичия грипен вирус подтип А/H5N1/ у нас. За периода януари – март2006 г. в лабораторията по “ Грип и ОРЗ” са изследвани 26 суспектни пациентив тесен контакт с болни или умрели птици и последваща симптоматика нареспираторно заболяване. Приложени са специфични тестове за доказване наптичия вирус А/H5N1/-бърз антиген откриваща тест GeNet Bio и RT-PCR кит нафирма Sacace. За вирусна изолация са използвани два лабораторни модела-клетъчна линия MDCK и кокоши ембриони. Вирусът не беше доказан катоетиологичен причинител в нито един от изследваните пациенти.В три от пациентитебе изолиран човешки грипен вирус идентифициран чрез РЗХА и RT-PCR катоподтип А/H1N1/.
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V17
ДИАГНОСТИЧНИ ПРОУЧВАНИЯ ВЪРХУЕТИОЛОГИЧНАТА РОЛЯ НА РЕСПИРАТОРНО-СИНЦИТИАЛНИЯ И ГРИПНИТЕ ВИРУСИ ПРИ
ХОСПИТАЛИЗИРАНИ ДЕЦА
С. Павлова, Т. Хаджиолова, Р. КоцеваОтдел Вирусология, НЦЗПБ
Ролята на РСВ като причинител на тежки заболявания на дихателната системав малки деца е приоритетен проблем в световен мащаб. Ежегодното епидемичноразпространение на грипните вируси също дава отражение върху заболеваемосттав тази възрастова група. През периода октомври 2005г.-март 2006г. влабораторията по “Грип и ОРЗ” са проведени сравнителни изследвания за РСВ игрипни вируси на общо 359 проби от хоспитализирани деца до 5 г. възраст.Няма паралелизъм между изследванията при едни и същи деца. Привирусологичното изследване (изолация, бързи имуносорбетни тестове и ИФМ)на 174 проби 14 (8,04%) са положителни за РСВ, а 10 (5,7%) за грипни вируси типА. При изследванията по ELISA IgG на 185 единични проби серуми са получени42 (22,7%) положителни резултата за РСВ и 52 (28,1%) за грипни вируси тип А.Получените резултати потвърждават литературните данни за едновременнатациркулация на РСВ и грипните вируси сред посочената възрастова група.Изследването обогатява диагностичните проучвания за етиологичната роля натези вируси в детска възраст със съвременни данни и поставя началото на добраколаборация между вирусолози и педиатри.
V18
ДИАГНОСТИЧНИ АСПЕКТИ НА ХЕМОРАГИЧНИТЕТРЕСКИ
Д. ВелчеваНЦЗПБ, отдел Вирусология, София
Показани са основните диагностични методи за диагностика на КонгоКримска хеморагична треска и Хеморагична треска с бъбречен синдром, коитосе използват в лаборатория „Арбовируси” – изолация, типизация и серологичнадиагностика, както и диагностичната стойност на всеки методл Проследена езаболеваемостта за периода 1997-2005 година. Сравнена е и заболеваемостта от
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Конго Кримска хеморагична треска преди и след въвеждането на специфичнаваксинопрофилактика.
V19
STUDIES ON CCR5, CXCR4 AND CCR2 GENETIC POLY-MORPHISM IN HIV-INFECTED BULGARIANS
K. Borisov1, A. Savov3, I. Kremensky3, S. Raleva1,L. Froloshka1, R. Markova2, V. Terzieva2, K. Kostov4, R. Argirova1
1Laboratory for Retroviruses, Natl. Center of Inf. And Paras. Dis.,2Dept. of Immu-nology, Natl. Center of Inf. And Paras. Dis., 3National Lab. of Genetics, Med.Uni.,Sofia, 4Inf. Dis. Hospital, Sofia
Although intensive studies of chemokine receptor genes have been done globally , itwould be of interest to study genetic polymorphism of co-receptor genes in HIV-infectedBulgarians, as well as the link to clinical course of HIV-infection. Here we present 177HIV(+) Bulgarians infected within 1986 – 2004 studied for genetic polymorphism of CCR5.63 out of them were also studied for CCR2 and SDF-1 (CXCR4) genetic polymorphism. Theanalyses were performed by PCR (CCR5) and restriction analysis + PCR using bloodspots on Gutrie cards with preliminary DNA extraction. Concerning the course of thedisease 9 out of all persons studied were long-term non-progressors (LTNPs) and the resthad moderate course of HIV-infection. Three LTNPs showed CCR5 heterozygous pattern,the other 6 LTNPs and 174 progressors had wild type CCR5. Separately, the frequency ofCCR2-64I in healthy Bulgarians as control was measured (6,9%) – one of the lowest inEurope. No clear link between CCR2-64I genotype and disease progression has beenfound. In 100 non-infected Bulgarians SDF-1-3’A frequency has been studied – 26,5% -one of the highest in Europe. There was no significant difference in SDF-1-3’A frequenciesin healthy and HIV(+) individuals. In LTNPs SDF-1-3’A homozygosity was significantlyhigher (44%) compared to the group with moderate progression to AIDS (5,5%). The dataobtained show that CCR5 heterozygotic pattern was not the only one linked to LTNPstatus. In conclusion, multiple polymorphism of genes coding for chemokine receptorswere observed. CCR5, CCR2 and SDF polymorphism frequencies alone and in combinationsuggest a number of HIV-infected individuals should be genetically tested to predict thecourse of infection, the prognosis and the response to highly active anti-retroviral therapy(HAART).
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V20
AN EXPERIMENTAL STUDY OF HIV-1 EPITOPE STRUC-TURE CHANGES UNDER INHIBITION OF
GLYCOSYLATION
R. Gavazova1, S. Ivanov1, D. Ivanov1, P. Genova3, S. Raleva2,L. Froloshka2, D. Dundarova3, R. Argirova2
1Section of Biochemistry, Institute of Experimental Pathology and Parasitology,Bulgarian Academy of Sciences, G. Bonchev Str.,Building 25, 1113 Sofia, Bulgaria; 2Laboratory for Retroviruses, 3Laboratory forCell Culture, Dept.of Virology, National Center of Infectious and Parasitic Diseases,44A Boul.,Stoletov, 1233 Sofia, Bulgaria
To further understand changes of epitope structures conferring viral escape in HIV-infected individuals we studied the Sgp profile of HIV-1LAI in MT-2 infected cells treatedwith tunicamycin (Tu) – a well known inhibitor of glycosylation, as well as correspondingST activies. A study of N-acetyl-[C14]-mannosamine (14C-NAcMan) incorporation incytosols of HIV-1LAI acutely infected and uninfected MT-2 cells treated with Tu (0,5µg/ml) was carried out. Fractions derived after isoelectrofocusing (IEF) were measured for14C-incorporation, protein content and reverse transcriptase (RT) activity (Cavidi, Sweden).ST-activity was detected by Serafini-Cessi F. method. Higher incorporation of 14C-NAcManfor MT-2/HIV + Tu compared to MT-2 + Tu was observed. MT-2 IEF profile compared tothat of MT-2 + Tu showed a shift of the latter to the basic zone and only two common pIpeaks. On the contrary, a number of common pI peaks in MT-2/HIV and those treated byTu were seen. ST activity was reduced by 33% for MT-2 + Tu compared to MT-2 v. 20,6 %for MT-2/HIV + Tu compared to MT-2/HIV (inhibition of glycosylation 27,8 and 14,2%resp.).
As a result from these experiments it could be stated that the higher inhibition ofglycosylation by Tu in MT-2 cells compared to MT-2/HIV probably confers higherinhibition of sialylation and the changes in Sgp profiles of Tu treated cells. The differingeffect of equal concentrations of Tu on glycosylation in MT-2 and MT-2/HIV underexperimental conditions might at least partially explain already described viral escape inHIV-infected persons by changes in glycosylation.
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V21
РАЗПРОСТРАНЕНИЕ НА ГЕНЕТИЧНИТЕ ФОРМИ НАHIV-1, ЦИРКУЛИРАЩИ В БЪЛГАРИЯ
Пенева М.1, Бешков Д.1, Алексиев И.1, Георгиева В.1, Костов К.2, Еленков И.2,Върлева Т.3, Еленков И.И.4
1Национална Потвърдителна Лаборатория по HIV, НЦЗПБ, София; 2СБАЛИБ„Проф. И. Киров”, София; 3Министерство на Здравеопазването, София; 4СУ„Св. Климент Охридски”, Биологически факултет, София
Известна е извънредно голямата генетична разнородност на HIV, както инепрекъснатото възникване на нови HIV варианти. Голямото разнообразие отразлични форми на HIV може да има влияние върху диагностиката,антиретровирусната терапия, прогресията до СПИН, предаването на вируса исъздаването на ваксина срещу него.
Целта на настоящето изследване беше да установим разпространенитесубтипове и циркулиращи рекомбинантни форми (CRF) на HIV-1 в България.Нуклеотидните последователности, използвани за субтипирането, бяха полученичрез секвениране на RT-PCR продуктите на регионите PR и RT от гена pol наHIV-1 чрез диагностичните набори TrugeneTM и ViroSeqTM. Субтипирането бешеизвършено според база данните на Standford University, CA, USA; Los AlamosNational laboratory, CA, USA и National Center for Biotechnology Information(NCBI), NIH, MD, USА.
Бяха изследвани 80 пациента, инфектирани с HIV-1, които представляват 13%от регистрираните HIV серопозитивни лица в България. Установихме, че най-разпространен е субтип В, който циркулира в 41 (51.25%) от тях, следван отCRF01_AE в 24 пациента (30%), субтип C – в 6 (7.5%), субтип H – в двама (2.5%),CRF02_AG (2.5%) – в двама, субтип F – в двама (2.5%), субтип A – в един (1.25%),G – в един (1.25%) и D – в един (1.25%) пациент.
Това изследване представя първи данни за разпространението нациркулиращите генетични форми на HIV-1 в България. Беше установеноприсъствието на девет генетични форми на HIV-1, от които преобладават субтипB и CRF01_AE. Установеното голямо разнообразие на генетични варианти наHIV-1 в България вероятно се дължи на географското разположение на странатаи засилените миграционни процеси през последните 20 години.
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V22
ПРОУЧВАНЕ НА РАЗПРОСТРАНЕНИЕТО НА ЧОВЕШКИТ-ЛИМФОТРОПНИ ВИРУСИ HTLV-І И ІІ СРЕД
БЪЛГАРСКОТО НАСЕЛЕНИЕ
Алексиев И.1, Бешков Д.1, Георгиева В.1, Пенева М.1, Бакалова С.2, ГеноваМ.2, Атанасова М.3, Eленков И.4
1Национална Потвърдителна Лаборатория по НІV, НЦЗПБ, София;2Национален Център по Хематология и Трансфузиология, София ; 3МедицинскиУниверситет – Пловдив; 4СУ “Св. Климент Охридски”, Биологически Факултет
Цел на настоящото изследване е да се проучи разпространението на HTLVсред различни групи от българското население, както и да се оценинеобходимостта от въвеждане на скрининг за HTLV при кръводарители и донорина тъкани и органи. HTLV–I и II са човешки ретровируси, които могат да причинятонкохематологични и неврологични заболявания. Те се предават по кръвен, полови вертикален път. Понастоящем скрининг на кръводарители за HTLV се прави вЯпония, САЩ и някои европейски страни.
Материали и методи: Проведоха се серологични изследвания за наличиена анти-HTLV I и II антитела с ELISA тестове на Murex и Bio-Rad, както ипотвърдителни изследвания с Western blot на Genelabs Diagnostics. Бяхаизследвани 1446 кръвни проби от различни групи от населението: кръводарители- 380, пациенти с болести предавани по полов път - 180, венозни наркомани - 99,пациенти с хематологични заболявания: таласемия - 20, хемофилия А и В - 70,левкози и лимфоми - 21, лица от КАБКИС - 676.
Резултати: Серологичните изследвания на 1446 серумни проби показаха,че 11 от тях (0,76%) са реактивни в ELISA тест. Нито една от тях не бе потвърденав Western Вlot.
Изводи: В настоящото проучване не бе установено нито едносеропозитивно лице за HTLV І и ІІ. Имайки предвид циркулацията на тези вирусив съседните ни страни, както и свободното движение на големи групи хора, такивасерологични проучвания следва да се провеждат периодично. По този начин щесе осигури мониторинг на тази инфекция и актуализиране на показанията заскрининг на донорската кръв за HTLV в България.
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V23
HIV-1 VARIANTS (MUTANTS) AFTER LONG-TERM PAS-SAGING IN PRESENCE OF NEWLY SYNTHESIZED
4-HYDROXICOUMARINS (4-HC)
S. Raleva1, A. Yordanova3, Y. Gradinarova3, A. Savov3, L. Froloshka1, P. Genova2,I. Manolov4, D. Dundarova2, R. Argirova1
1Lab. for Retroviruses, 2Lab. for Cell Culture, Dept. of Virology, National Center ofInfectious and Parasitic Diseases, Sofia, 3 National Lab. of Genetics, Med. Uni.,Sofia4 Faculty of Pharmacy, Med. Uni., Sofia, Bulgaria
A number of studies has been published about the anti-HIV potency of some 4-hydroxycoumarins (4-hc) (Zhao, H. et al., 1997). Stimulated by these findings mostlytargeting HIV-1 integrase, in Faculty of Pharmacy, Med. Uni – Sofia 19 novel 4-hc weresynthesized by I. Manolov and 3 out of them – IM-7, IM-8 and IM-10 showed anti-HIVeffect in MT-2 cells using HIV-1LAI. Further, no effect on both early (reverse transcription)and late (protease, budding and release) phases of HIV-1 replication has been observed.To target the anti-HIV-1 activity of studied 4-hc, serial passages (20 – 30) to yield mutantHIV-1 variants in presence of increasing concentrations of 4-hc were undertaken. Culturesupernatants were concentrated 20x by Polyethyleneglycol (PEG) method. Supernatantswere tested for reverse transcriptase (RT) activity and the viral concentrates – for linearcDNA (integrated) and 2-LTR rings by routine PCR. The results obtained showed anabundance of 2-LTR rings compared to linear and unintegrated DNA. The latter finding istypical for HIV-1 integrase mutants and was observed in viral concentrates after the 20thpassage of HIV-1LAI in presence of increasing concentrations of IM-7. The mutation(s)are not reversible after the next 17 passages without IM-7 pressure. The mutant(s) obtainedare subjected to further analysis.
V24
АНТИРЕТРОВИРУСНА РЕЗИСТЕНТНОСТ НА HIV-1 СРЕДБЪЛГАРСКИ СЕРОПОЗИТИВНИ ПАЦИЕНТИ (2002-2006)
Бешков Д.1, Алексиев И.1, Пенева М.1, Георгиева В.1, Костов К.2, Еленков И.2,Върлева Т.3, Еленков И.И.4
1Национална Потвърдителна Лаборатория по HIV, НЦЗПБ, София, 2СБАЛИБ„Проф. И. Киров”, София, 3Министерство на Здравеопазването, София, 4СУ„Св. Климент Охридски”, Биологичестки факултет, София
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Високоактивната антиретровирусна терапия (HAART) е въведена в Българияпрез 1999 г. За първи път се съобщават резултати от изследвания заантиретровирусна резистентност (АРВР), като са обхванати пациенти срегистиран неуспех при провеждане на HAART и наивни за HAART пациенти.
За периода 2002 г.- април 2006 г. са проведени изследвания на 101 кръвнипроби от 80 български ХИВ-1 серопозитивни лица, като 65 от тях са получавалиHAART, а 15 са наивни пациенти. При всеки пациент вирусът е генотипиран,чрез сенвениране на RT-PCR продуктите на регионите RT и PR на pol гена наHIV-1. Използвани са генотипиращите тестове TrugeneTM и ViroSeqTM заустановяване на мутации, отговорни за АРВР.
При 49 (75,38%) от 65 пациента, показали неуспех в хода на приложение наHAART е установена АРВР към трите групи препарати по отделно и в различникомбинации от тях. Към NRTI тя е 69.39%, към NNRTI-36.73% и към PI -59.18%.Към трите групи препарати АРВР е установена при 10.20% от пациентите, къмNRTI и NNRTI при 14.29%, към NRTI и PI при 24.50% и към NNRTI и PI при6.12%. АРВР само към една група лекарствени средства е устновена както следва:към NRTI при 20.41%, към NNRTI при 6.12% и към PI при 18.37% от пациентите.Изследванията при 15 наивни пациенти не показаха данни за АРВР.
Получените резултати показват, че при 75.38% АРВР е отговорна за неуспехапри HAART. Тези данни корелират с прилаганата терапия. Това налагарутинното приложение на изследванията за АРВР, имащи основна роля при изборана нови терапевтични режими.
V25
DIAGNOSTICS OF THE VIRAL HEPATITIS -ACHIEVEMENTS AND PROBLEMS
P. Teoharov
Infection of the liver with hepatotropic viruses is a serious public health problem. Theburden of disease from acute and chronic viral infections is staggering. Each of the fivehepatotropic viruses belongs to different virus family and three of them - HAV, HBV andHCV are the leading cause of the acute and chronic liver diseases in Bulgaria.
A combination of biochemical, serological and virological tests and histological featureshave been used to diagnose and classify infections from hepatotropic viruses. Assaysfor HAV, HBV and HCV antigens and antibodies are widely available and standardized.There are a highly specific and sensitive kits for detection of viral markers. The mostimportant method for laboratory diagnosis of the viral hepatitis is immuno-ensyme assay/EIA /, but molecular techniques for determination of nucleic acids become widely usealso. Different molecular assays have been developed with different sensitivity and range
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of linearity. There are some disadvantages, mostly connected with the specificity of themore sensitive molecular methods based on amplification of the viral nucleic acids. Someof the techniques, like determination of the genotype of HBV remains a research tool, butgenotyping of HCV is important step from the therapy. The polymerase chain reaction /PCR/ is now accepted as a gold standard for detecting nucleic acids and it has become anessential tool in the research laboratory. Real-time PCR has wider acceptance due to itsimprived sensitivity, rapidity, reproducibility and the reduced risk of carry-overcontamination.
V26
МОЛЕКУЛЯРНИ ХАРАКТЕРИСТИКИ ИПАТОГЕНЕТИЧЕН ПОТЕНЦИАЛ НА ЧОВЕШКИТЕ
ПОЛИОМНИ ВИРУСИ
Зл. КълвачевЛаборатория по молекулярна вирусологияНационален Център по Заразни и Паразитни Болести (НЦЗПБ)
Наскоро (2000 г.) формираното семейство Polyomaviridae обединява 13 вирусапаразитиращи по различни млекопитаещи, вкл. човек, някои видове примати,гризачи, зайци и птици. Огромният научен и медицински интерес, койтопредизвикват тези вируси се дължи на високият им онкогенен потенциал ивъзможните последствия за човек. За някои неврологични заболявания(прогресиращата мултифокална левкоенцефалопатия) се счита за доказанаетиопатогенетичната роля на човешкия полиомен вирус JC. Освен товапациентите, на които се извършват тъканни или органни трансплантации сеподлагат на имуносупресивна, при което често се активират инфекции,предизвикани от другият човешки полиомен вирус - BK. Молекулярнитемеханизми, по които се развиват тези инфекции са в процес на интензивнипроучвания. Представеният материал обобщава съвременната информацияотносно основните биологични свойства на човешките полиомни вируси BK иJC, както и данни за епидемиологията, клиниката, диагностиката и възможноститеза терапията при тези инфекции.
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V27
БЪРЗА ИДЕНТИФИКАЦИЯ НА POLYOMAVIRUSHOMINIS-1 (ВКV) В УРИНАТА НА ПАЦИЕНТИ С
БЪБРЕЧНА ТРАНСПЛАНТАЦИЯ
С. Славов1, И. Ненков1, А.Петрова1, Л. Христова2, П. Симеонов2, Д. Димова3,З. Кълвачев1
1Национален Център по Заразни и Паразитни Болести, Отдел по вирусология,Лаборатория по молекулярна вирусология; 2Медицински Университет-София,Университетска болница-„Александривска”, Клиника по нефрология итрансплантации; 3САГБАЛ-„Шейново”, Лаборатория по патологичнахистология
Polyomavirus hominis-1, по-известен като BK вирус (сем. Polyomaviridae) епатоген с широко разпространение сред човешката популация. Инфекцията сепридобива още в ранна детска възраст, като остава латентна в генито-уринарниятракт. Реактивация се наблюдава често,особено при пациенти с бъбречнатрансплантация. Поради подтиснатите имунни механизми те развиват симптомикато хеморагичен цистит, уретрална обструкция, ретинит, хепатит, като често сестига до отхвърляне на трансплантанта. Поради това, своевременното откриванена вируса е от съществено значение при прогнозата от трансплантацията исъставянето на подходяща терапевтична схема.
За бързо диагностициране на вируса в урина на пациенти с бъбречнатрансплантация бяха използвани уринарно-цитологичен метод и полимеразно-верижна реакция (PCR) в реално време (TaqMan real-time PCR). Изследвани бяхаобщо 50 проби. В 36 от тях, бяха наблюдавани характерните за полиомавируснатаинфекция клетки с вирусни включвания, известни като „Decoy cells”. От седиментана всичките 50 проби беше екстрахирана ДНК, която изследвахме чрез TaqManreal-time PCR. Наличие на специфични вирусни геномни секвенции бяха доказанипри 48 проби. Обсъждат се клиничните и лабораторните находки, което е въввръзка с прогнозата на състоянието след трансплантация. Използваният от насвариант на PCR e подходящ за скриниране на BK полиомната инфекция припациенти с бъбречна трансплантация, и би послужил като допълнение къмкласическите цитологични изследвания.
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V28
POLYOMAVIRUS HOMINIS-2 (JCV) В УРИНАТА НАПАЦИЕНТИ С БЪБРЕЧНА ТРАНСПЛАНТАЦИЯ
И. Ненков1, С. Славов2, А. Петрова2, Л. Христова3, П. Симеонов3 , З.Кълвачев2
1Софийски университет ,,Св. Климент Охридски”, Биологически факултет,катедра Биохимия; 2Национален Център по Заразни и Паразитни Болести;Лаборатория по молекулярна вирусология, 3Медицински Университет - София;УМБАЛ „Александровска”, Клиника по нефрология и трансплантации
Polyomavirus hominis-2 или JC вирусът (JCV) принадлежи към сем.Polyomaviridae. Според литературните данни, около 80% от населението по светаима антитела към този вирус. Счита се, че става още в ранна детска възраст, ноинфекцията протича безсимптомно. По време на първичната инфекция сенаблюдава виремия и вирусът се транспортира до мозъка, бъбреците и/или другиоргани, където остава да персистира в латентно състояние. Приимунокомпроментирани индивиди (такива с трансплантации или другиимунодефицитни състояния) се наблюдава реактивация на вируса и вирурия.
JCV е невротропен вирус и е причинител на прогресивната мултифокалналевкоенцефалопатия (progressive multifocal leucoencephalopathy, PML). Прибъбречно-трансплантирани пациенти може да се развият тежки усложнения и дорида се стигне до отхвърлянето на трансплантанта. Поради това, своевременнотооткриване на вируса е критично за прогнозата от трансплантацията иназначаването на подходяща терапевтична схема.
Ние изследвахме 50 уринарни проби получени от пациенти с бъбречнатрансплантация и с помощта на полимеразно-верижната реакция (PCR) проведенав реално време (TaqMan real-time PCR) доказахме наличието на JCV при 43 оттях. TaqMan real-time PCR e ефективен метод за бързо скриниране на JCV инфекцияпри пациенти с бъбречна трансплантация. Установено беше, че най-подходящаза изследване е втората сутрешна урина. Обсъжда се връзката между полученитерезултати, клиничните данни и вероятният бъдещ ход на състоянието.
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V29
КЛИНИЧНИ ОСОБЕНОСТИ И СЕРОЛОГИЧНАВЕРИФИКАЦИЯ НА ОСТРАТА ЕВV ИНФЕКЦИЯ
А.Гоцева1,Т.Кузмова1, Д.Велчева21УМБАЛ ”Св.Иван Рилски”, София2НЦЗПБ, отдел Вирусология, София
Проследени са 24 пациенти с мононуклеозен синдром, лекувани в Клиника поинфекциозни, паразитни и тропически болести- УМБАЛ ”Св.Иван Рилски”, Софияи изследвани в лаборатория “Херпесни вируси “към НЦЗПБ.Всичките бяхаизследвани чрез ELISA за наличие на антитела от клас IgM към капсидния антигенна ЕВV(маркер за активна инфекция).При 17 от тях бяха доказани специфичниантитела , докато при останалите 7- резултатът беше негативен. П р ивсички пациенти основни клинични прояви бяха интоксикацията,фебрилитета,лимфоаденопатията, възпалителните промени в гърлото и хепатомегалията. При17 от тях ( всички позитивни за anti EBV VCA IgM) клиничната картина сеутежняваше от наличието на лекарственоиндуциран екзантем, ранен и преходендвустранен оток на горните клепачи, обструкция на въздухоносните пътища,спленомегалия и повишени стойности на чернодробните ензими.При останалите7 лица описаните клинични симптоми не бяха регистрирани, което съответствашена липсата и на серологично потвърждение.
V30
ПТИЧИ ГРИП (H5N1)-НОВАТА ГЛОБАЛНА ЗАПЛАХА:ЕПИДЕМИОЛОГИЯ, РАЗПРОСТРАНЕНИЕ И ВЪЗМОЖНО
ИЗПОЛЗВАНЕ КАТО БИОЛОГИЧНО ОРЪЖИЕ
Красимир МекушиновВирусологична лаборатория, Военномедицинска академия, София-1606,ул. Г. Софийски, 3
Цел: Да се опише специфичната епидемиология, разпространение и главнитеособености на вируса, които го определят като потенциален агент за Биологичнооръжие:1.Начин на предаване: заразена вода, храна и почва2.Клинично протичане: къс инкубационен период, внезапно начало, тежкапневмония, респираторен дистрес синдром
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3. Вирулентност: смъртността при заразените хората е над 50%, а у чувствителниптици може да достигне до 100%, причинявайки огромни икономически загуби.
V31
EFFECTS OF SOME ANTIVIRALS AGAINST RHINOVIRUSH-14
Irina L. Georgieva, Angel S. GalabovDepartment of Virology, The Stephan Angeloff Institite of Microbiology, BulgarianAcademy of Sciences, 26 G. Bonchev St., 1113 Sofia
Human rhinoviruses (HRVs) are the predominant cause of viral respiratory tractinfections, particularly common cold, as well as acute otitis media, sinusitis, bronchitisetc. A great number of picornavirus replication inhibitors in vitro have been described butuntil now there are no specific antiviral chemotherapy to prevent or treat diseases causedby rhinoviruses.
Here are presented data of a pilot study on the effect of several antiviral substanceswith different mode of action on the replication of HRV-14. Monolayer cultures of humancervical carcinoma (HeLa Ohio-I) cells in 96-well tissue culture plates were used. The viralCPE inhibition test was applied. The neutral red uptake (NRU) assay was done to quantitatethe antiviral activity and the cytotoxicity of the compounds. Absorbance values of thedrug-treated samples were expressed as percentage of the untreated controls and thedose-response curves of each compound were drawn. The compound action at variousviral inoculation doses was studied. The following compounds have been tested: ribavirin(large-spectrum viral inhibitor, mostly of RNA viruses), arildone, disoxaril, S7, PTU-23,HBB and oxoglaucine (a newly characterized in our laboratory antiviral efficient againstenteroviruses).Two of these compounds - HBB and oxoglaucine show highest activity,characterized with: (i) lowest values of IC50; (ii) highest values of selectivity ratio and (iii)effectivity against all viral inoculation doses tested. Ribavirin and disoxaril occupyintermediate position by their antiviral effect followed by PTU-23, active against 100 and1000 CCID50. Arildone and S7 manifested a border-line effect.
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V32
STUDY OF VIRUS RNA SYNTHESIS IN CELL CULTURESINFECTED WITH BOVINE VIRAL DIARRHEA VIRUS (BVDV)
USING HYPERTONIC NACL SOLUTIONS
Angel S. Galabov, Yurii P. Abashev, Lucia Mukova and Lilia WassilewaThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Sofia, Bulgaria; E-mail: [email protected]
Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus, member of thefamily Flaviviridae, genus Pestivirus. It causes infections in cattle with significant economiclosses. BVDV is often used as a surrogate hepatitis C virus in screening tests for anti-HCVantivirals. The exact study on the mechanism of action of such antivirals insists the wellknown synthesis of viral RNA and proteins. The problem of pestiviral RNA synthesisstudy is the lack of a significant shut-off of the cellular RNA and protein synthesis in thecaurse of pestivirus infection until the later step when the cytolytic changes are evident.In order to overcome this problem the antibiotic actinomycin D in concentration of 5ìg/ml is usually applied. Unfortunately, addition of actinomycin D in suchconcentration to some cell lines (e.g. calf trachea cell line) even at short time intervalresulted in a great degree of cell toxicity. The hypertonic NaCl solution used for the firsttime by Nuss et al. (1875) blocks selectively the host cell RNA and protein syntheses. Ourexperiments using hypertonic NaCl solution (0.15 – 0.3 M/l) and calf trachea (CT) cell lineshowed a significant (98.9% inhibition of cellular RNA and 98.0% inhibition of the cellularprotein synthesis. Addition of the same NaCl concentration to CT cells infected withBVDV, CC strain, with multiplicity of infection 10 resulted in a well documented viral RNAsynthesis with a maximum at 9th hour after virus adsorption in parallel with a marked shut-off of the cellular RNA synthesis. The maximum BVDV infectious titre (107.5 CCID50) wasfound at 18th hour after viral adsorption.
V33
БИОЛОГИЧНА АКТИВНОСТ НА ЕКСТРАКТ ОТORTHOSIPHON STAMINEUS BENTH.
Стоян Шишков1, Калина Костова1, Елена Георгиева1, Венета Капчина-Тотева1, Женя Йорданова1, Валентина Чипева2, Станислава Трандева2,1 Биологически факултет, СУ “Св. Кл. Охридски”, бул. Драган Цанков №8,София 11642 НБПМКК, бул. “Цариградско шосе” № 125, бл. 2, София 1113
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Проучен за биологична активност е етанолов екстракт от азиатскотомедицинско растение Orthosiphon stamineus Benth. Растението е отгледано вусловия in vitro. Установена е ясно изразена инактивираща активност спрямоекстрацелуларен вирус херпес симплекс тип 1 (ВХС-1). Инфекциозният вирусентитър е понижен с 2 log (99%) само след 5 минутен контакт на вирионите секстракта. Ефектът се запазва за целия интервал на изследване. Вируснатарепликация не се повлиява при апликиране на изследвания извлек.
С прилагане на дифузионен метод е изследвана антибактериалната активност.Резултатите показват наличие на активност спрямо референтни бактериалнищамове от Bacillus, Staphylococcus, Klebsiella. Изледван е ефектът и върху широкнабор от клинични грам-отрицателни и грам-положителни бактериални изолати.
V34
ВИРУСОЛОГИЧЕН НАДЗОР НАД ОСТРИТЕ ВЯЛИПАРАЛИЗИ В БЪЛГАРИЯ ПРЕЗ ПЕРИОДА 2001-2006
ГОДИНА
Н. Корсун, Сн. Гюрова, З. МладеноваНационална референтна лаборатория по Ентеровируси -Национален център по заразни и паразитни болести, София
В педиода на близка ерадикация на полиомиелита опасността от възникванена паралитични заболявания, причинени от внос на диви полиовируси отостаналите четири полиоендемични страни, както и от появата на вируси дериватина живата полиоваксина, все още съществува. Надзорът над острите вялипарализи е ключов елемент в контрола над полиомиелита, поради което тазисистема трябва да продължи да функционира до пълно прекъсване на циркулациятана диви полиовируси в глобален мащаб. Висококачественият, отговарящ насертификационните стандарти надзор гарантира бързо откриване на вирулентниполиовируси и предприемане на спешни противоепидемични мерки.
Националната референтна лаборатория по Ентеровируси към НЦЗПБ, Софияе част от Глобалната лабораторна мрежа на СЗО и като такава осъществявавирусологичен надзор над случаите на остри вяли парализи при деца на възрастпод 15 години в цялата страна. В доклада са представени резултатите отизвършените вирусологични изследвания в лабораторията в това направлениепрез периода 2001-2006. След епидемичния взрив от полиомиелит у нас през2001г., причинен от вносен див полиовирус І тип, у нас диви и ваксино-дериватниполиовируси не са изолирани. Представени са и актуалните данни относноситуацията по отношение на полиомиелита по света.
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V35EFFECT OF THIOSEMICARBAZONE ON HSV-1 AND HSV-2
REPLICATION IN VITRO
N. Vilhelmova1, M. Gacovska1, S. Shishkov1, P. Souza2
1University of Sofia”St. Kl. Ohridski”, Sofia 1164, Bulgaria, 2UniversidadAoutonoma de Madrid, Madrid 28049, Spain
Thiosemicarbazones are big group of organic molecule with prove activity againstHSV. We tested the effect of two bisthiosemicarbazone - H2L
1 and H2L2 and theirs complexes
with Zn(II), Pd(II) and Cd(II) on HSV-1 (Victoria) and HSV-2 (BJ) in cell culture MDBK. Wedetermined maximal nontoxic concentration (MNC) and concentration required to inhibitcell viability by 50% (CC50) of compounds tested. The complexes with Zn(II) and Pd(II) arewith the same value of MNC such as its ligands - 1x10-8 µM. The cadmium complexes inrespect to cell viability are most cytotoxic: Cd2/H2L
1 – MNC=1x10-10µM and Cd/H2L2 -
MNC=1x10-9µM.The ligands and theirs zinc complexes expresse activity against HSV-1 and HSV-2.
Toward strain Victoria Zn2/H2L1 is most active with effective concentration required to
inhibit virus yield by 50% (IC50) - 0.001x10-8µM. The ligands and Zn/H2L2 indicate the
same activity - IC50b= 0.01x10-8 µM. Against strain BJ the strongest effect exhibit H2L2 and
Zn2/H2L1 - IC50 = 0.001x10-8 µM both. IC50 of H2L
1 and Zn/H2L2 have value 0.01x10-8 µM. The
most highly selectivity toward both strains have H2L1 и Zn2/H2L
1.
V36
СЕРОЛОГИЧНО-ЕПИДЕМИОЛОГИЧНИ ПРОУЧВАНИЯНА ГРИПА В ГРАД ВАРНА ЗА ПЕРИОДА 1990-2004 г.
В. Русев, Л. Иванова, А. Щилянова, Р. Иванова, Бр. Шматов
Серологично-епидемиологичните проучвания са от голямо научно-теоритичнои приложно значение. За периода серологично-епидемиологичните проучванияса от голямо научно – теоретично и приложно значение.
За периода 1990-2004 година ние изследвахме общо 16 000 единични пробисеруми от здрави лица от различни възрастови групи от град Варна и региона.
Проучванията ни са проведени по РЗХА с 4 антигена от щамове на грипенвирус тип А с антигенна формула H1N1 и H3N2.
Основната цел на това проучване е определянето на средните геометричнититри на антихемаглутинините към различните щамове на грипен вирус тип А.При проследяване динамиката на средните геометрични титри на антителатакъм изследваните щамове грипни вируси бе установено, че титрите варират от 2
90
до 20. Тези резултати в едни случаи отразяват епидемичната обстановка– привирусите А (H3N2), а в други – се тълкуват като резултат на анамнестични реакциикъм по-стари щамове, под влияние на заболявания, причинени от по-нови щамове(H1N1).
V37
INFLUENCE OF OSMOTIC SWELLING ON THEDHPC LIPID BILAYERS CONTAINING NDV GLYCOPRO-
TEINS
Petrana Borissova, Vassil Neitchev, Lyuba Doumanova1
Institute of Biophysics, 1 The Stephan Angeloff Institute of Microbiology, BulgarianAcademy of Sciences, 1113 Sofia, Bulgaria, e-mail: [email protected]
The osmotic stress is one of the ways to achieve high membrane tension which canresult in strong adhesion between membranes of fixed volume. There is increasingly dataindicating a possible effect of osmotic stress on membrane fusion. The NDV (Newcastledisease virus) glycoproteins play a critical role in limiting membrane lipids in the virus-cellfusion. It has been found that they both interact with the lipid bilayers (liposomes) tochange the spontaneous membrane curvature, as well as the lipid hydration and surfacetension, with minimum osmotic work required for the lipid phase transition. Theseglycoproteins, when associated with phosphatidylcholine liposomes, may affect theosmotic permeability of lipid bilayers under positive osmotic (higher inside) gradientsacross the membrane. Lipids thus participate in this process, and their structure should beconsidered of interest for examination of membrane fusion.
We investigated the influence of the osmotic stress on the structure of 1,2 dihexadecyl-sn-glycero-3-phosphatydilcholine (DHPC) vesicles under transition from lamellar gel tofluid state in the presence of both HN and F glycoproteins in the vesicles. Although thelamellar bilayer structures do not promote the lipid fusion, it is of interest to examine howthese structures are influenced in such conditions. Our assumption is that the lamellarinterdigitated gel structure of DHPC membranes is more sensitive to external osmoticstress than the normal gel phase. The last is also influenced by the presence of viralglycoproteins in the membrane which yield strikingly different molecular shapes of thelipid. X-ray study was performed in this report to observe the changes in the structure ofHN-F/lipid associations under osmotic stress conditions.
The results obtained show the appearance of a swollen gel phase in pure DHPCvesicles at the boundary between the normal gel phase and liquid crystalline phase. Thenature of the swollen phase is similar to the anomalous swelling reported in literature. Theaddition of HN-F to swollen vesicles suppresses the formation of any gel phase attemperature below 20 0C. Above Tm (42.3 0C) the presence of HN-F leads to formation ofdifferent lamellar and non-lamellar structures which can exist simultaneously. The structural
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changes due to the presence of HN-F in the vesicles are almost limited to the outerhydrophilic regions of the bilayer structure. This infers that the hydrophobic chains areonly peripherally influenced by the forces acting on the polar headgroups. Probably, theHN-F molecules occupy the free water layer, where their location is thermodynamicallymore favourable. This effect is more pronounced in the liquid crystalline phase. Theprocess is assisted by the positive osmotic gradient across the lipid membrane.
V38
DETECTION OF CANINE PARVOVIRUS SEROTYPE 2 INFECAL SAMPLES FROM DOGS BY POLYMERASE CHAIN
REACTION
Chavdar Filipov1, Petar Grozdanov2, Zahari Raikov2, Haralambi Haralambiev1
and Angel S. Galabov2
1 Faculty of Veterinary Medicine, University of Forestry, Sofia, 2 The StephanAngeloff Institute of Microbiology, BAS
There are two serotypes of parvoviruses, circulating among canine populations andthe most dangerous of them, causing diarrhea, is canine parvovirus serotype 2 (CPV-2). Inthis study we have worked out parameters of polymerase chain reaction (PCR) for canineparvovirus detection. A reference strain of CPV-2 was used as a control. It was found thatthe samples tested contained reaction inhibitors, possible reasons for false negativeresults. Therefore, we have used two pairs of specific for CPV-2 primers following thestandard procedure for DNA isolation from fecal samples. The optimal temperatureconditions for each pair of primers were determined. Four PCR positive out of the total 27fecal samples tested of dogs with enteritides were recorded with both pairs of primers. Weconsider this method is very sensible and precise for detection of parvoviral infection indogs.
Key words: canine parvirus serotype 2, polymerase chain reaction (PCR), fecal samples
V39МОЛЕКУЛЯРНА ДИАГНОСТИКА НА СИНДРОМ НА
LEWANDOWSKY-LUTZ (ЕPIDERMODYSPLASIAVERRUCIFORMES)
Б. Бонева1, М. Сайедж1, Г. Матеев2, З. Кълвачев1
1Национален център по Заразни и Паразитни Болести, Отдел по Вирусология,лаборатория по Молекулярна Вирусология;
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2Медицински Университет-София, Университетска Болница” Александровска”,Клиника по Дерматология и Венерология, Централна Лаборатория по Микози
Открити са повече от 100 типа човешки папиломни вируси (HPV). Около 30типа се предават при сексуален контакт със заразен парнтньор и инфектиратгениталните области, като вагина, вулва, цервикс, скротум, пенис и ректум. Другитипове HPV се предават при орален секс и причиняват поява на брадавици вустна кухина и фаринкс. Симптомите при папиломната инфекция се появяват отняколко седмици до месеци или години след първична инфекция и започват съссърбеж или парене около половите органи, дискомфорт и болки при полов контакт.Ако не се лекуват, гениталните брадавици се разрастват гроздовидно ипричиняват усложнения.За диагностиката HPV вируса се изолира отповърхността или вътрешността на ректум, гърло, уретра и вагина. Синдромана Lewandowsky-Lutz (Epidermodysplasia verruciformes) е автозомно-рецесивнаболест, която отслабва Т-клетъчния и хуморален имунитет към HPV антигени.Открити са около 15 вирусни типа, като HPV 5, 8, 9, 12, 14, 15, 17, 19, 25, 36, 38,47, 50.Това заболяване започва в детството и се среща в 10 дo 20 % от пациентите,обикновено млади индивиди. Води до злокачествена трансформация, сравнимацитологично с плоскоклетъчен карцином. Лечението е хирургично или схимиотерапия с Acitretin (25 mg/j).
Case report: В лаборатория по Молекулярна вирусология бе изследванматериал от пациент със синдром на Lewandowsky-Lutz. По метода наконвенционалната полимеразно верижна реакция (PCR) бе открит HPV-6, катобяха използвани праймерни системи за HPV- 6, 11, 18, 16 и 33. HPV-6 не се считаза етиологичен агент при съответния синдром, но това изследване показва, че ветиопатогенезата на епидермодисплазията може да участват и други HPV- типове.
Key words : Epidermodysplasia verruciformes, HPV, PCR
V40ВАРИЦЕЛА ЗОСТЕР ВИРУС (VZV) И БРЕМЕННОСТ
L.IvanovaDept of Microbiology and Virology, University hospital St Marina, MU - Varna ,BULGARIA
Варицела и херпес зостер по време на бременността могат да прчинятконгенитални дефекти, неонатална варицела, херпес зостер на новороденото дете.Цел на настоящото проучване е да се определят последиците за новороденитедеца от майки, контактни на варицела по време на бременността. Изследванапопулация: 56 бременни жени с несигурна анамнеза за преболедуване от варицелав детството, в тесен контакт с болни от варицела деца, бяха изследвани заопределяне на специфични IgG анти VZV антитела в CFT и ELISA. Единадесет
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(20%) бременности бяха усложнени с варицела и 4 (7%) – с херпес зостер.Интраутеринна варицела зостер вирусна инфекция беше идентифицирана поклинични критерии (синдром на конгенитални аномалии, остра варицела прираждането, херпес зостер на новороденото дете) въз основа на история нараждането. Четири деца с неонатална варицела бяха изследвани серологично всравнителен аспект с техните майки. Резултати: Серонегативни в CFT бяха 19(34%) от бременните жени и 10 (18%) в ELISA. Синдром на конгенитални дефектии херпес зостер на новороденото дете не бяха регистрирани. Новородените децаот майки с херпес зостер нямаха клинични данни за заболяване. Три деца снеонатална варицела бяха родени от майки с варицела в трети триместър набременността и ниски титри на антитела спрямо VZV. Едно дете от майка,контактна на варицела непосредствено преди раждането, серонегативна в CFTи ELISA почина от неонатална варицела. Заключение: Варицела по време набременността, като резултат от първична инфекция с VZV може да предизвикаувреждания на развиващия се плод и заболяване на новороденото дете.
V41DEMONSTRATION OF RABBIT MYXOMA VIRUS
IN CELL CULTURES BY DIRECTIMMUNOPEROXIDASE METHOD
R. Bostandjieva1, Zl. Dimitrova2, R. Peshev1
1Central Diagnostic and Research Veterinary Institute – Sofia, 2National Center forContagious and Parasitic Diseases –Sofia
A direct immunoperoxidase assay was developed for direct and quickly indication ofthe rabbit myxoma virus /VMZ/ in cell cultures. The method was modified on the basis ofthe peroxidase conjugate VMZ for ELISA obtained.
he viral reproduction of cytopathic VMZ strains in the stable RK13 cell line wasstudied.
V42EVALUATION OF THE EFFICACY
OF THE BROAD-SPECTRUM DISINFECTANT ON SOMEVIRUSES PATHOGENIC FOR THE FISHES
Darinka IlievaNational Diagnostic Research Veterinary Institute “Prof. Dr. D. Pavlov”
The antiviral activity of Aldekol Des FF “in vitro” against a field strain of the Infectious
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pancreatic necrosis (IPN) of Salmonids and a reference strain of the Spring viremia of carp(SVC) have been evaluated. The experiments were made under laboratory conditions withvarious concentrations and expositions.
The treatment of tested viral suspensions with the disinfectant at the lowestconcentration 1:3200 resulted for 90 min complete inactivation of the two viral strains.
V43СРАВНИТЕЛНИ ПРОУЧВАНИЯ НА КИТОВЕ ЗА
ДИАГНОСТИКА НА НЯКОИ ВИРУСНИ ЗАБОЛЯВАНИЯПО РИБИТЕ
В. Чикова, Д. ИлиеваНационален диагностичен научноизследователски ветеринарномедицинскиинститут, София
Проведено е сравнително изследване на стандартни китове за откриване иидентификация на VHSV, SVCV, IHNV и IPNV. Установено е, че използванитетърговски китове на база индиректен имунофлуоресцентен тест (IFAT) са с по-ниска специфичност и чувствителност за доказване на VHSV и IPNV. Китоветене дават възможност за отдиференциране на серотипове IPNV - Sр и IPNV - Ab,които се характеризират с различна патогенност. По аналогичен начин SVCкитовете не позволяват диференциране на SVCV и PFRV. Предлаганите ELISAкитове и имунопероксидазни тестове се характеризират с висока чувствителности специфичност и могат да бъдат прилагани с успех за бърза диагностика наразлични вирусни заболявания по рибите.
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MEDICAL MICROBIOLOGY
MM1ПОЛИФАЗЕН ИДЕНТИФИКАЦИОНЕН АНАЛИЗ НАКЛИНИЧНИ ИЗОЛАТИ ОТ BURKHOLDERIA CEPACIA
COMPLEX (BCC)
Мария Средкова1, Сашка Михайлова1, Edward Moore2
Медицински университет – 1 Плевен, 2 Culture Collection University Goteborg
Цел на проучването е идентифициране на клинични изолати от BCC до нивовид или геномоварова група чрез използване принципите на полифазнататаксономия. Двадесет и един щама, изолирани в УМБАЛ – Плевен, се изпитахачрез конвенционални фенотипни методи (57 теста), използване на автоматизиранасистема (32 теста), секвениране на гените за 16S рРНК и анализ на recA гена(ПВР със специфични праймери и секвениране). Резултатите от конвенционалнитефенотипни тестове позволиха всички изолати да се идентифицират до ниво BCCи да се определят като сборна група I/III/VII/VIII геномовар. Автоматизиранатасистема идентифицира изолатите като B. cepacia (геномовар I). Частичнотосеквениране на гените за 16S рРНК доказа принадлежност на изолатите къмкомплекса (степен на сходство с типови щамове 98-100%). Деветнадесетсеквенции показаха най-близко родство с щам BCC с неизвестна геномоваровапринадлежност, а две секвенции имаха най-изразено подобие с щам от геномоварIX (B. pyrrocinia). Анализът на recA гена потвърди отнасянето на изолатите къмкомплекса. В допълнение се установи, че те не принадлежат към нито един отизвестните геномовари. Изолатите от Плевен се обособиха в нова група BCC –тип АТ, която изисква по-нататъшно таксономично уточняване. Класическитефенотипни тестове позволяват идентифициране на изолати от BCC до ниво групаот геномовари. Секвенирането на гените за 16S рРНК е подходящ метод заразграничаване на бактериите от комплекса от BCC подобните микроорганизми,но не притежава достатъчна дискриминираща сила за видово диференциране.Изследването на recA гена дава възможност за по-прецизен таксономичен анализ.Необходими са нови методи в полифазния подход за окончателно идентифициранена изолатите.
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MM2STUDY OF VAGINAL LACTOBACILLI FROM BULGARIAN
WOMENG. D. Stoyancheva, S. P. Dimitonova, P. M. Petrova, R. N. Aleksandrova, I.Tzvetkova, S. T. DanovaDepartment of Microbial Genetics, Institute of Microbiology, Bulgarian Academyof Sciences, 26, Acad. G. Bontchev, str, 1113 Sofia, Bulgaria,e-mail: [email protected]
The human vagina is a complex dynamic ecosystem containing an abundance ofmicroorganisms. In women of childbearing age this system is dominated by Lactobacillusspp., producing a variety of metabolites including lactic acid which maintains the acidicconditions (pH<4.0), hydrogen peroxide (H2O2), bacteriocins and other antimicrobialsubstances as important inhibitors of the pathogenic organisms in the vagina. The use ofprobiotics to control certain infections and to reestablish the human bacterial microbiotais gaining acceptance as an alternative to conventional antibiotic therapy.
The present study summarised results from tree-years research project onLactobacillus microflora of sixty reproductive-age Bulgarian women. Forty eight strainsisolated from collected vaginal samples were determined as Lactobacillus sp. Antagonisticactivity of all vaginal lactobacilli were evaluated by different in vitro methods. Activestrains inhibiting the growth of pathogens were selected. Qualitative tests for H2O2 andexopolysaccharides production was made. Some of these exopolysaccharides promotedLactobacillus adhesion to epithelial cells and it is important for vagina colonization. Inorder to revealed the biodiversity of studied Lactobacillus strains six molecular methods(ribotyping, ARDRA, rep-PCR, PCR with species-specific primers, hybridization withspecies-specific probes and sequencing analysis) were applied. The predominant speciesin the studied group was L. fermentum. Three strains, possessing high antimicrobialactivity were studied by sequencing analysis of 16S rDNA. After additional research,these strains could be used as therapeutic agents for the treatment and prevention ofurogenital infections. This work was financially supported by National Council forScientific Research of Republic of Bulgaria. (Grant МУ-Л-1406/2004).
MM3ЧЕСТОТА НА ОТКЛЮЧВАЩАТА ИНФЕКЦИЯ ПРИ
ПАЦИЕНТИ С РЕАКТИВЕН АРТРИТ ИНЕДИФЕРЕНЦИРАН ОЛИГОАРТРИТ И
НЕОБХОДИМОСТТА ЗА ТЯХНОТО ИЗСЛЕДВАНЕ
Р. СтоиловМУ, София – МБАЛ “Св. Иван Рилски”Клиника по ревматология
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Реактивният артрит, предизвикан от някои грам-негативни бактерии (Chlamidiatrachomatis, Yersinia enterocolitica, Salmonella, Shigella, Campylobacter jejuni) е смного висока степен на асоциация с HLA-B27 молекулата от клас І на главниякомплекс за тъканна съвместимост. Въпреки че тези микроорганизми иматинтрацелуларен начин на живот, отделни техни фрагменти могат да бъдатпредставени чрез HLA-B27 молекулата. Цитотоксичните Т клетки разпознаваттези бактериални пептиди или други, индуцирани от самия гостоприемник катореагират кръстосано със собствените пептиди, представени в ставата. По тозиначин се отключва автоимунна възпалителна реакция, чиято клинична изява есиновита.
Реактивният артрит (РеА) е основна диференциална диагноза при пациенти снедиференциран олигоартрит. В 55 – 60% от случаите при болните, отговарящина клиничните критерии за РеА може да бъде идентифициран отключващияпатоген. При изследване на пациенти с недиференциран олигоартрит, липса наклинични симптоми за чревна или урогенитална инфекция и изключено другоревматично заболяване в 45 – 50% от случаите може да бъде доказана инфекция.Това означава, че приблизително в половината от пациентите с вероятен иливъзможен РеА може да бъде доказана отключващата инфекция в случай, че тебъдат изследвани и тестовете са достатъчно надеждни.
MM4MICROBIOLOGICAL INVESTIGATION OF IMPORTED
BRUCELLA INFECTION AMONG BULGARIAN CITIZENS
R. Nenova, T. Kantardziev, I. Ivanov, I. Tomova, B. Popov, Vl. Novkirishki
Brucellosis in Republic of Bulgaria has been officially eradicated since 1958. Despitethis, the closeness of the Mediterranean region, endemic for this zoonotic infection andalso the yearly presence of human cases in the neighbor countries creates a real possibilityof importing this infection in Bulgaria.
The aim of this study is the microbiological investigation of clinical samples fromsuspected for brucellosis bulgarian citizens, who have been worked in a neighbor countrywhich proved to be endemic for the disease.
We use methods in consistent with the guidelines of FAO/WHO Expert Committee ofBrucellosis and the CDC Basic Diagnostic Tseting Protocols for the PresumptiveIdentification of Brucella species.
529 investigations of 164 materials from 83 patients are made – 21 haemocultures, 429serological and 79 genetic. All haemocultures are negative (45days of cultivation)According to the serological tests there are three groups of sera samples:
- Negative- Serological data for active Brucella infection- Serological data for chronic Brucella infection
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In 28 of the patients Brucella DNA was found in the sera samples.The golden standard for proving the infection is the isolation of Brucella from the sera
samples. In our case the haemocultures were negative because of the delayed time fortesting the blood and the previous antibiotic therapy.
Detection of a four-fold or greater change in the serologica titres against Brucella
MM5CHARACTERISTICS OF THE CULTIVATION OFBIFIDOBACTERIUM BIFIDUM 1 IN MEDIA WITH
PALATINOSED. Blazheva, Z. Denkova, A. Krastanov
The cultivation of Bifidobacterium bifidum 1 was investigated in nutrient media,containing palatinose, which was obtained through the transformation of sucrose withimmobilized cells of Serratia plymuthica ATCC 15928.
It was determined, that Bifidobacterium bifidum 1 assimilates palatinose as a singlecarbohydrate source.
It is shown, that during the cultivation of this Bifidobacterium strain in a laboratorybioreactor with constant stirring in a medium with palatinose, a higher concentration ofviable cells was reached for a shorter period of incubation and a moderate titratable acidity.
The redox potential of the medium (Eh) decreased during the lag-phase (from +26 to -11) and during the transition to the stationary phase increased (from -11 to +42).
MM6КОНТАМИНАЦИЯ НА ХОТЕЛИ ПО БЪЛГАРСКОТО
ЧЕРНОМОРИЕ С РАЗЛИЧНИ КЛОНОВЕ ЛЕГИОНЕЛИ-ЕКОЛОГИЧНИ ПРЕДПОСТАВКИ ЗА КОЛОНИЗАЦИЯ
И. Томова, Ю. Хелбиг, В. Левтерова, Р. Ненова, И. Иванова
През последните три години зачестиха сигналите от Надзорната мрежа наЕвропейската Работна Група по Легионелни Инфекции за Легионерската болестпри пътуване (ЛБП) в България. В тази връзка бяха изследвани води отводоснабдителни системи (ВС) на хотели по черноморието за легионелни бактерии(Лб) и се събраха данни за особеностите на изследваните ВС, отговорни занамножаване на Лб. Проучването се извърши по методите описани в
Европейското методично указание за превенция на ЛБП. Обследвани бяха общо12 хотела в различни курортни комплекси. Контаминацията им с култивируемиЛб варираше от <101 до 104 CFU/L. Изолатите, принадлежащи към вида Legionella
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pneumophila бяха субтипирани, при което се установиха серогрупи: L.р.Sg1 (смоноклонални субтипове Allentown, Knoxville, Philadelphia и Oxford), Sg 3, Sg5, Sg6,Sg8 и единадесет добре разграничени (независимо от серогруповатапринадлежност) генетични варианти. Физико-химичните показатели на ВСварираха в широки граници: t0C студена H2O от 9- 29.70C , t0C топла H2O от 38.6-610C, остатъчен Cl2 <0.05-0.28 mg/L. Осем от обследваните хотели бяхановопостроени, с ВС на 4 месеца до 3 години. При всички хотели ВС бяха сразнороден дизайн и материали. Липсата на нормативна база и информация попроблема на ЛБП бе причината за проектантски грешки, използване нанеподходящи материали и устройства, неправилната им експлоатация, неволносъздаване на слепи точки във ВС, отсъствие на профилактика в мъртвите точки ипрограми за превенция на ЛБП. Комбинацията от изброените фактори е в основатана създаването на подходящи за намножаване на Лб екологични ниши в хотелскитеВС. Резултатите доказват необходимостта от мониториране на антропогеннитеводни екосистеми за Лб и спазване на международните норми за превенция наЛБП.
MM7NORMAL FLORA IS FOUND IN HUMAN BLOOD
E. Kalfin
Abstract: Normal flora microorganisms, living in human’s blood, are unknown toscientific literature. They were isolated only because instead of sheep blood, 10% ofnative human blood was added to the nutrient medium, and the cultivation period continuedfor 5 months, instead for 7 days. Routine cultivation requires a 3-4-week period, at 37°C.The newly discovered microorganisms have no cell nuclei. They have no Gram-positive-or Gram-negative microorganisms cell walls. Their surface is covered with very fine pili,clearly expressed in young cells, as demonstrated by electron microscopy During theirreproductive cycle, they penetrate through erythrocyte walls and settle inside theerythrocytes as in nests. Some formation resemble EB -/elementary bodies/ Outsideerythrocytes, they show well-expressed mimicry, but differ in size-0.3µm-2.6µm /microorganisms / versus 3.5µm-7.5µm / erythrocytes / and pili presence. Pure cultures ofCLM was isolate in BAS and in NCIPD, but DNA analysis failed in both institutions
MM8Q-FEVER – AN INSUFFICIENTLY KNOWN AND UNDERES-
TIMATED INFECTION IN BULGARIA.
V. Serbezov
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The Q-fever is cause by Coxiella burnetii – ricketsiosis which is spread all over theworld. It is known on the Balkan Peninsula and in Bulgaria since the World War II.
Our investigations in the 90-ties of the XXth century indicated that the social andeconomic changes in Bulgaria during the last 15-17 years (the vast areas of deserted land,the small farms, the primitive stock-breeding, the increase of goat-breading, lower levelsof veterinary service, etc.) lead to increase in the natural and anthropurgical focuses of theCoxiella.
From the clinical forms of the acute human Q-fever in our country is diagnosticated asQ-fever only the one which presents like athipical pneumonia during endemic outbreaks.
The most frequent – influence-like, also the hepatitis-like and other forms of the disease,including the variety of sporadic forms usually are not recognized. The inappropriatetreatment leads to chronization of the infection. Almost nothing is known and presentedabout the Q-fever in childhood.
Severe chronical forms like: endocarditis, hepatitis and so on, are also not familiar tothe general practitioners, despite that in 1.5 to 5% of the people who had passed the acuteforms of Q-fever the infection becomes chronic.
The unsterile immunity of the Q-ricketsiosis has negative effect on the pregnancy ofwomen who had been ill with Q-fever: late abortions, premature birth, still-born children.
In spite of the bad demographic situation in our country this problem is seriouslyunderestimated.
In the moment there is discussion about the problems and the situation with Q-fever inour country.
MM9КЛИНИЧНИ АСПЕКТИ ПРИ МИКОТИЧНИ ИНФЕКЦИИ
НА БЕЛИТЕ ДРОБОВЕ
В. Пенчева* , Д. Петрова*, О. Георгиев*, Т. Кантарджиев**, Ц. Мондешки*,Ц. Терзиева*, Д. Вълев****Катедра по Пропедевтика на Вътрешните болести, УМБАЛ”Александровска”; **Национален институт по заразни и паразитни болести-София;***СБАЛББ”Св.София” - бронхологично отделение
С широкото използване на антибиотичните средства в амбулаторната иболнична практика се създават условия за увеличаване на микотичните инфекции.При 73% от пациентите постъпили в клиниката е било приложено пероралноантибиотично лечение в амбулаторни условия. След последващатахоспитализация в 8% от случаите персистират общите оплаквания, без динамикав рентгеновия образ и лабораторните показатели. При всички тези пациенти секасае за подостро протичане на заболяването с водещи симптоми суха дразнещакашлица, фебрилно- интоксикационен синдром, бронхиална обструкция и в частот случаите придружаваща патология - ХОББ, бронхиална астма, захарен диабет,
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белодробна туберкулоза. С оглед диагностичното уточняване бяха приложенидопълнителни неинвазивни и инвазивни методи на диагностика - ФБС /фиброоптична бронхо скопия/ с БАЛ /бронхоалвеоларе лаваж/, катетърбиопсия,фиброщипкова биопсия, серологични проби, компютър томография. В резултатна проведените изследвания се позитивираха резултати за налични микотичниинфекции. При 22 пациента се изолира Candida spp.,при 5 пациента Аspergillusspp., при 1 болен Coccidiomycosis. След антимикотична терапия оплакваниятаотзвучаха, лабораторните показатели се нормализираха. В 36% от случаитерентгенологичните промени претърпяха обратна резорбция, като при трима отболните се наложи последващо хирургично лечение.
Микотичните инфекции на белите дробове се увеличават по честота, коетосъздава значителни диагностични проблеми по отношение изолиране наетиологичния причинител. Използването на инвазивни техники на изследване вкомбинация със серологични тестове в значителна степен повишава възможносттаза верификация на инфекциозния агент и последващ правилен терапевтиченподход.
MM10PCR-БАЗИРАНИ МЕТОДИ ЗА ДИАГНОСТИКА НАСИСТЕМНИ МИКОЗИ И ГЕНОТИПИРАНЕ НА
ПАТОГЕННИ ГЪБИЧКИ
П. Ангелов, Т. Кантарджиев, В. Левтеровa, Е. Замфирова, М. Лесева, Р.Вачева, Е. Шопова, Е. Бобчева
Честотата на системните микози нараства непрекъснато през последните 20години, представлявайки сериозен проблем за съвременната клиничнамикробиологична диагностика.
На базата на изследването на 40 клинични материала от пациенти със системнимикози, чрез разработен от нас PCR-базиран генетичен метод беше доказанаспецифична за патогена ДНК, като методът би могъл с успех да се използва заранното установяване на микозен сепсис и назначаването на подходящаантимикотична терапия.
Установяването на сродство между различните щамове гъбички, причинителина инфекции посредством генетични методи (генотипиране), се характеризира свисока дикриминативност и е от основно значение за ефикасния епидемиологиченнадзор на различните типове микози. Чрез използуване на различни генетичнимаркери е проведено генотипиране посредством техниките AFLP, RAPD-PCR, PCR-RFLP.
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MM11ЕТИОЛОГИЧНА СТРУКТУРА НА ЛЕКАРСТВЕНА
РЕЗИСТЕНТНОСТ И КОНСУМАЦИЯ НА АНТИБИОТИЦИВ БЪЛГАРИЯ
Т. Кантарджиев, М. Петров, Ц. Велинов, А. Бъчварова, П. АнгеловОтдел по микробиология – НЦЗПБ, София
На базата на данните от BulSTAR за изминалата година се прави анализ наетиологичната структура на вътреболничните инфекции, инфекции, придобити вобществото, пневмонии, сепсиси и уроинфекции, като получените данни сасравнени с тези, получени при аналогични проучвания в Европа и САЩ.
Въз основа на данните от 150 микробиологични лаборатории в страната иизвършени 200 000 антибиограми се прави анализ на лекарствената резистентностна най-важните патогенни микроорганизми.
Анализира се лекарствената резистентност на основните причинители набактериални менингити, пневмонии, уроинфекции и сепсиси – S. aureus, S.pneumoniae, H. influnzae, E. coli, K. pneumoniae, E. fecalis, както и данни за тяхнатаантибиотична резистентност
На базата на данните за надзора и консумацията на антибиотици в болничнатаи извънболничната помощ, се правят изводи и препоръки за национална стратегияи научно обоснована антибиотична употреба в клиничната медицина.
MM12INVESTIGATION OF THE VIRULENCE FACTORS AND
ANTIBIOTIC RESISTANCE OF MORAXELLACATARRHALIS
I. Mitov1, R. Gergova1, V. Ouzunova1, R. Markovska1, D. Kuncheva2
Chair of Microbiology, Medical University of Sofia, Bulgaria1; Microbiologylaboratory, Capital City Regional Inspectorate of Public Health Protection andControl, Sofia, Bulgaria2
During the last decade in Bulgaria the etiology role of Moraxella catarrhalis hasincreased. omp B2, omp CD and omp E genes, which M. catarrhalis demonstrates in vivoas outer membrane proteins (OMPs), as well as bro1 and bro2 genes coding beta-lactamaseproduction were detected in 103 clinical isolates and 4 control strains from CCUG and CIPcollections by PCR. The first group of 52 strains was isolated from patients with pulmonaryinfections. The second group of 41 isolates M. catarrhalis comprises patients withotorhinolaryngological infections. The third group of 10 strains consists of healthy children.The strains with the OMP factors in the first group were more than 60%, with prevalence
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of omp B2 and omp E. The role of OMP B2 is important to human serum resistance. Itenhances pulmonary clearance and inhibits iron acquisition from lactoferin and transferin.omp E is coding OMP E – major adhesin, which plays role in serum resistance and transportof fatty acids porin. In the second group the strains having the three OMP factors werenearly 30%. The prevailing OMP factors in this group were omp CD and omp E – 86% and96%, respectively. Adhesin coding omp CD gene binds to middle ear mucin, It is connectedwith resistance towards complement killing. 50% of the strains from healthy childrenpossessed only omp E and in 40% no factors were detected. Since 1996 more than 90% ofclinical strains in Bulgaria possess bro1 gene determining high level resistance to penicillin,aminopenicillins without inhibitors and first generation cephallosporins. This resistancemechanism and virulence factors studied by PCR in M. catarrhalis isolates are reason forthe increase of the infections caused by this microorganism.
MM13ВИДОВА ПРИНАДЛЕЖНОСТ И АНТИБИОТИЧНАРЕЗИСТЕНТНОСТ НА КЛИНИЧНИ ИЗОЛАТИ ОТ
ХЕМОКУЛТУРИ
Руканова Д., Е. Стайкова, К. Рачкова, И. Дукова, Х. Дженева, М. Бойчева, Г.ЛазароваТракийски университет, Медицински факултет, катедра Микробиология, СтараЗагора, България
Изследването на хемокултури е от изключителна важност за диагнозата,терапията и прогнозата на редица инфекциозни заболявания и инфектни процеси.
Целта на настоящото проучване е да установи видовото разпределение наизолати от хемокултури и честотата на фенотиповете на резистентност.
Материали и методи: За периода 2002-2005 година са изследвани хемокултуриот болни, лекувани в Университетска болница-Ст.Загора. Използвана е систематаBactec, BBL, както и среди за хемокултури на Бул Био,София. Идентификациятае извършена чрез автоматизирани системи Sceptor и Crystal и рутинни методи, аантибиотичната чувствителност е определяна по дифузионния-дисков метод наBauer-Кirby.
Резултати: Положителните хемокултури са 10.9%. Преобладават Грам-положителните бактерии-61.8%, срещу 37.0% Грам-отрицателни и Candidaalbicans 1.2%. Най-чести са CNS (32.8 %), E. coli (13.9%), S .aureus (11.3%), P.aeruginosa (10.1%) и E. faecalis (7.5%).
Устойчиви на Oxacillin са 51.3% от CNS и 28.6% S. aureus. Ampicillin-резистентни са 5.6% E. faecalis и 60.0% E. faecium. HLAG резистентност е намеренапри 11.1% E. faecalis и при 80.0% E. faecium. Не са регистрирани PNSSP иVancomycin–устойчиви Грам-положителни бактерии. ESBLs cа открити при 23.7%от сем. Enterobacteriaceae. Резистентността на P. aeruginosa се движи в широки
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граници: 7.1% към Imipenem, 25.0% към Ceftazidime, 30.0% към Amikacin, 52.9%към Piperacillin и 60.9% към Ciprofloxacin.
MM14ПРОУЧВАНЕ НА ВИДОВАТА ПРИНАДЛЕЖНОСТ ИАНТИБИОТИЧНА ЧУВСТВИТЕЛНОСТ НА ЩАМОВЕ,ИЗОЛИРАНИ ОТ УРИНА НА ПАЦИЕНТИ С ХРОНИЧЕН
ПИЕЛОНЕФРИТ
В. Григорова 1, В. Тодоров 2, В. Едрева 1, К. Драгоев 1
1 МДЛ по Микробиология и вирусология,2 Клиника по нефрология; „УМБАЛ д-р Г. Странски” Плевен, ЕАД, България
Въведение:Хроничният пиелонефрит е една от най-честите причини зарецидивиращи инфекции на уринарния тракт.
Цел: Да се проучи видовата принадлежност и антибиотичната чувствителностна микроорганизми, изолирани от урина на пациенти с хроничен пиелонефрит.
Материали и методи: проведени бяха микробиологични изследвания на уриниот 218 пациенти с хроничен пиелонефрит, лекувани в Клиниката по нефрологияпри „УМБАЛ д-р Г. Странски” Плевен, ЕАД за едногодишен период (2005). От116 пациенти със сигнификантна бактериурия и пиурия бяха изолирани 213 щамауропатогени, идентифицирани с конвенционални методи. Щамовете E.coli бяхаскринирани за продукция на ESBLs. Чувствителността към антибиотици бешеопределена чрез дисково-дифузионен метод на Bauer-Kirby, съгласно NCCCLS.
Резултати: Най- често изолираните микроорганизми бяха: E.coli (44.1%),E.faecalis (26.8%), и P.aeruginosa (10.8%). Продуценти на ESBLs сред щамоветеE.coli бяха 18.3%. Щамовете E.coli продуценти на широкоспектърни â-лактамази(ESBLs) бяха резистентни към gentamicin (72.7%), ciprofloxacin и TMP/SMZ. ЩамоветеE.faecalis бяха резистентни към ampicillin (61.4%), gentamicin (70.5%) и ciprofloxacin(77.3%). Щамовете P.aeruginosa бяха резистентни към gentamicin и ciprofloxacin(33.3%). Всички щамове E.coli и P.aeruginosa бяха със запазена чувствителносткъм imipenem и meropenem, а E.faecalis към vancomycin и teicoplanin.
Лечението на пациенти с хроничен пиелонефрит е необходимо да се провеждасъобразно чувствителността на изолираните микроорганизми към антимикробнисредства, за да се постигне максимален терапевтичен ефект и да се ограничиразпространението на полирезистентни щамове.
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MM15ANTIMICROBIAL SUSCEPTIBILITY OF URINE
PATHOGENES ISOLATED FROM PATIENTS WITH BENIGNPROSTATIC HYPERPLASIA TREATED WITH UROSTIM .
V. Grigorova 1, S. Stratev 2, F. Shargabi 2, N. Kolev 2, P. Panayotov 2, R. Kostov 2,O. Mihaylov 2, Ts. Georgiev 2, V. Dunev 2, B. Atanasov1 Laboratory of microbiology,2 Clinic of Urology, University Hospital - Pleven,Bulgaria
Introduction:Benign prostatic hyperplasia (BPH) is one of the most frequent reasons for recurring
infections of the urinary tract.Aim:
To determine the antibiotic susceptibility of microorganisms isolated from urine ofpatients with BPH and the results of immunotherapy with urostim.
Material and Methods:A total number of 126 urine samples of patients with BPH treated in the Clinic of
Urology during 2005, were examined. Isolated strains were identified by conventionalmethods. E. coli and K. pneumoniae strains were screened for ESBLs production.Susceptibility to antibiotics was determined by using disc diffusion method of Bauer-Kirby, according to NCCLS. Immunotherapy with urostim for a 3-month period was appliedto 36 patients and the urine was monitored on a monthly basis.
The patients were divided into two groups: Group I – 12 patients with asymptomaticbacteriuria treated with urostim only, and Group II – 24 patients with symptomatic bacteriuriatreated with urostim and antibiotic.
Results:Fifty strains were isolated from 40 patients with significant bacteriuria and pyuria.
The most frequently isolated microorganisms were: E. coli (42.0%), K. pneumoniae (18.0%)and E. faecalis (12.0%). Producers of ESBLs among E. coli strains were 61.9%, and amongK. pneumoniae - 77.8%. The E. coli and K. pneumoniae strains remained susceptible toimipenem and meropenem. The E. faecalis strains were susceptible to vancomycin andteicoplanin.
After conducted treatment liquidation of the uroinfection were registered in 10(83.3%) patients from Group I and in 18 (75.0%) patients from Group II. The bacteriuriaremained persistent in 2 (16.7%) patients from Group I and in 6 (25.0%) patients fromGroup II.
Conclusions:The immunotherapy with urostim does not exclude treatment with antibiotics in
order to achieve highest therapeutic results
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MM16ЧУВСТВИТЕЛНОСТ НА БОЛНИЧНИ ИЗОЛАТИ S.
PNEUMONIAE КЪМ НЯКОИ АНТИБИОТИЦИ
К. Божкова, Т. Стоева, В. Калудова, В. Каменова, В. Русев
Проучени са общо 69 етиологично значими щамове S. pneumoniae, изолираниот различни клинични материали (секрети от горни и долни дихателни пътища,ликвор, хемокултури) в Лабораторията по клинична микробиология ивирусология на УМБАЛ “Св. Марина” – Варна за периода 01.01.2005 – 1.06.2006.Определена е чувствителността им in vitro към няколко антимикробни препарата(penicillin, erythromycin, clindamycin, tetracyclin, rifampin, vancomycin, linezolid,trimethoprim/sulfamethoxasol). От изследваните изолати делът на пеницилин-нечувствителните S. pneumoniae, определен чрез дисково-дифузионния метод е76,8% (53 щама). Определянето на MIC, с Е тест, установява, че 95,4% от тях сарезистентни на пеницилин. Относително резистентни с МIC 0.12 до 1 µg /ml са44,2% (23 щама), а 55,8% (29 щама) са абсолютно резистентни. Резистентносттакъм останалите антибиотици е както следва: V, Lzd – 0%, R – 4%, Cli – 10%, Ery, T, ST– 22%. Един от изолатите демонстрира MLS индуцибелен фенотип нарезистентност.
Предвид нарастващата резистентност към penicillin, определянето на MIC заизолати S. pneumoniae, особено при тежки инвазивни инфекции, е от критичнаважност за правилно терапевтично поведение.
MM17ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF A
POLYPHENOL-RICH EXTRACT FROM GERANIUMSANGUINEUM L.
Medine Gulluce1, Fikrettin Sahin2, Atalay Sokmen3, Ani Teodosieva4, JuliaSerkedjieva4
1Atatьrk University, Faculty of Art and Science, Department of Biology, Erzurum,Turkey, 2Atatьrk University, Biotechnology Application and Research Centre,Erzurum, Turkey, 3Cumhuriyet University, Faculty of Art and Science, Departmentof Biology, Sэvas, Turkey, 4Institute of Microbiology, Bulgarian Academy ofSciences, Sofia, Bulgaria
With the appearance of bacterial and viral variants, resistant to “classical”chemotherapeutic agents, the search of microbial inhibitors of plant origin becomes apromising approach in the development of new drugs. A polyphenol extract (PC) has beenobtained from the aerial roots of the Bulgarian medicinal plant Geranium sanguineum L.
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The extract inhibited the reproduction of a range of viruses (influenza, herpes simplex,vaccinia, HIV-I) in cell cultures. Its anti-influenza effect was most pronounced; PC reducedthe infectivity of various influenza virus strains in vitro and protected mice in theexperimental influenza infection. The present study sought to evaluate the antibacterialand antifungal properties of the preparation using routine assays. PC was tested againsta panel of microorganisms including a total of 56 microbial cultures, belonging to 32bacteria and 24 fungi and yeast species. However the preparation showed a markedantifungal potential. Five strains - Fusarium pedis, Alternaria solani, Trichophytonrubrum*, Trichophyton mentagrophytes* and Aspergillus niger* were resistant to thePC-antifungal activity. Most susceptible to the inhibitory action of the extract wasRhizoctonia solani (MIC 15.62 µg/ml). There was no activity in terms of bacteria tested,the only exception being Staphylococcus aureus 209 (MIC 1000 µg/ml). Based on theresults from the present and previous investigations we believe that the Geraniumsanguineum extract possesses some interesting pharmacological properties. Itsphytochemical analysis revealed the presence of flavonoids, catechins, gallotannins andcarboxilic acids; some of them were identified by chromatographic methods (PC, TLC,HPLC).
MM18INVESTIGATION OF THE INHIBITORY EFFECT OF LACTICACID BACTERIA ON THE CELLS OF ESCHERICHIA COLI
NBIMCC 8739
P. Nedelcheva, Z. Denkova, R. Nikolova
The inhibitory effect of growing cultures of Lactobacillus plantarum 226-15,Lactobacillus casei subsp. casei C, Lactococcus lactic subsp. lactis l4 and Pediococcuscerevisiae 2P on the cells of Escherichia coli NBIMCC 8739 was investigated during theirmixed cultivation in skimmed milk at temperatures 23±1°С and 37±1°С.
It was established, that during the first 6 h the viable cell counts of both the lactic acidbacteria and Escherichia coli NBIMCC 8739 increased. During the next 12-24 h the growthof Escherichia coli NBIMCC 8739 gradually slowed up and held up. This effect was moredistinct during the mixed cultivation of lactic acid bacteria and E. coli at 37°С.
MM19PROBIOTICS AND PROBIOTIC FOODS
IN THE PROTECTION OF HUMAN HEALTH
I. Murgov, Z. Denkova
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A series of probiotic preparations ‘Enterosan’ was developed on the basis of selectedLactobacillus and Bifidobacterium strains from the national gene fund, which survive theconditions in the stomach and the intestines, exhibit high antimicrobial activity andresistance to most of the applied in the clinical practice antibiotics.
Tested in leading clinics in Bulgaria and abroad th eprobiotics ‘Enterosan” are appliedsuccessfully in the complex therapy of diseases of the digestive, endocrine, secretory,vascular, bone, neural and other systems.
The necessity was reasoned for the inclusion of probiotic foods and drinks in the dietof the contemporary human.
MM20СЕПСИС, СВЪРЗАН С ЦЕНТРАЛНИ ВЕНОЗНИ КАТЕТРИ,
ПРИ БОЛНИ В КРИТИЧНО СЪСТОЯНИЕ –ПРИЧИНИТЕЛИ И ЧЕСТОТА
Димитър Терзийски, Николай Петров, Николай МладеновКАРИЛ, ВМА, София, е-mail: [email protected]
УВОД: Системните нфекции, свързани с централни венозни катетри (ЦВК), сасред най-тежко протичащите вътреболнични инфекции и причина за значителнасмъртност и увеличени разходи при лечението на болни в критично сътояние.
ЦЕЛ: Да се установят честотата и причинителите на сепсис, свързан с ЦВК,при болни в критично състояние.
МЕТОД: Болни: Проспективно изследване на болни, лекувани в КАРИЛ,ВМА, за периода март 2001 – април 2004 г. ЦВК: метод на въвеждане, престой,локализация и условия на поставяне. Микробиологични методи: полу-количествено изследване на катетера по метода на Maki; определяне надиференциално време до позитивиране на хемокултури с кръв, взета през ЦВК иот периферна вена.
РЕЗУЛТАТИ: Болни: 95 (от 18 до 81год) и ср.сбор по APACHE II – 20 т. Значимаколонизация на ЦВК – 52 (44 %); локална инфекция, свързана с ЦВК – 26 (22 %);сепсис, свързан с ЦВК – 25 (21 %). Микробиологични изследвания : (1) свързани сЦВК – изолирахме 89 щама, грам-положителни –53 щама, грам-отрицателни 31щама, Candida spp – 5 щама; (2) несвързани с ЦВК изследвания – изолирахме 221щама, грам-отрицателни – 161 щама, грам-положителни коки 44 щама и Candidaspp – 16.
ИЗВОДИ : Установената от нас висока честота на сепсис, свързан с ЦВК,може да се дължи на спецификата на изследваните болни, както и наполивалентната употреба на катетрите. Най-чест причинител на сепсис, свързанс ЦВК, е S. aureus, следван от K. pneumoniaе. Броят на щамове S. epidermidis,S.aureus и Enterococcus spp, изолирани от катетрите и от другите области е сходен,което показва значимата им роля за възникване на сепсис, свързан с ЦВК, при
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изследваните болни.
MM21МОЛЕКУЛЯРНА ДИАГНОСТИКА НА ПРИЧИНИТЕЛИ НА
АТИПИЧНА ПНЕВМОНИЯ
Надя Бранкова, Тодор Кантарджиев, Виктория Левтерова, Елина Георгиева,Искра Томова и Стефан ПанайотовНационален център по заразни и паразитни болести, Отдел „Микробиология”,Янко Сакъзов 26, София - 1504
Coxiella burnetii, Chlamydophila pneumoniae, Legionella pneumophila иMycoplasma pneumoniae причиняват остри респираторни инфекции като атипичнапневмония придобита в обществото, хронични бронхити, астма и с по-малкачестота инфекции на горните дихателни пътища. Настоящето изследванепредставя обобщение на разработени съвременни методи за диагностика на C.burnetii, C. pneumoniae, Legionella pneumophila и M. pneumoniae в различниреспираторни материали. Методите са разработени на базата на видово-специфични IS1111A, omp1, mip и P1 гени. Всички клинични материали бяхаанализирани чрез метода на полимеразно-верижна реакция. Бяха потвърдениепидемични взривове на Ку-треска в гр. Ботевград (2004) и атипична пневмониясред деца, причинена от M. pneumoniae в с.Ключ (2002) и гр. Павликени (2005).Положителните резултати са от носогърлени секрети и храчки. PCR методите сеотличават с висока специфичност и чувствителност и могат да бъдат използванив рутинната лабораторна диагностика на C. burnetii, C. pneumoniae, Legionellapneumophila и M. pneumoniae.
MM22Emm SEQUENCE TYPING OF CLINICAL ISOLATES
STREPTOCOCCUS PYOGENES RECOVERED IN BULGARIA
A. Decheva , V. Karjeva , D. Beshkov , I. AlexievNational Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
Streptococcus pyogenes (group A streptococci – GAS) is an important bacterialpathogen for humans. Its surface M protein is the main pathogenic factor with antiphagociticactivity. More than 120 M-serotypes have been registered until now. Production of rabbittypespecific antisera has become quite expensive and time consuming. Sequence typingof the emm gene encoding the M protein has proved to be more specific, sensitive, quickand convenient method for epidemiological typing of S. pyogenes. We performed emmsequence typing of 69 clinical isolates (48 from upper respiratory tract, 15 from skin lesions,
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1 vaginal isolate, 3 from invasive infections and another 2 from noninvasive wound infectionbut epidemiologically related with the three invasive isolates). The group of upperrespiratory tract isolates was heterogenous but the most frequent genotypes were emm 12and emm 1. The group of skin isolates was also heterogenous with emm 1, emm 44/61 andemm 117 being most common. Overlapping of genotypes was observed for these twogroups of isolates. The three invasive isolates and two wound isolates were recoveredfrom epidemiologically related cases. All of them expressed emm 65 which confirmed therelation. To our knowledge emm 65 has not been reported in association with invasiveinfections until now.
MM23RECOMBINANT BORRELIA BURGDORFERI PROTEINS AS
ANTIGENS FOR SEROLOGIC DIAGNOSTICS OF LYMEBORRELIOSIS
I. Christova1), M. Lesseva2), G. Miloshev2)
1) National Center of Infectious and Parasitic Diseases; 2) Institute of MolecularBiology, Bulgarian Academy of Sciences
Application of purified or recombinant antigens, instead of whole-cell Borreliaburgdorferi antigens, has been suggested to increase specificity of serological diagnosticsof Lyme borreliosis. In addition, using recombinant antigens, dynamics and variation ofthe main serological markers in the course of the disease could be investigated. Theproblem is, that at least three B. burgdorferi species – B. burgdorferi sensu stricto, B.garinii and B. afzelii, cause Lyme disease in Europe and respectively in Bulgaria. In anattempt to obtain recombinant B. burgdorferi proteins, which could be used to study onspecificity of humoral immune response in Lyme disease, we made a comparative analysisof genes for the major outer surface protein antigens, OspA and OspC, as well as for thegene of immunodominant flagellar antigen, p41, of the three species Borrelia that causeLyme disease. The highest level of homology was detected in the gene FlaB – 93-94%,lower but also high was detected in the genes OspA and OspC, respectively 87-88% and83-88%. Next step was cloning and expression of these proteins. The proteins obtainedwere confirmed by immunoblot with specific monoclonal antibodies. Applied as antigensfor ELISA, OspA showed higher sensitivity in the late stages of the disease, OspC -higher sensitivity in the early stages, and FlaB – higher sensitivity but at the same timelow specificity. The optimal combination of recombinant antigens for detection of differentstages of Lyme disease, should be elucidated.
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MM24DETECTION OF BORRELIA BURGDORFERI SENSU LATO,ANAPLASMA PHAGOCYTOPHILUM AND FRANCISELLA
TULARENSIS IN WILD RODENTS FROM AN ENDEMICFOCUS OF TULAREMIA IN BULGARIA
Gladniska T., Christova I., Taseva E., Nenova R.Microbiology Department, National Center of Infectious and Parasitic Diseases
Lyme disease, tularemia and human granulocytic anaplasmosis (HGA) are among themost common tick-borne bacterial infections in Bulgaria. Reservoirs of the infections arewide spectrum of wild and domestic mammals. The most important reservoirs among wildmammals are rodents – Mus musculus, Rattus rattus, Apodemus agrarius. The aim of thisstudy was to establish prevalence of infections with Borrelia burgdorferi sensu lato,Anaplasma phagocytophilum and Francisella tularensis, the etiological agents of Lymedisease, HGA and tularemia, in wild rodents from an endemic focus of tularemia in westBulgaria, Slivnitza region. Serologic methods (ELISA, agglutination) detected infectionwith B. burgdorferi in 14% of the rodents and in 21% infection with F. tularensis. Geneticmethods (PCR) established infection with B. burgdorferi in 26% of the rodents, with F.tularensis in 21% of the samples and with A. phagocytophlim in 7% of the rodents. Thehigher percentage of infected rodents, detected by PCR is due to the higher sensitivity ofthe methods. This is also probably the explanation of the discrepancy detected betweensome of the serologic and PCR – positive results in different stages of the diseases.Contemporary PCR methods are adequate tools for fast and reliable surveillance ofcirculations of different tick borne pathogens among rodent populations. Combination ofPCR and serologic methods reveal current incidence of rodent infections.
MM25НОВОУСТАНОВЕНО ОГНИЩЕ НА ТУЛАРЕМИЯ В
СЕВЕРОИЗТОЧНАБЪЛГАРИЯ ПРЕЗ 2004 – 2005 ГОД.
Н. Готев, К. Младенов, Ц. Цветанов, Е. Пенков, Н. Коруджийски,Сн. Иванова, В. Дойчева
През 60-те години на миналия век беше наблюдавано огнище на туларемия врайона на гр. Силистра – езерото Сребърна, а след 1997 год. и до сега сенаблюдава такова в района западно от София.
През 2004-2005 год. са установени данни за “ново” огнище в района на гр.Русе – Разград – Търговище. От намерен умрял заек е изолиран причинителя натуларемията – Francicella tullarensis и у двама заболели е потвърдена положителна
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за туларемия серологична проба.Тези данни говорят за наличие на ензоотична туларемия сред дивите зайци и
разпространенние на инфекцията и сред хората в този район.Фенотипната характеристика на изолираната култура Francicella tullarensis е
характерна за тип В на този бактерий, както в установените преди това две огнища.Тези данни показват, че е необходимо д0а се провеждат системно наблюдения
предписани в Националната програма за борба с кърлежо-преносимите инфекции.
MM26ФЕНОТИПНА ХАРАКТЕРИСТИКА НА ПРИЧИНИТЕЛЯНА ТУЛАРЕМИЯТА – FRANCICELLA TULLARENSIS
ИЗОЛИРАНИ В БЪЛГАРИЯ ПРЕЗ 1961 – 1965И 1998 – 2005 ГОД.
Н. Готев, К. Младенов
Досегашните лабораторни изследвания показват, че причинителя натуларемията F. tullarensis проявява характерни фенотипни белези, което позволяванеговата индентификация и разграничаване на две разновидности – тип А и типВ.
Тип А е установен досега в Северна Америка, а тип В и в Европа и Азия.В нашата страна бяха изолирани голям брой щамове на F. tullarensis в
наблюдавани огнища през периода 1961 – 65 год. в района на езеро Сребърна,Силистренско и 1998 – 2004 год. западно от София.
Щамовете са изолирани от различни източници – от гризачи (ондатри, зайци,мишевидни), артроподи, вода и човек.
Фенотипната характеристика на всички изследвани щамове съответства за F.tullarensis – тип В, разпространен в Европа.
MM27ЛЕЧЕНИЕ НА ЕКСПЕРИМЕНТАЛНА ТУЛАРЕМИЙНА
ИНФЕКЦИЯ
К. Младенов1, Х. Найденски2, Ц. Цветанов1, Н. Готев1
1Военномедицинска академия, София, България2 Институт по микробиология “Стефан Ангелов” - БАН
Туларемията е зоонозна инфекция, към която човек показва високавъзприемчивост. Заразяването е възможно по всички пътища на попадане навъзбудителя в организма – през дихателния тракт, слизестите обвивки,
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конюнктивата, през увредена кожа и парентерално.Туларемийният бактерий има висока инфекциозност по отношение на човека
и редица дребни и едри гризачи, което го прави подходящ за използване катоагент за биологично поразяване.
Целта на разработката е да се проучи чувствителността на възбудителя къмнякои антибиотици и продължителността на третиране на модела наекспериментални животни.
Използвани са опитни животни, отнасящи се към I група по отношението сикъм инфекцията – морски свинчета, чрез парентерално заразяване в доза 1000микробни клетки на туларемийния щам “Сребърна-19”.
Третирането е извършено с два антибиотика: доксициклин (Doxycyclin), в доза6 mg на един прием с продължителност 5, 10 и 15 дни per os.
Tetracyclin – depo, в денонощна доза 15 и 30 mg, дадени на два приема спродължителност 10 дни.
Установени са положителни резултати и очистване на организма отпричинителя на инфекцията. Данните от количественото натрупване иперсистиране на бактериите в различните органи са определени в динамика и придвата антибиотика.
Пероралното третиране с Doxycyclin – еднократно-6 mg иTetracyclin-depo –двукратно - 30 mg, осигуряват добър защитен ефект на експерименталнитеживотни от туларемийната инфекция.
MM28RAPID IDENTIFICATION AND BIODIVERSITY OF LACTO-
BACILLUS SPECIES IN VAGINAL SAMPLES
S. Dimitonova1, P. Grozdanov2, A. Galabov2, B. Bakalov3, R. Aleksandrova1, G.Stoyancheva1 and S. Danova1
1- Department of Microbial Genetics and 2- Department of Virology, Institute ofMicrobiology, Bulgarian Academy of Sciences, 26, Acad. G. Bontchev, str, 1113Sofia, Bulgaria.3- Department of General and Applied Microbiology, Biological faculty, SofiaUniversity, 8, Dragan Tzankov, bvd, 1113 Sofia, Bulgaria
Lactobacilli are commonly present in the human vagina and critically important towomen’s vaginal health. They have received a considerable attention as a result of theirsignificant properties. The application of modern genomic analyses has advanced theircorrect species identification and taxonomy. In this study the Polymerase chain reaction(PCR) was demonstrated to be suitable tools for the analysis of Lactobacillus communityof reproductive age Bulgarian women. It allow the detection of species very quickly andreliable. The results showed the presence of L. fermentum, L. crispatus, L. acidophilusand species from L. plantarum group in vaginal smears collected from healthy volunteers
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and patients with HPV. We did not find a correlation between HPV infection and disturbanceof Lactobacillus microbiota, by PCR and classical microbiological analysis. Combinedmolecular analyses revealed the presence of two or three different Lactobacillus speciesoriginated from a single vaginal sample. The study accomplishes the scarce informationexisted in Bulgaria on the real identity of Lactobacillus species in relation of women’shealthy status.
MM29VIRULENCE DETERMINANTS OF AEROMONAS SPP. ISO-
LATED FROM FOOD, DRINKING WATER AND PATIENTS INBULGARIA
P. Orozova, I. Abrashev
Bulgarian Academy of Sciences, Institute of Microbiology, Department of MicrobialBiochemistry, 26 Acad. G. Bonchev str., Sofia 1113, Bulgaria
In the present study 45 Aeromonas spp strains isolated from food, drinking water andpatients in Bulgaria were tested for pathogenicity by studding its hemolysis, neuraminidaseactivity and cytotoxicity. The drug resistance profiles were also evaluated and all strainsdisplayed multiple drug resistance.
Hemolysis, tested on sheep erythrocytes, was more frequently seen with water isolatesand human isolates than with food isolates. Neuraminidase activity was detected in allisolates and it was the highest to Aeromonas hydrophila strains. Cytotoxicity, evaluatedon Vero cells was frequently observed with food (90%), with water isolates (70%) andwith human isolates (62%). It was found that negative pyrazinamidase activity wasassociated with Aeromonas sobria and positive pyrazinamidase activity was associatedwith Aeromonas hydrophila and Aeromonas caviae. All isolates exhibited multiple drugresistance.
These results indicate that the occurrence of Aeromonas spp. in the environmentrepresents a potential risk for humans. These results also suggest that Aeromonas speciesare potential enteric pathogens in our geographical region.
MM30ОПРЕДЕЛЯНЕ СРОК НА ГОДНОСТ НА АНТИМИКРОБНИ
ДИСКОВЕ ЦЕФОКСИТИН И ЦЕФТРИАКСОН ИВЪВЕЖДАНЕТО ИМ В ПРОИЗВОДСТВО
Даринка Чанкова, Даниела Пенчева“Бул Био – НЦЗПБ” ЕООД
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Цефокситинът принадлежи към втора генерация цефалоспорини. Отличава сес висока стабилност към ß-лактамазите и подчертана активност към облигатнитеанаероби и гонококите. При изпитване ин витро чувствителността намикроорганизмите се налага залагането му като отделен диск. Според CLSI/NCCLS2006 докладването на оксацилиновата резистентност се извършва на базата нацефокситиновия диск. Препоръчва се да се използва за изпитване резистентносттакъм пеницилиназа стабилните пеницилини при S.aureus и S.lugdunensis.Цефокситиновият дисков тест има по-голяма специфичност и еквивалентначувствителност от оксацилиновия дисков тест към коагулаза негативнитестафилококи.
Цефтриаксонът има най-широк спектър на действие в сравнение с останалитецефалоспорини от трета генерация, като към него са чувствителни и S.pneumoniae,стрептококи група А и С, H.influenzae, гонококите (включително ß-лактамазапродуциращите), менингококите. От всички цефалоспорини неговият полуразпаде най-бавен, което позволява да се прилага веднъж на 24 часа. Според CLSI/NCCLS 2006 е задължително залагането в рутинните антибиограми нацефтриаксоновия диск при наличие на резистентност на семействоEnterobacteriaceae и Acinetobacter към антимикробните дискове от група А.
Точното определяне срока на годност на антимикробните дискове е от голямозначение за получаване на достоверни и възпроизводими резултати отантибиограмите, а от там и за правилното лечение на пациентите.
MM31LABORATORY METHODS FOR DRUG SUSCEPTIBILITY
TESTING OF MEDICALLY IMPORTANT YEASTS ANDMOULDS
Kouzmanov A., T. Kantardjiev, Ivanova Z., L. BoyanovaNational Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
Introduction: During the last two decades the number of patients with serious fungalinfections has increased dramatically. The risk group includes: immunocompromizedpatients, especially those infected with HIV, those receiving immunosuppressive therapyfor organ transplantation and cancer. The long-term use of broad-spectrum antifungalsfor prevention and therapy of mycosis has led to the detection of increased drug resistanceof fungal pathogens. Therefore there is a prominent necessity of standard susceptibilitytesting methods.
Aim: Determination of Fluconazole susceptibility of strains from genus Candida withthe use of standard disk diffusion method M44-A. Additional testing of preliminary chosenresistant to Fluconazole strains and determination of their susceptibility to five antifungals:Itraconazole, Ketoconazole, Voriconazole, 5-Fluorocitosine, Amphotericine B
Materials and Methods: In the study were used clinical isolates collected by the Referent
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Mycology Laboratory of National Center of Infectious and Parasitic Diseases, Sofia. Theyare strains from genus Candida and some moulds. All were identified by conventionalbiochemical methods API 20C AUX, AUXACOLOR 2 or automated system Vitek anddirect microscopy combined with the fungal culture. For the susceptibility testing wereused 5 methods: disk diffusion method (DDM) NCCLS M44-A; reference microdilutionmethod NCCLS (currently CLSI); agar dilution E-test; commercial kit ATB FUNGUS 2 INTand MICRONAUT-AM (Merlin).
Results: We detected high pro cent of resistant strains, witch can be explained withtheir isolation from patients with HIV, under a long-term azole therapy. High resistancewas detected in the strains C. glabrata and C. krusei. There is a good correlation betweenthe 5 methods in the testing of Fluconazole resistance. Voriconazole showed goodeffectiveness but the results received for Itraconazole and Ketoconazole were not sogood. For the determination of 5-Fluorocitozine were used ATB FUNGUS 2 INT andMICRONAUT-AM with only one resistant strain detected. There was a sufficientcorrelation between the results received for Amphotericine B.
Conclusions: The most important benefit of antifungal susceptibility testing is thedetermination of the minimal inhibitory concentration of a certain drug. This allows anaccurate dozing and adequate therapy of the patient. The introduction of standard methodsfor antifungal susceptibility testing can stop the aimless use of drugs.
MM32STUDY OF HYPOCHOLESTEROLIC EFFECT OF HIGHER
BASIDIOMYCETES
Popov A.1, Panchenko A.2, O. Chistova1, Petrishchev N.2, Denisova N.3, ShamtsyanM.1
1. St. Petersburg State Technological Institute (Technical University), Moskovskyprospect, 26, St. Petersburg, Russia. E-mail: [email protected]. I.P. Pavlov State Medical University, 6/8 L.Tolstoy Str., 197022 Saint Petersburg,Russia3. V.L. Komarov Botanical Institute, Russian Academy of Sciences, ul. ProfessoraPopova, 2, St. Petersburg, 197376, Russia
It is known, that hypercholesterolemia increases the risk of heart disease. Elevatedlevels of circulating cholesterol cause deposits to form inside blood vessels. Thesedeposits can result in a disease process called atherosclerosis, which can cause bloodclots to form that will ultimately totally stop blood flow. If this happens in the arteriessupplying the heart, a heart attack will occur. If it happens in the brain, the result is a strokewhere a portion of brain tissue dies. Atherosclerosis causes more deaths from heart diseasethan any other single condition. The most common cause of elevated serum cholesterol iseating foods that are rich in saturated fats or contain high levels of cholesterol.
Cholesterol has been divided into two major categories: low-density lipoprotein (LDL),
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the so-called “bad” cholesterol, and high-density lipoprotein (HDL), the so-called “good”cholesterol.
We studied the influence of mushroom additives to hypercholesterolic diets on thelevels of total cholesterol and high-density lipoproteins in rats. The obtained results aredemonstrating, that the addition of mushrooms to hypercholesterolic diets is significantlydecreasing the levels of LDL, leading to increase of HDL, and almost normalizing thecoefficient of atherogenity.
MM33RESISTANCE AND GENOTYPIC DIVERSITY OF
MULTIDRUG RESISTANT ACINETOBACTER BAUMANII INUNIVERSITY HOSPITAL
Vatcheva-Dobrevski*, Rossi, Savov* Encho, Bernards** Alexandra, van denBarselaar** M., van den Broek** Peterhans, Dijkshoorn** Lenie*Military Medical Academy, Dept. Clinical Microbiology, Sofia BULGARIA**Laiden University Medical Center, Dept.Medical Microbiology, Leiden,THENETHERLANDS
OBJECTIVE: To evaluate the frequency, antimicrobial resistance evolution andappearance of carbapenem-resistant Acinetobacter baumanii isolates. To investigategenotypic diversity during reccurrent epidemic episodes.
MATERIALS&METHODS: Between 2000-2004 a total of 326 multidrug resistant(MDR)A.baumanii isolates, investigated by Vitek 2 System(Bio Merrieux ,France) wereanalysed. The MICs of imipenem(Imp),meropenem(Mp), amikacin(Amk), cefoperasone/sulbactam(Cps),ciprofloxacin(Cl),ceftazidime(Caz),and metallo-beta lactamase productionwere determined by E-test(AB,Biodisk,Sweden). The genotype method was AFLP genomicfingerprinting.
RESULTS: The isolates were recovered from bronchial aspirates(42,6%), surgicalsite(18,3%),catheters(12,7%),bloodcultures(8,1%)in ICU.The susceptibility to antimicrobialsdecreased as follow from/to: ticarcillin 76%-59%,piperacillin 73-42%,Caz 52%-32,4%,Cp46%-23%,tobramycin 96%-81%.The most common MDR patterns for 2000-2002:isolateswere resistant to multiple antibiotics,but most were susceptible to Tob and colistin,allsusceptible to Imp and Mp.From 2003-2004 very common MDR patterns wereobserved:i.ImpR,MpR, CpsS(11%),ii.CpsS,TobS,AmkS ImpR,MpR(17%),iii.only ImpS,MpS(56%).The resistance to carbapenems increased from 0% to 16%.
CONCLUSIONS:The Acinetobacter baumanii nosocomial isolates during last five yearsin our hospital presents a high level antimicrobial resistance. Four different genotypeswere distinguished, one of which (type 1) was observed among 13 isolates indicating theprevalence of an epidemic strain.The emergence of MDR isolates require controlledcarbapenem prescription and infection control measures for prevention of untreatableinfections by Acinetobacter baumanii.
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MM34IMMUNOMODULATING AND ANTITUMOUR ACTION OF
HIGHER FUNGI
V. Spiridonova(a), P. Tsvetkov(a), A. Panchenko(b), A. Korchmaryova(a),N. Petrischev(b), M. Shamtsyan(a)
(a)St. Petersburg State Institute of Technology (Technical University), Russia, St.Petersburg, 198013, Moskovsky prospect, 26 E-mail: [email protected](b)St. Petersburg State I. P. Pavlov Medical University , Russia, St. Petersburg,197089, Leo Tolstoy str., 6/8
Aqueous extracts from fruit bodies and submerged mycelia of various higherBasidiomycetes were studied in search for reliable biological effects. In vitro and in vivoexperiments were conducted.
The results showed that the aqueous extracts demonstrated various types of markedbiological actions: an increased production of reactive oxygen forms by neutrophil cellsof human peripheral blood; a significant mitogenic activity in a wide range of concentrations;stimulation on production of inflammatory cytokines. Administered orally mixed with dailyfood they cause a decrease in average tumor size in mice with transplanted melanoma B16and Ehrlikh’s ascit carcinoma and a prolongation in the survival rate of such mice.
MM35ANTIOXIDANT PROPERTIES OF HIGHER MUSHROOMS
N. Dubyago,, I. Shugaley, E. Tozik. M. ShamtsyanSt. Petersburg State Technological Institute (Technical University), Moskovskyprospect, 26, St. Petersburg, Russia. E-mail: [email protected]
Ageing of an organism, influence of negative environmental factors, development ofmany pathological processes, are connected with formation of oxygen active forms insuperfluous quantities, that leads to infringements in functioning of living systems, so-called “free-radical pathologies”, to processes of the peroxide damages of proteins, lipids,DNA, carbohydrates.
One of the prospective ways to control related diseases as well as, for their prophylaxisis use of antioxidants. However, correction of each type of pathology by means ofantioxidants requires detailed studies of suggested preparations, determination of dozesand introduction methods. At the same time it must be considered, that supposedantioxidants are far from being universal and at different disorders of free-radical processes,their influence is not identical.
Food antioxidants possessing curative - prophylactic action are thought to be themost preferable and perspective ones. Substances isolated from higher basidiomycetesare also representing a perspective groups of natural antioxidants.
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Our studies show that fruit bodies, mycelia and cultural filtrates of variousbasidiomycetes possess pronounced antioxidant activity. For some of edible or non toxicbasidiomycetes SOD-like activity was also detected.
Antioxidants of mushroom origin can be used in various fields, such as pharmacy,food industry and cosmetics. Being added to common, daily food products they will alsocontribute in preservation of food from spoiling, and thus increasing food safety.
MM36ИНДИРЕКТЕН ИМУНОФЛУОРЕСЦЕНТЕН МЕТОД ЗАОТКРИВАНЕ НА HELICOBACTER PYLORI ДИРЕКТНО В
СТОМАШНИ БИОПСИИ
К. Иванова1, Цв. Илиева1, М. Марина1, И. Митов3, Б. Владимиров2, Й.Чурчев2
1Национален център по заразни и паразитни болести; 2Клиника погастроентерология, ДУБ“Царица Йоанна”, София; 3Медицински университет,София
Проучени са възможностите на индиректен имунофлуоресцентен метод /ИИФМ/, с използването на четири моноклонални антитела /собственопроизводство/, за откриване на
Helicobacter pylori директно в стомашни биопсии на хора.Изследвани са 95 стомашни биопсии, взети от пациенти с различни оплаквания
от страна на гастроинтестиналния тракт. Чувствителността и специфичносттана ИИФМ беше сравнена с културелния метод, директен микроскопски препарати бърз уреазен тест.
Чувствителността и специфичността на ИИФМ беше съответно 93% и 95% всравнение с другите методи, което го прави сравнително точен метод задиагностика на H.pylori.
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PRESENTATIONS OF FIRMS:
ZEU-INMUNOTEC
PCR-DIAPOPS METHOD FOR DETECTION OFLEGIONELLA IN WATER
Dr. Elena Domínguez- ZEU-INMUNOTEC, Maria de Luna 11, Zaragoza, Spain
Legionella is naturally found in environmental water sources. Hot water systems,cooling towers, and water distribution systems found in large buildings, such as hotelsand hospitals, are susceptible to legionella colonization. The presence of highconcentrations of Legionella in aerosols from contaminated waters, are associated withrespiratory diseases that can cause deaths. Since the first identification in 1977 numerouscases have been described in different countries causing loss of lifes and economicaldamages. Therefore, monitoring of susceptible water systems is of a paramount importance.
Traditionally, microbiological techniques have been used for Legionella identificationin water samples. However, these methodologies can be tedious and time consuming,taking over a week to generate results. ZEU-INMUNOTEC presents a molecular biologytest kit -Microline-Legionella- for screening of Legionella in water.
Microline-Legionella is based on a polymerase chain reaction (PCR) connected withan immunoenzymatic assay for colourimetric detection of the amplificated product(DIAPOPS). The test allows a qualitative detection of Legionella spp and/or Legionellapneumophila in water samples within 24 hours, allowing quick decisions in events ofoutbreak.
BIOSYSTEMS LTD., SOFIA, BULGARIA
REAL-TIME PCR IN MOLECULAR MICROBIOLOGY
Velichka Kardjeva, Biosystems Ltd., Sofia, Bulgaria
In the past ten years molecular biology techniques became a routine for identificationof pathogen and non pathogen viruses, bacteria and fungi. Widely applied technologiesare PCR, real-time PCR and sequencing. Genotyping and mutation analysis are bothpowerful tool for identification of any microorganism and discovery of resistant strains toantibiotics, different substances and physical factors.
Applied Biosystems provides the latest innovation in TaqMan assays for genotyping/ subtyping and quantitation of different microorganisms, which are applicablenot only togenomics studies but also in medicine for detection, identification and quantification ofpathogens, in ecology, taxonomy and etc. Real-Time PCR instruments from Applied
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Biosystems are well known in Bulgaria.Stay tuned with the latest developments in molecular genetics.Visit us at www.appliedbiosystems.com, or contact [email protected]
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GENERAL AND APPLIED MICROBIOLOGY
GAM1MYCOLOGICAL STUDIES IN CONTINENTAL
ANTARCTICA: AN OVERVIEW
Solveig TosiSezione di Micologia, Dipartimento di Ecologia del Territorio e degli AmbientiTerrestri, Universitа di Pavia, via S. Epifanio 14, 27100, Pavia, [email protected]
The geographic isolation and the relatively low anthropogenic impact are two of themain reasons why the study of the Antarctic microbiota involved a great interest since thebeginning of the XX century. The majority of papers dealing with mycoflora publisheduntil today were carried out in Victoria, Wilkes, Princess Elizabeth, McRobertson, andEnderby Lands, areas that can be considered biologically “rich” thanks to periodicallyunfrozen sites, presence of mosses and freshwater habitats, lichens, and animal remains.After more then one century of investigations it is possible to identify a continentalAntarctic mycoflora mainly composed of anamorphic fungi (filamentous and yeasts, 66%),some Zygomycetes, and very few species of Ascomycetes and Basidiomycetes. Incontinental Antarctica 1634 fungal or pseudofungal records, belonging to 146 genera and253 species and infraspecific taxa, were reported. They are present as psychrophiles,psychrotrophs, thermotolerants and thermophiles . These last two ecological groups werefound mainly in heated sites on active volcanoes. The Italian Program for AntarcticResearch (PNRA) has investigated on fungi since 1987 and several studies have beenconducted in the Victoria Land concerning the mycoflora of different substrates (mosses,lichens, soil, rocks, feathers, dung) as well as taxonomy, ecology, adaptations to extremeconditions, production of antimicrobial compounds, extracellular enzymes, and molecularbiology of the fungal isolates. Particular attention has been paid to the fungal species thatare supposed to be endemic for the Continental Antarctica such as those belonging to thecryptoendolithic community and the new taxa described from Antarctic sites. The mainresults of these researches are presented here.
GAM2MICROBIOLOGICAL CONTROL OF DETOXIFICATION
BIOREMEDIATION TECHNOLOGIES
Y. Topalova*, R. Dimkov*, C.V.Keer**, C. Y.Cheng***C.Y.Cheng***, M.Nunes****Biological Faculty, Sofia University, Dragan Tzankov str. 8,
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1164 Sofia, Bulgaria, Email: [email protected]**KAHO Sint-Lieven, KIHO, Dept. Biochemistry, Gebr. Desmetstraat 1, 9000Gent, Belgium*** University of Porto,Faculty of Engeneering, 4200-465,Porto, Portugal, Email:[email protected]
The microbiological indicators are important element of the control and managementof the bioremediation technologies. In the model conditions several bioremediationtechnologies are accomplished – aerobic, anaerobic, two-step anaerobic/aerobic, hybridtechnologies for the detoxification of xenobiotics of the group of penthachlorophenols(PCP). In parallel a microbiological pre-bioremediation research of the adaptive ponds ofLuck-Oil, Bourgas has been carried out. The sediments of these ponds are heavilycontaminated with crude oil and polycyclic aromatic compounds (PAHs). The specificdesign of microbiological control has been elaborated according to the type of technologyand features of the microbial communities.
The key taxonomical and physiological groups have been selected. The microscopicand electronic microscopic analyses have been applied for diagnostic of the structuralchanges of the biological systems in the course of the bioremediation technologies. Theobtained results from one hand connect the effectiveness of detoxification with quantitativemicrobiological indicators and biodiversity, but from the other – with the localization ofthe various processes occurring in bioremediation niches. As a third statement our resultsadditionally reveal new intermicrobial relationships on the base of microorganismsrelationships like co-metabolism, modulation and synthrophy.
GAM3DIVERSITY OF SYNECHOCOCCUS COMMUNITY
IN FRESHWATER LAKE GEORGE, NY
Dilnora E. GouliamovaRensselaer Polytechnic Institute, Department of Biology 110 8-th street Troy, NewYork 12180.e-mail: [email protected]
Members of the genus Synechococcus together with Prochlorococcus contributesignificantly to the primary production in aquatic ecosystems. In the present studyphylogenetic relationships of Synechococcus clones retrieved from Lake George andSynechococcus clones and strains isolated from marine and freshwater environmentswere studied. The results shown that Synechococcus clones from Lake George (USA),Lake Loosdrecht (The Netherlands), Lake Biwa (Japan) and Synechococcus strains fromLake Zurich (Switzerland) all group outside of the marine picoplankton clade and form aseparate freshwater clade. The clade is subdivided into two clusters. The first clusterincluded: the clones from Lake George (LGTI1, LGBH2, LHBH3, LGBH4, LBP1),Synechococcus rubescens and Synechococcus strain BO8807. The second cluster
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included: strains of Synechococcus PCC6307 and Synechococcus PCC7001 and clonesLGTI5, LGBH6, LD9. Sequence similarity between clones of the first cluster (LGTI1, LGBH2,LHBH3, LGBH4, LBP1) ranged from 97.9-99.4%; sequence similarity between clones of thesecond cluster (LGTI5, LGBH6, LD9) ranged from 99.7-99.9%. Sequence similarity betweenthe clones of the two clusters ranged from 96.6-97.5%. The differences in 16S rRNAsequences of 2.5-3.4% could correspond to ecologically significant physiological diversityas it was previously demonstrated for isolates of Prochlorococcus. Our results showexistence two genetically close freshwater populations of Synechococcus which are globallydistributed despite the geographic distance separating Lake George, Lake Loosdrecht,Lake Biva, and Lake Zurich.
GAM4MICROBIAL STATUS OF 7 THRACIAN VAULTS NEAR TO
KASANLAK, BULGARIA AND METHODS FORPREVENTATION
Tzvetanka Groudeva, Ana DoychevaSofia University ‘St. Kl. Ohridski”, Faculty of Biology, 8 Dragan Tzankov ,Department of General and Industrial Microbiology, Sofia, Bulgaria
As one of the most active deteriogens, microorganisms have a significant role indestruction of cultural monuments. They act in different ways, causing a variety ofdamages. The interactions between microorganisms (bacteria, actynomycetes and fungi)and monuments can be direct or indirect (production of different microbial metabolites).
The object of this research is the investigation of the microbial community of sevenThracian vaults near to Kasanlak in Bulgaria.
Determination of the prevalent physiological groups of microbial community as well astheir eventual role in the process of destruction was under investigation.
Eight physiological groups have been examined, considered that they are potentialbiodeteriogenes. Different taxonomical and biochemical tests have been carried out usingpure cultures, isolated from the predominant physiological groups. Classical taxonomywas used for investigation of the most important isolates.
The results obtained will be good basis for development of effective program forconservation of each vault.
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GAM5TAXONOMICAL IDENTIFICATION OF ARSENIC
RESISTANT AND ARSENIC TRANSFORMING SULFATE -REDUCING BACTERIA
Krasimira S. Krumova, Veneta I. GroudevaSofia University ‘St. Kl. Ohridski”, Faculty of Biology, 8 Dragan Tzankov ,Department of General and Industrial Microbiology, Lab. ”Geomicrobiology”,1164 Sofia, Bulgaria
Twenty eight bacterial strains, which are able to transform arsenic compounds, wereisolated and characterized in the laboratory of ”Geomicrobiology” at the Department of“General and Industrial Microbiology”, SU “St. Kliment Ohridski”. It was determined, that11 of these isolates are able to efficiently oxidize the high mobile and high toxic arsenite[As (III)] to less mobile and less toxic arsenate [As (V)]. The others 17 strains are able toreduce arsenate [As (V)] to arsenite [As (III)].
Оn the basis of the determination via classical methods, the isolated bacteria wererelated to different genera within the big group of sulphate-reducing bacteria.
The confirmation of the taxonomical belonging of these strains was done on basis ofthe DNA amplification with SRB specific primers for each genus.
GAM6LOW-TEMPERATURE BIODEGRADATION OF PHENOL
Rosa Margesin, Institute of Microbiology, Leopold Franzens University,TechcnikerstraЯe 25, 6020 Innsbruck, Austria
Phenol and phenolic compounds are widely distributed in nature and as environmentalpollutants. In cold climatic regions, wastewater temperature can decrease to 10°C andbelow. The activity of mesophilic degraders is limited at these temperatures. It was theobjective of this study to investigate the potential of cold-adapted bacteria and yeasts todegrade phenol at low temperatures. Psychrophilic and cold-tolerant bacterial and yeaststrains were isolated from various alpine habitats (soils, caves, glacier cryoconite) andwere characterized with regard to their growth temperature profile and their ability todegrade high amounts of phenol.
Using fed-batch cultivation in mineral medium with phenol as the sole carbon source,high amounts of phenol were degraded at 10°C. Bacterial strains were representatives ofthe genera Rhodococcus, Arthrobacter (including the novel species A. psychrophenolicus)and Pseudomonas; they utilized up to 12.5 mM phenol as the sole carbon source. Allyeast strains investigated were basidiomycota (Cryptococcus, Rhodosporidium,Rhodotorula, Mastigobasidium, Sporobolomyces, Trichosporon); they degraded up to15 mM phenol. Investigations on the toxicity of phenol and phenol-related monoaromatic
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compounds showed that Rhodotorula creatinivora strains were characterized by higerIC50 values than other species, while Sporobolomyces roseus was the most sensitivespecies. Yeasts were characterized by a substantially lower optimum temperature for growthand phenol biodegradation compared to the bacteria.
GAM7IN SITU BIOREMEDIATION OF SOIL POLLUTED
WITH CRUDE OIL AND HEAVY METALS
V.I. Groudeva 1, A.S. Doycheva 1 and S.N. Groudev2
1 – Department of General and Industrial Microbiology, Faculty of Biology,University of Sofia, Sofia Bulgaria2- Department of Engineering Geoecology, University of Mining and Geology,Sofia, Bulgaria
An experimental plot of soil contaminated with crude oil and heavy metals (cooper,zinc and cadmium) was subjected to in situ bioremediation using the activity of theindigenous soil microflora, which contained different oil-degrading and metal-solubilizingmicroorganisms. The oil was light, rich in paraffins and with a very low content ofasphaltene-resinous substances. The heavy metals were present mainly in formssusceptible to biological leaching.
The microbial activity was enhanced by suitable changes in the levels of some essentialenvironmental factors such as water, oxygen and nutrient content in the soil. This wasachieved by mixing the top soil horizon with solid biodegradable organic substrates (cowmanure, plant compost, straw), by applying irrigation with water solutions of organicsources of carbon and energy (lactate and acetate) and by adding zeolite saturated withammonium, phosphate and potassium ions. The soil was subjected to periodical flushingby means of the above-mentioned water solutions to remove the products from the oildegradation and dissolved heavy metals.
As results of such treatment the oil content in the soil was decreased from the initial 14g/kg dry soil to 1.70 g/kg within a period of 8 months. Simultaneously, the contents ofcopper, zinc and cadmium were decreased below the relevant permissible levels for soil ofsuch type.
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GAM8COMPARATIVE CHARACTERISTICS OF OIL-DEGRADING
ACTIVITY OF MICROORGANISMS WITH DIFFERENTTAXONOMIC STATUS.
Veneta Groudeva1, Iliana Ivanova 1, Ana Doycheva 1, Mihail Dumitru2, Anca-Rovena Vasculesku2
1 - Department of General and Industrial Microbiology,Faculty of Biology, University of Sofia, Sofia, Bulgaria2 - National Research and Development Institute for Soil Science,Agrochemistry and Environmental Protection, Bucharest,Romania
The decontamination of soil polluted with oil, petroleum products and petroleumresidues could be realising through different physical and chemicals and very expensivemethods, reasons for these are inaccessible to economic agents, which are responsible tosoils pollution. The alternative is in situ bioremediation because of their relative low costand not disturbing the natural soil structure in the process time. Among microorganisms,bacteria and fungi contain the enzymatic equipment necessary petroleum hydrocarbonsbreak down in soil. For applying of bioremediation techniques is necessary to know verywell the fate and the effects of pollutant compound on the ecosystems, how could beoptimise the biodegradation process and, mainly, finding some microorganisms with highdegrading abilities.
The main objective of this work is to establishing a efficient methodology for laboratorytesting of the capacities for oil degradation as well as to compare the degrading ability ofdifferent isolates from polluted soils with different taxonomic status.
GAM9COMPARATIVE CHARACTERISTICS OF TOXICITY
ASSESSMENT OF KEAVY METAL POLLUTED SOILS WITHDIFFERENT MICROORGANISMS
Iliana Ivanova, Damiana Dimova, Ana Stojanova,Veneta GroudevaSofia University “Snt.Kl.Ohridski”, Faculty of Biology, Dept. of Microbiology8 Dragan Tzankov, 1164 Sofia, BulgariaE-mail: [email protected]
Two samples from heavy metal polluted soils in West part of Bulgaria were investigated.Toxicity assessment was processed with two microorganisms: Bacillus cereus andPseudomonas putida. International standard with growth multiplication inhibition [ISO10712, 1994] was used. Two bacterial tests with Bacillus cereus - growth inhibition anddehydrogenase activity inhibition for toxicity evaluation of environmental samples were
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used. The 5-th hour of cultivation was the most suitable for measuring the growth inhibitionof Bacillus cereus. Dehydrogenase activity inhibition was measured at 6th h. This way theBacillus cereus tests are much shorter than international standard with Pseudomonasputida growth inhibition [ISO 10712, 1994], which duration is 10-16 h and had highersensitivity.
GAM10ИЗСЛЕДВАНИЯ ВЪРХУ ВЪЗМОЖНОСТИТЕ ЗА
ИНТЕНЗИФИКАЦИЯ ПОЛУЧАВАНЕТО НА БИОГАЗ ОТОТПАДЪЦИ В СЕЛСКОТО СТОПАНСТВО И
ХРАНИТЕЛНТА ПРОМИШЛЕНОСТ
Данка Гълъбова, Димитър Каракашев, Асен Мирков,Людмил Николов, Иван СимеоновИнститут по микробиология “Aкад. Ангел Стефанов”, БАН
Биодеградацията на органичната материя, образуваана вследствие човешкатапроизводителна дейност, има тристранен ефект: опазването на околната средаот много вредни змърсители, получаването на възобновяем енергиен източник(биогаз) и производстево на естествен органичен тор. В практично отношениетова е известен биотехнологичен процес с множество реализации в голям мащаб.Въпреки сравнителната нестабилност на метановата ферментация, пораждащасе от сложните взаимодействия на голям брой различни видове микроорганизми,интензификацията и стабилизацията на процеса могат да подобрят значителноикономическите покзатели на някои от приложенията й.
Целта на проведените изследвания в лабораторни биореактори с различнивидове субстрат (говежда тор, суроватка и спиртна шлемпа) е да се проследивлиянието на вида на органичните отпадъци и техни комбинации върху растежаи хидролитичната ензимна активност на анаеробнте бактериални култури.Изследвано е също влиянието на вещества стимулиращи микробния метаболизъмс оглед интензификацията на получаването на биогаз. Тези субстанции могат дабъдат определени в две основни групи: растежни фактори (дрождев екстрат,пептон) и повърностно активни вещества (тритон 100-Х и биосърфактант PS-17).С оглед намаляване степента на замърсеност на отпадните води от процеса сапроведени опити в каскада от два анаеробни биорактори.
Натрупан е ценен експериментален материал, който може да послужи заоснова за математическо моделиране на процеса след обработване нексперметалите даннни. Направени са важни изводи за практиката.
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GAM11MOLECULAR BIOMARKERS OF OXIDATIVE STRESS
IN FILAMENTOUS FUNGI
M. AngelovaThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences
Aerobic metabolism entails the production of reactive oxygen species (ROS), includingsuperoxide anion radical ( O2
-), H2O2 and the hydroxyl radical. These ROS cause oxidativedamage to important biomolecules, such as lipids, proteins, and DNA. Under normalconditions, ROS are cleared from the cell by the action of superoxide dismutase (SOD) andcatalase (CAT). Oxidative stress is imposed on cells as a result of increase in ROSgeneration, decrease in antioxidant protection, or failure to repair oxidative damage. Astate of oxidative stress can be induced by a number of environmental factors, includingchemical compounds, heavy metals, temperature treatment etc. The scientific validity ofthis hypothesis should be confirmed. Present study was designed to evaluate the effectsof paraquat, hydrogen peroxide, copper toxicity, as well as heat- and cold-stress treatmenton fungal cells by biomarkers, reflecting specific toxicity mechanism. Several oxidativestress biomarkers were validated: cyanide-resistant respiration; direct assessment of ROSproduction; oxidative damaged proteins; synthesis of reserve carbohydrates and changesin antioxidant enzyme defence (SOD and CAT). As a model microorganisms were usedfungal strains belonging to the genera Humicola, Aspergillus and Penicillium.
Our results indicate that exposure of fungal spores or mycelia to different stressorspromoted oxidative stress, as evidenced by stimulation of cyanide-resistant respiration,overproduction of ROS, accumulation of oxidative modified proteins, and acceleration oftrehalose and glycogen synthesis. Cell responses include enhanced expression of SODand catalase, however, the extent was different: treatment with PQ, Cu ions and temperatureincreased mainly SOD, whereas exogenous H2O2 led to enhanced CAT. We also foundthat glucose-6-phosphate dehydrogenase has a relevant role in the mechanism of protectionagainst superoxide and peroxide stresses.
GAM12NEW MITOCHONDRIAL CATALASE IN YEAST
SACCHAROMYCES CEREVISIAE
Ventsislava Petrova
In all eukaryotic cells, peroxisomes and mitochondria share a great variety of enzymaticreactions that are catalyzed by isoenzymes present in both organelles. As catalase andsuperoxide dismutase are essential enzymes in the decomposition of intracellular ROS(reactive oxygen species), their activities have been explored in the yeast Saccharomycescerevisiae during batchwise growth experiment. A mitochondrial fraction from three type
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strains of Saccharomyces cerevisiae has been isolated. Then the catalase, peroxidase,Mn and Cu/Zn superoxide dismutase activities were evaluated in the mitochondrial fraction.PAGE separations allowed identifying a new mitochondrial catalase which differed fromthe known catalase A and T in S. cerevisiae. A positive correlation between the activity ofmitochondrial catalase and Mn superoxide dismutase have been proved. To identify whichof the two catalase isoenzymes in yeast cell contributes to this mitochondrial activity aCta1p co-targeting was studied in a catalase A null mutant. A Cta1p–GFP (green fluorescentprotein)-fusion protein or a Cta1p derivative containing either a c-Myc epitope (Cta1pmyc)or a SKF-extended tag (Cta1pmyc-SKF) was constructed and their expression was followedup after growth of S. cerevisiae on different carbon sources. In the present study wedemonstrated that Cta1p can also enter mitochondria, although the enzyme lacks a classicalmitochondrial import sequence. Efficient Cta1p import into peroxisomes was observedwhen cells were cultivated under peroxisome-inducing conditions (i.e. growth on oleate),whereas significant co-import of Cta1p–GFP into mitochondria occurred when cells weregrown under respiratory conditions that favor oxygen stress and ROS accumulation withinthis organelle.
GAM13PROTEOMICS IN TARGET-SPECIFIC ANTIBACTERIAL
DRUG DISCOVERY BASED ON UMP KINASE
Neli Slavova-Azmanova, Cecile Evrin, Liliane Assairi, Hristo Najdenski, OctavianBвrzu and Anne-Marie Gilles
Bacterial uridine monophosphate (UMP) kinases are homohexamers whose primarystructure diverges from that of other nucleoside monophosphate kinases and fromeukaryotic UMP/CMP kinases. They are rather specific for UMP as substrate and areallosterically regulated by GTP (activator) and UTP (inhibitor). Being essential for bacterialcell survival, UMP kinases are appropriate candidates for designing new antimicrobialagents.
The aim of the present study was to identify common characteristics and/or differencesbetween UMP kinases from Gram-positive and Gram-negative bacteria regarding somepathogenic microorganisms. The pyrH genes encoding UMP kinase in S. pneumoniae andH. influenzae were cloned into pET expression vectors and overexpressed in E. coli. Therecombinant proteins were purified and studied with respect to kinetic characteristics,temperature and chemical stability. Despite several common properties including activationby GTP and inhibition by UTP, important differences between these enzymes werediscovered. It was found that activation by GTP in H. influenzae UMP kinase occurs bythe increase of Vm and K0.5, whereas in S. pneumoniae activation is due both to a decreasein K0.5 and to an increase in Vm. In the case of H. influenzae and E. coli the inhibition byUTP was strongly dependent on MgCl2 and ATP concentrations. The strongest activationof UMP kinase from H. influenzae was achieved by cGMP, whereas the S. pneumoniae
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enzyme was activated to the highest level by 3’-antraniloil-3’-dGTP (Ant-dGTP).UMP kinases from S. pneumoniae and H. influenzae are a reliable model for examination
of activity’s regulation. The initial characterization of the biochemical properties of theseenzymes is an important step towards target-specific drug design in the era of increasingmicrobial resistance.
GAM14HEAT SHOCK RESPONSE OF STREPTOCOCCUS
THERMOPHILUSINDUSTRIAL STRAINS
Penka M. Petrova, Dilnora E. Gouliamova and Galina D. StoyanchevaDepartment of Microbial Genetics, Institute of Microbiology, Bulgarian Academyof Sciences, 26, Acad. G. Bontchev Str, 1113 Sofia, Bulgaria, e-mail:[email protected]
Streptococcus thermophilus is the most frequently used in dairy industry speciesamong all lactic acid bacteria (LAB). It takes part not only in the production of yogurt, butalso in the cheese making (Mozzarella, Cheddar). During different stages of industrialfermentations S. thermophilus is subjected to the harsh heat treatment - up to 50-70ЪС.That is why heat resistant starters would be preferable in use under “industrial stress”conditions.
The response of S. thermophilus strain ST2980, containing 3.3 kb plasmid and itsplasmid free derivative (ST2980*) to heat-shock was studied. The percentage of survivedcells was determined by counting of colony forming units. Significant differences in survivalbetween strain variants were observed, especially when the cells were transferred from42°C directly to 62°C. After 1 hour only 1% of ST2980* cells were still alive, compared to50% of ST 2980. The prior incubation at 52°C (pre-shock) enables the plasmid-cured cellsto survive more efficiently. However, after 2 hours of exposure to the increased temperature,about 7% of ST 2980 cells were alive, compared to 0% of ST2980*. The analysis of growthcurves allows suggesting that the role of small hsp is the fast reaction to changes in theenvironment. It is possible that the 16.4 kDa Hsp works as a signal molecule, inducing theexpression of other (60 – 100 kDa) heat-shock proteins.
This work was financially supported by National Council for Scientific Research ofRepublic of Bulgaria. (Grant K 1307/2003).
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GAM15ВТОРА ГЕНЕРАЦИЯ ГЛЮКООЛИГОЗАХАРИДИ СПРЕБИОТИЧНИ СВОЙСТВА, СИНТЕЗИРАНИ
ПОСРЕДСТВОМ ГЛИКОЗИЛТРАНСФЕРАЗИ ОТ LEU-CONOSTOC MESENTEROIDES LM 286
И. Илиев1, Т. Василева1 и И. Иванова2
1- Катедра “Биохимия и микробиология”, Биологически факултет, ПловдивскиУниверситет, ул. “Цар Асен” № 24, стая 17, 4000 Пловдив, България,[email protected] Катедра “Обща и индустриална микробиология” Биологически факултет,Софийски Университет, бул. “Драган Цанков №8”, 1113 София, България
Неразградимите олигозахариди са обект на интензивни научни изследванияпоради приложението им като пребиотици. Потенциалът на олигозахаридите катопребиотици, представен като пребиотичен индекс, включва свойства катобифидогенен ефект, синтез на късоверижни мастни киселини, рН-устойчивост,имуностимулатор.
В представените изследвания се демонстрира възможността за ензимен синтезна глюкоолигозахариди от гликозилтрансферази, получени от мутантен щамLeuconostoc mesenteroides Lm 286. Изследваният мутант Lm 286 секретира дватипа гликозилтрансферази – декстранзахараза и алтернанзахараза и допълнителнолеванзахараза. Гликозилтрансферазите, предварително изолирани и частичнопречистени, синтезират олигозахариди с преобладаващи á-(1,3) и á-(1,4)гликозидни връзки. Синтезираните олигозахариди показаха висока степен нарезистентност при хидролиза с декстраназа и амилоглюкозидаза, съответно заглюкоолигозахаридите 47% и за изомалтоолигозахаридите 36%. Следдопълнително тестуване ин витро на пребиотичните свойства на синтезиранитеолигозахариди със силно разклонена верига се установи подчертан бифидогененефект, както и стимулиране на растежа на някои пробиотични щамове L.bulgaricus, проявяващи антибактериален ефект срещу E. coli и L. innocua иувеличена продукция на късоверижни мастни киселини.
GAM16ИЗОЛИРАНЕ И СВОЙСТВА НА ИЗВЪНКЛЕТЪЧНА
В-КСИЛОЗИДАЗА ПРОДУЦИРАНА ОТ ASPERGILLUS NIGER B03
Георги Добрев, Иван Пищийски, Лидия Колева
Изолирана е извънклетъчна â-ксилозидаза, продуцирана от Aspergillus niger
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B03. За целта е използвана колонна хроматография с Sephadex G 75 и Sephadex G100. Определени са оптималните рН и температура за действие на ензима,съответно рН 3.5 и t= 70 °C. Изследвани са рН и температурната стабилност напречистеният ензим. Определени са кинетичните параметри на изолирания ензимспрямо субстрат ñ-нитрофенил-â-D-ксилопиранозид, съответно Km= 0.35 µmol/ml иVmax= 3.03 µmol/(ml.min). Изследвано е влиянието на някои метални йони върхуактивността на изолираната â-ксилозидаза.
GAM17PECULIARITIES OF BIOFILM SYSTEM IMPLEMENTATION
IN MICROBIAL BIOTECHNOLOGIES
Ludmil N. NikolovBiological Faculty of Sofia University “St. Kl. Ohridski”
Implementation of the biofilms in microbial biotechnologies leads to rationalization ofthe industrial line structure, which reflects to serious economical advantages and makeseasier the maintenance of production systems. The efficiency of this implementation isbased on the manifold increase of the bioagent concentration in the bioreactor workingzone, rationalization of the separation processes and realization of the bioprocess in highperformance bioreactors.
In the presented study is shown that these attractive advantages have to be usedhaving in mind the properties and the structure of the biofilms as self-organizing livingsystems. Special attention is paid to the specificities of the biofilm applications like theirhigh sensitivity to the intensification of hydrodynamics in the vicinity to the biofilmsurface, the influence of the biofilm thickness on the bioprocess development as well asthe changes in biofilm properties during the bioprocess course. Experience is shared inthe use of this knowledge for development of suitable bioprocess systems both for biofilmstudies as self-organizing living systems in laboratory scale and realization of theirpotentialities in microbial biotechnologies in industry. On the concrete examples it isshown the increasing of the biofilm bioprocess systems productivity in comparison withthe suspended culture cultivation of the same microorganisms. Some considerations aboutthe applicability of the intensity working regimes in the high performance biofilm reactorslike two and three phase fluidization in the bioprocess systems with fixed films are discussed.
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GAM18EVOLUTION MODELING OF BACTERIAL OXIDATION OF
FERROUS IONS IN BIOFILM SYSTEM
L. Nikolov1, E. Petrova2 , V. Mamatarkova1, Kl. Mladenov2, St. Stoytchev2
1 Biological Faculty, Sofia University, “St. Kl. Ohridski”, 2 Institute of Mechanics, BAS
At recent time the biofilm systems are also studied using mathematical modeling,which is the subject of the present work. This research presents the development of theauthors previous experience in the problems of formulation and improvement of themathematical model of a biofilm system, formed by Acidithiobacillus ferrooxidans, basedon information about the subsystems and on verbal model of oxidation of ferrous ions inbiofilm reactors. The mathematical description of the bioprocess system consists of fiveordinary differential equations, at the following assumptions: oxidation proceeds throughhomogenous-heterogenity mechanism; all the processes proceed without diffusionlimitation; the kinetics of the sedimentation of the oxidated ferrous ions is of zero order;the economical coefficients of the biofilm and in the swimming cells are one and the same;as well as the maximal specific velocities of the biomass growth. Our investigations arerealized by evolution strategy, which consists in gradual complicating of the mathematicaldescription through adding some equations and members. The model sensitivity is testedwith respect to its parameters.
The numerical experiments show that the dimension of the identification task increasessignificantly during the model complication because of increasing the number of thekinetic constants. Here an attempt is made of decreasing the dimensions of the identificationtask using experimental data, obtained by homogeneity cultivation. The oxidizing dynamicsin fed–batch culture is investigated in parallel. The obtained results show that the usedsoftware is effective and helps to get a better understanding of the experimental results,which come from the starting phases of forming and functioning of biofilm systems atdifferent bioreactors.
GAM19POLYHYDROXYALKANOIC ACIDS (PHA) – INTERESTING
BACTERIAL POLYMERSAND ASPECTS OF THEIR PRODUCTION
Jцrg- Uwe Ackermann1, Gisela Mothes2
1University of Applied Sciences Dresden (HTW), Department of Chemical Engineer-ing, F.-List-Platz 1, D-01069 Dresden, Germany2Saxon Institute of Applied Biotechnology (SIAB), Permoserstr. 15, D-04318Leipzig, Germany
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PHAs (polyhydroxyalkanoid acids) are intracellular thermoplastic reserve polyestersoccurring in a wide variety of bacteria. These polymers became commercially attractivedue to their special physico-chemical properties together with biodegradability andbiocompatibility. Because PHA can be produced from renewable carbon sources they arealso interesting from the viewpoint of sustainability. Currently different applications,especially in the fields of pharmacy and medicine, are in the focus of interest. A widespectrum of polymer properties can be adjusted by the possibility to synthesize copolymersand, additionally, by the ability of post-biosynthetic modifications.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), (P(3HB/4HB)), is one of the mostsuitable absorbable materials for implantable medical applications, e.g. for the use asscaffolds. In contrast to the multitude of potential producers of polyhydroxybutyric acid(PHB) only a few bacteria are able to accumulate copolymers of P(3HB/4HB). Some resultsof production with Delftia acidovorans will be presented.
The overall process of PHA production comprises three stages, the synthesis of cells,the synthesis and accumulation of the polymer and the isolation of the product from thebiomass. The biosynthetic steps are commonly performed as batch or fed batch processes.However, continuous regimes of PHA production have several advantages and, moreover,a two-stage continuous process is more favourably than a single chemostat, especiallyfor the production of copolymers. For a better usability and understanding of the processwe developed mathematical models.
GAM20PRECONDITIONS FOR PRODUCING OF ORGANIC FOOD IN
MACEDONIA
A. Kuzelov; O. Kirovska CigulevskaMIK Sv. Nikole; Sveti Nikole, MacedoniaZavod za zdravstvena zastita – Skopje; Treta Makedonska Brigada 18, 1000Skopje, Macedonia; e-mail: [email protected]
Our purpose in this work will be to explain conditions and preconditions for producingorganic food in Macedonia. The main reason for that kind of farm – producing is topurchase helthier food without presence of food contaminants. It will be very complexprocess because we have to avoid pollutions from the air, water and soil to get healthyorganic food.
Our government is preceding a low for organic food producing and in that low will takeplace preparing, producing, marketing, labeling and inspection of organic food products.But, to get organic meat products, we have to purchase:
• healthy organic row material – meat from healthy animals,• organic way of animal farming,• marking of healthy animals,• using of organic food for cows feeding,
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• human way of transporting and sacrificed cows,• sanitary and veterinary inspection of animals (row materials)• using proposed additives for that kind of producing – organic meat products.
GAM21HETEROGENEITY OF L. PLANTARUM ISOLATES FROM
ARTISANAL WHITE BRINED CHEESE
R. Aleksandrova1, S. Dimitonova1, I. Ivanova2and S. Danova1
1- Dept of Microbial Genetics, Institute of Microbiology, Bulgarian Academy ofSciences, 26, Acad. G. Bontchev, str, 1113 Sofia, Bulgaria.2- Dept. of General and Applied Microbiology, Biological faculty, Sofia University,8, Dragan Tzankov, bvd, 1113 Sofia, Bulgaria
L. plantarum is ubiquitous, commonly isolated from foods of both animal and plantorigin, and is one of the main non-starter lactic acid bacteria (NSLAB) contributing to thefinal sensorial properties of different kind of cheeses. In this study 30 strains were isolatedfrom Bulgarian artisanal white brined cheese (WBAC) after a 2 months ripening period inbrine with 10% NaCl. A phenotypically homogenous group of 21 isolates were identifiedas L. plantarum according to polyphasic taxonomy. The repetitive PCR analyses allowedstudying the species and intra-species polymorphism within the phylogenetic group.With Rep PCR analysis we achieved a confirmation of correct species identification andalso a very good discrimination between closely related species L. plantarum, L. pentosusand L. paraplantarum. Obtained DNA polymorphic profiles using Rep, Eric and Boxprimers, revealed the heterogeneity of L. plantarum strains. The most discriminative andpromising for the strain typing was Box-PCR. The results present new information ongenetic diversity of NSLAB microflora of Bulgarian fermented milk products.
GAM22
СРАВНЕНИЕ НА ФЕРМЕНТАТИВНИЯ КАПАЦИТЕТ НА L.BULGARICS ПРИ КУЛТИВИРАНЕ НА ХРАНИТЕЛНИ
СРЕДИ С ОЛИГОЗАХАРИДИ
Ц. Игнатова1, И. Иванова2 и И. Илиев3
1- Катедра “Функционална биология”, Факултет “Природни науки”, ШуменскиУниверситет, ул. “Университетска №115”, 9712 Шумен, България2- Катедра “Обща и индустриална микробиология” Биологически факултет,Софийски Университет, бул. “Драган Цанков №8”, 1113 София, България3- Катедра “Биохимия и микробиология”, Биологически факултет, Пловдивски
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Университет, ул. “Цар Асен” № 24, стая 17, 4000 Пловдив, България,[email protected]
Неразградимите олигозахариди са обект на интензивни научни изследванияпоради приложението им в симбиотични продукти. Потенциалът наолигозахаридите като пребиотици, представен като пребиотичен индекс, включвасвойства като бифидогенен ефект, синтез на късоверижни мастни киселини, рН-устойчивост, имуностимулатор.
В представените изследвания се демонстрира възможността за ензимнадеградация на глюкоолигозахариди, галактоолигозахариди ифруктоолигозахариди от пробиотични щамове L. bulgaricus. Установена ещамова специфичност при ферментирането на тестуваните олигозахариди.Потенциалът на олигозахаридите да стимулират растежа на пробиотични щамовеL. bulgaricus е щамово детерминиран. Доказана е щамова специфичност и поотношение метаболизирането на олигозахаридите и продуцирането на органичникиселини и късоверижни мастни киселини. Доказан е индуциращ ефект натестуваните олигозахариди при секрецията на á-глюкозидаза, леванзахараза иâ-галактозидаза.
GAM23AMINO ACIDS
USE AND PRODUCTION - CURRENT STATUSAND PROSPECTS
Alexander RatkovInstitute of Microbiology, Bulgarian Academy of Sciences
As the building blocks of life, amino acids have long played an important role in bothhuman and animal nutrition and health maintenance. In the 1950s Corynebacteriumglutamicum was found to be a very efficient producer of L-glutamic acid. Since this timebiotechnological production processes have been used for industrial production of aminoacids. At present these processes are among the most important in terms of tonnage andeconomical value. Market development has been particularly dynamic for the flavor-enhancer glutamic acid and the animal feed amino acids L-lysine, L-threonine, and L-tryptophan. The growing market for amino acid led to significant improvements inbioprocess and downstream technology as well as in molecular biology. During the lastdecade big efforts were made to increase the productivity and to decrease the productioncost.
This article gives an overview of the world market for amino acids. Improvements inbioprocess technology, i.e. repeated fed batch and continuous production are summarized.Attempt and achievements in investigation and development of the technology for aminoacids production at the Institute of Microbiology, Bulgarian Academy of Sciences is
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summarized too.
GAM24MATHEMATICAL IDENTIFICATION
OF L-VALINE FED-BATCH FERMENTATION PROCESS
K. Todorov* I. Dimov*, Tz. Georgiev**, J. Kristeva*, V. Ivanova*, Al. Ratkov ** - Institute of Microbiology, Bulgarian Academy of Science** -Technical University, Faculty of Automatics,
The paper deals with identification problems of fermentation processes. Theidentification problem of biotechnological processes is threefold problems:(a) determinationof generalised stoichiometric reactions, (b) identification of the underlying reaction network,(c) identification of the kinetics-structure and parameters of reaction rates. This stages arediscussed based on experimental results from amino acids production. In practical case,biotechnological process for L-valine production by fermentation is used for elucidationof approach for solving the identification problem. The approach in this article includedthe following steps:(a) chose a set of generalised stoichiometric reactions, (b) chosenreactions are validated by extended autoregressive models (ARX). The adequacy of thismodels proved equations within reactions, (c) structural and parametric identification ofthe specific rates for discussed process using linear and nonlinear regression, (d)optimisation of unstructured dynamic model of L-valine production using constrainedoptimisation procedure. The adequacy of the proposed modes is proved throughsimulation researches.
GAM25CARCINOGEN-INDUCED TRANSPOSITION OF SACCHARO-
MYCES CEREVISIAE TY1 TRANSPOSON DEPENDS ONMITOCHONDRIAL FUNCTION
T. Stoycheva, P. Venkov, Ts. TsvetkovInstitute of Cryobiology and Food Technology, Department of Molecular Ecology,53A”Cherny vrah”Blvd., Sofia, Bulgaria, [email protected]
Ty1 is a Saccharomyces cerevisiae retrotransposon with life cycle and structure verysimilar to the known oncoviruses. In previous studies was found that Ty1 transpositionto new places in the genome is increased by carcinogens but not by mutagens that are notcarcinogenic (Pesheva et al., 2005). The molecular mechanisms underlying the specificresponse of Ty1 transposition to carcinogens are unknown. Recently, data accumulatingin the literature evidenced that carcinogens increased the cellular level of reactive oxygen
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species (ROS). Since the mitochondrial oxidative phosphorylation is the main source forROS production, we studied the role of mitochondria in the carcinogen-induced Ty1transposition. We first found that carcinogen-induced Ty1 transposition does not occurin S.cerevisiae cells deprived of mitochondria, indicating the role of mitochondria in theprocess. Next, we disrupted SCO1 – a nuclear gene involved in mitochondrial oxidativephosphorylation, and found that such cells do not respond to carcinogen treatment withenhanced Ty1 transposition. This result evidenced that oxidative phosphorylation, butnot another mitochondrial function, is involved into carcinogen-induced Ty1 transposition.In order to study in details the role of oxidative phosphorylation we treated cells with twoinhibitors: 1) antimycin which increases ROS production by inhibiting the electron transferalong the respiratory chain and 2) CCCP inhibiting mitochondrial ATP synthesis withouthaving effect on ROS formation. The results obtained showed that carcinogen-inducedTy1 transposition increases after treatment with antimycin while CCCP has no effect onTy1 transposition.
Our results indicate the key role of cellular ROS level in the carcinogen-inducedTy1 transposition. The significance of the results obtained will be discussed in the light ofthe cellular response to carcinogens.
GAM26PHENOL HYDROXYLASE DEPENDANCE ON THE
VARIOUSE HYDROXY PHENOLS UTILYZED AS SUB-STRATES BY TRICHOSPORON CUTANEUM R57
Z. Alexieva, M. Gerginova, B. AtanasovThe Stephan Angeloff Institute of Microbiology, BAS
The ability of the mono hydroxylated aromatics compounds to affect the level ofintracellular FAD-dependent phenol 2-monooxigenase (phenol hydroxylase, EC 1.14.13.7),catalyzing the initial step of phenol mineralization in Trichosporon cutaneum strain R57was studied. When Trichosporon cutaneum was grown on 1 g/l phenol, catechol, resorcinolor hydroquinone as a sole carbon source in the medium, the highest level of specificactivity of the investigated enzyme was attend with hydroquinone (2 U/mg protein). Theintracellular specific activities of phenol hydroxylase in grown on phenol (0.5 g.l-1) cells ofTrichosporon cutaneum R57 strain with different mono-hydroxyl phenol derivatives(catechol, resorcinol and hydroquinone) used as substrates in the reaction mixture fordetermination of the enzyme activity were measured, too. The data obtained showed thatthe investigated enzyme was capable of hydroxylating all applied aromatic substrates.The level of specific phenol hydroxylase activity in experiments with hydroquinone (1 U/mg protein) was equal to the data measured with phenol as a substrate in the sameconditions. The enzyme capacity to oxidize catechol or resorcinol in these experimentswas significantly lower (50 – 60 %). All enzyme activities were determined in Triton X100permeabilized cells. The comparison of all results obtained in both sets of experiments
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showed that investigated aromatics compounds are easily utilizable and strong inducersof phenol hydroxylase. Nevertheless, the better expression of the enzyme activity in cellsinitially grown on medium comprising each one of tested hydroxylated phenols wasobserved.
This work was supported by of the NCSR of the Bulgarian Ministry of Education andScience under project N K 1205/02.
GAM27CREATING OLIGONUCLEOTIDE PRIMERS FOR PCR
ANALYSIS AND PHYA GENE SEQUENCING IN TRICHOS-PORON CUTANEUM R57 STRAIN
Y. Manasiev, T. Primov, M. Gerginova, N. Peneva, Z. AlexievaThe Stephan Angeloff Institute of Microbiology, BAS
The methods for discovering new catabolic genes, as well as genes involved in thebioaugmentation process of the environment demonstrate the advantages of unique andeasily identified molecular markers for the investigation of natural microbial populations.The metabolism of aromatic compounds, phenol and its derivatives especially, is beinginvestigated very intensely in prokaryotic microorganisms The available in the scientificliterature data concerning catabolic genes in yeast species, in particular concerning genescoding for enzymes of the phenol degrading pathway is quite insufficient. The aim of thiswork is to contribute to more exact and complete information about the diversity andspecificity of yeast genes, as a specific response to the growing environmental pollutionwith toxic aromatic compounds.
Based on the sequence of the gene encoding phenol hydroxilase synthesis in T.cutaneum ATCC 46490 strain (TORPHD, GI = 170524, NCBI), the oligonucleotide primersfor phenol degrading yeast were designed using Primer 3 algorithm. Three pairs of primerswere used in accomplished PCR-reactions. The DNA fragments obtained were sequenced.With a purpose to create primers with a maximum specificity to the products obtained bythe first set of primers we also designed a set of degenerative primers. The initial sequenceTORPHD and PCR products sequences were aligned by using ClastalW algorithm. Twopairs of primers were selected on the basis of homology (not less then 80%) with TORPHDsequence.
This work was supported by of the NCSR of the Bulgarian Ministry of Education andScience under project N MU-K 1402/04.
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GAM28DOT-BLOT ANALYSIS BY BIOTIN LABELED PROBE FOR
IDENTIFYING PHYA GENE IN MICROBIAL STRAINSM. Gerginova, Y. Manasiev, P. Petrova, A. Krastanov*, Z. AlexievaThe Stephan Angeloff Institute of Microbiology, BAS,*University of food technologies, Plovdiv
Phenol and its various derivatives, as well as many other aromatic compounds, areknown as hazardous pollutants. Various phenol-degrading prokaryotic microorganismshave been extensively studied but only some members of yeast genera that can metabolizephenolic compounds as a sole carbon and energy source are described in the literature.
The utilization of molecular techniques can be useful not only to identify species, butto discover new genes involved in catabolism of aromatics for the purpose to innovateand improve the technological processes of biodegradation.
In an attempt to estimate the occurrence of phenol hydroxylase – related gene sequencesin different microbial strains we performed a Dot-blot analysis with DNA from phenolutilizing eukaryotes. The used oligonucletide was homologous to the 5’ end of phyA genein Trichosporon cutaneum ATCC 46490. As a negative controls were used incapable todegrade phenol microbial strain – Lactobacillus acidophilus 4356.
The results of this investigation showed that both strains Trichosporon cutaneumR57 as well as Trametes versicolor 1 may carry phyA genes of the high degree of similarityto the phyA gene from Trichosporon cutaneum ATCC 46490. The Lactobacillusacidophilus 4356 strain’s DNA used as negative control in the experiments did not revealany sequence similarity to the phyA gene under the conditions tested.
This work was supported by of the NCSR of the Bulgarian Ministry of Education andScience under project N MU-K 1402/04.
GAM29INFLUENCE OF TOXIC PHENOLIC COMPOUNDS ON THEPHENOL HYDROXILASE ACTIVITY IN TRICHOSPORON
CUTANEUMM. Gerginova, Y. Manasiev, N. Shivarova, Z. AlexievaThe Stephan Angeloff Institute of Microbiology, BAS
The phenol-degrading strain Trichosporon cutaneum R57 utilized various aromaticand aliphatic compounds as a sole carbon and energy sours. The intracellular specificactivities of phenol hydroxylase [EC 1.14.13.7] in grown on phenol (0.5 g.l-1) andpermeabilized cells of Trichosporon cutaneum R57 strain are measured. Different toxicphenol derivatives (cresols, nitro-phenols and Cl-phenols) were used as substrates in thereaction mixture for determination of the enzyme activity. The data obtained showed thatthe investigated enzyme was capable of hydroxylating all applied aromatic substrates. It
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should be point that some of them (o-cresol, p-nitro-phenol) are non-growth substratesbut others have different maximal growth concentrations. The measured specific activityof phenol hydroxylase with phenol as substrate was 1.0 Umg-1 protein. The analyses ofdata from experiments with o-, m- and p- cresols showed high degree of similarity to thedata obtained in experiments with phenol. The same effect could be observed in experimentsdone with o-, m- and p-Cl-phenols as well as with o-nitro-phenol. The established specificenzyme activities with both m- and p- nitro- phenols were rather lower (0.6 Umg-1 protein)than described above. All results demonstrated in this work confirmed the wide enzymespecificity of phenol hydroxylase in Trichosporon cutaneum R57 cells. Otherwise weshould suppose the action of more than one hydroxylating enzyme towards differentphenols. The enzyme activity obtained in the experiments with non-growth substratesindicated the existence of different cause for cell’s inability to assimilate them.
This work was supported by of the NCSR of the Bulgarian Ministry of Education andScience under project N K 1205/02.
GAM30MICROFLORA IN A NATURAL WETLAND USED FPR
TREATMENT OF ACID MINE DRAINAGE
V.I. Groudeva 1, S.G. Bratkova 2 and S.N. Groudev 21 – Department of General and Industrial Microbiology, Faculty ofBiology,University of Sofia, Sofia Bulgaria2- Department of Engineering Geoecology,University of Mining and Geology, Sofia,Bulgaria
Acid mine drainage waters generated in a polymetallic ore deposit and polluted withradionuclides, heavy metals and arsenic were treated by means of a natural wetland locatedin the deposit. The wetland was characterized by abundant water and emergent vegetationand a diverse microflora. The water flow rate through the wetland varied in the range ofabout 5-20 m3 / 24 h.
An efficient removal of pollutants was achieved within the wetland during the differentclimate seasons, even during the cold winter months at temperature close to the waterfreezing point. The removal of pollutants was due to different mechanisms but the microbialdissimilatory sulphate reduction and biosorption played the main role. The microflora inthe wetland was characterized by a rich variety of sulphate-reducing bacteria and othermetabolically independent microorganisms. As a result of their activity the heavy metalsand arsenic were precipitated mainly as the relevant insoluble sulphides and uranium wasprecipitated manly as uraninite (UO2). The wetland effluents contained no pollutants inconcentrations higher than the relevant permissible levels for waters intended for use inthe agriculture and/or industry.
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GAM31БИОДЕГРАДАЦИЯ НА СМЕСЕНИ ФЕНОЛНИСЪЕДИНЕНИЯ С МИКРОБНА АСОЦИАЦИЯ ОТ
ASPERGILLUS AWAMORI И THERMOASCUSAURANTIACUS
И. Стоилова, А. Кръстанов, Х. Буи, В. СтанчевУниверситет по хранителни технологии, Пловдив
Изследвана е биодеградацията на четири смеси от фенолни съединения катовъглеродни източници с микробна асоциация от Aspergillus awamori и Thermoascusaurantiacus: фенол + 2,6-диметоксифенол, фенол + 2,4-дихлорфенол, фенол +катехол, фенол + дифениламин. Общата концентрация на фенолните съединениявъв всяка смес е 0,4%: 0,2% за фенола и 0,2% за втория фенолен дериват. Насреда съдържаща фенол + 2,6-диметоксифенол се установи положителновзаимодействие между 2-те култури, проявяващо се в увеличен брой конидии всмесената култура превишаващ броя на конидиите в монокултурите, синергичнапродукция на лакказа в смесената среда и 1,7 пъти по-висока биодеградация нафенолната смес в сравнение с тази в монокултурата T. aurantiacus и 2,48 пътиповече от A. awamori. На среда съдържаща фенол + 2,4-дихлорфенол двата видапоказаха неутрален тип на взаимодействие, на останалите среди двете микробнипопулации проявиха конкурентни взаимодействия.
Постигната е 64% деградация на 4,0 g/l смес от фенол и 2,6-диметоксифенол смикробна асоциация от A. awamori и T. aurantiacus
GAM32BENZONITRILE AND 4-CYANOPIRIDINE DEGRADATIONBY IMMOBILIZED CELLS OF BACILLUS SP. UG-5B IN A
COLUMN BIOREACTORL. Kabaivanova1, E. Dobreva1, E. Emanuilova1,B. Samuneva2, G. Chernev2
1 Institute of Microbiology, BAS- Sofia, Bulgaria2 University of Chemical Technology and Metallurgy- Sofia, Bulgaria
Nitrile compounds are one of the most dangerous pollutants of the environment releasedby different industrial processes. The aim of the present investigation is to carry out abiodegradation process of benzonitrile and 4-cyanopiridine by immobilized cells of Bacillussp. UG-5B with thermostable nitrilase activity. Cell suspension with concentration of 30mg/ml cells and nitrilase activity of 2.25 U/ml was used in the immobilization procedures.The matrix was synthesized by the sol-gel method at room temperature and 5mol% of theinorganic precursor (tetramethylortosilicate) was replaced by polyacrylamide gel.
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The structure of obtained hybrid nanocomposites has been studied by IR Spectroscopy,XRD, BET, SEM, AFM and Roughness Analysis. The results proved that all samples areamorphous having pores with average diameter about 1 – 1.6 nm. A self- organizednanostructure have been observed by AFM. From the AFM images were calculated RMSroughness and average height of the particles and aggregate on the surface. Afterentrapment of the whole bacterial cells in the hybrid matrix, the obtained biocatalyst wasapplied in a continuous degradation process in a column bioreactor at 55°C. This factcould make possible the treatment of hot waste waters, containing nitriles. The processwas followed at three different flow rates of the substrate solution- 0.5 ml.min-1; 0.75ml.min-1 and 1.0 ml.min-1. The concentration of benzonitrile and 4-cyanopyridine in thephosphate buffer with pH=7.2 was 20 mM. Degradation of benzonitrile-16.07 mM and 4-cyanopiridine-38.5 mM was achieved at the optimal flow rate of 0.75ml.min-1 for five hours.
GAM33BIOLOGICAL PROPERTIES OF BIOSURFACTANT-COM-
PLEX FROM PSEUDOMONAS SP. PS-17
A. Sotirova, D. Spasova, E. Vasileva-Tonkova, D. GalabovaBulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology,Acad. G. Bonchev str., bl.26, 1113 Sofia, Bulgaria
Although rhamnolipids have been studied for 50 years little is known about theirinteraction with bacterial cells and their potential for use in biomedicine. The biosurfactantisolated from bacterial strain Pseudomonas sp. PS-17 contains an unique natural complexof a biosurfactant and a biopolymer-alginate with high surface and emulsifying activities.The low parameters for their surface and interfacial tensions as well as their criticalconcentrations for micelle formation (CMC), indicate high surface activity. For successfulapplication of biosurfactants in biomedicine their effect on the microbial surface needs tobe known.
Our research investigates antimicrobial properties of biosurfactant complex and itseffects on bacterial membrane of Gram positive bacteria, using Bacillus subtilis as a modelsystem. The product showed excellent antimicrobial properties. Antimicrobial activitywas evaluated according to the minimum inhibitory concentration (MIC), the lowestconcentration of an antimicrobial agent that inhibits development of visible microbialgrowth. Antimicrobial activity of biosurfactant-complex against Bacillus subtilis wasobserved (55 µg/mL),
Our results demonstrated that the exposure of B. subtilis cells to non-lethalconcentration (50 µg/mL) of biosurfactant-complex did not affect the protein componentof bacterial membrane but caused quantitatively changes in phospholipid headgroup.This changes lead to a higher proportion of negatively charged phospholipids.Ultrastructural studies revealed that biosurfactant-complex effects was directed not onlyon cell surface structures and on inner cell structures of bacterial cells.The results suggest
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that the biosurfactant-complex could be used in designing new more effective antimicrobialpreparations.
This work was financially supported by of the NCSR of the Bulgarian Ministry ofEducation and Science under project K 1206/02.
GAM34DECOLORIZATION OF THE ACID ORANGE 7 BY RESTING
ALCALIGENES FAECALIS AND RHODOCOCCUSERYTHROPOLIS CELLS: A COMPARATIVE STUDY
T. Avramova, L. Stefanova, B. Angelova and S. MutafovThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad G. Bonchev St., 26, 1113 Sofia, Bulgaria, e-mail: [email protected]
The presence of azo group in synthetic dyes renders these compounds resistance tobiological attack. Presented study aims at elucidation the role of cell surface in the microbialdecolorization of the Acid Orange 7 (AO7) carried out by resting Alcaligenes faecalis6132 and Rhodococcus erythropolis 24 cells. The effect of some surfactants on the dynamicsof the decolorization process was investigated and a preliminary evaluation of the energyof activation of the decolorization reaction was presented.
The process of the microbial decolorization carried out by resting cells of the twostrains started with an immediate adsorption of the AO7 on the cell surface and proceededaccording to the model of the first order decay typical for processes where surfacephenomena like adsorption took place. The degradation constants calculated were ë= 0.152 h -1 and ë = 0 .191 h -1 for A. faecalis and R. erythropolis, respectively.
The presence of cationic and anionic surfactants influenced the rate of thedecolorization process. In concentration above the critical micelle concentration (CMC),the anionic surfactant sodium N-laurosyl-sarcosine (SLS) inhibited the reaction ofdecolorization. The cationic surfactant hexadecyltrimetylammonium bromide (HTAB) inconcentrations above and below CMC accelerated the binding of the AO7 by the cellscausing a rapid staining of the biomass and complete decolorization of the reaction medium.In this, the colour of the A. faecalis cells faded faster compared to the decolorization of theR. erythropolis cells, which was most probably a result from the surface peculiarities ofthe R. erythropolis.
This study was supported by the Foundation for Scientific Investigations, Ministry ofEducation, Science and Technology, Grant B1311/03
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GAM35PH – RELATED EQUILIBRIUM STUDY ON COPPER
BIOSORPTION BY PENICILLIUM CYCLOPIUM
Maria Ianisa , Kolishka Tsekovaa, Dessislava Todorovaa and Pencho Marinovb
a Department of Microbial Ecology, Institute of Microbiology, Bulgarian Academyof Sciences,b Central Laboratory for Parallel Processing, Bulgarian Academy of Sciences
PH – related equilibrium and modeling study on copper biosorption in single metalsystems by resting cells of Penicillium cyclopium was performed. The equilibrium of theprocess (at different pH values and biomass concentrations) is well described by theclassical Langmuir sorption isotherm and their modifications. It seems that the processwas a chemical, equilibrated and saturable mechanism which reflected the predominantlysite – specific mechanism on the cell surface. The curves of Scatchard transformationplots obtained are nonlinear, indicating presence of multiple nonequivalent metal bindingsites on the fungal biomass. The maximum copper uptake was calculated to be 49,84 mg/gdry cells, when pH of the solution and biosorbent concentration were 4,5 and 1 g/l resp.
GAM36IMMOBILIZATION OF PENICILLIUM CYCLOPIUM CELLS
IN PVA HYDROGELS FOR HEAVY METAL IONSBIOSORPTION APPLICATIONS
Darinka Christova*1, Kolishka Tsekova2, Sijka Ivanova1, Maria Ianis2, SoniaGaneva3
1 Institute of Polymers – Bulgarian Academy of Sciences, 1113 Sofia, Bulgariaphone: +359 2 979 22 85; e-mail: [email protected] Institute of Microbiology – Bulgarian Academy of Sciences, 1113 Sofia, Bulgariaphone: +359 2 979 31 73; e-mail: [email protected] Faculty of Chemistry – Sofia University, 1164 Sofia, Bulgariaphone: +359 2 8161277; e-mail: [email protected]
The purpose of this work was to develop hybrid hydrogels by entrapping P. cyclopiumcells into poly(vinyl alcohol) (PVA) network and to characterize the ability of the systemfor Cu2+, Co2+ and Fe3+ ions uptake from aqueous solutions. The advantage of the createdsystem is in the proper combination of an organic polymer with metal binding functionalgroups and fungal cells containing effective metal binding groups on the wall surface.
Hybrid hydrogels of P. cyclopium cells immobilized in hydrophilic polymer networkhave been obtained in situ by crosslinking the PVA aqueous solutions with glutaraldehydewhen dispersing dried powdered biomass in the media. The influence of the reaction
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conditions as well as the components’ ration on the water content of the hydrogels hasbeen investigated. Metal binding abilities of the hybrid hydrogels for Cu2+, Co2+ and Fe3+
ions were determined by using atom absorption spectroscopy. The performance of theimmobilized biosorbent was evaluated by sorption kinetics, sorption capacities for differentmetal ions as well as mechanical stability in a range of pH.
GAM37BIOSORPTION OF BINARY MIXTURES
OF COPPER AND COBALT BY PENICILLIUMBREVICOMPACTUM
Kolishka Tsekova1, Maria Ianis1, Vera Dencheva1and Sonya Ganeva2
1Department of Microbial Ecology, Institute of Microbiology, Bulgarian Academyof Sciencesphone: +359 2 979 3173; e-mail [email protected] of Chemistry, Sofia Universityphone: +359 2 8161277; e-mail [email protected]
This work reports on a study of the biosorption of copper and cobalt, both singly andin combination (in equimolar concentrations), by the resting cells of Penicilliumbrevicompactum. Equilibrium batch sorption studies were carried out at 300C and pH 5,0,for a contact time of 1 hour to guarantee that equilibrium was reached. The equilibriumdata were analyzed using the Langmuir and Freundlich isotherms. The adsorption ofbinary mixtures of heavy metals solution on the fungal biomass was found to be ofcompetitive type where the adsorption capacity for any single metal decreased in thepresence of the others. The cobalt ions showed a greater affinity for Penicilliumbrevicompactum than the copper ions.
Financial support: The Grant B 1407/2004 allocated by the National Fond for ScientificResearch to the Bulgarian Ministry of Education and Science supported this work.
GAM38SENSITIVITY OF SACCHAROMYCES CEREVISIAE YEAST
TO ARSENATETatina Todorova1, 2, Stephane Vuilleumier2, Anna Kujumdzieva1
1Sofia University, Department of General and Applied Microbiology, 8 DraganTzankov, 1164, Sofia, Bulgaria2Universite Louis Pasteur, UMR 7156 Genetique moleculaire, Genomique etMicrobiologie, 28 rue Goethe, F-67083 Strasbourg Cedex, France
Arsenic is a toxic metalloid present in natural and polluted industrial environments. It
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is a human carcinogen but is also used in treatment of acute promyelocytic leukemia andprotozoan parasitic diseases. When mammals are exposed to arsenate, it is reduced toarsenite either by the PNP arsenate reductase or MMA(V) reductase, a member of omegaclass of glutathione S-transferases (GST). The later enzyme catalyzes the conjugation ofthe electrophilic toxic compounds with the –SH group of glutathione (GSH), thereforeplaying a critical role in xenobiotic elimination. Based on sequence, substrate specificity,structure and immunological properties, GSTs have been grouped into eight distinctfamilies. In contrast with mammals, plants and even bacteria, little is known about GSTs ofyeasts and fungi but they seem to be especially diverse both structurally and functionally.
In this study we have taken a systematic genetic approach to study the potential roleof GSTs in arsenate toxicity in Saccharomyces cerevisiae. A search in SaccharomycesGenome Database reveals the presence of 11 genes and ORFs with homology to GST,which single disruption mutants were tested for their sensitivity to arsenate. The mutant,disrupted for the gene TEF4, coding for translation elongation factor EF1ã (GSThomologue) was studied. A hypersensitivity of this mutant to AsV has been found,indicating a possible participation of Tef4 protein in arsenate detoxification.
GAM39ТРАНСПОРТ НА АРСЕНАТ В ДРОЖДЕВИ КЛЕТКИ
Стефан Иванов ШилевКатедра „Микробиология и екологични биотехнологии”Аграрен Университет – Пловдив, бул. Менделеев 12, Пловдив 4000, Е-mail:[email protected]
Известно е, че в голяма степен почвеното плодородие и разграждането наразлични замърсителите се дължи на микробиалната дейност. Понастоящем,замърсяването с метали и металоиди е един от най-важните екологични проблеми,като за неговото разрешаване все по-голямо внимание се обръща набиотехнологичните подходи. Нови изследвания показаха, че някои щамоведрожди изолирани от почвата притежават висока толерантност към наличие наметали в хранителната среда.
Целта на настоящето изследване е охарактеризиране на транспорта на арсенатв дрождевите клетки, като причина за толерантността към металоидния йон.
Резултатите от изследванията показаха, че в замърсените с арсен почви сесъдържат дрожди с висока толерантност към изследвания металоид.Толерантността към наличие на арсен в хранителната среда (Na2HAsO4, 50, 100, 500mg l-1) е обусловена от системите за транспорт в микробната клетка. Порадиструктурното сходство между арсенатния и фосфатния йони, съществениконцентрации As постъпиха в клетките. След приемане на вътреклетъчниконцентрации близки до критичните, концентрацията на металоида намалявазначително благодарение на задействане на механизмите за извеждане на
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токсичния йон извън клетката, както и на вътреклетъчното му изолиране.В заключение, толерантността към As при различни видове дрожди е
обусловена от способността на клетките да намаляват вътреклетъчнатаконцентрация на свободните арсенатни йони.
GAM40ISOLATION, IDENTIFICATION AND SELECTION OF AR-
SENIC AND CADMIUM RESISTANT YEAST
Nikolay Krumov1, Velitchka Gotcheva1, Tsonka Hristozova2, Clemens Posten3,Angel Angelov1
1Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd,4002 Plovdiv, Bulgaria;2The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,26 Maritza Blvd, 4002 Plovdiv, Bulgaria;3Department of Bioprocess Engineering, MVM-Institute, University of Karlsruhe,Kaiserstr. 12, D-76131 Karlsruhe, Germany
In the present scientific work, yeast strains capable of growing in the presence of highconcentrations of arsenic (As) and cadmium (Cd) were isolated and identified, and strainsshowing highest As- and Cd-resistance were selected. For the purpose, yeast strains fromthe collection of the Institute of Microbiology – Bulgarian Academy of Science (IM-BAS)and strains isolated from nature were first verified. 144 morphologically different isolateswere obtained from soil samples taken from the surroundings of Umicor Med – Pirdop andNon-Ferrous Metal Smelter (KCM SA) - Plovdiv. The majority of them were fungi – 73 andactinomycetes – 43, not being an object of the present research. Based on morphologicaland cultural characterization, 28 pure yeast cultures were isolated. Within this group, 3strains Schizosaccharomyces pombe and 1 strain Saccharomyces cerevisiae were identifiedby physiology-biochemical methods. Seven strains showed resistance in concentrationsof arsenic of 50 mmol: Saccharomyces cerevisiae 0505 and 0533, Saccharomycesellipsoideus 0112 and 0010, Rhodotorula rubra 3032, Torulopsis alba 2962 and strain S.pombe NK05/2 isolated from KCM surroundings. These strains will be further used inresearch on the production of cadmium-sulfide nanobioparticles and in exploring the cellresponse in extreme conditions and the activation of cell’s self-defense enzyme system.
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GAM41CHARACTERISATION AND IDENTIFICATION
OF BACTERIAL COMMUNITY ISOLATED FROMMETALWORKING FLUIDS
Stela Bakalova, Petia Hristova, Raycho Dimkov, Veneta Groudeva
Metalworking fluids (MWFs) are widely spread and used for cooling and lubricatingduring the machining process. They typically are formulated to include chemicals thatinhibit metal corrosion and microbial activity (biocides), whilst lubricating and cooling themetal cutting process. It can be an extreme environment with a high alkalinity and hightemperature when the MWFs are in-use. Users of MWFs are faced with the problem witha microbial contamination during their exploitation witch requires approaches that suppressmicrobial growth. On the other hand, the disposal of MWFs needs to develop methodsthat promote the biodegradation of the operationally exhausted products. Concerning thefactors mentioned above, the research into the microbiology of metalworking fluids isvery important. In this study samples from different machines operating with metalworkingfluids were taken. 60 bacterial isolates as pure cultures were obtained. Morphological,cultural and biochemical methods were applied for their identification, as well as sequencingof 16S rDNA gene. The bacterial isolates were identified as Stenotrophomonas maltopilia,Agrobacterium larrymoorei, and as Micrococcus sp. Stenotrophomonas maltopiliaconsisted the dominant group.
GAM42TAXONOMIC IDENTIFICATION OF XENORHABDUS
(ENTEROBACTERIACEAE) SPECIES BY RESTRICTIONANALYSIS OF PCR-AMPLIFIED 16S RDNA GENES
Georgieva1, J.H., Groudeva1, V.I., Shishiniova2, M.D.1.Department of Microbiology, Faculty of Biology, University of Sofia, DraganTzankov 8 Blvd., Sofia, Bulgaria2.Department of Zoology and Anthropology, Faculty of Biology, University ofSofia, Dragan Tzankov 8 Blvd., Sofia, Bulgaria
The entomopathogenic nematodes from family Steinernematidae (Rhabditida) aresymbiontically associated with bacteria from genus Xenorhabdus. The bacteria belong tofamily Enterobacteriaceae.
Twenty-one bacterial strains isolated from five nematode species (Steinernema spp.,S.feltiae, S.kraussei, S.intermedium and S.carpocapsae) belong to genus Xenorhabdusaccording to the classical taxonomy metodes we used. They were typed by analyzing 16SrDNA gene restriction patterns obtained after digestion of PCR-amplified 16S rDNAs.
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Seven restriction endonucleases were required: Cfo I, Hin fI, Dde I, Sau 3AI, Alu I, Hae IIIand Msp I.
Our results indicate that amplified 16SrDNA restriction analyses in an accurate tool foridentifying endonucleases entomopathogenic nematode bacterial symbionts
GAM43PRELIMINARY INVESTIGATIONS OF THERMAL SOURCES
BIODIVERSITY FROM RUPITE REGION
A. Terziyska, R. Mandeva, D. Lyutzkanova, M. Stoilova-Disheva, G. Radeva, M.Kambourova
In the present work we initiate examination of phylogenetic biodiversity in Bulgarianthermal sources. Soil and water samples with temperature gradient 45оС - 70 оС were takenfrom two hot springs in Rupite field.
Molecular methods based on 16S rDNA analysis were used for evaluation of the realmicroorganism variety. Genomic DNA isolated from the samples and purified byNucleobond cartridges was used as template for PCR. The optimal PCR parameters for 16SrDNA amplification with universal and group specific primers for different bacteria wereestablished. PCR products ready for cloning were obtained.
The samples were screened for new bacterial and actinomycetes producers ofcarbohydrases on twelve AZCL substrates. Four strains belonging to Bacillus groupwere found to be carbohydrate active. One of them produced cellulase, gluconase andxylogluconase, two – arabinoxylanase and one – amylase. The taxonomic affiliation of theenzyme producers was determined by phylogenetic analysis.
GAM44BIODIVERSITY OF CARBOHYDRATE DEGRADING
CULTIVABLE BACTERIA FROM BACILLUS GROUP,ISOLATED FROM BULGARIAN HOT SPRINGS
Anna Derekova1, Carsten Sjøholm2, Rossica Mandeva1, Margarita Kambourova1
1The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Science,acad. G. Bonchev str. 26, 1113 Sofia, Bulgaria2Novozymes, Krogshoejvej 36, 2880 Bagsvaerd, Denmark
The diversity of culturable bacteria from genus Bacillus and related genera in Bulgarianhot springs was investigated. A total of 77 thermophilic and facultative thermophilicstrains were isolated under aerobic conditions at 60°C. Seventy six of them belonged toeight species in four genera from Bacillus group. Based on phylogenetic analysis (<98%
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sequence similarity) eleven isolates represent potentially three novel species or genera.Producers of carbohydrases, degrading 11 from tested 12 substrates were isolated. Betweenthem, producers of biotechnologically valuable enzymes like starch and pectin degradingenzymes were isolated. Producers of both, exo- and endo- amylolytic acting enzymes werescreened. Some of the enzymes were between first reported thermostable enzymes in theirgroups, like curdlan lyase, gellan lyase and cellulase.
GAM45SPATIAL PATTERN OF MICROBIAL NUMBERSIN THE FREE WATER OF THE SREBARNA LAKE
Svetlana Naumova and Anastassia PetrovaCentral Laboratory of General Ecology, BAS
A microbiological investigation of the water of the Srebarna Lake was carried outaiming to test the representativeness of the biomonitoring site in the center of the free ofmacrophytes area. The spatial dynamics of the total bacterial count, the reproduction rate,and the numbers of carbophilic and ammonifying organotrophs were studied in July andOctober 2004. The change of bacterial numbers with depth was examined in the center ofthe free water at the surface, at the lower edge of the transparent water column (Secchidepth), and at the bottom. The horizontal distribution of the parameters values was surveyedin integral samples from the whole water column in three points of a South North transectof the free water: at Varban Bozu, in the center, and at the entrance of the Dragaykachannel.
The microbiological parameters characterized the water column in the center of the freeof macrophytes area as fairly homogeneous. The total bacterial count and the reproductionrate changed with the depth insignificantly, which is normal for a shallow lake with anactive water circulation. The slight vertical dynamics of the carbophils could be related tothe development of phytoplankton, and the numbers of ammonifyers followed the oxygenconcentration. No substantial differences were disclosed by the microbiological indicatorsamong the three points along the transect. Again, the total bacterial count and thereproduction rate were conservative, representing the transitory state of the water bodyin the two observations. The numbers of the two groups of organotrophs were confinedin the limits of an order.
The spatial uniformity of the microbiological pattern proved the adequacy of the sitein the center of the free water to the objectives of the biological monitoring of the SrebarnaLake.
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GAM46COMPARISON OF BACTERIOPLANKTON DEVELOPMENT
BETWEEN CONTROL AND FERTILIZED FISH PONDS
H. Kalcheva, D. Tersiisky, R. Kalchev.Institute of Zoology, BAS, Sofia
Two treated with manure of cattle origin and two control carp fish ponds wereinvestigated for bacterioplankton development by direct counting of bacteria stainedwith erythrosine (Razoumov,1932 in its contemporary modification). The experiment wascarried out from the beginning of May till the end of September 2005 at the Institute forfishery and aquaculture, Plovdiv branch, Bulgaria. General physical variables like watertransparency, temperature and others were measured, accompanied by simultaneoussamplings of nutrients, bacterioplankton and chlorophyll a at biweekly intervals. Bacterialnumber and biomass of free living and on detritus attached bacteria were determinedseparately. The results of control and treated fish ponds were compared by means ofrepeated measure variance and by correlation analyses for significant differences andrelationships.
GAM47ADAPTIVE STRESS RESPONSE OF HUMICOLA LUTEA 103
TO COPPER EXPOSURE
E. Krumova1, Y. Gocheva1, A. Dolashki2, P. Dolashka2, S. Stefanovic3, W. Voelter3,M. Angelova1
The Stephan Angeloff Institute of Microbiology1, Institute of Organic Chemistry2,Bulgarian Academy of Science, Sofia, Bulgaria and University of Tuebingen3, Germany
Copper is an essential element for fungal metabolism, but can be toxic to microorganismsat high concentrations. It is widely considered that copper ions exert its effect at thecellular level hrough induction of oxidative stress. Adaptive response to heavy metaltreatment refers to the ability of cells to better resist the damaging effects of the toxicagent when first preexposed to a lower dose. It is a widespread phenomenon that has beenobserved in prokaryotes and eukaryotes. In this study, the effect of pretreatment of thefungal cells of Humicola lutea 103 with copper salts on growth and antioxidant defensewas investigated.
The changes of biomass production, protein carbonyl content, synthesis of reservecarbohydrates and antioxidant enzyme activities in the experiments with pretreatment ofH. lutea cells in comparison to the stress conditions were analyzed. Pretreatment with 70µg/ml Cu2+ resulted in enhanced resistance of the conidiosperes and mycelia taken fromexponential growth phase to higher doses of the heavy metal. Adaptive cell response
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included restoration of the normal growth, decrease in oxidative damaged proteins andaccelerate in glycogen and trehalose production. Induced copper-tolerance in adaptiveexperiments corresponded with enhanced activity of the Cu/Zn-superoxide dismutasedue to de novo protein synthesis. The correlation between immunoprotective protein andCu/ZnSOD was confirmed by Western blot analysis. The primary structure of this fungalenzyme has been determined by Edman degradation of peptide fragments derived fromproteolytic digest. A single chain of the protein, consisting of 152 amino acid residues,reveals a very high degree (74-85%) of structural homology in comparison to the aminoacid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H.lutea Cu/ZnSOD, measured by MALDI-MS (15935 Da) and calculated by its amino acidsequence (15716 Da), is attributed to the carbohydrate chain of one mole of N-acetyl-glucosamine, attached to the N-glycosylation site Asn23-Glu-Ser.
This work was supported by grant K-1302/03 from the NCSI of the Ministry of Educationand Science, Bulgaria.
GAM48PHYSIOLOGICAL RESPONSE OF ANTARCTIC FUNGI TO
LONG-TERM TEMPERATURE STRESS
Y. Gocheva, E. Krumova, L. Slokoska, J. Miteva, M. AngelovaThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad. G. Bonchev 26, 1113 Sofia, Bulgaria
Harsh living conditions of the continent of Antarctica differ substantially from thoseof almost all other parts of the Earth’s biosphere, and its microbial inhabitants have had tobecome specifically adapted to the severe conditions. This adaptation includes differentstrategies, among which is the activation of antioxidant enzyme defense. The aim of thisstudy was to obtain new data about the role of oxidative stress and antioxidant enzymesin cold-adaptation of the filamentous fungi. We compared the effect of different temperatures(10- 30°C) on cell response of two Antarctic Penicillium sp. strains (Penicillin sp. p14and Penicillium sp. m12) with European temperate Penicillium sp t35. The changes ofbiomass production, protein carbonyl content, synthesis of glycogen and trehalose andantioxidant enzyme activities in long-term temperature stress conditions were analyzed.
The main finding from this study is that fungal strains from Antarctic region, and thatfrom temperate European region, may have evolved a specialised physiology allowingthem to survive prolonged low temperatures, and have enzyme systems that function atlow temperatures. Our results demonstrated that growth at low temperature does clearlyinduce oxidative stress events in all fungal strains tested, namely enhanced level of oxidativedamaged proteins, accumulation of reserve carbohydrates and increased activity ofsuperoxide dismutase and catalase. Compared to temperate mesophilic Penicillium sp t35,Antarctic strains (psychrophilic Penicillium sp p14 and mesophilic Penicillium sp m12)demonstrated adaptations, which permit survival in low-temperature conditions. Cell
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response of psychrophilic strain to temperatures above 15-20°C may be characterized asa heat-shock response, whereas a decreased antioxidant defense in temperate mesophilicstrain at low temperatures may be explained by more profoundly changes in metabolism atcold temperatures than under heat shock conditions.
This work was supported by grant B-1309/03 from the NCSI of the Ministry of Educationand Science, Bulgaria.
GAM49CELLULAR RESPONSE AND ANTIOXIDANTS ENZYMES IN
ASPERGILLUS NIGER STRAINAGAINST TEMPERATURE STRESS
Abrashev R1, Dolashki A4, Stevanovic S3, Dolashka P2, Voelter W4, Stefanova L1,Pashova S1, Hristova R2, and Angelova M1
The Stephan Angeloff Institute of Microbiology1, Institute of Organic Chemistry2,Bulgarian Academy of Science, Sofia, Bulgaria and University of Tuebingen3,Germany
Temperature is among the most crucial of factors affecting the growth and survival ofmicroorganisms. Exposure to elevated temperatures (“heat shock”, HS) has been found toincrease oxidative damage in microbial cells, possibly by accelerating the formation ofreactive oxygen species (ROS) and/or by increasing there activity. Fungal cell responseagainst temperature stress is not very well known.
This work was designed to study some of the physiological and biochemical eventsthat accompany survival of the fungal strain A. niger 26 under conditions of short-termheat shock. Exposure of mycelia taken from exponential growth phase to the differenttemperature (30 – 45oC) for 6 h caused an enhancement in cyanide-resistance respirationand ROS generation by temperature dependant manner. As a result of raise in oxidantlevel, a significant increase in the amount of oxidative damaged proteins was established.Our results suggest that the heat treatment dramatically increased the concentration oftrehalose and glycogen and induced expression of antioxidant enzymes, superoxidedismutase (SOD) and catalase (CAT). Two isoenzyme forms of SOD (Cu/Zn- and Mn-containing enzyme) were found in crude extract of the fungal cells. The complete aminoacid sequence of Cu/Zn-SOD, determined by a combination of automated Edmandegradation, MALDI-TOF analysis, and sequence alignment was shown in comparisonwith SODs from other eukaryotic sources.
This work was supported by grant K-1401/03 from the NCSI of the Ministry of Educationand Science, Bulgaria.
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GAM50BIOSYNTHESIS OF PROTEOLYTIC ENZYMES FROM
ANTARCTIC ACTINOMYCETE STRAINS
Darina Bl. Dimitrova, Petar D. Dorkov, Blagovesta T. GochevaBiological Faculty of Sofia University “St. Kliment Ohridski” Sofia, Bulgaria
Seventeen actinomycete strains, isolated from Antarctic soil samples of the LivingstonIsland, are screened in reference to their biosynthetic proteolytic abilities. These strainssynthesize extracellular nonconstitutive proteolytic enzymes, differing from one anotherin their activity. Tendencies for considerable differences are registered between the specificand general proteolytic activities. The highest specific proteolytic activity is peculiar toStreptomyces sp. 39 - 1430.8943 KU/mg, followed by Streptomyces sp. 35 (818.6874 KU/mg) and Streptomyces sp. 36 (698.7757 KU/mg). The general proteolytic activity is maximalfor Streptomyces sp. 35.
The pointed three strains are selected as most perspective for further investigationsas a result of the screening.
GAM51BIOSYNTHESIS OF ANTIMICROBIAL SUBSTANCES FROM
ANTARCTIC ACTINOMYCETE STRAINS
Darina Bl. Dimitrova, Petar D. Dorkov, Blagovesta T. GochevaBiological Faculty of Sofia University “St. Kliment Ohridski” Sofia, Bulgaria
After screening of 17 actinomycete strains, isolated from soil examples of theisle Livingston – Antarctida for producing antimicrobial substances active against Bacillussubtilis 6633 ATCC, the three most perspective ones are selected and their biosyntheticabilities are studied microbiologically. The strains synthesize antibiotic substances againstthe studied test microorganisms with the exception of Escherichia coli and Candidaalbicans. The two strains Streptomyces sp. 35 and Streptomyces sp. 36 are active against41.66% of the analyzed cultures and the strain Streptomyces sp. 39 – against 33.33%. Thestrain Streptomyces sp. 35 is remarkable for its considerable antifungal activity againstAspergillus, Fusarium and Penicillium. The antibiotic complex of the strain Streptomycessp. 36 is active against Gram-positive and Gram-negative bacteria, and that of the strainStreptomyces sp. 39 – against phytopathogenic bacteria (Erwinia amylovora andPseudomonas syringae patovar syringae). There is a correlation between the biosynthesisof proteolytic enzymes and antimicrobial substances within the three Actinomycetes strains.
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GAM52BIOSYNTHESIS OF PROTEINASE INHIBITOR FROM
ANTARCTIC ACTINOMYCETE STRAINS
Darina Bl. Dimitrova, Petar D. Dorkov, Vasil N. Atanasov, Blagovesta T. GochevaBiological Faculty of Sofia University “St. Kliment Ohridski” Sofia, Bulgaria
A prescreening of 17 actinomycete strains isolated from soil samples of the isleLivingston – Antarctida for biosynthesis of extracellular proteinase inhibitor proteins isdone. The most perspective three of them, remarkable for their considerably high activity- Streptomyces sp. 35, Streptomyces sp. 36 and Streptomyces sp. 39, are selected. Maximumspecific proteolytic inhibitory activity is registered for Streptomyces sp. 36 (1544.6384ТIU/mg). Some qualitative and quantitative methods for detection of the activity arecompared. It is proved that polypeptones have a strongly inducive effect over theproduction of proteinase inhibitors. The dynamic in accumulation of inhibitors is studiedand their biosynthesis is registered to begin after the 48-th hour; their maximal activity isdetermined to be at the late exponential phase (96-th hour of the cultivation). So it isestablished that these substances are not of primary significance for the growth of cells,but probably their appearance is attended with regulation of proteolytic activity. Probablythe strains inhabiting extreme conditions need protease inhibitors by reason of the strongregulative systems for their physiological processes. The protease inhibitor of Streptomycessp. 36 is isolated and partially purificated by using appropriate methods. The electrophoreticprofiles of the obtained isolate exhibit homogeneity and molecule weight about 14 kDa.
GAM53CYCLODEXTRIN GLUCANOTRANSFERASE PRODUCTION
BY MAGNETICALLY RESPONSIBLE BACILLUSCIRCULANS ATCC 21783 CELLS
M. Safarikovaa, N. Atanasovab, V. Ivanovac, S. Engibarovb, I. Safarika, A. Tonkovab
aDepartment of Biomagnetic Techniques, Institute of Systems Biology and EcologyAS CR, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic, bDepartment ofExtremophilic Bacteria, Institute of Microbiology, BAS, 26 Acad. G. Bonchev str.,1113 Sofia, Bulgaria, cDepartment of Organic Chemistry and Microbiology,University of Food Technologies, 26 Maritza str., 4002 Plovdiv, Bulgaria. E-mail:[email protected]
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is a unique enzyme convertingstarch into cyclodextrins (CDs). These compounds possess the ability to form inclusioncomplexes with many organic and inorganic molecules, changing their physical andchemical properties, which determines their wide applications in environmental protection,
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medicine, food, cosmetic, pharmaceutical and chemical industries.Four types of magnetic nano- and microparticles were used for the immobilization of
Bacillus circulans ATCC 21783 cells: magnetite microparticles (1-5 ìm), entrapped inagar gel beads with bacterial cells (AM); silanized magnetite (SM, 20-40 nm) covalentconnected on the cell surface; and alkaline and citrate ferrofluids (FFs, 10-20 nm),attached on the cell wall by ionic interaction.
The highest CGTase production was achieved after 96 h semi-continuous processwith cells immobilized on SM when the CGTase specific activity was 8.4-fold higher comparedto free cells. The enzyme synthesized by all kinds of magnetically immobilized cells was18-20% more thermostable than that of free cells. The magnetically responsible Bacilluscirculans ATCC 21783 cells demonstrate convincingly the effect of the used magneticcarriers for a significant enhance of the CGTase yield, specific enzyme activity and thermostability.
This work was supported by a bilateral grant between the Bulgarian and CzechAcademies of Sciences, the Grant Agency of the Czech Academy of Sciences and ISBEIntention No AV0Z60870520.
GAM54MOLECULAR ANALYSIS OF A THERMOSTABLE GELLAN
LYASE BY MECC/HPLC
Miroslava Atanassova1, Jose Ignacio Garabal Sanchez2, Anna Terziiska1, AnnaDerekova1, Rositsa Mandeva1, Margarita Kambourova1
1 Department of Extremophiles, Institute of Microbiology “Stefan Angelof”, Bulgar-ian Academy of Sciences, Sofia, Bulgaria2 Centro de Investigaciуns Agrarias de Mabegondo (CIAM), Apdo. 10, 15080 ACoruсa, Galicia, Spain
A scheme for the purification to homogeneity was developed for a thermostable gellanlyase, produced by a Geobacillus stearothermophilus 98 strain after continuousfermentation. Stepwise ammonium sulphate precipitation, hydrophobic interactionschromatography, anion exchange chromatography and size-exclusion chromatographywere applied. The final purity of the enzyme was confirmed both by SDS-PAGE and C18reverse-phase chromatography on HPLC. Its amino acid composition was obtained by theWaters AccQ•Tag method. Free-solution capillary electrophoresis with SDS containingBGE (MECC) of the newly isolated molecule showed that the gellan lyase had very lowmobility at alkaline pH, with tendency to precipitate, therefore, the best conditionsestablished for CE analysis were 0,2M BGE at pH 2 containing 0,1 % SDS, in untreatedfused-silica capillary of 92 cm/75ìm i.d./375 ìm o.d., 22°C and 25 kV. The enzyme’sisoelectric point and molecular weight were studied.
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GAM55PURIFICATION AND CHARACTERISATION OF
KERATINOLYTIC PROTEASES PRODUCEDBY STREPTOMYCES ALBIDOFLAVUS
Vania Stefanova1,3, Nicolay Kirilov1,3, Kirill Tsiroulnikov 2, MichиleDalgalarrondo3, Jean-Marc Chobert3, Iskra Ivanova1 and Thomas Haertlй3.1Department of Microbiology, Faculty of Biology, Sofia University “St. KlimentOhridski”, Sofia, Bulgaria2 Laboratory of Proteolytic Enzyme Chemistry, Shemyakin & Ovchinnikov Instituteof Bioorganic Chemistry of RAS, Moscow, Russia3FIPL, BIA, Institut National de la Recherche Agronomique, Nantes, France
Streptomyces are known to produce multiple proteases into the culture medium. Someof these proteases are well characterized. In the recent years, production of proteases withstrong keratinolytic activity by different Streptomyces sp has been investigated (Bressollieret al.,1999; De Azaredo et al. 2006).
In this study the exogenous keratinase produced by the strain of Streptomycesalbidoflavus strain ATCC25422 was purified and characterized. The strain was cultivatedin mineral medium supplemented with porcine hair 5 g/l as sole carbon and energy source.At least two proteases with powerful keratinolytic activity were obtained in threepurification steps – anion exchange chromatography than cation exchange chromatographySP Sepharose Fast Flow gel followed by gel filtration on Sephacryl S 300 HR. Two bandswere observed by zymogram on SDS-PAGE. The molecular weights of these two bandsestimated by SDS-PAGE were 54 kDa and 64 kDa. Substrate specificity of keratinases wasdeterminated with different synthetic amino acid derivates – Suc-Ala-Ala-Pro-Phe-pNA,Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Lys-pNa. The proteases exhibited activitywith Suc-Ala-Ala-Pro-Phe-pNA and Suc-Ala-Ala-Pro-Leu-pNA. However, no hydrolysiswas detected with Suc-Ala-Ala-Pro-Lys-pNa. The studied proteases were inhibited byPMSF, which is known serine proteinase inhibitor. EDTA and pepstatin have no influenceon enzyme activity. The studied proteases are stable and active at 50 °C and at pH valuesbetween 7.5 – 9.
GAM56BNMPK: A WEB BASED SUITE OF BACTERIALNUCLEOTIDE MONO PHOSPHATE KINASES
Ajay Bikumandla, Sai Guduru, Paulina Daalova, Alexandra Shosheva, PetyaChristova, Petras Kundrotas and Emil Alexov
The web server is a collection of models of 3D structures of all Bacterial Nucleoside
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Mono Phosphate Kinases (BNMPK), their pre-computed properties and relevantinformation. Nucleotide Mono Phosphate (NMP) kinases convert mono-phosphatenucleotides to di-phosphate nucleotides. The reaction involves binding of both ATP(adenosine tri-phosphate) and mono-phosphate nucleotide, post-sequential transfer of aphosphate from ATP to mono-phosphate nucleotide and then release of ADP (adenosinedi-phosphate) and di-phosphate nucleotide. The web server provides information in regardto sequence identity, 3D structure, Enzyme Commission (EC) number, pKa, desolvationpenalty and interaction energy with permanent dipoles of all BNMPKs. We also haveimplemented a search engine using Java script, MS Frontpage and PERL. The BNMPKdatabase has a search part where the user can search the proteins based on the family,name, seqID, pKa, polarity and desolvation energy and has an option to download all orpart of the search results. There are five different families of NMP kinases, categorized bythe nucleotide they bind, each family containing approximately 100 known members. Theseinclude adenylate (AMPK, 118 members), guanylate (GMPK, 87 members), uridylate(UMPK, 73 members), cytidylate (CMPK, 71 members) and thymidylate (TMPK, 107members) kinases. However while many sequences exist, only few 3D structures wereexperimentally determined. The dearth of 3D evidence is illustrated most markedly in theUMPK family, which does not have a publicly available representative 3D structure. Thus,our goal is to predict the structures of all members of these families and to use thestructures to calculate specific Biophysical properties. The results are publicly availablethrough the Internet link: http://www.ces.clemson.edu/compbio/databases/kinases.
GAM57СКРИНИНГ НА ДРОЖДИЕВИ ПРОДУЦЕНТИ НА ФИТАЗА
Данаил Георгиев1, Стоянка Гаргова2
1ПУ ,,Паисий Хилендарски” – катедра ,,Биохимия и микробиология” –Пловдив2Университет по Хранителни Технологии – катедра ,,Биотехнология” –Пловдив
Фитазата е една фосфомоноестераза (КНЕ 3.1.3.8), катализилаща постапаловиден механизъм дефосфорилирането на фитиновата киселина и нейнитесоли с получаване на различни миоинозитол фосфати и фосфорна киселина.
Проведена е двуетапна скрининг процедура на дрожди, принадлежащи къмродовете Saccharomyces, Zygosaccharomyces, Candida и Pichia, за продуциране надефосфорилиращия ензим фитаза. В първата стъпка, осъществена върху 4 видатвърди хранителни среди, съдържащи като селекциониращ фон калциев фитат саизследвани 118 култури. Установено е, че върху PSM среда почти всички щамовепоказват зони на просветляване още на двадесет и четвъртия час от развитиетоси. Във втората стъпка на скрининга, проведена в течна хранителна среда иколичествено определяне на фитазната активност, са селекционирани няколко
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щама, показващи значителен потенциал за продуциране на ензима. При най –добрия щам част от ензима се секретира в културалната среда. В следващитепроучвания ще се търсят условия за по – пълна екскреция на ензима, коетопредставлява значителен интерес в технологичен и практически аспект.
GAM58МУТАГЕННО ТРЕТИРАНЕ НА ЩАМ ASPERGILLUS
AWAMORI K-1 ПРОДУЦЕНТ НА ЕНЗИМА КСИЛАНАЗА
Яна Евстатиева, Светла Илиева, Диляна Николова, Валентин Савов, АтанасАтевБиологически факултет, Софийски Университет “Св. Климент Охридски”,катедра Биотехнология
Широкото приложение на ензимите определя необходимостта от получаванена продуценти с повишени биосинтетични възможности. Методите на индуцираниямутагенез все още са широко използвани, тъй като са лесно приложими иефективни. Отборът на мутантните култури се осъществява по коефициента К,изразяващ съотношението между диаметъра на микробната колония и диаметърана хидролизната зона на ксилан съдържащата скринираща среда.
Проведена е мутагенеза на родителски щам Aspergillus awamori K-1 с химичниямутаген N-метил-N’-нитро-N-нитрозогуанидин (НГ) с концентрация 250 ìg/ml.След мутагенното третиране са изолирани мутантни щамове, които показват по-висока активност на ензимите от ксиланолитичния комплекс при дълбочиното имкултивиране в колби. Проучени са морфологичната, физиолого-биохимичната ибиотехнологичните характеристики на секвенираните мутантни култури.Изолираните мутантни щамове показват над 1.5 пъти по-висока ендоксиланазнаактивност в сравнение с родителския щам Aspergillus awamori K-1, пълен наборна индивидуални ензими разграждащи ксилановите субстрати и с 12 часа по-късферментационен процес от изходната култура.
Благодарности: Настоящата експирементална работа е осъществена сфинансовата подкрепа на Фонд Научни Изследвания при МОН, по проект №ВУ-Б-7/О5
GAM59STUDYING OF BIOACTIVE METABOLITES FROM ARCTIC
COLD-ADAPTED STREPTOMYCETES
D. Lyutskanova, M. Stoilova-Disheva, , M. Kolarova, K. Alexieva, V. Peltekova, V.Ivanova
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In the course of investigation of soil samples from Spitzbergen, Arctic, 6 strains wereisolated. Microscopically and morphologically they were identified as Streptomycetes. Itis of great scientific interest to identify taxnomically the isolated strains , to study theirbiological characteristics, to perform a screening for some active metabolites produced bythem and reveal their biological action. Hydrolitic activity of the isolated strains wasinvestigated at 4ºС. The enzymes â-gluconase, arabinoxylanase, celulase, amylase,xylogluconase and arabinase were identified to present.
From the mycelium and the supernatant of one of the strains a 4 component antibioticcompex was isolated. It is active against Gram (+), Gram (-) bacteria and yeasts. Thecomplex has been separated by TLC in liquid fase butanol : acetic acid : water. The 4compounds show a typical colour reaction with ninhydrin solution.
GAM60ИМОБИЛИЗАЦИЯ И СВОЙСТВА НА ПЛЕСЕННА
ЦЕЛУЛАЗА ВЪРХУ ПОЛИАМИД
Гинка Делчева, Иван Пищийски, Георги Добрев
В настоящата работа е представена имобилизацията на плесенна целулаза отксиланолитичен комплекс, продуциран от Aspergillus niger B03. Като носител еизползван синтетичен полимер полиамид директно или след активиране съссвързващ агент глутаров алдехид и хексаметилендиамин. Определени сатемпературен и рН оптимум, температурна и рН стабилност на свободния иимобилизиран ензим. Изследвани са кинетичните параметри Km и Vmax на дветеформи на ензима.
GAM61PRIMARY CHARACTERIZATION OF A NEWLY
DISCOVERED BACTERIOCIN-LIKE SUBSTANCE DURACINPRODUCED FROM ENTEROCOCCUS DURUM M-3
Svetoslav G. DimovSofia University “St. Kliment Ohridski”, Faculty of Biology, Department of Genet-ics, e-mail: [email protected]
Bacteriocins are ribosomally synthesized bacterial toxins acting on the target cells asmembrane depolarizing and pore forming agents. Many bacteriocins are produced bylactic acid bacteria isolated from dairy products which inhibit the growth of pathogenmicroflora thus preventing spoilage.
Here is described a new bacteriocin-like substance - duracin, produced by Enterococcus
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durum M-3 isolated from traditional Bulgarian yellow cheese “kashkaval”. The researchwas emphasized on the purification of the bacteriocin molecule, determination of thespectrum of antibacterial activity, and the characteristics of the producer strain. It wasdetermined that the duracin molecule represents a few kilodaltons peptide which wasproduced at maximum at the end of the exponential phase of growth and having relativelybroad range of cytotoxic activity among Gram-positive bacteria.
GAM62NOVEL BACTERIOCIN FROM ENTEROCOCCUS FAECIUM3587 – SPECTRUM OF ACTIVITY AND SOME MOLECULAR
CHARACTERISTICS
Viktoriya M. Peeva, Pavlina M. Ivanova and Svetoslav G. DimovSofia University “St. Kliment Ohridski”, Faculty of Biology, Department of Genet-ics, e-mail: [email protected]
Many lactic acid bacteria produce bacteriocins – small peptide bacterial toxins inhibitingspecies in most cases closely related to the producer thus helping them in the concurrencefor the occupation of the ecological recesses.
In this study we focused our investigation on the following points: 1) characteristicsof the production of the bacteriocin-like substance during growth; 2) determination of thespectrum of antibacterial activity of the produced bacteriocin; 3) purification of thebacteriocin; and 4) preliminary characteristics of the bacteriocin molecule.
It was found that the maximum activity was observed at the end of the exponentialphase of growth and the bacteriocin production was not associated with plasmids henceno plasmids were isolated from the producer strain. The molecule was purified by preparativePAGE and it was active only against closely related enterococci but not against otherGram-positive or Gram-negative bacteria.
GAM63LACTOCOCCUS LACTIS SUBSP. LACTIS HV219 –
A PROBIOTIC?
SD Todorov1, M Botes (nee Brink)1, ST Danova2 and LMT Dicks1
1Department of Microbiology, University of Stellenbosch, 7600 Stellenbosch, SouthAfrica2Department of Microbial Genetics, The Stephan Angeloff Institute of Microbiology,Bulgarian Academy of Science, 1113 Sofia, Bulgaria
Probiotic bacteria have to resist a number of intestinal stress conditions, such as bile
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salts, acids and pancreatic juice to survive passage through the gastro-intestinal tract.Adhesion to intestinal epithelial cells and production of antimicrobial substances areadditional criteria used to select probiotic strains. In this study, a strain isolated fromvaginal secretions was selected based on antimicrobial peptide (bacteriocin) production,antibiotic resistance, hydrophobicity, resistance to bile salts and growth at low pH. Growthwas monitored in MRS broth, modified to contain 0.3%, 0.6%, 0.8%, 1.0%, 3.0% and 5.0%bile, respectively. MRS broth was adjusted to pH 3.0, 4.0, 5.0, 7.0, 9.0, 11.0 and 13.0,respectively. Lactococcus lactis subsp. lactis HV219 showed good adhesion to humanCaco-2 cell-lines.
Bacteriocin HV219, produced by Lactococcus lactis subsp. lactis HV219, is activeagainst Escherichia coli, Enterococcus faecalis, Lactobacillus casei, Listeria innocua,Proteus vulgaris and Pseudomonas aeruginosa. Activity was lost when treated withproteolytic enzymes, SDS, Triton X-114 and Triton X-100, but not at pH 2.0 to 10.0 or after20 min at 121 °C. The mode of activity is bacteriolytic, as confirmed by atomic forcemicroscopy. The strain harbours a plasmid of 3.0 kb. Growth of Lactococcus lactis subsp.lactis HV219 was not significantly affected in the presence of increasing bile concentrations.Low hydrophobicity (4.20%) was observed for L. lactis subsp. lactis HV219.
GAM64EFFECT OF MEDIUM COMPONENTS ON PRODUCTION OFBACTERIOCINS JW3BZ AND JW6BZ BY LACTOBACILLUS
PLANTARUM ISOLATED FROM BOZA
JW von Mollendorff, SD Todorov and LMT DicksDepartment of Microbiology, Stellenbosch University, 7602 Stellenbosch, SouthAfrica
Bacteriocins JW3BZ and JW6BZ, produced by Lactobacillus plantarum JW3BZ andJW6BZ, respectively, inhibits the growth of Lactobacillus sakei, Enterococcus mundtii,Enterococcus faecalis, Lactococcus lactis subsp. lactis and Lactobacillus casei. Thegrowth of Streptococcus caprinus was only inhibited by bacteriocin JW6BZ and that ofListeria innocua LMG 13568 by bacteriocin JW3BZ. Treatment with Proteinase K,trypsin and pronase inactivated the bacteriocins. No change in activity was observedin the presence of á-amylase. Maximal activity (25 600 AU/ml) was recorded forbacteriocin JW3BZ and JW6BZ in MRS broth, adjusted to pH 5.0 to 6.5. Optimalproduction of bacteriocin JW3BZ was recorded in the 20.0 g/l glucose and 20.0 g/l maltose.In the case of bacteriocin JW6BZ, maximal activity was recorded in the presence of 20.0 g/l glucose and 20.0 g/l mannose, respectively. Modified MRS medium, containing tryptoneand meat extract (1:0.6), yielded activity levels of 25 600 AU/ml for bacteriocins JW3BZand 51 200 AU/ml for bacteriocin JW6BZ. Increased concentrations (5.0 g/l) of K2HPO4doubled bacteriocin JW3BZ activity (51 200 AU/ml). Concentrations of 2.0 g/l to 20.0 g/lKH2PO4 doubled the activity of bacteriocin JW3BZ. In the case of JW6BZ, concentrations
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of 2.0 g/l to 20.0 g/l KH2PO4 doubled the bacteriocin activity. A reduction in bacteriocinactivity was recorded when strains JW3BZ and JW6BZ were grown in the presence ofglycerol. The inclusion of selected vitamins in the growth media led to different levels ofbacteriocin JW3BZ and JW6BZ activity. The optimal concentration of tri-ammonium citratefor production of bacteriocins JW3BZ and JW6BZ was 5.0 g/l. Low activity levels forboth bacteriocins were recorded in BHI broth, M17 broth and skim milk (20.0 g/l).
GAM65FACTORS AFFECTING THE ADSORPTION OF
BACTERIOCIN ST194BZ TO LACTOBACILLUS SAKEI ANDENTEROCOCCUS FAECIUM
SD Todorov1, M Meincken2 and LMT Dicks1
1Department of Microbiology and 2Department for Forest and Wood Science, SPMunit, University of Stellenbosch, 7600 Stellenbosch, South Africa
Lactobacillus plantarum ST194BZ, isolated from boza, a cereal based fermentedbeverage from Bulgaria, produces two bacteriocins, viz. ST194BZ(a) of 3.3 kDa andST194BZ(b) of 14.0 kDa. A combination of the two bacteriocins inhibits the growth ofLactobacillus casei, Lactobacillus sakei, Lactobacillus delbrueckii subsp. bulgaricus,Enterococcus faecalis, Escherichia coli, Enterobacter cloacae and Pseudomonasaeruginosa. A maximum total bacteriocin activity of 12 800 AU/ml was recorded in MRSbroth. Bacteriocins ST194BZ(a) and ST194BZ(b) withstand 20 min at 121oC and incubationat pH 2-10, but is inactivated when treated with proteolytic enzymes such as pepsin,papain, á-chymotrypsin, trypsin and Proteinase K.
Images obtained by atomic force microscopy showed clear signs of membrane damageof Lactobacillus sakei, accompanied by the leakage of DNA and â-galactosidase.Adsorption of the bacteriocin to cells increased when the cells were treated with buffersat pH values above neutral. An increase in bacteriocin ST194BZ adsorption to cells ofEnterococcus faecium and L. sakei was observed with an increase in incubation temperature.Treatment of the latter two species with various inorganic salts and solvents gave differentresults regarding the adsorption of bacteriocin ST194BZ. In general, pre-treatment of thetwo sensitive cells with Triton X-100, Triton X-114 and chloroform increased the adsorptionof bacteriocin ST194BZ. Treatment of E. faecium and L. sakei with Na-EDTA and SDS ledto a decrease in the adsorption of bacteriocin ST194BZ. Variable results were recordedwith inorganic salts.
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GAM66DEFORMATION OF BACTERIAL CELLS AS A RESULT OFBACTERIOCINS PRODUCED BY LACTIC ACID BACTERIA
M Meincken2, SD Todorov1, JW von Mollendorff1 and LMT Dicks1
1Department of Microbiology and 2Department for Forest and Wood Science, SPMunit, University of Stellenbosch, 7600 Stellenbosch, South Africa
The interaction between bacteriocins and target cells was studied using differentmethods, such as atomic force microscopy (AFM), cell lysis, and extracellular leakageof DNA and â-galactosidase. Most methods used to detect bacteriocin-cell interactionsare based on the observation of indirect reactions or cellular changes. AFM reveals thedirect effect of a bacteriocin on a sensitive cell. This includes deformation of the cellsurface, vesiculation and leakage of the cytoplasm.
AFM images were obtained in air and with tapping mode on a Multimode AFM fromVeeco. A silicon non-contact cantilever from Nanosensors (Neuchatel, Switzerland) witha resonance frequency of 160 kHz and a spring constant of approximately 50 N/m wasused. The results clearly showed changes in cell morphology, such as collapse of theapical ends or the cell center, signs of cytoplasm leakage and vesiculation. Differencesobserved among the bacteriocins suggests different mode of actions, such as the barrelstave model and the toriodal model, which describes the formation of pores in the cellmembrane or the carpet model, which leads to a vesiculation of the outer cell membrane.
GAM67PARTIAL CHARACTERIZATION OF A BACTERIOCIN PRO-
DUCED BY LACTOBACILLUS CURVATUS ISOLATEDFROM CERVELAT SALAMI
A van den Worm, SD Todorov and LMT DicksDepartment of Microbiology, Stellenbosch University, Stellenbosch 7602, SouthAfrica
A bacteriocin-producing strain of Lactobacillus curvatus (AW42CS) was isolatedfrom Cervelat salami collected at a local food store (Stellenbosch, South Africa). BacteriocinAW42CS inhibited the growth of Gram-positive bacteria (Streptococcus caprinus ATCC700066, Streptococcus spp. TL2W and TL2R, Lactobacillus sakei DMS 20017, Listeriainnocua LMG 13568, Listeria ivanovii N29, Listeria monocytogenes N22 and N26,Lactococcus lactis subsp. lactis HV219, and Enterococcus faecalis HKLHS, E90, E88).The bacteriocin is approximately 2.5 kDa in size as determined by tricine-SDS-PAGE and isproduced at 1600 AU/ml after 21 h of growth in MRS broth. Activity was lost aftertreatment with proteolytic enzymes and when heated for 20 min at 121 ÚC. Treatment
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with á-amylase, lipase, SDS, Tween 20, Tween 80, urea and EDTA, or incubation atpH 2 to 12 did not inactivate bacteriocin AW42CS. Cells of L. sakei DSM 20017, Linnocua LMG 13568 and E faecalis treated with the bacteriocin did not lyse, suggestinga bacteriostatic mode of action. The peptide did not adsorb to producer cells. BacteriocinAW42CS was partially purified by precipitation with 60% ammonium sulphate andseparation in a SepPakC18 column.
Treated cells of L. sakei DSM 20017 collapsed after contact with bacteriocins JW11BZand JW15BZ produced by L. fermentum JW11BZ and JW15BZ. Leakage was observedafter treatment of exponentially growing cells of L. sakei DSM 20017 with bacteriocinsAMA-K and JW6BZ, and L. innocua LMG 13568 treated with bacteriocin ST8KF.Vesiculation was observed for cells of L. sakei DSM 20017 treated with plantaricin 423 andbacteriocin ST23LD produced by L. plantarum
GAM68SANIONINS: ANTIINFLAMMATORY AND ANTIBACTERIAL
AGENTS WITH WEAK CYTOTOXICITY FROM THEANTARCTIC MOSS SANIONIA GEORGICO-UNCINATA
Veneta Ivanovaa, Klaus-Juergen Dornbergerb, Albert Haertlb, Ute Moellmannb,Hans-Martin Dahseb,Mariana Kolarovaa, Krasja Aleksievaa, Nesho Chipevc
aThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,26 Acad.Georgy Bonchev St., 1113 Sofia, BulgariabLeibniz Institute for Natural Product Research and Infection Biology-Hans-Knoell-Institute, Beutenbergstrasse 11a, D-07745 Jena, GermanycCentral Laboratory of General Ecology, Bulgarian Academy of Sciences, 2Gagarin St., 1113 Sofia, Bulgaria
Sanionins A (1) and B (2) were isolated from the moss Sanionia georgico-uncinata,collected on the Antarctic Livingston Island. The compounds 1 and 2 were purified bysolvent extraction, silica gel column chromatography and preparative HPLC consecutively.The structures of the both compounds were elucidated by 1 and 2D NMR experiments andmass spectrometric investigations. Both substances sanionin A (1) and sanionin B (2) forthe first time were isolated from the antarctic moss Sanionia georgico-uncinata and canbe classified into cinnamoyl bibenzyls.
These compounds showed activity against important Gram-positive pathogens likemycobacteria, multiresistant staphylococci and vancomycin resistant enterococci. Thisactivity is combined with antiinflammatoric activity and low cytotoxicity. The sanionin A(trans-isomer) was with better biological activities that sanionin B (cis-isomer).
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GAM69MALONYL,4-5-DIHYDRONIPHIMYCIN: NEW POLYOL
MACROLIDE ANTIBIOTIC, PRODUCED BYSTREPTOMYCES HYGROSCOPICUS
Veneta Ivanova, Mariana Kolarova and Krasja AleksievaThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad. G. Bonchev-Str. 26, 1113 Sofia, Bulgaria
Malonyl,4-5-dihydroniphimycin was isolated as a new antibiotic from the mycelium ofa Streptomyces hygroscopicus. The chemical constitution was elucidated from the physico-chemical properties, NMR techniques and mass spectrometry to be a 36-membered macroliderelated to non-polyenic antibiotics. Malonyl,4-5-dihydroniphimycin is a new antibiotichaving a 36-membered ring, a two malonate and a monomethylguanidino groups. Theposition of the two malonyl residues is at C-23 and C-19 and displayed activity againstGram-positive bacteria and filamentous fungi.
GAM70DIPHENYLETHER AND MACROTRIOLIDES OCCURRINGIN A FUNGAL ISOLATE FROM THE ANTARCTIC LICHEN
NEUROPOGON
Veneta Ivanova1, Udo Graefe2, Brigitte Schlegel2, Mariana Kolarova1, KrasjaAleksieva1
1The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad. G.Bonchev Str. 26, 1113 Sofia, Bulgaria2Hans-Knoll-Institute of Natural Products Research, Beutenbergstrasse, 11a, D-07745 Jena, Germany
The fungus Tritirachium sp. 0317 was isolated from the Antarctic lichen Neuropogonsp. Fermentation of this strain, extraction of the culture broth and preparative separationof produced compounds furnished 4-carboxy-5,5’-dihydroxy-3,3’-dimethyldiphenylether(1), macrosphelide A (2) and macrosphelide J (3). The structures were elucidated on thebasis of MS and NMR measurements and display structural features such as diphenyletherand lactone groups, which were found too in the depsidones as typical products of thislichen. Macrosphelides A and J displayed interesting activities as cell adhesion inhibitorsand moderately cytotoxic agents.
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GAM71MICROBIAERATIN, A NEW NATURAL INDOLE ALKALOID
FROM A MICROBISPORA AERATA STRAIN, ISOLATEDFROM LIVINGSTON ISLAND, ANTARCTICA
Veneta Ivanova1, Udo Grдfe2, Hans-Martin Dahse2, Mariana Kolarova1, KrasjaAleksieva1, Hartmut Laatsch3
1The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad. G. Bonchev-str. 26, 1113 Sofia, Bulgaria2Leibniz Institute for Natural Product Research and Infection Biology-Hans-Knoell-Institute, Beutenbergstrasse 11a, D-07745 Jena, Germany3Department of Organic and Biomolecular Chemistry, University of Gцttingen,Tammannstrasse 2, D-37077 Gцttingen, Germany
A new sulphur-containing natural alkaloid named microbiaeratin (1a) was isolatedtogether with the known microbiaeratinin (2) from the culture filtrate of Microbisporaaerata strain. The organism was isolated from penguin excrements, collected on theAntarctic Livingston Island. The structure was elucidated by one- and two-dimensionalNMR experiments and mass spectrometric investigations. The structure of 1a was finallyelucidated as N-3-β-indolylethyl-15-α-acetylamide-ethylthiazole-12-carboxamide. A lowantiproliferative and cytotoxic effects of 1a was determined with L-929 mouse fibroblastcells, K-562 human leukemia cells (GI50 >50 µg/ml) and HeLa human cervix carcinoma (CC50>50 µg/ml). Microbiaeratin (1a) did not show antimicrobial activity.
GAM72AN INVESTIGATION ON THE OPPORTUNITIES FOR IN-CREASING STREPTOMYCES AMBOFACIENS BIOSYN-
THETIC ACTIVITY THROUGH INDUCED MUTAGENESIS
Nataliya Hristova, Venelin Baloutzov, Stanislava Karafizova*University of Sofia “St. Kliment Ohridski”,Faculty of Biology, Department of Biotechnology,8 Dragan Tzankov blvd., Sofia 1164*postgraduate student – MS programme
The present research is related to studying the possibilities for an increase inStreptomyces ambofaciens ATCC 15154 (a spiramycin producer) biosynthetic activity.
Once having determined the optimal regime of mutagen treating by statistical analysisof experimental data, the strain has been subjected to both the single and combined actionof N-methyl – N’-nitro – N-nitroso- guanidine and ã-rays.
The biosynthetic potency of the isolated active varieties has been defined by the agardiffusion method, after the varieties were grown through submerged cultivation.
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GAM73ПЕРМЕАБИЛИЗИРАНЕ НА ДРОЖДЕВИ КЛЕТКИ ЗА
ПРОДУКЦИЯ НА ΑΑΑΑΑ-КЕТО КИСЕЛИНИ
Донка Д. Костова, Анна В. КуюмджиеваСофийски университет „Св. Климент Охридски”Катедра по „Обща и промишлена микробиология”1164 София, бул. „Драган Цанков” №8
Получаването на високо активни дрождеви клетки за продукция на α-кетокиселини, чрез трансформация на D-амино киселини, е ефективна алтернатива наизползването на чисти ензимни препарати. Селекционираният термотолерантен,лактозоусвояващ щам Kluyveromyces marxianus 903 притежава висока специфичнаD-аминооксидазна активност (60 U/g белтък), която е повишена деветкратно (545U/g белтък) чрез подбор на хранителна среда и условия за ефективна индукция наензима.
С помощта на химични (третиране с катионния детергент цетилтриметиламониев бромид - СТАВ) и физични (лиофилизация) подходи за пермеабилизиране,D-аминооксидазната активност на цели клетки от селекционирания щам еповишена многократно. Изследвани са детайлно условията за пермеабилизацияпри химично третиране (концентрация на СТАВ, продължителност натретирането, температура, рН) и различни режими на лиофилизация за постиганена най-добро пермеабилизиране на клетките. Установено е, че третиране наклетките с 0,2% СТАВ в 100 mM калиево-фосфатен буфер за 20 минути при 22ЪС,увеличава 43 пъти D-аминооксидазна активност на клетките, а лиофилизирането- 26 пъти.
GAM74ИЗСЛЕДВАНЕ НА КИСЛОРОДНИЯ МАСООБМЕН
ВЪРХУ БИОСИНТЕЗА НА ЕКЗОПОЛИЗАХАРИД P -45
А.В. Атанасова, К. Павлова, Г.Щ. Атанасова, А.И. Тончев, В.В. Лосев, В.Г.Назаров
От особенно значение при получаването на екзополизахарида Р-45 еосигуряване на контролируем синтез на полимер и получаване на продукт свъзпроизводими показатели. Важен параметър при синтеза на Р–45 представляватстойностите на скоростта на консумирания кислород и скоростта на отделениявъглероден диоксид по време на процеса. Представените експериментални даннипоказват, че степента на използване на кислорода може да послужи като критерийопределящ добива на екзополизахарида в крайния продукт. Анализът на кислороди въглероден диоксид се осъществява с помоща на газ- анализатор Tandem на
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фирмата Adaptive Biosistems. На базата на този вид анализ е определенреспираторния коефициент на микробната култура. Тенденцията на изменениена този физиологичен параметър показва специфичното ниво на синтез наекзополизахарида. Това е един от възможните механизми за промяна на степентана полимризация, състава и качествата на синтезирания екзополизахарид.
GAM75МАЩАБЕН ПРЕХОД ЧРЕЗ ИЗПОЛЗВАНЕ НА
КИСЛОРОДНИЯ МАСООБМЕН ПРИ БИОСИНТЕЗА НААМИНОСИСЕЛИНИ
А.В.Атанасова, А.И.Тончев, В.В.Лосев, С.Б.Петков, В.Г.Назаров
Един от основните проблеми при получаването на биопродукти чрездълбочинно култивиране се явяват лимитиращите масообменни процеси. Важенкритичен параметър при култивирането им представлява скоростта наконсумиране на кислород и скоростта на отделяне на въглероден диоксид повреме на процеса. Добивът на много първични метаболити, получени по тозиначин е предопределен от кинетиката на растежа на клетките, която обикновеннопредхожда началото на биосинтеза на продукта.
Изследвана е зависимостта между степента на трансформацията на субстрати количественото изменение на интензивността на кислородния масообмен вбиореактора. Анализирането на съдържанието на кислород и въглероден диоксидв изходящите ферментационни газова се осъществява с помоща на газованализатор Tаndem на фирма Adaptive Biosistems. Тези on-line пресметнатипараметри се корелират емпирично със степента на трансформацията на субстратав активна субстанция. Получените експериментални резултати показватизменението на стойностите на респираторния коефициент в различните фази напроцеса. Експериментите са проведени на 20L и на 3m3 биореактори. Сравненитепоказатели илюстрират създаването на модел на биосинтетичния процес, набазата на който се придобива допълнителна информация за мащабиране напроцеса.
GAM76КОЛИЧЕСТВЕНО ОПРЕДЕЛЯНЕ НА ГЕНТАМИЦИН В
ПРОБИ ОТ КРЪВНА ПЛАЗМА ЧРЕЗМИКРОБИОЛОГИЧЕН МЕТОД
П. Янкова, Ан. Варсанова
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Настоящия метод е разработен с цел количествено определяне съдържаниетона гентамицин в проби от кръвна плазма, получена от телета третирани i.m.(интрамускулно) с гентамицин сулфат инжекционен разтвор еднократно в доза4mg/kg маса.Същността на метода се основава на дифузия на антибиотика вагарова среда, инокулирана с подходящ тест микроорганизъм. Имайки предвидвъзможността в някой от интервалите за вземане на кръвни проби съдържаниетона гентамицин да варира под 100 ng/ml, важно бе да се определи такъв тестмикроорганизъм и условия за провеждане на анализа, чрез които да се постигнемаксимална чувствителност на метода. За целта са тествани тримикроорганизъма: B. subtillis, B. pumilus и B. mycoides в различни концентрации ина различни среди,като най-подходящ се оказа B. mycoides. Установена еграницата на количествено определяне на гентамицин – 60 ng/ml и границата наоткриване на гентамицин - 20 ng/ml. Определен е срока на годност на изходния иработните сравнителни разтвори на стандартното вещество, съответно 20 дни и2 дни, съхранявани при температура 4-80С.
Разработеният метод е валидиран по отношение на линейност, прецизност иточност.
GAM77DESIGNING A WHOLE-CELL BIOTRANSFORMATION
DOUBLE-PHASE OXIDATION SYSTEM WITHSTREPTOMYCES ROSEOCHROMOGENES
Boris Marinov , D. Koleva, I. Kostova
Whole cells of Streptomyces roseochromogenes, contains a cytohrome P450 which,in conjunction with two indigenous electron transfer proteins, roseoredoxin androseoredoxin reductase, hydroxylates exogenous mevastatin to pravastatin. In intact cellsthe natural electron donor is NADH. Trying to design a whole-cell biotransformationdouble-phase oxidation system with Streptomyces roseochromogenes was used a knownhydroxylation of mevastatin to pravastatin. In case to design organic : aqueous systemwas overcome through the implementation of mevastatin lacton which is low water soluble.The prolonged productivity of such a system depends on cell viability, since viable cellsare naturally able to regenerate the co-factor.
Cyclohexane, n-Decane, n-Hexadecane, Glycerin-tributyrat, Glycerin-1-monooleat, Soyabean oil were the solvents that allowed cell activity and viability. The most adequatephase ratio was 1:4 but it was possible to be used phase ratio 1:1. For all other testedorganic solvents was found to be toxic, causing cell death at any concentration level.
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GAM78БИОФИЗИЧНИ ХАРАКТЕРИСТИКИ НА БАКТЕРИАЛНИ
БИОСЪРФАКТАНТИ
Емилия Стоименова, Eвгения Василева-Тонкова*, Михаела Иванова,Христина Петкова**, Албена Йорданова***, Анна Сотирова*, ДанкаГълъбова*, Здравко ЛалчевБиологически Факултет, СУ “Св. Климент Охридски”, 1164, София* Институт по Микробиология, БАН, 1113, София**Химически факултет, СУ “Св. Климент Охридски”, 1164, София***Институт по Биофизика, БАН, 1113, София
Биосърфактантите са разнообразни по химичен състав повърхностно активнивещества - нискомолекулни гликолипиди и липопептиди, както и високомолекулнилипопротеини и други. Особено актуална група биосърфактанти сагликолипидите, към които принадлежи класа на рамнолипидите, продуцирани отразлични бактерии, най – вече от различни видове на рода Pseudomonas.
Напоследък замърсяването на околната среда с органични вещества се явявасериозен екологичен проблем и все по-често се налага използването на безвреднии сигурни биологични методи за тяхното отстраняване. Основните организми,които разграждат въглеводородите по естествен начин и така спомагат запречистването и поддържането на баланса на екосистемите са микроорганизмите.Въглеводород-разграждащите микроорганизми могат да продуциратбиосърфактанти, които понижават повърхностното и междуфазовото напрежениеи улесняват емулгирането на хидрофобните субстрати, при което значитено сеувеличава достъпността им за разграждане.
Целта на настоящото изследване е да се охарактеризират и сравнятбактериални биосърфактанти, секретирани от щам на Pseudomonas fluorescensпри култивиране върху различни органични субстрати. Направен ефизикохимичен анализ на повърхностните свойства на биосърфактантите, като епроследена промяната на повърхностното напрежение на културалната течностпри различно рН и различна солева концентрация на NaCl. Микроорганизмите сакултивирани в минерални среди с различни въглероден източници-хексадекан,керосин, маслинено олио и др. с цел намиране на подходящо практическоприложение на новоизолираните биосърфактанти.
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GAM79КОМБИНИРАНЕ НА МЕТОДИ ЗА ВИДОВА
ИДЕНТИФИКАЦИЯ НА LACTOBACILLUS DELBRUECKIISSP. BULGARICUS
Савова Т., Уршев З., Спасова М., Петрова И., Ишлимова Д., Алексиева П.ЕЛБИ Булгарикум ЕАД, София, ул. „Малашевска” 12АЕ-mail: [email protected]
Поради необходимостта от бърза идентификация на културите Lactobacillusdelbrueckii ssp. bulgaricus (L. bulgaricus), съхранявани в LBB колекцията (EЛБИБулгарикум ЕАД), беше възприета полифазна система за видова идентификация,включваща три достъпни и икономически обосновани метода. Установяванетона биохимичните характеристики на изследваните култури по отношение наусвояването на различни въглехидратни източници с помощта на API-50CHL (API,Bio-Merieux) комбинирахме с определянето на оптичната форма на образуванатамлечна киселина с ензимен кит (Roche) и анализ на EcoRI рестриктния профил на16S рДНК (ARDRA). В зависимост от получените резултати, при анализа на 155култури L. bulgaricus, изолирани от домашно приготвено кисело мляко в периода1968-1972 г., както и на щамове, получени от единични колониии придопълнителното пречистване на културите, се оформиха 6 групи.
Първата група определихме категорично като L. bulgaricus, като и тритеизбрани от нас метода дадоха съвпадение с типовия щам L. bulgaricus ATCC 11842.Втората група, съгласно данните от ARDRA анализа, беше определена като L.bulgaricus, но профилът на захарите, получен с API тест кита, показа 53.6-87.1%идентичност с L. lactis. Допълнителните изследвания на щамовете от тази група спомощта на макрорестрикционнен анализ (пулсова електрофореза) показахаголямо генетично сродство между отделните щамове, от които най-добре проученае културата LBB.B144. По-ранни изследвания с анализ на тоталните клетъчнибелтъци с денатурираща ПА гел-електрофореза категорично определиха щамLBB.B144 като L. bulgaricus (Ж. П. Димитров, непубликувани резултати). С тезидопълнителни данни, приехме работната хипотеза, че втората група лактобацилиса от вида L. bulgaricus, които обаче допълнително усвояват N-ацетилглюкозамини трехалоза, което води до подвеждащи резултати с API-теста.
В културите от третата група установихме едновременното присъствие нащамове с фенотипната характеристика от първите две групи и приехме, че енеобходимо допълнително пречистване на съответните изходни култури.Четвъртата група лактобацили генотипно определихме като L. bulgaricus, но притях не се наблюдаваше развитие при API-теста. Тъй като същите култури серазвиват добре на MRS-бульон, предположихме, че индикаторът бромкрезолпурпур има инхибиращо въздействие. Смяната на индикатора с хлорфенол ред непромени резултата. В останалите две групи установихме съществени отклоненияв съдържанието на D(-) млечна киселина. Изолираните единични колонии от тези
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групи съгласно ARDRA анализа отнесохме към L. bulgaricus и L. helveticus.В заключение, подбраните от нас три идентификационни метода дадоха
достатъчно надеждни резултати при потвърждаване на принадлежността наизследваните култури към вида L. bulgaricus. В случая с щамовете, генетичноблизки до LBB.B144, беше необходимо използуването на данни и от други анализи.Единичните случаи на присъствие на L. helveticus бяха категорично отчетени отвъзприетата идентификационна методика.
GAM80СЕЛЕКЦИОНИРАНЕ НА ЩАМОВЕ STR.THERMOPHILUSЗА УСЪВЪРШЕНСТВАНЕ НА СТАРТЕРНИ КУЛТУРИ ЗА
ДИРЕКТНО ПРИЛОЖЕНИЕ
Пашова-Балтова К., Нинова Н., Уршев З., Михайлова М..ЕЛБИ Булгарикум ЕАД, София ул.”Малашевска 12А”Е-mail: [email protected]
Като основен производител на стартерни култури, ЕЛБИ Булгарикум ЕАДтърси нови възможности за оптимизиране параметрите на симбиотичните закваски,наложили се от години в производството на българските традиционни продукти,но използувани досега главно за получаване на производствени закваски впредприятията.
Съвременното развитие на млечния отрасъл все повече налага използванетона закваски за директно приложение./DVS/. Те изместват от приложениепроизводствените закваски, утвърдили се в продължение на дълги години припроизводството на българско кисело мляко и бяло саламурено сирене.
При разработването на закваски за директно приложение, се вземат предвидмного по-строги изисквания към лиофилизираната култура, най-вече по отношениена нейната активност, а също и по достигане на подходящи технологичнипараметри –киселинообразуване по време на технологичния процес, нископосткиселинообразуване по време на съхранение, осигуряване на добраконсистенция на продукта.
Приложението на DVS формите налага и нови изисквания към технологичнитекачества на щамовете. Една възможна стратегия за усъвършенствуване назакваските за DVS приложение е обогатяването им със селекционирани щамовеStr.thermophilusл.
Нови 150 щама Str.thermophilus бяха изолирани от домашно приготвеникисели млека от различни райони на страната и детайлно охарактеризирани. Втехнологичната характеристика на проучените щамове са включени важнитехнологични параметри –динамика на киселинообразуване до 16 час и 48 час,при различни температури на инкубиране-430С, 370С, 340С, натрупване набиомаса, осмотичен толеранс към висока концентрация на въглехидрати,
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кинематичен и роторен вискозитет, посткиселинообразуване при различнитемператури.
Като култури, които в значителна степен могат да допринесат за подобряванесвойствата на DVS закваските, от натрупаните база данни бяха селекционирани4 щама с къса лаг фаза на развитие, както и 2 структуроподобряващи щама.
Получените нови комбинации стартерни култури, обогатени с подбранитещамове Str.thermophilus, приложени като DVS показаха по-добри параметриотносно време на коагулация, структура и консистенция на готовия продукт,посткиселинообразуване.
GAM81ОЦЕНКА НА ПРОТЕОЛИТИЧНАТА АКТИВНОСТ НАКУЛТУРИ LACTOBACILLUS DELBRUECKII SSP.
BULGARICUS
Уршев З., Фачикова Н., Пашова-Балтова К., Петрова И.ЕЛБИ Булгарикум ЕАД, София, ул. „Малашевска” 12АЕ-mail: [email protected]
Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) е широкоизползуванамлечнокисела бактерия при производството на кисело мляко и бяло саламуреносирене. Общо 153 култури L. bulgaricus съхранявани в LBB Колекцията на ЕЛБИБулгарикум ЕАД бяха охарактеризирани по протеолитичната им активност (ПА)в мляко при култивиране в продължение на 16 часа при две температури по методпрепоръчан от IDF Стандарт 149А:1997. По този метод ПА се дефинира катоколичеството образувани Фолин-позитивни аминокиселини и пептиди по времена ферментационния процес, а резултатите се представят като тирозиновеквивалент (mg% Tyr).
Разпределението на стойностите на ПА при инкубиране при 42оС и 37оСпоказа изключителна нехомогенност на културите от този бактериален вид потози показател (от 0.3 до 10.7 mg% Tyr). Получените стойности (средно 6.4 ± 2.1mg%Tyr за 42оС и 6.2 ± 2.1 mg% Tyr за 37оС, n = 153) по правило бяха значителнопо-високи от стойностите, установени за щамове Streptococcus thermophilus (0.8 ±0.8 mg% Tyr, n = 62) и по-ниски от тези, определени за щамове Lactobacillus helveticus(10.1 ± 2,5 mg% Tyr, n = 16). За изследваните култури L. bulgaricus беше установенаслаба статистически значима положителна корелация между изменението наактивната киселинност (“pH) и стойностите на ПА, определени в 16 час и придвете температури (корелационен коефициент r = 0.34, p<10-4 за 42оС и r = 0.40,p<10-6 за 37оС). При по-ниската температура на инкубиране във ферментиралитес културите L. bulgaricus млека “pH беше статистически достоверно по-малко оттова в млеката ферментирани при 42оС (средна разлика от 0.24 ± 0.25 pH единици),докато разликата в ПА на културите, определена на 16 час съответно при двете
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температури, не беше статистически достоверна.Варирането на температурата на инкубиране (37оС-42оС) влияе много силно
върху темпа на киселинообразуване на културите L. bulgaricus, но не и върхуобщото количество натрупани продукти на протеолизата, като едно възможнообяснение е наличието на широк температурен интервал на оптимално действиена протеолитичните ензими на L. bulgaricus. Значителната вариация на ПА наизследваните култури е добра предпоставка за селекцията на щамове L. bulgaricusс ниска/висока ПА в зависимост от приложението на съответните стартерникултури.
GAM82АКТИМИКРОБНА АКТИВНОСТ НА МЛЕЧНО КИСЕЛИ
БАКТЕРИИ,ИЗОЛИРАНИ ОТ БЪЛГАРСКИ РЪЖЕНИ КИСЕЛИ ТЕСТА
Любомира Йочева1, Гергана Добрева2, Радка Василева1 и Стефка Антонова-Николова2
1 – Институт по криобиология и хранителни технологии2 – Катедра “Обща и промишлена микробиология”, СУ “Св. Кл. Охридски”
Млечно киселите бактерии са сред най-изучаваните микроорганизми, порадидоказаната си способност да синтезират различни биологично активни вещества– бактериоцини, биологично активни пептиди, екзополизахариди.
От друга страна, в резултат на метаболитната си активност, някои от тяхразграждат съдържащи се в млякото или в брашното белтъци с алергеннисвойства, с което повишават хранителната им стойност.
Разнообразните възможности за приложение на млечно киселите бактериинасочва изследователите към проучване и на други екологични ниши, освенмлечно киселите продукти, с цел получаване на изолати с желани биологичнисвойства.
При проучване на микрофлората на кисели теста, получени чрез спонтаннаферментация на българско ръжено брашно от различни реколти, са изолирани 88щама млечно кисели бактерии, на които са изучени различни метаболитниактивности.
Културалните, морфологичните и физиологичните свойства на изолатите гиотнасят към родовете Lactobacillus и Pediococcus. При изследване антимикробнатаим активност спрямо патогенни или причиняващи развала на хляба тестмикроорганизми беше установено, че най-голям е броят на млечно киселитебактерии, потискащи в различна степен развитието на Bacillus cereus,предизвикващ картофена болест по хляба, и Staphylococcus aureus. Един отизолатите показва слаба активност спрямо Listeria sp. Не бяха установенищамове, инхибиращи тест-микроорганизма Escherichia coli.
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Изследването е финансирано с договор 47/2006г от Фонд “Научниизследвания” към СУ “Св. Кл. Охридски”.
GAM83MILK-CLOTTING ENZYMES FROM SUBMERGE CULTI-
VATED BASIDIOMYCETES
T. Dmitrieva, I. Feist, M. ShamtsyanSt. Petersburg State Technological Institute (Technical University), Moskovskyprospect, 26, St. Petersburg, Russia. E-mail: [email protected]
Milk clotting enzymes from higher basidiomycetes are a promising source to substituterennin in cheese making. The aim of our studies was a screening of the producers of milkclotting enzymes among various species of higher basidiomycetes.
The quality of cheese significantly depends from enzyme preparations used for milkclotting. For this purposes animal enzymes extracted from rennet are usually used. Althoughvarious microbial coagulants are developed as inexpensive substitutes for animal enzymes,but they does not found wide utilization in the countries of developed cheese making,were are used mainly for the production of cheddar type cheeses.
The fruit bodies, submerged mycelium, grown on various media, as well as the culturebroth of several higher basidiomycetes were studied for milk clotting activity. Mushrooms,which fruit bodies, mycelium or culture broth posses high milk clotting and low proteolyticactivities were selected. Growth media composition was optimized to achieve higherenzymatic activity.
Due of separation of enzymes the ratio of milk clotting and general proteolytic activitiesin the preparations highly increased.
Milk clotting enzymes were purified and characterized.
GAM84INTENSIFICATION OF YEAST BIOMASS ACCUMULATION
AND ETHANOL FERMENTATION PROCESSES
I. Koltyga, T. Dmitriyeva, M. ShamtsyanSt. Petersburg State Institute of Technology (Technical University), Russia, St.Petersburg, 198013, Moskovsky prospect, 26 E-mail: [email protected]
К факторам, вызывающим замедление брожения, относят: условияброжения, ис-ходный состав сусла (содержание сбра-живаемых сахаров,возможный недо-статок витаминов и азотистых веществ), наличие в средекомпонентов, отрицательно влияющих на дрожжи (тяжелые металлы, остатки
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пе-стицидов и т. д.), технологические при-емы при переработке сырья иполупро-дуктов, накопление в сбраживаемой среде продуктов метаболизма(этиловый спирт, углекислый газ, жирные кис-лоты) и др.
Один из путей интенсификации про-изводства в пивоварении, виноделии,спиртовой и безалкогольной промыш-ленности — применение сорбирующихматериалов на различных технологичес-ких стадиях. Однако, из всего разнообразияприменяемых сорбирующих материалов лишь мень-шая часть пригодна дляиспользования на стадии брожения, еще меньше спо-собны сорбировать наиболеетоксичные для дрожжей компоненты сусла — жир-ные кислоты с короткой цепью,токсичные вещества (микотоксины). Боль-шинство сорбентов, обладающихспо-собностью удерживать жирные кисло-ты, вместе с токсичными компонентамивыводят из среды вещества, определяю-щие сортовые характеристики готовогопродукта — вкус и аромат. Наилучшим средством для решения проблемнедоброда были бы сами дрожжевые клетки или те их составляющие, которыесорбируют жирные кислоты.
В наших исследованиях изучалась сорбционная способность клеточных стеноквысших грибов (хитинглюканового комплекса, глюканов). Установлено чтоисследуемые вещества интенсификацировали выделения спирта в процессеброжения. путем сорбции компонентов оказывающие негативное воздействие наэтот процесс.
Полученные результаты могут быть использованы доля решения различныхзадач в пивоварении, виноделии, спиртовой и безалкогольной промышленности.
GAM85БАЛИСТИЧНА ДЕЗИНТЕГРАЦИЯ НА БИОМАСА ОТ
LACTOBACILLUS DELBRUECKII SUBSP. BULGARICUSBTCC 50
Диляна Николова, Валентин Савов, Яна Евстатиева, Светла Илиева, ПенчоДалев, Атанас АтевБиологически факултет, Софийски Университет „Св. Климент Oхридски”,катедра Биотехнология
Пробиотиците имат редица доказани клинични и други полезни ефекти,благодарение на които могат да се използват както с превантивна цел, така икато активни агенти с терапевтични свойства. Изследванията върхупробиотичните свойства на млечнокиселите бактерии, показват участието накомпонентите на бактериалната клетъчна стена в реализирането на тези полезнисвойства. За изолирането на тези компоненти най-често се прилага дезинтеграцияна клетките с последващо фракциониране.
Балистичната дезинтеграция на микробни клетки като физичен метод се влияе
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от редица параметри - тип и количество биомаса; интензивност на разбъркване;времетраене; количество балистичен товар и др.
В настоящата работа са проведени експерименти, свързани с оптимизиранеусловията за балистична дезинтеграция на биомаса от щам Lactobacillusdelbrueckii ssp bulgaricus BTCC 50. Проучено е влиянието на възрастта накултурата млечникисели бактерии, съотношението между количеството биомасаи балистичен товар, времетраене и други параметри на процеса дезинтеграция.На базата на осъществената експериметална работа са установени оптималнитестойности на основни параметри на процеса на дезинтеграция на клетки от щамLactobacillus delbrueckii ssp bulgaricus BTCC 50 и е постигнат ефект над 17%.
Благодарности: Настоящата експериментална работа е осъществена сфинансовата подкрепа на Фонд Научни Изследвания при МОН по проект № ВУ-Б-7/05.
GAM86HYBRIDIZATION OF LACTOBACILLUS PLANTARUM226-15 AND LACTOBACILLUS CASEI SUBSP. CASEI C
A. Slavchev, I. Murgov, Z. Denkova
A successful hybridization of protoplasts of Lactobacillus casei subsp. casei C andLactobacillus plantarum 226-15 was accomplished.
It was shown, that as a result of the hybridization was obtained a heterogenic populationand amongst its clones were selected strains with high reproductive ability, resistance tothe conditions in the stomach and the intestines, non-sliming, etc.
The recombinant clones Lactobacillus LPC 6T, Lactobacillus LPC 40 and LactobacillusLPC 52, which were distinguished for accelerated coagulation of milk, improved acid-forming and flavour-forming capabilities were selected.
GAM87INVESTIGATION OF THE INHIBITORY EFFECT OF LACTICACID BACTERIA ON THE CELLS OF ESCHERICHIA COLI
NBIMCC 8739
P. Nedelcheva, Z. Denkova, R. Nikolova
The inhibitory effect of growing cultures of Lactobacillus plantarum 226-15,Lactobacillus casei subsp. casei C, Lactococcus lactic subsp. lactis l4 and Pediococcuscerevisiae 2P on the cells of Escherichia coli NBIMCC 8739 was investigated during theirmixed cultivation in skimmed milk at temperatures 23±1°С and 37±1°С.
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It was established, that during the first 6 h the viable cell counts of both the lactic acidbacteria and Escherichia coli NBIMCC 8739 increased. During the next 12-24 h the growthof Escherichia coli NBIMCC 8739 gradually slowed up and held up. This effect was moredistinct during the mixed cultivation of lactic acid bacteria and E. coli at 37°С.
GAM88THE EFFECT OF DNA REPLICATION ON THE REPAIR OF
DAMAGEDPLASMIDS IN SACCHAROMYCES CEREVISIAE
Miglena Koprinarova, Anastas Gospodinov, George Russev
One of the mechanisms of repair of DNA and maintenance of genome integrity is thehomologous recombination. For homologous recombination repair there should be anundamaged sequence used as a template. The obvious time period for searching andfinding such an undamaged sequence is the S-phase of cell cycle during which theeukaryotic DNA is doubled. The close bond between DNA replication and repair is logicalbut not firmly established.
The subject of our work was in vivo examination of the repair of plasmids damagedwith trioxalen using yeast Saccharomyces cerevisiae as a host system. Trioxalen causesinterstrand cross-links in the molecule of DNA for the repair of which homologousrecombination and NER (Nucleotide excision repair) is required in yeast.
We transformed previously damaged centromere plasmid (YCp50) in temperature-sensitive degron (td) mutant strain (YJT18) Saccharomyces cerevisiae. That was a heat-inducible Cdc45 degron mutant that promotes rapid degradation of Cdc45p at the restrictivetemperature of 37°C. The Cdc45p plays a central role in both initiation and elongationphases of chromosomal DNA replication. We used a wild-type strain with the samebackground as a control. At certain intervals we took aliquotes of yeast culture, isolatedDNA and examined the rate of repair of previously damaged plasmids by CompetitivePCR.
The results showed 50% decrease in the repair capacity of the temperature-sensitivedegron mutant strain in comparison with the wild-type strain indicating that the processof homologous recombination repair might be coupled to DNA replication in the yeaststrains Saccharomyces cerevisiae.
GAM89AGEING IN BREWING YEAST
Tamara Ginova, Silvia MilevaInstitute of cryobiology and food technology, Sofia
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Yeast ageing include changes that occur during cell lifespan leading to senescenceand death. Brewing yeast Saccharomycs cerevisiae ageing refers to some distinctphysiological states : stationary phase, stored yeast, serially used yeast population,individual age of the cell. Yeast ageing is accompanied by several modifications –morphological, metabolic, physiological. Cells in extended stationary phase are referredas aged cultures because the population are composed of individual cells with young andaged phenotype. Aged cells are alive and in different rate metabolically active, but notreproductive. Survival in stationary phase during fermentation depends on environmentalstresses. Stored yeast that are exposed to some stress factors (nutrient starvation, lowtemperature) and yeast that are used for several successive fermentations are also studied.We report an extensive changes in cell size, surface wrinkles, granular appearance andincreases of generation time that characterized aged phenotype in age-heterogeneouspopulation. Mortality in stored yeast increases with diminished of cell glycogen contentImpact of ageing on flocculation properties and fermentation performance of the yeast isdiscussed.
GAM90GROWTH INHIBITORY PROPERTIES OF CHALCONES
AGAINST VARIOUS YEAST SPECIES
K. L. Lahtchev1, D. I. Batovska2, St. Parushev2, V. Bankova2
1The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Acad. G. Bonchev Str. Bl. 26, Sofia 1113, Bulgaria2Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academyof Sciences, Acad. G. Bonchev Str. Bl. 9, Sofia 1113, Bulgaria
Chalcones are natural or synthetic flavonoid compounds with a common structural skeletonof 1,3-diphenyl-2-propen-1-one. They possess various biological properties including antifungalactivity. For deeper insight of its antifungal mechanisms we first synthesized a series of 19chalcones with diverse combinations of substituents in ring B and further examined theirstructure-activity relationships. The chalcones were tested for their growth inhibitory activityagainst 32 strains belonging to baker yeast Saccharomyces cerevisiae (13 strains),methylotrophic yeast Hansenula polymorpha (16 strains) and lactic Klyveromyces lactis (3strains). All the strains employed were with defined ploidy and carried specific mutations ineither the biosynthetic pathways or defence systems. The MICs determination revealed thatthe most active chalcones were DB1, DB9, DB12, DB24 and DB48 while the rest of thecompounds exhibited antifungal activity at very high concentrations only. The strainsbelonging to Kl. lactis appeared to be the most sensitive among the species tested. S.cerevisiae strains were more resistant to the parent chalcone DB1 then H. polymorpha, butthe opposite was true for DB24. The MICs depended on both the chalcone concentrationsand the strain cell density. At high chalcone concentrations and low cell density a strongkilling effect was observed. Clear differences were observed between the sensitivities/
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resistances of the strains belonging to the same species. These differences depended on thestrain genotypes and presence of specific mutations. While no effect was evident for themutations controlling purine, pyrimidine and some amino acids biosynthetic pathways wefound that H. polymorpha strains carrying mutation gsh1 (impairment of glutathionesynthesis) conferred increased sensitivities to some of the chalcones. This result suggeststhat glutathione is important defence against chalcone action.
GAM91ELECTRO-OPTICAL METHOD AS A NEW APPROACH OF
INVESTIGATION AND DISCRIMINATION OF TWO STRAINSESHERICHIA COLI
A. Gurova-Chausheva1, E.Velichkova1, R. Aleksandrova2, S. Danova2, S. P. Stoylov1
1Institute of Physics Chemistry, Bulgarian Academy of Science, Bulgaria, Sofia1113, Akad. G. Bonchev Str. Bl.112Department of Microbial Genetics, Institute of Microbiology, Bulgarian Academyof Science, 26, Acad.G. Bontchev Str. , 1113 Sofia, Bulgaria
In present study two physico-chemical methods (electro-optics andmicroelectrophoresis) were used to investigate Gram negative bacteria Esherichia coli.The used electro-optic method is based on light scattering by biological particles underthe influence of electrical field, which orients them depending on the intensity andfrequency of the electric field, surface electrical, optical and geometrical properties ofcells. The target strains HB 101 and XL-1-blue (the last one was obtained from anotherstrain treated by Ca2+, undergone electroporation and so it has become capable to acceptan alien DNA) were comparatively investigated .Our purpose was to evaluate thediscriminative power of the above mentioned methods. Special attention was paid on therecording of the detectable and reproducible changes.
Considerable differences in the surface electrical, optical and geometrical properties oftested strain’s cells were established. Thus the differences in characteristics of so calledelectro-optical pulses, their amplitudes and relaxation times, were large and significantand probably may be used for correct further identification and discrimination. Moreoverthe electro-optical pulse in question can be masured in seconds, because the speed is oneof the advantages of the electro-optic method. In conclusion the applied electro-opticalmethod might be considered as a new, promising approach to the investigation andidentification of different microbiological objects from Gram negative taxons.
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GAM92APPLICATION OF CO-POLYMER MICROPARTICLES FOR
IMMOBILIZATION OF TRYPSIN
Todor Ivanov1, Michail Kamburov1, Viara Ivanova2*, Jordan Hristov3
1Department of Biotechnology and 3Department of Chemical Engineering, Univer-sity of Chemical Technology and Metallurgy, 8 Kl.Ohridsky Blvd., 1576, Sofia,Bulgaria2Department of Organic Chemistry and Microbiology, University of Food Technolo-gies, 26 Maritza Blvd., 4002, Plovdiv, Bulgaria
Co-polymers were synthesized on the basis of acrylonitrile and maleinic anhydride.From these co-polymers were formed spherical particles by extrusion. Fine and coarsepowder supports were obtained by grinding of the dry co-polymers. Trypsin wasimmobilized on the three supports. From initial mixtures, containing the monomers inproportion 72% AN - 28% MA and 68% AN – 32% MA, were obtained two co-polymersPANMA1 and PANMA2, containing 2.4 % and 3.7% maleinic acid, respectively. The polymeryields were between 70% and 90%. After the polymerization step, the co-polymers werefiltered, washed with water and methanol and dried at 50 ºC. Finally, the particles werefragmentized and sieved by standard sieves and were separated in three main groups –particles with size from 100 to 200 ìm, from 200 to 500 ìm and from 500 up to 1000ìm. The IR-spectrum of the polymers showed sharp peaks at 2200 cm-1, specific for thenitrile íCa”N group, at 1700 cm-1 - specific for the carbonyl group íC=O, and for the specificabsorption for íOH, in the region of 3000 cm-1, specific for the carboxylic group (3200 – 3300cm-1). Particles with size between 100 and 200 ìm were more suitable for trypsinimmobilization. Up to 34.0 mg trypsin/g wet support were immobilized on theseparticles. The bounded enzyme quantity was two times lower on supports with size > of500 ìm and only 4 mg/g on extruded spherical particles with diameter between 500and 1000 ìm.
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GAM93IMMOBILIZATION OF TRYPSIN ON CO-POLYMERS OF
ACRYLONITRILE AND MALEINIC ANHYDRIDE
Todor Ivanov1, Michail Kamburov1, Viara Ivanova2*, Jordan Hristov3
1Department of Biotechnology and 3Department of Chemical Engineering, Univer-sity of Chemical Technology and Metallurgy, 8 Kl.Ohridsky Blvd., 1576, Sofia,Bulgaria2Department of Organic Chemistry and Microbiology, University of Food Technolo-gies, 26 Maritza Blvd., 4002, Plovdiv, Bulgaria
Abstract. Optimization of conditions for covalent coupling of trypsin on co-polymersof acrylonitrile and maleinic anhydride was achieved by variation of pH and trypsin initialconcentration. The maximal immobilized amounts of enzyme (as protein per g support) onco-polymers PANMA1 and PANMA2 at pH 4.0 were about 50.0 and 34.0 mg/g wet support,respectively. The relative amidase activity (substrate BAPNA) represented 17 and 19% ofthe corresponding enzyme activity in native state. Optimum protein concentration in theimmobilization solutions was determined to be about 12.5 mg/ml, and at this concentration47.0 and 28.0 mg trypsin/g wet support were bounded on these two polymers. At pH 7.5,the maximal relative activities of immobilized enzyme on these co-polymers reached 23 and14%, but at lower bounded protein quantities - 4.0 and 8.0 mg coupled protein/g wetsupport. The maximal bounded protein quantities were 14.0 and 27.0 mg trypsin/g wetsupport. Trypsin activity was also determined using macromolecular substrates, such ascasein. Immobilized on PANMA1 trypsin with 17% relative amidase activity retained only5% of its proteolytic activity. The immobilized trypsin preparations were tested forapplication in affinity chromatography. About 40% of the coupled enzyme was capable tobind specifically aprotinine.
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VETERINARY MICROBIOLOGY
VM1DEVELOPMENT AND APPLICATION OF RABBIT
POLYCLONAL MONOSPECIFIC AFFINITY PURIFIED ANTI-BODY AGAINST SYNTHETIC SEQUENCE FROM PEPTIDE
P32 TO DETECT SHEEP POX VIRUS BY IMMUNOHIS-TOCHEMICAL POLYMER STAINING TECHNIQUE ON
FROZEN TISSUE SECTIONS
Bumbarov V., Eligulashvili R., Yadin H.Kimron Veterinary Institute, Department of Virology, Beit Dagan, Israel
Conventional laboratory diagnosis of Sheep pox disease is based on transmissionelectron microscopy inspections, virus isolation in cell culture and confirmation by virusneutralization. However virus isolation is a lengthy procedure up to few weeks.
A new Rabbit Polyclonal Affinity Purified Monospecific antibody against syntheticsequence from peptide P32 of Sheep pox virus was elaborated and developed for rapiddiagnosis of Sheep pox infection.
In this report is discussed advantage at usage of Polymer Staining Techniques ofImmunohistochemistry on cryostat tissue sections using Rabbit Polyclonal Affinity PurifiedMonospecific Antibody against synthetic sequence from peptide P32 of Sheep pox virusas a primary antibody. The interpretation of results is very simple. All of procedure is upto 2 hours.
VM2НАХОДКИ И СЕРОТИПИЗИРАНЕ НА LISTERIA
MONOCYTOGENES В МЕСО ОТ ПИЛЕТА БРОЙЛЕРИ
Румен КараколевРегионален диагностичен ветеринарномедицински институт - Велико Търново
За петгодишен период (2000 – 2005 г.) са изследвани 464 проби от пилетабройлери - 224 от охладени и 240 от замразени пилета. Общо положителни за L.monocytogenes са 65 материала или 14,0 % от изследваните. От охладени пилетаса получени 42 изолата L. monocytogenes (18,75 %), а от замразени пилета – 23изолата от същия вид (9,58 %). След извършеното серотипизиране, 57 изолата саотнесени към серогрупа І и 8 – към серогрупа ІІ. От изследваните пилета саполучени и 9 изолата L. innocua.
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VM3ОПТИМИЗИРАНЕ НА ПВР-ДГГЕ ЗА ДИРЕКТНОДОКАЗВАНЕ И МОЛЕКУЛЯРНО ТИПИРАНЕ НА
CAMPYLOBACTER JEJUNI И CAMPYLOBACTER COLI ВЦЕКАЛНИ ПРОБИ ОТ ПТИЦИ
Х. НайденскиИнститут по микробиология “Стефан Ангелов” - БАН
Методът на ПВР-ДГГЕ (полимеразноверижна реакция - дегенеративнаградиентна гел електрофореза) е оптимизиран и успешно приложен за директнодоказване и типиране на Campylobacter jejuni и Campylobacter coli в цекални пробиот бройлери без предварително набогатяване.
27 щама Campylobacter jejuni и 1 щам Campylobacter coli бяха изследванипосредстом ПВР-ДГГЕ, използвайки праймерите WM12/WM22, произхождащиот 3’ края на flaA гена и 3’ края на интергенния участък, разделящ тандемноориентираните гени flaA и flaB. Получените ДНК фрагменти бяха разделени в 13групи, което е пряко доказателство за значителното разнообразие от щамовесред популацията от вида Campylobacter jejuni. Освен това, посредством ПВР-ДГГЕ бяха доказани бактерии от вида Campylobacter jejuni в изкуственоконтаминирани цекални проби, както и бактерии от видовете Campylobacter jejuniи Campylobacter coli в естествено контаминирани проби. Оптимизираниятпротокол на ПВР-ДГГЕ позволява надеждна и бърза детекция и идентификацияна Campylobacter jejuni и Campylobacter coli в цекални проби от птици - бройлерии носачки, с граница на позитивиране от 1 х 104 кое/мл.
VM4COMPARATIVE RESEARCHING OF PH IN SOME MUSELS
FROM SACRIFIED ANIMALS
A.Kuzelov; O. Kirovska CigulevskaMIK Sv. Nikole; Sveti Nikole, Macedonia e-mail; [email protected] ZZZ – Skopje; 1000 Skopje, Macedonia, e-mail; [email protected]
Some of the most important factor for identifying meat quality is pH; witch is about 7in the very first moment from sacrificing animals. That very first moment is known asmoment for starting physical, biochemical and structural changes in the meat structures.
Intensity of those changes depends of animal condition before sacrificing, geneticallycode, stress exposure of animals and muscle structure post mortem. The main processafter sacrificing animals is relieving of glucose and milk acid in the muscles. That is thereason for decreasing of pH factor and getting a PSE meat.
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In the read meat muscles that glucose relieving process has a low intensity, whichconsequence is low pH. Both of the processes are decreasing for the meat quality.
Our researching for PSE and DFD meat in some special kind of pork legs (yorkshire andlandrace). Researching was improved in muscle M. quadriceps femoris and M.semitendineus after 30 minutes and 24 hours from sacrificing of female animals. Theseresearching show us that in 13% from male animals had PSE meat and 12% from female hadto. DFD meat had only 2% from the male animals.
PSE meat (low quality meat) had bigger percentage that DFD meat in exploring dadanimals.
VM5ЕКОЛОГИЧЕСКИ АЛТЕРНАТИВИ НА
АНТИБИОТИЧНАТА ПРОФИЛАКТИКА И ТЕРАПИЯ
Христо Арнаудов1, Румен Караколев2, Пенка Калчева3
1ВТУ “Св.Св. Кирил и Методий” - Велико Търново,2РДВИ – Велико Търново,3СД „СИНАПС & ЕСКЛ” – Велико Търново
След триумфа на антибиотиците и сулфонамидите от средата на 20-ти век, всветовната литература напоследък се появи тенденция на повишено вниманиекъв естествените лечебни източници и преди всичко към билките.
В настоящата статия се прави преглед на предлагани от различни фирмилечебни и профилактични продукти на билкова основа, за ветеринарнатамедицина. Прави се оценка на тяхната ефикасност и безвредност. Изтъква се, чеизползвайки традиционните познания върху дрогите и съвременните научнипостижения /ултразвукова техника, биотехнологии и пр./ могат да се получатефикасни и безвредни конкуренти на антибиотиците от етерични растителни масла,полигликани, алкалоиди и др.
Авторите дават пример с получен от тях продукт от етерично масло отOriganum vulgaris и микробиялният полигликан Xanthan gum. Препаратът еефикасен срещу десетки патогенни микроорганизми, като същевременно неиндуцира появата на антибиотикова резистентност.
В заключение се препоръчва да се извърши цялостен скрининг на богататабългарска флора, който ще подпомогне създаването на широка гама отмедикаменти, биостимулатори, нутритиви и пр., като алтернатива на антибиотици,сулфонамиди и химически стимулатори.
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VM6ЛЕКАРСТВЕНА УСТОЙЧИВОСТ НА БАКТЕРИИТЕ ОТ
РОД MORAXELLA, ИЗОЛИРАНИ ОТ ЗАЙЦИ СПНЕВМОНИЯ
С. Иванова1, Н. Коруджийски1, Т. Гълъбинова2 Б. Митов1
1Национален Диагностичен Научноизследователски Ветеринарно МедицинскиИнститут- гр. София,2Институт за Контрол на Ветеринарни Продукти – гр. София
По методите на микробиологията са изследвани бели дробове от 174 домашнизайци с пневмония. Изолирани са и са определени таксономично 37 щамове отгрупа Moraxella.
Обсъжда се значението на тези щамове за пневмонията и тяхната лекарственачувствителност във връзка с терапията.
Провеждат се опити по метода на двукратните серийни разреждания вгеометрична прогресия.
Установява се, че щамовете Moraxella са устойчиви на В- лактами,тетрациклинии някои аминогликозиди., но са чувствителни спрямофлуорохинолони, което е особено важно за профилактиката и терапията напневмониите.
VM7ДЕЗИНФЕКЦИЯ НА ОБЕКТИ В ПЧЕЛАРСТВОТО,КОНТАМИНИРАНИ С PAENIBACILLUS ALVE И
ASCOSPHAERA APIS
Калинка Гургулова*, Даринка Илиева*, Йордан христов*, Стоил Караджов*,Емил Илиев**
*Национален диагностичен научноизследователски ветеринарномедицинскиинститут, София**Национална ветеринарномедицинска служба, София
Изпитана е биоцидната активност на Vircon S® срещу 3 щама на Paenibacillusalvei – причинител на европейския гнилец и срещу 3 щама на Ascosphaera apis –причинител на аскосферозата, като ефикасността е доказана с неутрализиращразтвор. Бактерицидното и фунгицидното действия бяха установени по методана количествено разреждане на суспензиите и чрез контаминирани тест-носители.Естериментите бяха извършени в лабораторни условия с различни концентрациии експозиции.
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Установено е, че 0,5 % Virkon S® показва гермицидна активност срещу P.alvei за 30 min и 1% Virkon S® е ефективен срещу A. apis за 15 min.
VM8МИКРОБИОЛОГИЧНИ ПРОУЧВАНИЯ ПРИ
ПСЕВДОТУБЕРКУЛОЗАТА ПО ОВЦЕТЕ И КОЗИТЕ
Н. Коруджийски, С. Иванова, Б. Гюров, Д. Тодоров, Цв. ДиковаНационален Диагностичен Научноизследователски Ветеринарно МедицинскиИнститут – гр. София
Проучванията са проведени при дедуктивни условия с 45 овце и 15 кози съссъмнение за псевдотуберкулоза.
Постмортално при овцете или при проведения Stemping out са взетиматериали от абсцедирани, казеозни лимфни възли и вътрешни органи.
При козите от живи животни са изследвани, абсцедирали долночелюстни иингвинални лимфни възли и форункули от млечната жлеза.
Получените микробиологични резултати показват, че псевдотуберкулозатапри овцете и козите е полибактериална инфекция при която се изолиратпредимно представители на род Corynebacterium / C. pyogenes, C.pseudotuberculosis, C.bovis /, род Streptococcus / групи С и Д / и най- честоStreptococcus група А.
VM9СРАВНИТЕЛНИ ПРОУЧВАНИЯ ВЪРХУ МЕТОДИТЕ ЗА
ДОКАЗВАНЕ НА MYCOBACTERIUM BOVIS ПРИЖИВОТНИ
Магдалена Боновска, Ст. Милашки, А. Абасс, Т. Савова, Е. ГюроваЦентрален Научноизследователски Ветеринарномедицински Институт, София
Използваните рутинни диагностични методи (патологоанатомичен,бактериологичен и биологичен), са сравнени с въведения в лаборатория“Туберкулоза” към НДНИВМИ молекулярно биологичен метод (PCR). За целтав продължение на 4 години са изследвани тъканни суспенсии от лимфни възли(111 броя проби), бял дроб (32), черен дроб (19), сърце (10), млечни проби (10),носни секрети (10) и кръв от реагирали съмнително и положително на туберкулинкрави от различни стопанства в страната.
От използваните методи най-ниска е чувствителността на микроскопскотоизследване (МИ) -средно 86%, по-висока на бактериологичното (БАК) - 94,8%,
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на патоанатомичното (ПА) - 97% и на PCR - 98,94%. Най-висока е чувствителносттана биологичния метод (БИО) - 100%, проведен с морски свинчета.
Средният процент на съвпадение между отделните стандартни методи и PCR есъответно: PCR/МИ –89,46%, PCR/БАК – 96,52%, PCR/ПА – 98,33%% и PCR/БИО– 100%.
Получените резултати показват, че по чувствителност PCR метода съвпада100% с биологичния метод и е по-чувствителен от останалите стандартни методи.Въвеждането на PCR може да отмени използването на биопроби. Като се вземепредвид и бързината на реакцията, убедително се доказва възможността завъвеждане на метода като дъпълнителен диагностичен тест, който да подобри иускори поставянето на диагноза туберкулоза.
VM10INFLUENCE OF KARBOFURAN ON CAPABILITY OF
FUSARIUM MONILIFORME TO PRODUCE FUMONISINSUPON MAIZE
Lidiya Borisova, Yuliana Tasheva, Velichka VrabchevaNacional Diagnostic and Research Veterinary Medical Institute “ Prof. dr GeorgePavlov “ – Sofia
It,s well known that mouldy of Fusarium species / Fusarium moniliforme /, producingfumonisins, are contaminators of maize in our country / Popova, T., 1984; Borisova, L.,2004 /. In fact that carbamate insecticide Karbofuran is widely used for treatment of maizeagainst storehouse,s pests, it will be interesting to investigate the influence of thisinsecticide upon fumonisins,s production in storage of grain.
The aim of our work was to investigate the influence of Karbofuran, especially the timeof adding of insecticide to contaminated substrate, on production of fumonisins.
The results show that effects of Karbofuran in dose 8 g/kg maize /concentracion usedin treated seed-corn/, as inhibitor of biosynthesis of fumonisins, reduse with increasingage of culture; on the 10 –th day there is no difference between control /non-treated/ andtreated culture; contents of fumonisins on the 3-th week after contamination withF.moniliforme both the samples are 12 ppm. The contents of fumonisins in maize, in whichKarbofuran was adding together with contamination of substrate is 4,12 ppm. The pesticideis most active inhibitor of production of mycotoxins as the Karbofuran is added 5 daysbefore contamination of substrate with F.moniliforme – the content of fumonisins is 0,76ppm.
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VM11DETERMINATION OF PSEUDOMONAS
AERUGINOSA IN BULL AND BOAR SEMENBY REACTION CO-AGGLUTINATION
Georgi VasilevRegional Research Institute of Veterinary Medicine, Stara Zagora, Bulgaria
Investigations were carried out on 118 samples of bull semen and 94 samples of boarsemen aimed at determination of P. aeruginosa by reaction co-agglutination. All sampleswith P. aeruginosa, determined by the conventional microbiological methods, manifestedpositive co-agglutination at 3 – 8-min intervals. All the control samples and all the samplesfree from pseudomonas microorganisms did not react. These results would encouragefurther investigations in search of other reliable methods for improvement of semen control.
Key words: Pseudomonas aeruginosa, Co-agglutination, immunization, semen.
VM12EXPERIMENTS OF INDUCTION OF TOXIGENOUS
MUTANTS OF CLOSTRIDIUM PERFRINGENS TYPE C
E. Iliev*, D. Ilieva**, M. Petkov*, N. Korudgiysky***National Veterinary Service, Sofia**National Diagnostic and Research Veterinary Institut “Prof. Dr. G.Pavlov, Sofia
Comparative studies are carried out to select Clostridium perfingens type C cultivatedon modified agar of Zeissler in six variants with different content of defibrinated blood oframs. As a result from the experiments the level of toxicity is studied on 4 generationsdaughter colonies of strain M. The isolated colonies of Cl.perfringens type C stabilized onliquid media with the highest blood content keep their toxicity for more than 75 days.
VM13ПРОМЕНИ В ПАТОГЕННИЯ ПОТЕНЦИАЛ НА YERSINIA
ENTEROCOLITICA ПРИ СЪХРАНЕНИЕ НАКОНТАМИНИРАНО СВИНСКО МЕСО
М. Илиев, Х. НайденскиИнститут по микробиология “Стефан Ангелов” – БАН
Патогенните серотипове на Yersinia enterocolitica и заболяванията, асоциирани
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с тях имат нарастващ социален и икономически ефект върху обществото. Зареализиране на техният патогенен потенциал са отговорни редица хромозомни иплазмидно детерминирани фактори на вирулентност.
Настоящето изследване има за цел да определи настъпващите промени впатогенния потенциал на Y. enterocolitica при различни температури и интервалина съхранение на контаминирано свинско месо, чрез мониторинг напреживяемостта и плазмидното носителство сред бактериалните клетки.
Разработена е селективна хранителна среда за мониторинг на фенотипнатаекспресия на плазмида на вирулентността (pYV), както и ПВР за генотипенмониторинг. Изследвани са промените в плазмидното носителство на 6 щама Y.enterocolitica от серотиповете O:3, O:8 и O:9.
Получените резултати разкриват изразена серотипна хетерогенност впреживяемостта и плазмидната дисоциация. Регистриран е спад впреживяемостта, детерминирана от дозата на инокулума, продължителността итемпературният режим на съхранение на месото. При съхранение на месото на+4oC тя е по-ясно изразена при щамовете от силно патогенния серотип O:8, всравнение с щамовете от по-слабо патогенните серотипове O:3 и O:9. При силнопатогенните серотипове се наблюдава по-слабо изразена плазмидна дисоциация(до 32%), за разлика от по-слабо патогенните серотипове при които загубата наpYV достига до 54% за O:9 и 63% за O:3. Подобен ефект се наблюдава присъхранение на месото при %20 oC. Паралелно провежданият мониторинг върхупреживяемостта и плазмидната дисоциация посредством ПВР потвърждавафенотипните изследвания и позволява бърза и надеждна детекция и диференциацияна pYV+ и pYV- бактериални клетки от популацията. След оптимизиране, ПВР сепозитивира в диапазона от 5х101 до 9х101 КОЕ/мл.
VM14ДОКАЗВАНЕ НА ПАТОГЕННИ СЕРОТИПОВЕ YERSINIA
ENTEROCOLITICAВ КОНТАМИНИРАНО ПРЯСНО МЛЯКО
М. Илиев, Х. НайденскиИнститут по микробиология “Стефан Ангелов” – БАН
Консумацията на контаминирани хранителни продукти представлява основенпът за иницииране на инфекция с Yersinia enterocolitica. Патогенните био/серотипове на вида са хетерогенна група, чийто патогенен потенциал екомплексно детерминиран от хромозомални и плазмидно локализирани фактори.Тяхното наличие и комбинирана детекция е важно условие за епидемиологичнатахарактеристика на контаминиращите щамове Y. enterocolitica.
Независимо от големият брой на регистрираните случаи на хранителниинфекции след консумация на мляко, контаминирано с Y. enterocolitica, значително
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по-често се изолират непатогенни щамове в сравнение с патогенните. За оценкана млякото и млечните продукти като потенциални рискови храни, е необходиморазработването на адекватна ПВР за детекция на Y. enterocolitica в изкуственоконтаминирани проби. Оптимизирането на реакцията включва избор на праймерии адекватна обработка на пробите, за отстраняването на възможните инхибиторина ПВР и съкращаване на времето за анализ. Като основни таргетни участъци отгенома са подбрани гените ail (хромозомно локализиран) и vir F (плазмиднолокализиран), разграничаващи патогенните щамове от серотиповете О:3, О:8 иО:9 на Y. enterocolitica от непатогенните, както и от други непатогенни видове народ Yersinia.
Изследвани са ефективността и детекционния лимит на ПВР в проби от прясномляко, изкуствено контаминирани с 6 плазмид-положителни (pYV+) щама Y.enterocolitica от серотиповете О:3, О:8 и О:9, съхранявани на +4oC за 24 часа и 72часа. Паралелно е провеждан фенотипен мониторинг на преживяемостта найерсиниите и плазмидното носителство на изолираните колонии чрез използванетона модифицирана хранителна среда. Оптимизиран е методът за обработка напробите и е постигнато значително намаляване на времето, необходимо заосъществяване на генетичния анализ. ПВР реакцията се позитивира в диапазонаот 1,1х101 до 1,5х101 КОЕ/мл при всички pYV+ щамове, което утвърждаваизползваната схема за директна детекция на патогенни щамове Y. enterocolitica вмляко.
VM15ВЗИСКАТЕЛНИ БАКТЕРИИ В ЕТИОЛОГИЯТА НА
ПУЕРПЕРАЛНИТЕ ЕНДОМЕТРИТИ ПРИ КРАВИТЕ ИВЪЗМОЖНОСТИТЕ ЗА ТЕРАПИЯ
Никола КоруджийскиНационален Диагностичен Научноизследователски Ветеринарно МедицинскиИнститут, София
Проведени са проучвания при крави с клиника на акутен катарално - гноенендометрит, при който от маточните изтечения не се изолират бактерии - порутинните микробиологични методи.
В нативни натривки от изтеченията на същите крави се установява присъствиена бактерии.
Култивирането на посевки от изтеченията върху специализирани хранителнисреди, в анаеробни условия в среда от 10% СО2 , позволява да се изолират щамовеBacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, при които савъзможни успешни терапевтични третирания с флуорохинолони и някоиаминогликозиди.
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VM16ЛЕКАРСТВЕНА УСТОЙЧИВОСТ НА БАКТЕРИИТЕ ОТ
РОД ALCALIGENES, ИЗОЛИРАНИ ОТ ЗАЙЦИ СИНТЕСТИНАЛНИ РАЗСТРОЙСТВА
Т. Гълъбинова1, Б. Митов2, Н. Коруджийски2, С. Иванова2, Л. Ангелов2
Институт за Контрол на Ветеринарни Продукти – гр. София1
Национален Диагностичен Научноизследователски Ветеринарно МедицинскиИнститут- гр. София2
Постмортално е изследвано чревно съдържание и вътрешни органи отдомашни зайци с акутен ентероколит, при което са изолирани 45 щамамонокултури на Alcaligenes faecalis.
Щамовете са проучени за чувствителност на химиотерапевтични средства пометода на двукратните сериини разреждания върху твърди хранителни среди.
Прави се заключение, че бактериите от вида Alcaligenes faecalis имат значениеза ентеритите при домашните зайци и се отличават с висока и широкоспектърналекарствена устойчивост, което е от интерес за профилактиката и терапията.
VM17НЕОПЛАЗИИ ПРИ ЕСЕТРОВИ РИБИ
Ваня Чикова, Вера Коларова, Ангел Цеков*Национален научноизследователски ветеринарномедицински институт – София;* Институт по рибарство и аквакултури - Варна
Значителното намаляване на естествените популации от есетрови риби гипревръща в атрактивен обект за аквакултура .
Тумори при риби от този вид са сравнително рядко срещани. Изследваниятаобхващат материал от 12 есетрови риби от вида Acipenser guldenstadti от 2рибовъдни ферми в страната. Описана е наблюдаваната клинична картина и сапредставени резултатите от патологоанатомично и патохистологично изследванена туморовидни образувания, разположени в основата на хрилните дъги.
Установени са също макроскопски и микроскопски промени в черен дроб,далак и сърце.
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VM18СЛУЧАЙ НА АЕРОМОНОЗА ПРИ ВЕСЛОНОС
Ваня Чикова, Таня Хубенова*, Вера Коларова, Райна Атанасова*, АнгелЗайков*Национален научноизследователски ветеринарномедицински институт - София* Институт по рибарство и аквакултури - Варна
При опитите за интродуциране на веслонос /Polyodon spathula / като нов видза аквакултура в страната е установена висока смъртност и е наблюдаванахарактерна клинична и патологоанатомична картина.При микробиологичнитеизследвания за доказване и идентифициране на етиологичния агент - Aeromonashydrophila са използвани стандартни микробиологични техники и експреснасистема за бактериална идентификация Мerck MCN6.
PRESENTATIONS OF FIRMS
ZEU-IMMUNOTEC
MICROBIOLOGICAL TESTS FOR SCREENING OF ANTIBI-OTIC AND SULFONAMIDE RESIDUES IN FOOD
Dr. Elena Domнnguez- ZEU-INMUNOTEC, Maria de Luna 11, Zaragoza, Spain
Veterinary medicines are commonly used for prevention or treatment proposes byproducers of animal and animal products across the world. Residues of these drugs cancarry over into foods such as fresh meat, meat or dairy products, fish and honey.
Veterinary residues pose a hazard to consumers by causing allergic reactions anddeveloping bacterial resistance. They also have an inhibitory effect on starter cultures,used in fermented products, causing financial losses in the food industry. In order toprevent from residues going to the food chain strict EU regulations are in force.
Food testing is strongly advised to ensure anti-microbiological residues are below theMaximum Limits of Residues (MLR) set in the European legislation.
Microbiological methods, based on the bacteria growth inhibition, are used for screeningof a broad range of antibiotic and sulfonamide residues. These methods are available ascommercial kits allowing food producers and farmers to run their own self-control. Studiesto check the performance of these test kits show good sensitivity to comply with theEuropean MRLs. They are also very easy to use and require minimum laboratory equipment.More than 90 samples can be screened in about 3 to 4 hours on site or in the lab.
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HYGIENA INTERNATIONAL LTD
HYGIENE MONITORING IN SUPPORT OF FOOD SAFETY -A REVIEW OF METHODS AND TRENDS
Ing. Frans Martens, Hygiena International Ltd., United Kingdom
Good Hygienic Practices are an essential to ensure food safety. They are required bylaw under national and international Food Hygiene Regulations and are frequentlyconsidered as pre-requisites to food safety systems based on Hazard Analysis such asHACCP. The presentation will discuss the availability of simple, user friendly and costeffective rapid methods for checking sanitation programs and day to day hygienemonitoring. These methods give immediate feedback to the quality of the cleaning andhave proven to be a powerful tool in food processing industries.
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INFECTIOUS IMMUNOLOGY
II1QUALITY OF ANALYSIS
IN CLINICAL MICROBIOLOGY
I. Smirnov, Moskow, Russia
II2IMPROVED POOLED IGG PREVENTS DEATH
IN EXPERIMENTAL SEPSIS
Tchavdar Vassilev, Jordan Dimitrov and Nina IvanovskaStephan Angeloff Institute of Microbiology, 1113 Sofia, Bulgaria
Sepsis and septic shock are one of the major causes for death worldwide. Despite thehigh numbers of patients involved, these medical conditions rarely make headlines. Thelife-threatening symptoms of septic shock are the result of a generalised, uncontrolledinflammatory reaction of the body to an invading microorganism (bacterium, virus or afungus). The high mortality in some emerging diseases (e.g. of avian flu) is also due to adysregulated inflammatory response. Currently available therapeutic strategies in sepsisare not satisfactory as far as efficacy is concerned. A better understanding of the molecularmechanisms that are implicated in the pathogenesis of sepsis has provided us withindications on the design of new class of immunomodulatory agents to combat thegeneralized inflammatory reaction in sepsis. The use of therapeutic immunoglobulin (Ig)preparations represents one such approach in the prevention and in the treatment ofsepsis and septic shock. Immunoglobulin preparations exert anti-inflammatory activity,mediated by their interactions with complement components, inhibitory receptors onimmune cells, down-regulation of T-, B-cell and dendritic cell functions and effect oncytokine networks. These anti-inflammatory activities do not result in immunosuppressionunlike high doses of corticosteroids. However, Ig preparations in their present form havenot been fully successful in preventing sepsis-related complications. Therefore, there isan urgent need for improving the currently available therapeutic strategies to counterthese problems. Conception and designing of next generation Ig preparations is a criticalnecessity.
We have recently developed a technology to produce improved pooled therapeutichuman immunoglobulin G with strongly enhanced anti-inflammatory activity. It is basedon the exposure of IgG to prooxidative ferrous ions or to reactive oxygen species. This
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treatment results in enhanced IgG paratope flexibility and hydrophobicity, leading toexpansion of the spectrum of recognized antigens, regulation of cell proliferation andprotection in experimental sepsis. The injection of a single dose (30 mg/kg) of Fe(II) ion-exposed pooled human therapeutic IgG, but not of native IgG, resulted in the preventionof death in an experimental model of sepsis. A beneficial effect was also seen when usinga dose of 150 mg/kg, but not of 6 mg/kg).
II3ЕФЕКТ НА YOPK ПРОТЕИНА НА YERSINIA PSEUDOTU-
BERCULOSIS ВЪРХУ ИНВАЗИН-МЕДИИРАНОТОПОГЛЪЩАНЕ ОТ HELA КЛЕТКИ
Е. Иванова1, Х. Найденски1, А. Веселинова1, F. Deleuil2, H. Wolf-Watz 21Институт по микробиология “Стефан Ангелов” - БАН, София, България2Университет Умеа, Швеция
Патогенните бактерии от род Yesinia (Y. pestis, Y. enterocolitica и Y.pseudotuberculosis) притежават тип III секреционен апарат, който експортираефекторни протеин, т.н. Yops (Yersinia outer proteins), кодирани от плазмида навирулентността (pYV). Част от тези протеини се транслоцират в еукариотнатаклетка, което води до възпрепятстване на фагоцитозата и подтискaне навъзпалителния отговор. Един от тези протеини – YopK, е необходим на Y.pseudotuberculosis да предизвиква системна инфекция, а нивото на експресия наYopK влияе върху нивата на транслоцираните Yops в гостоприемниковата клетка.
Целта на настоящето изследване е, да се определи ефекта на YopK върхуинвазин-медиираното поглъщане на Y. pseudotuberculosis от Hela клетки и далиекспресията на YopK протеина от трансфектирани с yopk гена Hela клетки могатда променят ефекта на хипертранслокация на други Yops при ÄyopK мутантeнщам. Получените резултати показват, че YopK протеинът не участва в процесана блокиране на инвазин-медиираната фагоцитоза. Наличието му в еукариотнитеклетки преди инфекция, не може да индуцира сигнален път в клетката, отговоренза контрола върху количеството транслоцирани Yops при ÄyopK мутантния щам,нито да блокира Yop транслокацията при дивия щам.
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II4RAPID IMMUNOHISTOCHEMISTRY METHOD FOR DE-TECTION OF TSE PRION PROTEIN ON FROZEN BRAIN
TISSUE SECTIONS
Lubashevsky E., Yadin H., Perl S., Adry N., Gorochov A., Bumbarov V.
Prion diseases are manifested as genetic, infectious or sporadic , lethalneurodegenerative disordes involving alterations of the prion protein (PrPc). PrPc isconstitutively expressed in normal adult brain and is sensitive to proteinase K digestion,while the altered PrP-scrapie (PrPsc) conformation is resistant to proteases. AbnormalPrPsc is found at high levels in the brains of animals affected by scrapie in sheep, BSE incattle and Creutzfelt-Jacob disease in humans.
Procedures that detect PrPsc, such as Western immunoblotting, ELISA technologyand immunohistochemistry (IHC) techniques, ara based on the ability to identify infectedanimals subclinically by detecting small quantities of PrPsc. The current IHC method –gold as standard – used to supplement or replace histophathology as the method ofchoice for diagnosing diseases ,to obtain reliable results.To maximize sensitivity of IHCrequired brain is usually preserved in the fixative paraformaldehyde, lysine, periodic acidand hydrated autoclaving before immunostaining. These procedures need a relativelylong time (5 days) and sometimes led to artifacts that affect tissue morphology. The aim ofthe study is the development of raped efficient HIS method for mass screening prionprotein.
In proposed IHC PrPsc detection is carried out in frozen tissue brain preparations.
II5“RESPIVAX” MODULATES THE EXPRESSION OF CD86 ON
DIFFERENT CIRCULATING APC SUBSETS
D.Stankulova1, A. Mihova1, H. Taskov1, Vl. Maximov2, M. Nikolova1
1Central Laboratory of Immunology, National Center of Infectious and ParasiticDiseases, Sofia, Bulgaria2Specialized Hospital for active treatment of Pulmonary Diseases “St Sofia’, Sofia,Bulgaria
Background: The oral immunomodulator “Respivax” (BulBio-NCIPD) has a wellestablished stimulatory effect on cellular immune responses. However, the underlyingmechanisms have not been studied in detail. The efficient activation of naïve T lymphocytesrequires the delivery of second signal by the antigen-presenting cell (APC), and CD86(B7.2) is one of the best characterized co-stimulatory molecules on human APC. Aim: Tostudy the effect of “Respivax” treatment on CD86 expression by different subsets of
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circulating human APC. Material and methods: Fifteen patients with recurrent non-specificrespiratory infections were subjected to a standard course of “Respivax” treatment.Heparinized whole blood samples were collected at baseline, day 20 and day 80. Multicolorflow cytometry (FACSCanto, B-D) was used to study CD86 expression on circulatingdendritic cells (DC, lin-HLA-DR+), monocytes (Mo, CD14+), and B Ly (CD19+). Accordingto the mean fluorescence intensity (MFI) of CD86 expressionCD86low and CD86high DC weredistinguished. The effect on B Ly was assessed after 24h in vitro stimulation of wholeblood with LPS. Results: “Respivax” treatment increased the CD86+ DC population from68.7% to 76.2%, (p<0.01) and promoted its maturation resulting in the increase of CD86high
subset as compared to baseline (a mean of 52% vs. 43%, p<0.05). This increase was moresignificant in a subgroup of patients with initially decreased CD86high DC level (a mean of56.8% vs.39%, p<0.01), and was already detectable at day20. Further on, CD86 MFI wassignificantly increased on Mo from patients with initially lower intensity of CD86 expression(1318 vs. 1071, p<0.01), already at day 20. At baseline 38.2% of the LPS -stimulated B Lyexpressed CD86 vs. 44.6% at day 20 (p<0.02), and 48.2% at day 80 (p<0.01). Conclusion:These results suggest that one of the mechanisms for stimulation of T cell immune responsesby “Respivax” is the increased co-stimulatory potential of circulating APC, and hence -the possibility for activation of naïve T-lymphocytes.
II6КОЖНИЯТ ТУБЕРКУЛИНОВ ТЕСТ В ОЦЕНКАТА НА
ПОСТВАКСИНАЛНАТА БЦЖ АЛЕРГИЯ . КАЧЕСТВА НАБЪЛГАРСКИЯ PPD ТУБЕРКУЛИН
Е. Сапунджиева, Е. Йорданова, БУЛ-БИО, НЦЗПБ, СОФИЯ
Кожната туберкулинова чувствителност, определена чрез теста на Mantouxсе използва много години за диагностика при туберкулозата, както и за оценкана постваксиналната алергия. Видът и размера на кожната реакция зависи отмного фактори, в това число: вида на щама и дозата на ваксината, възрастта наваксинирания, начина на хранене, годините изминали след ваксинация, честотатана кожния туберкулинов тест.
За стандартност при оценката на кожния тест от първостепенна важност еизползвания туберкулин и техниката при прилагане и отчитане на резултата.
Представен е анализ на данните от стабилността на българския PPD Tuberculin,при изследвания проведени съгласно Европейската фармакопея.
Препаратът при съхраняване на тъмно в хладилник при температура 20 – 80С,запазва своите химико-физични и имунобиологични свойства в продължение наповече от 3 години.
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II7ПОЛОВИНВЕКОВНА ИМУНОПРОФИЛАКТИКА НА
ТЕТАНУС С БЪЛГАРСКА ВАКСИНА
Е. Сапунджиева*, И. Тодорова*, Е. Йорданова*, Й. Христова*, М.Демирева**, Р. Мълчанова***БУЛ БИО-НЦЗПБ, София;** Столична хигиенно-епидемиологична инспекция (СХЕИ)
Тетанусът е остро спорадично ранево инфекциозно заболяване, протичащо свисок леталитет. И до днес няма специфично лечение, като прилаганата терапиясе свежда до опит за туширане на характерната неврологична симптоматика.
Единственото средство е специфична имунопрофилактика с тетаничентоксоид. В България тя е въведена през 1959 г.
В резултат на постепенното до пълно обхващане на населението,заболяемостта от 4.8%000 през 1945 г. е сведена до единични случаи приневаксинирани лица. През 2003 и 2004 г. заболяемостта е 0%000. Изследван еимунологичния статус на повече от 6 000 лица от различни възрасти (0 до 90години). Резултатите показват, че 96% от българското население е защитено оттетанус (титър на тетанични антитела ≥ 0.01 IU/ ml). Интерпретират се различиятав степента на защита в отделните възрастови групи. Нередовното обхващане среимунизации води до почти 50% незащитеност при лица в активна трудовавъзраст. Последното крие потенциален риск от заболяване при нараняване.
II8MONITORING OF CYTOTOXIC CD8 T LYMPHOCYTES IN
HIV-1+ PATIENTS SUBJECTED TO HAART
M. Muhtarova, M. Nikolova, S. Magaev, H. TaskovCentral Laboratory of Immunology, National Center of Infectious and ParasiticDiseases
Background: One of the goals of highly active antiretroviral therapy (HAART) isrestoration of the differentiation and functional potential of CD8+ cytotoxic T cells (CTL)in HIV patients. We have previously reported that the expression of CTL receptor CD160is increased in HIV patients and may predict a good therapy response.
Aim: To study the changes in the co-expression of the cytotoxic mediators granzymeB (GzmB) and perforin (Per) in HIV-1 patients subjected to HAART, in relation to the CD8T cell differentiation/functional subsets defined by CD160/CD27/CD28 co-expression.
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Material and methods: Heparinized whole blood from 9 treatment-naive HIV(+) patientswas analyzed at baseline and after 6 months of HAART in comparison with 11 age- andsex-matched non-treated HIV+ patients and 10 HIV(-) healthy controls. GzmB/Perintracellular co-expression and CD27/CD28/CD160 membrane co-expression on CD8+ Tcells were studied by multi-color flow cytometry (FACSCanto, B-D).
Results: At baseline, HIV+ patients were characterized with increased percentage ofintermediate (CD27+CD28-), late (CD27-CD28-) and CD160+ CTL as compared to HIV-controls. Although the percentage of functional GzmB+Per+ CTL was increased in HIV+patients (10.4 vs. 3.8, M-W p<0.05), immature GzmB+Per- CTL predominateddisproportionately (39.9 vs. 5.8, M-W p<0.001). At second examination, untreated patientshad no significant changes in any of the studied parameters. However, HAART induced asignificant increase of the GzmB+Per+ (functional) CTL subset (22.5 vs.10.4, Wilcoxon,p<0.05), Importantly, GzmB+Per+ CTL correlated with the subset of late CTL, expressingCD160 molecule (CD27-CD160+, Spearman R=0.7, p<0.02).
Conclusion: Expression of CD160 on CD8+ T cells in chronic HIV infection is importantfor monitoring the restoration of cytotoxic function in the course of HAART.
II9PROTECTION AGAINST WHLOOPING COUGH IN
CHILDREN BETWEEN 0-6 YEARS OLD
R. Alexiev1, K. Hadjiisky1, S. Malchanova2, V. Demireva2 and Pl. Nenkov1
1 BB - NCIPD Ltd. - Sofia, Bulgaria; 2 Hygiene Epidemiological Inspection - Sofia,Bulgaria
The specific immunoprophylaxis of humans with pertussis, as a component of combinebacterial vaccine leads to production of specific antibodies that is indicator of the whoopingcough prevention. For evolution of immunization procedures and the vaccine itself,antibody levels against pertussis are useful to show the immune status of the population.An enzyme-linked immunosorbent assay was used for measuring immunoglobolin Gpertussis antibodies in human sera. The ELISA involves the blinding of bacterial cells topolystyrene tubes. Results of the direct ELISA test are highly reproducible. It is believedthat a pertussis antibody level of 1:81 provides protection against disease. The titters ofantibody from 1:161 to 1:320 showed full protection of people against whooping coughand titters over 1:321 are used as a criteria of disease or used as a criteria of passeddisease. The results of estimation of immune status of population in Bulgaria were basedon the same criteria.
The assay was done in plastic plates coated with inactivated bacterial cells. Forinvestigation 279 human sera were tested in age group between 0 – 6. The tested serashowed that 54% of persons have full protection against whooping cough and in 42% ofchildren the obtained titters showed basic immunity. In 4% of patients the titter of antibodyis more than 1:321, which titter is used as a criteria of disease of human sera. All of sera
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have good level of protection against whooping cough and non-protected patients werenot found.
Present results indicate a good protection against pertussis in Bulgaria in children till6 years old. This fact is a result of specific immunoprophylaxis by pertussis vaccine, as acomponent of DIFTETKOK (DTP) combined bacterial vaccine, produced by BB – NCIPD,Ltd.
II10INVESTIGATION ON THE IMMUNE STATUS OF
THE POPULATION AGAINST DIPHTHERIA DURING THEPERIOD 2001 – 2005
R. Alexiev1, K. Hadjiiski1, S. Malchanova2, V. Demireva2 and Pl. Nenkov1
1 BB - National Centre of Infectious and Parasitic Diseases - Sofia, Bulgaria2 Hygiene Epidemiological Inspection - Sofia, Bulgaria
The specific immunoprophylaxis of humans with diphtheria toxoid, as a component ofcombine bacterial vaccines leads to production of specific antibodies that have main rolein diphtheria prevention. An enzyme-linked immunosorbent assay was used for measuringimmunoglobolin G diphtheria toxin antibodies in human sera. The assay was done inplastic plates coated with purified diphtheria toxoid. 4711 human sera in different agegroups were tested for investigation. The estimations showed that 67.8% of tested serahave full protection against diphtheria, 4.4% have basic immunity and in 27.8% lackprotection against disease.
The comparison of different groups of age showed that the best protection againstdiphtheria have in children and teenagers younger than 15 years old. In these groups ofage the sera without antibodies against diphtheria are significant. Aging of people leadsto reduction of the percent of protected persons and to increasing the percent of non-protected people. Despite this, the percent of protected population in group of age between16 to 65 years old is better than in some European countries and in the USA. The patientsover 65 years old had very low levels of antibodies against diphtheria and in about 83%lack protection against diphtheria.
Present results indicate a good protection against diphtheria in Bulgaria. This fact is aresult of specific immunoprophylaxis by diphtheria toxoid, as a component of combinedbacterial vaccines, produced by BB – NCIPD, Ltd.
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II11DETERMINATION OF MINIMAL SENSITIZING DOZE OFBCG VACCINE (SUBSTRAIN SOFIA SL222) IN GUINEA PIG
T. Stefanova1, M. Chouchkova1 , S.Nikolaeva2
1BCG Laboratory, BB-NCIPD Ltd., Sofia2Laboratory of Imminomorphology; NCIPD, Sofia
During the investigation of the allergenic potency of BCG strains it is advisable tovaccinate with decreasing doses of vaccine in order to estimate the lowest dose thatinduces tuberculin sensitivity, rather than to compare the effect of large doses of BCG.The impact of the BCG vaccine dose on the development of cell-mediated and protectiveimmunity in the guinea pig model has been intensively discussed in different studiesduring the last few years.
Although the biological test does not reveal the mathematical correlation of dose-effect, it is important to look for the determination of the minimal sensitizing dose for everyBCG vaccine. In this study twelve groups of six guinea pigs each were vaccinated withthree different doses (0.12 ng, 1.2 ng and 12 ng) of freeze-dried vaccine (Batch No 42,NCP=2.698x106/ml). Tuberculin tests were performed in different groups at the 30th, 60th,90th and 120th day after BCG injection. The negative tuberculin reactions converted topositive between 60th and 90th day. The dose of 1.2 ng elicited the largest tuberculinreactions in the animals. This dose contained only about 64-65 culturable particles andcould be regarded as the smallest effective sensitizing dose of the BCG vaccine producedfrom Working Seed Lot 7-93. The histological examinations demonstrated that a very lowinoculum (0.12 ng or 6 viable cells) is sufficient to induce a specific inflammation after BCGvaccination.
II12ЛИЧЕН ОПИТ ПО ОТНОШЕНИЕ НА ДИАГНОСТИКАТА ИДИФЕРЕНЦИАЛНА ДИАГНОСТИКА НА ПТИЧИЯ ГРИП
В.Люпке1, В. Йонкова2, В. Люцканова2
1 ВУ “Земеделски колеж”- Велико Търново;2 МУ – Варна, катедра Микробиология
Територията на Р.България се намира под трасета и сборни места на редицавидове прелетни птици, които могат да бъдат преносители на вируса на птичиягрип. Съществува реална опасност и риск от заразяването на хора и животни припо-тесен контакт с фецес, секрети и трупен материал.
Диференциалнодиагностично трябва да се изключват псевдочума по птиците,различни видове инфлуенцавируси и Хламидии.
В настоящето съобщение са представени прилаганите от нас лабораторно-
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диагностични методи за доказване и диференциране на миксовируси и някои другипричинители на респираторни заболявания. Предлага се създаване наспециализирани за тази цел лаборатории.
II13CHLAMYDIA TRACHOMATIS (CT) ANTIBODIES IN SERUM
AND GENITAL FLUIDS IN INFERTILE COUPLES
V. Yonkova1, V. Lyoutzkanova1, V. Savouleva2, Y. Yonkov3
1 Dept. of Microbiology and Virology, Medical University – Varna;2 Dept. of Obstetrics and Gynecology, Medical University – Varna;3 Dept. of Anatomy, Histology and Embryology, Medical University – Varna
Chlamydia Trachomatis (CT) infections of the genital tract of women and men, althougha major cause of infertility, are often asymptomatic and undetected. The majority of coupleswho are infertile have no history of a sexually transmitted disease or pelvic inflammatorydisorders. Since many infertile women now seek in vitro fertilization that is a problem ofpriority to detect or to exclude the relation between an unsuspected CT infection andinfertility. The most useful marker of that condition is the local antiCT sIgA, which isassayed to look for persistent antigenicity, potential subsequent disorders and partuer’scontamination.
By using conventional methods, materials (cervical mucus – 63, prostatic fluid – 38,semen – 55, sera – 100) of 55 men and 63 women were tested. All samples were free fromblood. The positives results to CT infection we detected are as follows: 38,1 % (21) of menand 47,6 % (30) of women. Differences in the prevalence of CT infection between thevarious outcome groups did not reach statistical significance (P = 121). AntichlamydialIgA was present in the cervices of 25,2 % of the women with endometrioses, 18,1 % ofwomen with tubal factor infertility, and 14,3 % of women whose infertility was due to poorsperm quality. In conclusion, women with evidence of a current or recent CT genital tractinfection had a poorer outcome in their in vitro fertilization cycles than did women with noevidence of this infection.
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II14IMMUNOBLOT ANALYSIS OF ANTIBODY RESPONSE
TO PLASMID ENCODED RELEASED PROTEINS OFYERSINIA ENTEROCOLITICA IN PATIENTS
WITH REACTIVE ARTHRITIS
Elica Golkocheva1, Hristo Najdenski1 and Rumen Stoilov2
1 Department of Pathogenic Bacteria, The Stephan Angeloff Institute of Microbiol-ogy, Bulgarian Academy of Sciences, Sofia; 2 Clinic of Rheumatology, The MedicalUniversity, Sofia
Yersinia enterocolitica has been identified as causative organism of reactive arthritisin humans. Antibodies developed against the Yersinia outer membrane proteins /Yops/,in cases of chronic enteritis and reactive arthritis, usually persist at high levels for longertime periods. Immunoblot analysis of anti-Yersinia antibody response of blood sera frompatients with reactive and rheumatoid arthritis was carried out. The assay was applied byusing plasmid encoded Yops as antigen. Seven strong bonds of the molecular weights: 26kDa - YopE, 33 kDa - YopN, 36 kDa - YopD, 41 kDa - V-ag, 43 kDa - YopB, 46 kDa - YopM and51 kDa - YopH were visualised. In sera from patients with reactive arthritis IgG antibodieswere detected against all the seven Yops. IgA antibodies were established against YopM,YopB, YopD, YopN and YopE. Sera from the patients with other rheumatic diseases weretested for the presence of anti-Yersinia antibodies too. These sera were negative for thepresence of anti-Yersinia IgA antibodies, but two of them were positive for IgG againstYop D. Antibodies from the classes IgA and IgG were not detected in the serum samplesfrom healthy people.
II15ATTENUATION AND PRESERVED IMMUNOGENIC
POTENTIAL OF YERSINIA PSEUDOTUBERCULOSISMUTANT STRAINS EVIDENCED IN ORAL PIG MODEL
H. Najdenski1, E. Golkocheva1, E. Ivanova1, V. Kussovski1, A. Vesselinova1, S.Garbom2, H. Wolf-Watz2
1 The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Sofia, Bulgaria;2 University of Umea, Umea, Sweden
Experimental oral infection of pigs with a wild type Yersinia pseudotuberculosis strainpIB102, serotype O:3 and two mutant isogenic strains – pIB155,”yopK and pIB44,”ypkAhas been carried out. Clinical findings, microbiological and immunological parameterswere examined in dynamics from day 7 to day 60 post infection (p.i.).
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All types of infections ran asymptomatically, without hyperthermia, loss of appetite,etc. Experiments on the blood parameters demonstrated a transient leukocytosis withlymphocytosis and monocytosis better expressed after pIB155"yopK infection. Eventhough pig is usually known as a reservoir of yersiniae, bacterial colonization was foundin tonsils and mesenterial lymph nodes on days 7 and 14 p.i. with wild type strain, andonly in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity ofmutants to the bactericidal effect of leukocytes and blood sera are the characteristicfeatures of attenuation in their pathogenicity, in comparison with the wild strain.Comparative in vitro experiments on the immune response and immunostimulating capacityof Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential,predominantly in case of pIB155"yopK. Immunomorphological rearrangements like ahyperplasia and strong activation of the lymphoid tissue of Peyer’s patches, mesenteriallymph nodes, spleen and peribronchial lymph tissue of pigs challenged with both mutantstrains were proved. The results obtained give the reason to claim that the geneticallyconstructed yopK null mutant strain is significantly attenuated but still immunogenic andhas the potential for a live vaccine carrier strain.
II16HEMOCYANINS AS IMMUNOSTIMULATORS
Reneta Toshkova 1, Emilia Ivanova2, Maria-Dorothea Nastke3, Lyudmila Velkova4,Stefan Stevanovic3, Rumyana Hristova4, Alexandar Dolashki4, Maria Gardeva4,Ivan Dimitrov4, Wolfgang Voelter5, Pavlina Dolashka-Angelova4
1 Institute of Experimental Pathology and Parasitology, BAS, Bulgaria;2 Institute of Microbiology, BAS;3 Department of Immunology, Institute for Cell Biology, University of Tьebingen,Germany;4 Abteilung fьr Physikalische Biochemie des Physiologisch-chemischen Instituts derUniversitдt Tьbingen, Germany;5 Institute of Organic Chemistry, Bulgarian Academy of Sciences, Bulgaria
Hemocyanin from the giant keyhole limpet Megathura crenulata has been a subject ofbiomedical interest because of its remarkable immunostimulatory properties in experimentalanimals and man. Molluscan Helix vulgaris (HvH) and Rapana venosa (RvH), andarthropodan Carcinus aestuarii (CaH) hemocyanins have been studied in order to evaluatetheir potential biochemical and medical applications.
It was established that the serum IL-2 production was better expressed in animalsimmunized by HvH and CaH then with the native molecule of KLH. Increased IL-2production in supernatants of in vitro cultivated lymphocytes was observed in animalsimmunized with native CaH and KLH. Spleen cells from the mice immunized with otherhemocyanins showed negligible stimulation. It was found that CaH causes increased
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specific and non-specific proliferation of spleen lymphocytes and Th1 associated cytokinproduction in BDF1 mice.
The effect of the molluscan Rapana venosa hemocyanin on the antibody-dependentcell cytotoxicity (ADCC) and mitogen responsibility of spleen lymphocytes from hamsterswith transplanted myeloid Graffi tumors was demonstrated. After treatment of animalswith KLH or RvH the spleen lymphocyte ADCC decreased during tumor progression,while ADCC of spleen lymphocytes against own tumor cells increased about twofold incomparison to that of lymphocytes from untreated tumor bearing hamsters (TBH). Thelymphocytes isolated from normal animals without treatment showed two times lowercytotoxic activity compared to those from RvH- or KLH- treated controls. RvH induced 3-5% higher ADCC compared to KLH in all combinations of sera and lymphocytes. Wesuggest that this action is caused by stimulation of the Th-1 and T-cytotoxic lymphocytepopulation as well as unspecific lymphoproliferative properties of Hc preparations.
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PARAZITOLOGY
P1EMERGING AND REIMURGING PARASITIC DISEASES IN
BULGARIA
Kurdova-Mintcheva R., D. Jordanova, T. Marinova, M. Ivanova, N. Tsvetkova, I.Marinova, R. Harizanov
The impact of emerging and reemerging parasitic diseases in Bulgaria and their healthinfluence have increased in recent years. Among them, toxoplasmosis, cryptosporidiosis,pneumocystosis, visceral leishmaniosis, blastocystosis, cystic echinococcosis andtrichinellosis are the leading diseases.
Seroprevalence of toxoplasmosis in the population during the past 10 years was 31.21%-47.7% with 40-50 cases of acute primary infection detected per year. Cases ofcryptosporidiosis and blastocystosis among travelers, immunocompromised persons andpatients with intestinal disorders were recorded. Indigenous visceral leishmaniasis is alsoon rise with endemicity in the southern part of the country. All protozoan infectionsmentioned above are often associated with AIDS.
Regarding human cystic echinococcosis, since 1993 there has been a marked rise inmorbidity - 8.47 per 100 000 population in 1996 and 8.32 per 100 000 population in 2002,with an average of 6.37 per 100 000 population for the last decade. In some regions (Sliven,Burgas, Chaskovo, Pazardjick, etc.), the morbidity is much higher (15-27 37 per 100 000population) than the country average. In Bulgaria, trichinellosis outbreaks and sporadiccases as well as an increased morbidity among the human population has been on the rise,too. During 1991-2005, the number of outbreaks registered was 139.
The situation regarding emerging and reemerging parasitic diseases in the countryclearly demands joint organized measures for their surveillance and control.
P2EPIDEMIOLOGICAL ASPECTS OF HUMAN
TRICHINELLOSIS IN BULGARIA (2001 – 2005)
M. Ivanova1, R.Kurdova1, D.Jordanova1, N.Tsvetcova1
National Center of Infectious and Parasitic Diseases, NCIPD, Sofia
Human trichinellosis is an important zoonosis, which is annually registered in Bulgaria.Among all ages and population groups, every year trichinellosis outbreaks and sporadiccases are reported. Worldwide different encapsulated and non-encapsulated Trichinella
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species and genotypes have been described. Some differences have been observed inthe signs, symptoms and clinical course of the disease, caused by various Trichinellaspecies.
The epidemiology of infection greatly varies depending on Trichinella species. Recentinvestigations discovered that in Bulgaria besides T.spiralis, cases of human trichinellosisare also associated with T.britovi. During 2001 – 2005 period, altogether 49 outbreaks and73 sporadic cases of human trichinellosis have been officially registered in the country.For this period 965 people were reported as consumers of infected meat products and 813(84,25 %) of them developed asymptomatic or clinical form of trichinellosis.
Basic epidemiological aspects of the disease, peculiarities in geographical distributionof the registered cases and tendencies, which were observed over the past years, regardingalso the source of infection and Trichinella genotypes involved, were revealed in thestudy.
P3STUDY ON THE DIAGNOSTIC VALUE OF SPECIFIC IGE
ANTIBODIES IN HUMAN ECHINOCOCCOSIS
I.Marinova, G.Nikolov, A.Mihova, R.Kurdova, B.Petrunov
Cystic echinococcosis is a widespread parasitic zoonosis, caused by the cestodeEchinococcus granulosus. In human, as intermediate host, develops the larval stage ofthe parasite and different antigens (cyclophilline, EgEF-1) lead to the formation of specificIgE antibodies. Although the contemporary immunologic methods detect them only in 42– 52 % of the patients, the specificity is very high – 95 – 100 %.
In the present study150 sera – 75 sera from patients with proved echinococcosis and75 sera from people without echinococcosis (blood donors, patients with nonparasiticdiseases and samples with false positive results for IgG in IHA or ELISA) were examined.Automatic system UniCap, based on ELISA, was used for detection of IgE antibodies.The level of the specific antibodies was determined quantitatively and the results werearranged in 7 patients groups. The examination of the sera from patients with echinococcosisrevealed IgE antibodies from negative to extremely high levels. Specific IgE antibodieswere not detected in the samples from people without echinococcosis – specificity of thereaction 100 %.
These data prove the high specificity of IgE antibodies, which makes them very usefulfor confirmation of the diagnosis in patients with doubtful results in the routinely appliedserological tests.
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P4DETECTION OF CROSS-REACTIVE BANDS IN CYSTIC
FLUID BY WESTERN BLOT ANALYSIS
I. Marinova, I. Rainova, A. Tchernov, R. KurdovaNatiоnal Centre of Infectious and Parasitic Diseases, Sofia
Presence of cross-reactive interactions among different helminthic antigens can leadto missinterpretation of positive results in serological tests. Detection of antibodies againstparticular parasitic proteins by Western blot (WB) is а way to overcome this issue. Theaim of the present investigations was to examine by WB with echinococcus antigens,sera from patients with parasitic diseases in order to reveal common bands indicatingcross-reactivity. Sera from patients with amoebiasis, cysticercosis, taeniarhynchosis,trichinellosis, alveococcosis, toxocarosis and a pool of human sera from healthy individuals.Electroforesis of cystic fluid was performed in 15% separating gel according to Laemmliand electrophoretic transfer of proteins from polyacrylamide gels to PVDF membrane wasmade according to Towbin. All samples revealed bands with high molecular weight. Serafrom patients with alveococcosis, cysticercosis and toxocarosis showed false positiveresults by reacting with the bands with diagnostic value – 12 kDa and 24-26 kDa. Samplesfrom the other parasitoses did not react with the bands with diagnostic value 8, 12 and 24-26 kDa, and were interpreted as negative results.
P5APPLICATION OF IGG АVIDITY FOR DIAGNOSIS OF
ACUTE TOXOCAROSIS
I.RainovaNational Center of Infectious and Parasitic Diseases (NCIPD), Sofia
Toxocarosis is a parasitic disease in humans, caused by larvae of dog ascarid Toxocaracanis. The infection is generally diagnosed by demonstration of specific immunoglobulinsto Toxocara excretory/secretory (E/S) antigens in sera of infected patients. The test thathas been clinically useful is the ELISA reaction with E/S antigens. This diagnostic methoddetects the antibodies for months or even years after infection and this is the reason whythe discrimination between chronic and recent infection is very difficult. That is why weuse assay with measuring IgG avidity to distinguish acute from chronic Toxocara infection.Sera samples tested in ELISA with E/S antigen and positive for toxocarosis were treatedwith urea and then tested in ELISA again. The index of avidity was calculated as the ratioof IgG values in sera treated with urea and the value of IgG in non-treated sera, multipliedby 100. An index up to 40 is considered as low avidity which means freshly acquiredinfection (36 to 40 borderline) and more than 40 is high avidity (chronic infection). From1184 patient sera, tested in ELISA, 129 were positive for toxocarosis and most of them
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showed high avidity. It means that predominantly patients are in the chronic stage ofinfection at the time of examination.
P6АНТИТЕЛА СРЕЩУ TOXOPLASMA GONDII В ЧОВЕШКИ
IG ПРЕПАРАТИ
И. Райнова1, Ю. Начева2, А. Чернов1
1Национален център по заразни и паразитни болести (НЦЗПБ) – София2Бул Био НЦЗПБ –ЕООД – София
Три партиди човешки Ig препарати, произведени в Бул Био НЦЗПБ - ЕООДбяха изследвани в реакциа пасива хемаглутинация (РПХА) и ELISA за наличиена антитела срещу Toxoplasma gondii., като във всички изпитвани препарати бяхаустановени антитела срещу паразита.
Получените резултати могат да бъдат обяснени с високия процентсеропозитивност за токсоплазмоза сред населението и трябва да се отчитавъзможността за наличие на антитела срещу T.gondii при пациенти, лекувани счовешки Ig препарати.
P7IDENTIFICATION OF FREE-LIVING AMOEBAE BY PCR.
N. Tsvetkova, R. KurdovaNational center of infectious and parasitic diseases, Sofia
Free-living amoebae (FLA) group include different genera, such as Acanthamoebae,Naegleria, Hartmannella, Vahlkampfia, Vannella etc., wide spread in the nature. Some speciesof the genera Acanthamoebae, Naegleria and Hartmannella have been implicated in humanpathology.
The aim of the present study was to apply PCR tests for detection and identification ofFLA in water samples.
Materials and methods. Different types of water habitats, including rivers, tap, well,spring, mineral water, a lake and wastewater treatment plant were examined for the presenceof cysts of FLA. DNA was isolated using DNAsy Tissue kit (Quigen). Culturing on 1.5%nonnutrient agar plates and 3 PCR tests were carried out. Reference amoebae strainsrepresentatives of the genera Acanthamoebae, Naegleria and Hartmannella were includedin the study. Separation through 1.5 % agarose gel and visualization under UV lightillumination of the amplification products was performed.
Results. Seven types of water habitats, including 41 sources with 126 samples were
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examined for the presence of cysts of FLA. Forty-six of the samples tested were foundpositive on 1,5% nonnutrient agar culture plates and 42 – PCR positive. Acanthamoebaespp. cysts were detected in 18/16 samples (culture/PCR); Hartmannella spp. cysts – in 17/17 samples and both Acanthamoebae spp. + Hartmannella spp. cysts in 11/10 samples.
Conclusion. Amoebae of the genera Acanthamoebae and Hartmannella were detectedwith high frequency in water samples from sources examined. Further, tests forpathogenicity have to be done aiming at study the potential of the amoebae to producediseases in animals and humans.
P8VISCERAL LEISHMANIASIS IN BULGARIA
R.Harizanov, G. Filipov, D. Yordanova, R. KurdovaNational Center of Infectious and Parasitic Diseases, NCIPD, Sofia
Visceral leishmaniasis is a protozoal infection spreading over large territories of theglobe. According to the WHO data, more than 350 millions of people in 88 countries areleaving at risk and more than 12 millions are already infected. Annually among 100 and 150thousands new cases are registered, and many others remain undiagnosed.
Foci of visceral leishmaniasis exist in every border Bulgarian country. In Turkey for1982 – 1991 period, over 500 clinical cases of human leishmaniasis were registered, andparasitological and serological surveys confirmed presence of the disease in dogs. InGreece endemic regions are situated in the south and peninsula parts of the countrymainly, and canine visceral leishmaniasis estimates to 22 %. Endemic regions exist also inSerbia and Montenegro, while in Macedonia sporadic cases are annually reported.
In the past a number of authors reported in their publications for the presence ofindigenous visceral leishmaniasis in Bulgaria, while the last described cases dated from1952.
At the end of eighties of the twentieth century, indigenous cases of visceralleishmaniasis reappeared, and their transmission occurred in the country. As in the past,nowadays the infection has the peculiarities of the Mediterranean clinico-epidemiologicalvariant and infected persons are not only children, but adults also. Domestic and wildanimals from the Canidae family are considered to be the reservoir and source of infection,and the main vectors for disease transmission are species of the genus Phlebotomus.During the 17 years observation period (1988 – 2005), 95 cases of visceral leishmaniasishave been registered - 55 cases (57,89%) in children between 0 to 18 years of age and 40cases (42,11 %) in persons over 18 years.
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P9ECHINOCOCCOSIS DISTRIBUTION AMONG CHILDREN
AND ADOLESCENTS IN BULGARIA (1990 – 2005)
D. Yordanova, R. KurdovaNational Center of Infectious and Parasitic Diseases, NCIPD, Sofia
Echinococcosis /hydatidosis/ is a serious health, social and economical problem forour country. During the past 15 years a sharp increase in the basic epidemiological indices(morbidity, mortality rate and lethality rate) has been observed.
This study was made on the basis of official notification data received for operatedpatients and those with PAIR, from the surgical units and clinics.
Since 1990 to 2005 year period a constant tendency of elevation in echinococcosismorbidity rate among the whole country population was established. Morbidity dynamicsin the age group of 0 – 19 years ranges between 0,53 %000 to 10,54 %000. Rise in the relevantproportion of echinococcosis cases in the young-adolescent age group was observed.This index is extremely important for the purposes of investigation of the parasitictransmission and intensity of disease transmission in the country.
Territorial distribution of echinococcosis cases in the country is uneven. The mostaffected territories are those of Plovdiv, Burgas and Sliven regions.
Organ localization of the hydatid cysts in children and adolescents most frequently isin the lungs, liver, both lungs and liver and other organ localizations (kidneys, spleen,brain, subcutaneous tissues).
The high echinococcosis morbidity index and the high relative proportion ofechinococcosis cases among children in the country, in comparison to other MediterraneanBalkan countries, led to elaboration of a national programme for control of this parasitosis.
P10CLINICAL FORMS AND CHEMOTHERAPY
OF TRICHINOSIS
D. Vuchev1, K. Anichina2, K. Eneva1, V. Blagoeva1, A. Russinova3, M.Darakchieva4, G. Stancheva3
1Medical University, Plovdiv, Department of Infectious Diseases, Epidemiology,Parasitology and Tropical Medicine;2Bulgarian Patent Department, Sofia;3Center for Disease Control, Plovdiv;4Center for Disease Control, Smolian
In the last two decades the number of registered trichinosis cases has been on therise. This fact made effective etiological therapy with benzimidazoles (thiabendazole,mebendazole and albendazole) particularly important. This study is aimed at outlining a
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treatment strategy for trichinosis with benzimidazoles and its clinical forms (asymptomatic,mild, moderate and severe) and stages (intestinal and muscular) as well as for itschemoprophylaxis. Experimental-laboratory and clinical data on disease outbreaks arepresented. Subsequent follow-up examinations are necessary in the convalescence period.
Key words: trichinosis, clinical, chemotherapy, benzimidazoles
P11EPIDEMIOLOGICAL FEATURES OF TRICHINOSIS IN CEN-TRAL SOUTHERN BULGARIA (PLOVDIV, PAZARDJIK ANDSMOLIAN REGIONS)
D. Vuchev1, K. Eneva1, V. Blagoeva1, A. Russinova2, M. Darakchieva3, G.Stancheva2, N. Paliiska4
1Medical University, Plovdiv, Department of Infectious Diseases, Epidemiology,Parasitology and Tropical Medicine;2Center for Disease Control, Plovdiv;3 Center for Disease Control, Smolian;4Center for Disease Control, Pazardjik
A surge in the number of human trichinosis cases has been noticed in the last decades.The aim of this study is to analyze morbidity rate and clinical features of trichinosis inPlovdiv, Pazardjik and Smolian regions (Central Sothern Bulgaria) over a 10-year period.These regions are among the most severely affected and disease outbreaks as well assporadic cases are registered annually. Trichinosis distribution and morbidity rate dynamicsin the different administrative and landscape zones are presented. Sources of the disease( boars and domestic pigs ) and the ratio of rural to urban population affected are analyzed.Analysis is based on epidemiological surveys, conducted by the Centers for DiseaseControl in Plovdiv, Smolian and Pazardjik as well as on clinical and laboratory observations.Main factors contributing to trichinosis management are: strict epidemiological andveterinary control, adequate clinical, laboratory and parasitological diagnosis, healthpromotion and education of population, especially in the regions where temporarysynantropic foci of infection exist.
Key words: trichinosis, epidemiology, distribution, prevention
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PLANT AND SOIL MICROBIOLOGY
PSM1SPREAD AND DETECTION OF PHYTOPLASMA DISEASES
Dimitrijka SakalievaBulgaria, 4000 Plovdiv, 12 Mendeleev str., Agricultural Universitye-mail: [email protected]
Phytoplasmas are associated with several diseases in flower crops, vegetables andweed, especially from Aster yellows group (16 Sr I). Sometimes they were detected inmixed infection with phytoplasmas belonging to other ribosomal groups, especially inwoody plants. Phytoplasmas of this group were often identified also in insects.
When cloned phytoplasma DNA probes were produced a large increase was noticed:their use shows clear evidence that phytoplasma differentiation was possible on the basisof their DNA sequences. Polymerase chain reaction with primers from sequences ofrandomly cloned phytoplasma DNA, from 16S rDNA, from ribosomal protein gene andothers opened the ‘modern era’ of phytoplasmology.
The use of molecular technologies has provided tools to differentiate phytoplasmasand preliminary results indicate that mixed phytoplasma infection are not uncommon,especially in woody host plants or insects. These findings have proven that the conceptthat each disease is caused by one type of phytoplasma is not generally applicable. Infact, phytoplasmas infecting flower crops or vegetables, result in belonging to differentsubgroups in the 16 Sr I group.
PSM2MANIFESTATION OF TOLERANCE TO SHARKA
(PLUM POX) VIRUS OF PLUM CULTIVARS IMPORTEDIN BULGARIA
Antoniy Stoev *, Pencho Iliev *** Plant Protection Institute, Kostinbrod 2230, Bulgaria** Experimental Station of Plum Growing, Dryanovo, Bulgaria
Several plum cultivars were determined as tolerant to sharka virus (PPV) under naturalconditions. This mode of reaction gives opportunity for an enrichment of plum productionvariety.
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PSM3PROPOSALS FOR IMPROVEMENT OF THE VARIETY
TESTING OF WHEAT AND BARLEY ACCORDINGLY THEIRREACTION TO THE MOST IMPORTANT IN BULGARIA
VIRUSES
Nonka Bakardjieva, Antoniy StoevPlant Protection Institute, Kostinbrod 2230, Bulgaria
The report substantiates the necessity of an addition of the variety testing system sothat the reaction of the cereal plants to barley yellow dwarf virus (BYDV) and wheat dwarfvirus (WDV) to be evaluated. The tasks specified as main for the improvement alreadymentioned are creation of infectious experimental plots and maintenance of viruliferousvectors.
PSM4MORPHOLOGIC AND CULTURAL VARIATION IN
PHOMOPSIS FOENICULI
R. Rodeva1, J. Gabler2
1 Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria2 Institute for Resistance Research and Pathogen Diagnostics, Federal Centre forBreeding Research on Cultivated Plants, 06449 Aschersleben, Germany
Phomopsis foeniculi Du Man. et Vegh. causes umbel browning and stem necroses onfennel (Foeniculum vulgare Mill.). The fungus was isolated from naturally infected umbelsand stems.
Twelve isolates were studied for variation of their morphological and cultural characterson five nutrient media each variant represented by five replications. The experiments werecarried out twice. The isolates showed great variability. They differed in colony colourand linear growth, the quantity of pycnidia and ability to produce teleomorph in vitro. Thebest growth of isolates was observed on V-8 agar and oat agar. Some of isolates scarcelyproduced pycnidia in vitro. The most abundant were pycnidia on potato dextrose and oatagar. They were black, superficial, scattered or aggregated in the colony center. Two typesof conidia were formed in the pycnidia: á- and â-conidia. Alpha conidia were lessabundant than beta conidia, oblong to fusiform, straight to slightly curved, 2-3 guttulate,5-11 x 2-4 ìm. Beta conidia were filiform, curved or sometimes hamate, eguttulate, 15-30 x 1-2.5 ìm.
The most suitable media for the sexual production were malt - yeast agar and oat agar.Ascomata developed as black patches on both nutrient media but only two isolatessucceeded to produce mature perithecia with ripe ascospores. The perithecia were globose
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to subglobose with long necks, often clustered and contained numerous asci with aconspicuous refractive apical ring and 8 biseriate ascospores. The ascospores wereunicellular, colourless, ellipsoidal, guttulate, papillate, with rounded ends, 10–15 x 3-4.5ìm.
PSM5IN VITRO AND IN PLANTA INTERACTIONS BETWEEN
THREE SCLEROTIAL PLANT PATHOGENS
R. Rodeva, R. PandevaInstitute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
An investigation was undertaken to assess in vitro and in planta interactions betweenBotrytis cinerea Pers. Fr., Sclerotinia sclerotiorum (Lib.) de Bary and Verticillium dahliaeKleb. The three fungi form sclerotia and are among the more devastating plant pathogens.The ability to survive in adverse conditions and remain viable for long periods in theabsence of hosts makes them difficult to control. They are soil inhabiting, have commonhost plants, similar requirements for resources and other niche overlaps. Their interactionsare potentially important determinant of spatial distributions and functioning of theirpopulations.
Strains obtained from two naturally infected host plants: pepper (Capsicum annuumL.) and caraway (Carum carvi L.), were included in the study. In vitro tests were made indual cultures. Plugs of mycelial inoculum with 5 mm diameter were removed from thegrowing margin of the colony and were placed 3.5 cm apart on agar plates in 9-cm petridishes, one pairing per dish and incubated in the dark at room temperature. Strains werepaired in all combinations - with itself and with each of the remaining ones. All treatmentswere replicated three times. Mycelial compatibility was macro- and microscopically recorded4 and 14 days after inoculation. Pairings were scored as compatible when the two strainsmerged to form one colony with no distinct interaction zone and as incompatible when thetwo strains failed to grow together and a thin mycelial-free space remained between themor when a dark interaction line was observed at the zone of contact. Most pairings showedincompatible mycelial interactions. All pairings of a strain with itself were compatible. Inplanta tests were made by inoculations of pairings on the stem of both host plants atdistance of 5 cm between the points of inoculation. All strains used in this study were insome way affected by the presence of another fungus.
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PSM6БИОЛОГИЧНИ СВОЙСТВА И ЛИОФИЛИЗАЦИЯ НА
ИЗОЛАТИ НА ВИРУСА НА ХЛОРОТИЧНИТЕ ЛИСТНИПЕТНА ПО ЯБЪЛКАТА (ACLSV) ОТ РАЗЛИЧНИ ОВОЩНИ
ВИДОВЕ В БЪЛГАРИЯ
Aнелия Борисова1, Анжела Йорданова2
1Институт по земеделие, Кюстендил 2500, България;2Националната банка за промишлени микроорганизми и клетъчни култури,София 1113, п.к. 239, България
Проучени са биологичните свойства на пет български изолата на Аpplechlorotic leaf spot virus (ACLSV), произхождащи от ябълки, череши и праскови,идентифицирани чрез ELISA.
Изолатите на вируса са пренесени от естествено инфектираните дървета върхупет вида дървесни индикатори чрез методa на двойното окулиране. Всички изолатипредизвикват сходни, слабо разграничаващи се симптоми, характерни засъответните индикаторни дървета - Cydonia oblonga (C7/1), Мalus sylvestris R12740-7А и Malus platycarpa. По листата на Prunus tomentosa и Аronia melanocarpaне се наблюдават признаци на вирусна инфекция.
Пет тревисти вида, чувствителни към ACLSV, са инокулирани механично съссок от инфектирани венчелистчета и листа на отделните изолати. При четири отизползваните индикатори се наблюдават локални симптоми, а Chenopodium quinoaреагира с локални лезии и системна инфекция.
Проведено е вакуумно-сублимационно сушене на листа, венчелистчета ифлоем от заразени дървета ябълка, череша и праскова. Инфекциозността етестирана на Ch. quinoa. Установено, е че вирусът се запазва най-добре влиофилизирани венчелистчета.
PSM7EFFECT OF INOCULATION WITH AZOSPIRILLUM AND
AM FUNGI ON THE PLANT BIOMASS OF WHITE THISTLE(SYLIBIUM MARIANUM L.)
E. Jonova, N. Kaloyanova“N. Poushkarov” Institute of Soil Science
A pot experiment with meadow-cinnamonic soil (luvisol) was carried out to study theeffect of single and double inoculation with Azospirillum brasilense sp. 36/24 and Glomusmossae on the growth and development of white thistle (Sylibium marianum L.). Theexperiment was carried out without fertilization, with two types of soil fertilization
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(N50P100K100, N100P200K100) and with leaf fertilization using Bioleaf.The plant shoots increase more with the mycorisal fungi when there is no fertilization.
The effect of double inoculation on the root mass is noticeably higher.The N50P100K100 variant showed the opposite - the inoculation decreases the yield from
the plant shoots. In this case the root mass increases only with the application ofAzospirillum bacteria whereas in the other variants it decreases. In comparison with thelower doses, the N100P200K100 variant decreases the weight of the plant shoots in the control,while the mixed inoculation increases it. The root mass increases only with singleAzospirillum inoculation.
Our experiment with leaf fertilization didn’t show any particular effect as a result fromthe inoculations.
PSM8ЕФЕКТ ОТ ИНОКУЛАЦИЯТА С МЕСТНИ ЩАМОВЕФОСФАТРАЗЛАГАЩИ БАКТЕРИИ ВЪРХУ РАЙГРАС
Костадинка НедялковаИнститут по почвознание “Н.Пушкаров”, София, ул. “Шосе Банкя” 7
Инокулацията с полезни почвени микроорганизми с цел подобряванехраненето на културите и повишаване на продукцията е предмет на многобройниизследвания през последните десетилетия.
В настоящата работа се изследва влиянието на инокулацията сфосфатразлагащи бактерии, изолирани от наши почви, върху продукцията набиомаса и усвояването на азот, фосфор и калий от райграс. Опитът е изведен въввегетационна къща. Почвата е наторена с N, P, K във всички варианти. Извършенае инокулация с четири бактериални щама, притежаващи фосфатразлагащаактивност. Проследен е ефекта върху количеството суха биомаса (зелена маса икорени) и съдържание на азот, фосфор и калий при три откоса на зелената маса.
В резултат на бактериалните инокулации количеството на зелената маса приI откос се увеличава незначително (3-5%). Чувствително повишение на биомасата(15%) е установено при II откос при инокулация с щам 67. При III откосколичеството на зелената маса намалява и ефект от бактериалните инокулациине се наблюдава. Под влияние на инокулациите се увеличава съдържанието нафосфор и калий в корените.
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PSM9TAXONOMICAL IDENTIFICATION OF ARSENIC RESIS-
TANT AND ARSENIC TRANSFORMINGSULFATE - REDUCING BACTERIA
Krasimira S. Krumova, Veneta I. GroudevaSofia University ‘St. Kl. Ohridski”, Faculty of Biology, 8 Dragan Tzankov ,Department of General and Industrial Microbiology, Lab. ”Geomicrobiology”,1164 Sofia, Bulgaria; e-mail: [email protected]
Abstract: Twenty eight bacterial strains, which are able to transform arsenic compounds,were isolated and characterized in the laboratory of ”Geomicrobiology” at the Departmentof “General and Industrial Microbiology”, SU “St. Kliment Ohridski”. It was determined,that 11 of these isolates are able to efficiently oxidize the high mobile and high toxicarsenite [As (III)] to less mobile and less toxic arsenate [As (V)]. The others 17 strains areable to reduce arsenate [As (V)] to arsenite [As (III)]. Оn the basis of the determinationvia classical methods, the isolated bacteria were related to different genera within thebig group of sulfate-reducing bacteria. The confirmation of the taxonomical belongingof these strains was done on basis of the amplification of their DNA with SRB specificprimers for each genus.
Key words: Sulfate-reducing bacteria, arsenite oxidizing and arsenate reducing bacteria;
PSM10ВЛИЯНИЕ НА ПРОТЕИНОВ ХИДРОЛИЗАТ ОТ
КЕРАТИНОВИ ОТПАДЪЦИ ВЪРХУ ПОЧВЕНАТАМИКРОФЛОРА
Мая Нусторова, 1 Адриана Гущерова2, Диана Брайкова 2, Евгения Василева2
1Лесотехнически университет –София2 БАН-Институт по микробиология
Кератин-съдържащите отпадни материали / вълна, четина, рога, перушина идр. / от редица индустриални производства са потенциален източник на ценнипродукти, богати на протеини и аминокиселини с възможности за широкопрактическо приложение. Перспективен метод за използване на кератиновитеотпадъци е трансформирането им в евтини и екологично безопасни протеиновихидролизати.
Изследвана е възможността за използване на протеинов хидролизат откератинови отпадъци като почвен подобрител чрез активиране на почвенатамикрофлора. Препаратът е изготвен на базата на алкална хидролиза наживотински отпадни продукти. Той съдържа 75 – 80% водонеразтворими
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съединения – пептиди, аминокиселини, соли, липиди, въглехидрати, пигменти,калиеви йони. Изследването е извършено в лабораторни условия – 8-месеченсъдов опит с три варианта на торене. Използвана е почва – чернозем-смолница,засята с райграс /lolium perenne/.В динамика са проучвани почвени,микробиологични и растителни показатели. Данните сочат трайна тенденция заповишаване на почвената биогенност, растежа и натрупването на растителнабиомаса и при трите варианта на торене. Определена е оптималната концентрацияна въздействие на препарата.
PSM11ИЗСЛЕДВАНИЯ ВЪРХУ ТОЛЕРАНТНИ КЪМ
ЗАСУШАВАНЕ ГЕНОТИПОВЕ СОЯ. І. ОТЗИВЧИВОСТКЪМ ИНОКУЛАЦИЯ С BRADYRHIZOBIUM JAPONICUM
Анна Маркова, Радка Алтимирска, Йорданка Киркова, Георги СтоименовИнститут по почвознание “Н.Пушкаров”, София
При селекция на соята е необходимо да се имат предвид не само стопанскитей качества, но и нейната способност да фиксира атмосферен азот.. В този аспектизследването на отзивчивостта на тази култура към инокулация със специфичнитеза нея азотфиксиращи бактерии от вида Bradyrhizobium japonicum е от особеноважно значение.
При условията на съдов и полски опити с излужена ливадно-канелена почваот с.Цалапица,Пловдивско са изпитани вирулентността и ефективността наBradyrhizobium japonicum при сухоустойчиви и отзивчиви на напояване сортове илинии соя. Установена е различна отзивчивост на соята към инокулация взависимост от генотипа и почвената влажност.
PSM12ИЗСЛЕДВАНИЯ ВЪРХУ ТОЛЕРАНТНИ КЪМ
ЗАСУШАВАНЕ ГЕНОТИПОВЕ СОЯ ІІ.МИКРОБИОЛОГИЧНА АКТИВНОСТ В РИЗОСФЕРАТА
НА СОЯ
Радка Алтимирска, Анна Маркова, Христо Стойков, Красимир ЧачевИнститут по почвознание “Н.Пушкаров”, София
Засушаването като важен абиотичен фактор е от особено значение както поотношение продуктивността на соята, така и по отношение на микробиологичната
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активност в почвата. Освен симбиотичните азотфиксиращи бактерии същественозначение за храненето на соевите растения има и ризосферната им микрофлора.
В условията на съдов и полски опити върху излужена ливадно-канелена почва/с.Цалапица, Пловдивско/, бяха изследвани основните физиологични групимикроорганизми, участващи в трансформацията на хранителните вещества вризосферата на различни сухоустойчиви и отзивчиви на напояване сортове илинии соя. Установените изменения в микробиологичната активност, както итези в добива варират в зависимост от генотипа на соята и изпитваната влажност.
PSM13ВЛИЯНИЕ НА ХЕРБИЦИДА РИЛЕЙ ВЪРХУ ОСНОВНИ
СВОЙСТВА НА BRADYRHIZOBIUM JAPONICUM
Радка Донкова – Институт по почвознание “Н.Пушкаров”, София
В условията на съдов опит с две почви (излужена смолница, Божурище иалувиално-ливадна, Цалапица) и соя сорт “Даниела” е проучено влиянието нахербицида рилей върху основни свойства на Br. japonicum. Хербицидът е изпитанв две концентрации – препоръчваната за употреба в практиката и двукратноувеличена – съответно 200 и 400 ml/dka за алувиално-ливадната почва и 280 и560 ml/dka за излужената смолница.
Установено е отрицателно влияние на рилей върху вирулентността,азотфиксиращата активност и ефективност на Br. japonicum. Наблюдаваният ефекте по-силно проявен при вариантите с високата концентрация на хербицида, прикоито е отчетено двукратно намаление на вирулентността, 30% по-малкофиксиран азот и окооло 20% по-нисък добив. Хербицидното действие показвазависимост от почвените свойства. То е по-силно проявено при алувиално-ливадната почва.
PSM14МИКРОБИОЛОГИЧНА ХАРАКТЕРИСТИКА НА ПОЧВИ В
РАЙОНА НА КЦМ- ПЛОВДИВ
Радка Донкова и Николай ДиневИнститут по почвознание „Н.Пушкаров”, София
Функциониращите в страната комбинати за цветни метали генерират проблемаза замърсяването на почвите с тежки метали и металоиди. Акумулирането на тезизамърсители в почвата би могло да окаже влияе върху състоянието ифункционирането на почвените микробиални съобщества. което да промени
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устойчивостта на почвената екосистема като цяло. Целта на настоящетоизследване е да се установи съдържанието на тежки метали в района на КЦМПловдив и влиянието им върху почвената микрофлора. Определени сасъдържанието на тежки метали в почвата, количеството на основни групи почвенимикроорганизми и общата биологична активност на проби , взети на различноотстояние от комбината по мониторингови точки. Установено е, че основнизамърсители за района са Cd, Pb и Zn, чиито концентрации в някои от изследванитеточки превишават пределно-допустимите с няколко порядъка. Наблюдава сепромяна в съотношението между отделните групи почвени микроорганизми инамаляване на общата биологична активност, което е указание за стесняванеспектъра на микробиологичната дейност и понижаване функционалнатаактивност на микроорганизмите.
PSM15ВЛИЯНИЕ НА КАДМИЯ ВЪРХУ
МИКРОБИОЛОГИЧНАТА АКТИВНОСТ НАКАРБОНАТЕН ЧЕРНОЗЕМ
Галина Петкова, Радка ДонковаИнститут по почвознание “Н.Пушкаров”, София
Тежките метали могат да предизвикат сериозни нарушения в биоценозите,почвеното плодородие и здравето на човека поради своята токсичност испособността им да се акумулират трайно в околната среда. От особено значениее влиянието им върху почвената микрофлора, от дейността на която зависифункционирането на почвата като жива система и поддържане на биологичнатай продуктивност.
В моделен опит в динамика е проучено влиянието на нарастващиконцентрации кадмий върху основни групи почвени микроорганизми и общатабиологична активност на карбонатен чернозем. Установено е, че кадмия имаотрицателно действие върху някои от проучваните групи микроорганизми приконцентрации по-ниски от ПДК. Най-чувствителни са микроорганизмитесвързани с азотната трансформация на органичното вещество. Сравнително най-устойчиви са актиномицетите. Въз основа на получените данни е направенаоценка на възможността за използване на проучваните показатели катоиндикатори за замърсяване на почвата с кадмий.
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ACTUAL PROBLEMS OF THE BIOSCIENCE
BS1SCIENCE FUNDING IN BULGARIA
Albena Vutsova, Ministry of Education and Science, Sofia
It is evident that science as a whole has a significant role to play in support of a societywhich has placed knowledge creation at the heart of its vision. What is more, science is ofprimary importance for the development of economy and for strengthening its structure(especially for the countries in transition). It is not by chance that we use the term“knowledge based economy”. The challenges bared by this term are building on the well-documented public interest in science, using all forms of communication to foster dialogueand interest, maintaining international competitiveness in high technology areas and, inaddition, providing scientific and technological competence as a precondition for globalcompetitiveness and the key to successful partnerships.
To address these challenges, one of the most important prerequisites for thedevelopment of a knowledge-based society is the funding of science. Scientific researchfunding is closely related to the adoption and implementation of scientific, technologicaland innovation policy – three different spheres of activity, however, incorporated within adynamic framework.
Bearing in mind that scientific and technological policy and scientific research funding areinterrelated, we should look at the sources of funding and the way funds are allocated.
Development of a stable, vital and competitive research system in Bulgaria,, contributingto the creation of knowledge-based economy depends on several factors, namely (but notexhaustively) :
- General increase of state expenditures for research and development- Mix of financial instruments – public funds, private sources and European funds- Allocation of funds on a competitive basis
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BS2БИОИНФОРМАТИЧНИ РЕСУРСИ В
МИКРОБИОЛОГИЯТА:ПРЕДИЗВИКАТЕЛСТВА И ПЕРСПЕКТИВИ
Димитър Василев, Агро Био Институт, София
През последните няколко десетилетия напредъкът на молекулярната биологияи наличното модерно оборудване за научни изследвания позволиха секвениранетона големи части от генома на множество видове организми. В действителност досега са секвенирани напълно геномите на няколко вида бактерии и на няколкоеукариоти (напр. хлебните дрожди). Човешкият геном също беше изцялосеквениран в рамките на мащабен международен проект. Популярни бази-даннисъс секвенции като GenBank и EMBL, ресурси за търсене и анализ на молекулярнаинформация (EMBOSS,), обектно ориентирани бази данни и софтуер за обработкасе създават с нарастваща скорост за съхраняване, анализ, визуализиране насъбраната информация. Този огромен приток на информация е необходимо дабъде грижливо съхраняван, организиран и индексиран. За тази целинформационните технологии и техните приложения в биологията създадоха еднанова, бързоразвиваща се научна област, наречена биоинформатика.
Най-елементарните задачи на биоинформатиката засягат създаването иподдържането на бази-данни с биологична информация. Последователности отнуклеинови киселини, получени след секвенирането им, както и протеиновипоследователности, получени от тях, представляват болшинството отинформацията на тези бази данни. Докато съхраняването и организацията намилиардите нуклеотиди е далече от обичайната дейност, изграждането на базатаданни и създаването на интерфейс, чрез който изследователите ще могатедновременно да имат достъп до информацията и да подават нови данни, е самоначалото.
Най-неотложната задача на биоинформатиката включва анализа насеквенираната информация. Този процес се осъществява от т.нар. изчислителнабиология, която включва: предсказване и откриване на гените в ДНКпоследователностите в различните организми; изработване на методи запредсказване на триизмерната структура и/или функция на новооткрититепротеини и структурни РНК последователности; групиране на протеиновитесеквенции в семейства от свързани секвенции и разработване на протеиновимодели; подреждане на сходни протеини и създаване на “филогенетични дървета”за изследване на еволюционните връзки.
Бързото навлизане и приложение на биоинформатиката вмикробиологията създава предпоставки за все по-мащабно и динамично развитиена огромни международни проекти засягащи особено вирусологията,изследването на не-малък брой социално значими заболявания: СПИН, хепатит,in silico моделирането и разработването на различни видове лекарства.
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Dimitrova D. Bl. GAM50, GAM51, GAM52Dimov Sv. G. GAM61Dmitrieva T. GAM83Dobrev G. GAM16Donkova R. PSM13, PSM14Dubyago N. MM35Dundarov S. V8
Evstatieva Y. GAM58
Filipov Ch. VP38
Galabov A. S. Tb1, V2Galabova D. GAM10Galabinova T. VM16Gavazova R. V20Georgiev D. GAM57Georgieva I. L. VP31Georgieva J.H. GAM42Gerginova M. GAM28, GAM29Ginova T. GAM89Gladniska T. MM24Gocheva Y. GAM48Golkocheva E. II14Gotev N. MM25, MM26Gotseva A. V29Gouliamova D. E. GAM3Grigorova V. MM14, MM15Groudeva Tz. GAM4Groudeva V. GAM7, GAM8, GAM30Gulluce M. MM17Gurgulova K. VM7Gurova-Chausheva A. GAM91
Hadjiolova T. V16Harizanov R. P8Hristova N. GAM72
Ianis M. GAM35Ignatova Tz. GAM22Iliev I. GAM15.
Abashev Y. P. VP32Abrashev R. GAM49Ackermann J- U. GAM19Aleksandrova R. GAM21Aleksiev I. V22Alexiev R. II9, II10Alexieva Z. GAM26Altimirska R. PSM12Andonov A.P. V13Angelov P. MM10Angelova M. GAM11Argirova R. V14Arnaudov H. VM5Atanassova M. GAM54Atanassova А. V. GAM74, GAM75
Bakalova St. GAM41Bakardjieva N. PSM3Beshkov D. V24Bikumandla A. GAM56Blazheva D. MM5Bojkova K. MM16Boneva B. VP39Bonovska M. Tb8, VM9Borisov K. V19Borisova A. PSM6Borissova P. VP37Bostandjieva R. VP41Brankova N. MM21Bumbarov V. VM1
Chankova D. MM30ChikovaV.VP43,VM15, VM16,VM17,VM18Chouchkova M. Tb2Christova D. GAM36Christova I. MM23
Decheva A. MM22Delcheva D. GAM 60Derekova A. GAM44Dimitonova S. MM28
FIRST AUTHOR INDEX - ABSTRACT NUMBER
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Iliev M. VM13, VM14Ilieva D. VP42Iovcheva L. GAM82Ivanov T. GAM92, GAM93Ivanova E. II3Ivanova I. GAM9Ivanova L. VP40Ivanova K. MM36Ivanova M. P2Ivanova S. VM6Ivanova V. GAM68, GAM69, GAM70, GAM71
Kabaivanova L. GAM32Kalcheva H. GAM46Kalfin E. MM7Kalvachev Z. V26Kantardjiev T. Tb6, MM11Karakolev R. VM2Koltyga I. GAM84Koprinarova M. GAM88Korsun N. VP34Korudjiisky N. VM8, VM15Kostova D. GAM73Kouzmanov A. MM31Krumov N. GAM40Krumova E. V12, GAM47Krumova Kr. GAM5, PSM9Kurdova-Mintcheva R. P1Kuzelov A. GAM20, VM4Kuzmin V. V3
Lahtchev K. GAM90Lozitsky V. V4Lubashevsky E. II4Lupke V. II12.Lyutskanova D. GAM59
Manasiev Y. GAM27Margesin R. GAM6Marinov B. GAM77Marinova I. P3, P4Markova A. PSM11.Meincken M. GAM66Mekouchinov K. V30
Mileva M. V12Minchev P. Tb5Mitov I. MM12Mladenov K. MM27Mladenova Z. V15Muhtarova M. II8Murgov I. MM19
Najdenski H. VM3, II15Naumova S. GAM45Nedelcheva P. GAM87, MM18Nedjalkova K. PSM8Nenkov I. V28Nenova R. MM4Nikolov L. GAM17, GAM18Nikolova D. GAM85Nikolaeva-Glomb L. V4Nustorova M. PSM10
Orozova P. MM29
Pashova-Baltova K. GAM80Pavlova S. V17Peeva V. M. GAM62Pencheva V. MM9Peneva M. V21Petkova G. PSM15Petrova P. GAM14Petrova V. GAM12Popov A. MM32
Rainova I. P5, P6Raleva S. V23Ratkov A. GAM23Remichkova M. V10Rodeva R. PSM4, PSM5Rukanova D. MM13Rusev V. VP36
Safarikova M. GAM53Sakalieva D. PSM1Sapungieva E. II6, II7Savova T. GAM79Serbezov V. MM8
230
Serkedjieva J. V9Shilev S. GAM39Shishkov S. VP33Simeonova L. V7Slavchev A. GAM86Slavov S. V27Slavova-Azmanova N. GAM13Smirnov I. II1Sotirova A. GAM33Spiridonova V. MM34Sredkova M. MM1Stankulova D. II5Stefanova D. Tb4Stefanova T. II11Stefanova V. GAM55Stoev A. PSM2Stoilov R. MM3Stoilova I. GAM31.Stoimenova E. GAM78Stoyancheva G. MM2Stoycheva T. GAM25
Teoharov P. V25Terziyska A. GAM43Terziiski D. MM20Todorov K. GAM24Todorov S.D. GAM63, GAM65Todorova T. GAM38Tomova I. MM6
Topalova Y. GAM2Toshkova R. II16Tosi S. GAM1Tsekova K. GAM37Tsvetkova N. P7Tumbarski J. D. V6
Urshev Z. GAM81
Valcheva V. Tb7Van den Worm A. GAM67Vasilev G. VM11Vassilev D. BS2Vassilev T. II2Vassileva-Pencheva R. V5Vatcheva R. MM33Velcheva D. V18Vilhelmova N. VP35von Mollendorff J.W. GAM64Vuchev D. P10, P11Vutsova A. BS1
Wild F. V1
Yankova Z. Tb1Yankova P. GAM76Yonkova V. II13Yonova E. PSM7Yordanova D. P9
231
232
ELEVENTH CONGRESS
OF THE BULGARIAN MICROBIOLOGISTS
with International Participation
PROGRAM AND ABSTRACTS
St. Constantine, Varna, October 5-7, 2006
233
ELEVENTH CONGRESS
OF THE BULGARIAN MICROBIOLOGISTS
with International Participation
PROGRAM AND ABSTRACTS
St. Constantine, Varna, October 5-7, 2006
