EXPERT TOPIC - SALMON

September | October 2013

EXPERT TOPIC - SALMON

The International magazine for the aquaculture feed industry

International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2013 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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36 | InternatIOnal AquAFeed | September-October 2013

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September-October 2013 | InternatIOnal AquAFeed | 37

Welcome to Expert Topic. Each issue will take an in-depth look at a particular species and how its feed is managed.

SALMONEXPERT TOPIC

World viewAccordingtostatisticsfromtheGlobalSalmonInitiative (GSI) approximately 60 percent ofthe world’s salmon is farmed. Figures showthat in 2011, wild-caught salmon reachedapproximately930,000tonnes.Adropintheocean compared to the 1,600,000 tonnesproducedbyaquaculture.

Salmon belong to a family of fish knownasSalmonidaeandbasedontheirdistribution,are further classified in to two main genera;Atlantic dwellers (Salmo) and Pacific Oceanbasedspecies(Oncorhynchus).Thedistinguish-ing factor in this classification is that unlikethe Salmo genus, species belonging to theOncorhynchusgenusdieafterspawning.

Duetocomplexproductionneeds,widewatertemperaturerangesandbiologicalconditions,farm-ingofAtlanticsalmon-themostpopularspeciesofSalmonidae-isdominatedbyjustahandfulofcountries.Currently,theEU,USAandJapanhavethelargestsalmonaquaculturemarkets.

Atlantic salmon is pisciverous and there-forerequiresadietrichinproteinandlipids.Farmed fishareusually fedacombinationoffishmealandfishoilandalthoughthewasteproducedfromfishprocessingcanbeusedincertain components of fishmeal production,theriskoftransferringdiseasemeansitcannotbeuseddirectlyinfishfeed.Thereison-goingresearch into supplementing fish feed withplant or microbe-based products, thoughcurrentlyno supplementhasbeen found forpisciverousspecies(FAO).

www.globalsalmoninitiative.orgwww.fao.org/fishery/en

1 IcelandThe Icelandic Ministry of Fisheries andAgriculture states that salmon farming firstbeganinIcelandatthebeginningofthe19thcentury,withthefirstattemptstorearsalmonfryoccurringin1961.

The first land-based salmon production

farmwasdeveloped in1978andbythe late1980sbiggerfarmswerebeingconstructed.

Between 1984 and 1987, salmon eggswere imported from Norway. At thistime,therewere large investments intheproduction of salmon smolts for export.Later,ocean ranchingandcageand land-based farming attracted interest amonginvestors.Oceanranchinginvolvesreleas-ing young reared smolts into rivers andstreams.Theyoungsmoltsusethecoastalenvironmenttomatureforaroundayearbefore returning at which point they areharvested.

The country owes much of its farmingcapabilities to itsclimate.Withunpollutedseas and an abundance of clear water,aquaculture conditions are regarded asamong the best in the world in Iceland.In 2007, 600 tonnes of Atlantic salmonwereexported.By2009,therewereabout45 registered fish farms on the island.Figuresshowthatofthese,about30wereproducing juveniles, mostly for Salmonidon-rearing.According to figuresproduced

36 | InternatIOnal AquAFeed | September-October 2013 September-October 2013 | InternatIOnal AquAFeed | 37

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21 3 4

5 6

by theDirectorateofFisheries,the facilities released around 6million salmon smolts with anannual return of around 500metrictonnes.

In Iceland, selective breedingandproductionofhealthyAtlanticsalmon eggs are supplied on ayear roundbasis. In recentyears,major fishery operations havemovedintoaquaculture,investingin both research and develop-mentand thesustainable farmingofsalmon.

www.fisheries.is/aquaculture/species/atlantic-salmon

2 Faroe IslandsDespiteitssmallsize,theFaroeseaquacultureindustryproducestopqualityAtlanticsalmon.

With steady ocean tempera-tures and strong currents, TheFaroe Islands is a prime locationfor premium salmon production.As a result of this, wild Atlanticsalmon from all over northernEurope make their way north oftheFaroeIslandstofeed.

Fish farming in the FaroeIslands began in the 1970s.Nowadays, Faroese fish farmingtoday,whichtakesplaceinocean-based fjords, consistsprimarilyofAtlanticsalmonandlargerainbowtroutandhasbecomeasignificantcomponentofFaroeseeconomicactivityoverthepasttwodecades(NASCO).

http://salmon-from-the-faroe-islands.com

3 ScotlandScotland,whichalongsideNorwaypioneered salmon farming in the1960s, is now second only toNorway in Europeanproduction.As smolts are not indigenous tothe Shetland Islands, they wereoriginally imported fromNorwaytoestablishfarms.

Atlanticsalmonaquaculture inScotland has increased from 14tonnesin1971to154,164tonnesin2010andScotland isnow thelargest farmed salmon producingcountry intheEU. It isestimatedthattheworldwideretailvalueofScottish farmed salmon is overGB£1billion.

TheUSAisthelargestexportmarket for Scottish farmed salm-on,closelyfollowedbyFrance.

www.scottishsalmon.co.uk

4 NorwayThe development of commer-cialaquacultureinNorwaybeganin 1970. At present, intensivefarmingofAtlantic salmon in thecountry is substantial, accountingformorethan80percentoftotalNorwegian aquaculture produc-tion(FAO).

According to the internationalenvironmental organisation, TheBellonaFoundation,anaverageof20 percent of the oil content infish feed in Norway comes fromvegetableoils.

www.fhl.no/english/norwegian-seafood-federation-article15-14.

html

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5 TasmaniaSalmon farming commencedin Tasmania in the mid-1980safter a report to theTasmanianFisheriesDevelopmentAuthorityconcluded that a salmon farm-ingindustrycouldbesuccessfullydevelopedontheislandstate.

As a result, in 1984 fertilisedAtlantic salmon eggs were pur-chased from the Gaden TroutHatchery, Jindabyne,NewSouthWales,Australiafromstockorigi-nallyimportedinthe1960sfromNovaScotia,Canada.Aseafarmwas then established at Doverin the south of Tasmania and ahatcherydevelopedatWayatinahinthecentralhighlands.

The first 53 tonne commer-cial harvest of Atlantic salmonoccurred between 1986 -1987.Nowadays,theTasmanianindus-trynowproducesalmost40,000tonnesperannum.

Nearly 93 percent ofTasmanian salmonid productionwassoldinthedomesticmarketin2006.(DPIW)

www.tsga.com.au

6 New ZealandNewZealandKingSalmon'splansformarinefarmsinMarlboroughSound, New Zealand, may getthe go ahead after the HighCourt dismissed an appealagainstthem.

The decision of the BoardofInquiry,reachedinFebruary2011, to approve four newsalmon farming sites in theMarlborough Sounds wasappealed by two parties andthat appeal was heard at theHigh Court in Blenheim inMay.

The news has been wel-comed by the government,“The impacts of these newmarine farms on the impor-tant recreation and conserva-tionvaluesof theMarlboroughSoundsaresmall.This isaboutuse of only six hectares ofmore than 100,000 hectaresofwater space in theSounds,”said Conservation Minister DrNickSmith.

“We are a BluegreenGovernmentthatwantsjobsanddevelopment but also wants toensure we look after our envi-ronment and great kiwi lifestyle.This decision confirms this bal-ancedapproach.”

“Primaryindustriesarevitalforeconomicgrowthinourregions,andaquacultureplays an impor-tant role in the Marlborougheconomy. I welcome the newsthat extra jobs will be createdas a resultof thesenew farms,”said Primary Industries MinisterNathanGuy.

“Thisdecisionisanotherstepforward for New Zealand KingSalmon in its plans to establishfour new farms, delivering anadditional $60 million a year inexportincomeandproviding200newjobs.”

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Global salmon farming industry joins forces with new sustainability initiativeby Alice Neal, Associate editor, International Aquafeed magazine

The Global Salmon Initiative (GSI)unites 15 global farmed salmonproducers committed to greaterindustrycooperationandtranspar-

ency,inordertoachievesignificantandcon-tinuousprogressinindustrysustainability.

Together, these 15 companies represent70percenttheglobalsalmonindustry,mean-

ingthe initiativecouldhaveareal impactonsalmonaquaculture.

ThemajorsalmonproducingcountriesofChile,Norway,Scotland,theFaroeIslandsandCanadaareallrepresentedintheGSI.

YgnveMyhre,CEO, SalMar,Norway andGSImember,said,“Whilewehavebeenmak-ingattemptsatsustainability,salmonfarmingisayoungindustryandwerecognisethatmoreneedstobedoneandwecandobetter.

“Weknow itwill take timeandwillbeacontinuousprocess,but through theGSIwehave committed to the significant improve-ment that is needed. This initiative is aboutsignificant improvement in sustainability. It isnotaboutsatisfactionwiththestatusquo.”

The GSI will achieve its aim throughglobal collaboration and research, poolingofresourcesandsharingknowledge.

“What is different is that as the GSI, thecompanies have committed to helping eachother towards improved sustainability. It’sabout cooperation, not competition,” saidMyhre.

AlfonsoMarquézde laPlata, chairof theGSIstandardscommitteeandCEO,EmpresasAquaChile S.A., Chile, said, “We cannotchoose between a healthy environment andhealthy food,weneedboth.This initiative isapracticalapproachtoachievingboth.Whilemeetingthestandardatagloballevelwillbeasignificantchallenge,thisisamajorcommit-ment from the salmon farming industry andwehope that throughGSI collaboration,wecangettheretogether.”

TheinitialimpetusfortheGSIcamefroma meeting in 2011 which was attended bya number of CEOs. At that meeting, theCEOsheardabout significantprogressotherindustrieshadmadeinsustainabilitybywork-ingtogether.ThatgroupofCEOsdecidedtomeetagainandinviteotherCEOsandinduecourseitwasagreedtoformtheGSI.

Currently,theGSI is focusingonbiosecu-

rity, feed and nutrition andmeeting industrystandards.

In terms of feed ingredients, the GSI iskeento findsources thatdonotput furtherstress onmarine resources. TheGSI is con-sidering utilizing by-products and is workingcloselywith theFAOtoassessavailabilityoftheseresources.

The GSI has chosen the AquacultureStewarshipCouncil(ASC)asitsaccreditationbodyandaimstohaveall itsmembersmeettheASCSalmonStandardby2020.

Chris Ninnes, chief executive, ASC said,“GSI’s commitment to significantly improvingthe sustainability of salmon farming mirrorsASC’saimto transformaquaculture towardsenvironmentalsustainabilityandsocialrespon-sibility.

“A commitment at this scale presentsan unprecedented opportunity to realise ameaningful reduction in the environmentaland social impact of the sector. It is a hugestatementofleadershipintenttotackletheseissues.”

The initiative ties in with ASC plans tolaunch a certified salmon to the market inearly2014.

“I consider it extremely positive that amajor proportion of the salmon farmingindustry is voluntarily seeking to becomeenvironmentally responsible and to do thisin a transparent way so that all can see thereductionofindustryimpact.

“Transparency is one of the corner-stones of ASC. The standards require anunprecedentedamountofpublicdisclosureof farm-leveldata fromcertified farms thatare currently not publicly available inmostcases. GSI members are aware of theserequirements. However, as an industry-ledinitiativeandbyworkingtogethermembersare well placed to meet them as theyachievecertification.”

Theinitiativehasbeenwarmlywelcomedby the aquaculture industry. Mary EllenWalling,executivedirector,BritishColumbiaSalmon Farmers’ Association, Canada said,“This initiative recognises that there is nolimit to how sustainable you can be. Youdon’treachthehighestlevelandstop;thereis always room for improvement, alwaysmore you can learn. This collaboration willbenefit the industry in BC and around theworld.”

GSI member companies includeAcuinovaChile;Bakkafrost;Blumar;Cermaq;CompañíaPesqueraCamanchaca;EmpresasAquaChile; Grieg Seafood; Lerøy SeafoodGroup; Los Fiordos; Marine Harvest;NorwayRoyalSalmon;SalMar;MultiexportFoods SA; The Scottish Salmon Company;ScottishSeaFarms.

More InforMatIon:www.globalsalmoninitiative.org

7

Ygnve Myhre, CEO, SalMar, Norway and GSI member, “While we have

been making attempts at sustainability, salmon farming is a young industry and

we recognise that more needs to be done and we can do better"

Chris Ninnes, chief executive, ASC, “GSI’s commitment to significantly improving the sustainability of salmon farming mirrors ASC’s aim to transform aquaculture towards environmental sustainability and social responsibility"


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notconducivetoeasydigestion.Thefinerthecrushinggranularity,thelargerthesurfaceareawhichcontactswiththedigestiveenzymethusthedigestibilityisincreased.

Raw materials come in different shapesand thicknesses. So if they are not groundbefore processing, the finished pellets canlack a balanced nutritional quality and havepoor stability in water. Table 1 shows therelationshipbetweenthegrindingfinenessandstabilityinwater.

Feed pellets have little viscosity whenground to a large particle size. The crushingfineness also has an effect on the followingprocessessuchasmixingandsteammodulat-

ingandthefinenessofpulverizationhasgreatinfluenceonstability.Whenthegrainfinenessis perfect, the raw materials can be fullymixedandtheswellingpropertyofmaterialsconvergemakingforgoodstability.

Finer particle sizes will have a largersurface area which can be fully modulated,makingbetter-formedpellets.Althoughfinerparticle size is conducive toproducing feedpellets with good stability, the grain sizeshould not be too fine otherwise the pel-leted feeds are fragile. The proportions ofcoarse grain, medium grain and fine grainshould be appropriate so that during thepelleting period the fine grain can fill the

spacebetweenthecoarsegrains.Thismeansthat the contact area between particles isincreasedandthepelletizingperformance isimproved.

Choosing the appropriate grinding equipment is crucial

Controlling the grinding fineness has adirectinfluenceonthestabilityofaquaticfeedpellet and the production cost. The impor-tanceofcost shouldnotbeunderestimated;electricity consumption during the grindingprocess accounts for 50-70 percent of thetotalpowerconsumption.

Choosing the appropriate grinding equip-ment isalsocritical.Differentaquaticanimalshavedifferent requirements in termsof par-ticle size of feed ingredients which requirescorresponding grinding equipment. Hammermillsarewidelyusedinthefeedindustryandinaquaticfeeds.Thehammermillconsistsofhammers, a rotor, the grinding surface andsieve.Thehammer is themainworkingpartwhoseshape,size,quantityandlinespeedhasagreatinfluenceonthegrindingefficiencyandgrindingfineness.Whenthelinearvelocityofhammerbladeisslower,thegrindingefficiencyandproductionefficiencyarelow.

A quicker line speed will improve grind-ing efficiency. However, too high a speedwill make the material speed fast, reduce

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Evaluation of prebiotic and probiotic effects on the intestinal gut microbiota and histology of Atlantic salmon by Mads Kristiansen, Einar Ringø Norwegian College of Fishery, Faculty of Bioscience, Fisheries and Economics, University of Tomso, Norway- Aquamedical Contract Research, Vikan, Norway; Daniel Merrifield, Aquaculture and Fish Nutrition Research Group, School of Biomedical and Biological Sciences, University of Plymouth, UK; Jose Gonzalez Vecino, EWOS Innovation AD, Dirdal, Norway and Reidar Myklebust, Molecular Imaging Center, Institute of Biomedicine, University of Bergen, Norway

Today it is generally accepted thatthethreemajorroutesofinfectionin fish are through: a) skin, b) gillsand c) the gastrointestinal (GI)

tract.TheGImicrobiota,includinglacticacidbacteria (LAB), have been suggested to beimportantinfishhealthandithasbeensug-gestedthattheautochthonousgutbacterialcommunitymayberesponsible forcontrol-ling thecolonizationofpotentialpathogensbyadhesioncompetitionandproductionofantagonistic compounds. If the GI tract isinvolvedasaninfectionroute,scientistshaveaddresswhetherprobioticbacteriaareabletoadheretoandcolonisemucosalsurfaces

and outcompete endogenous bacteria andpathogens.

Investigating these topics effectively in invivomodelscanbedifficultas theyare timeconsuming and costly. Furthermore, as theEU has recommend reductions of in vivoexperimentsandthenumbersofanimalsusedinexperiments (Revisionof theEUdirectivefor theprotectionofanimalsused for scien-tificpurposes [Directive86/609/EEC];8thofSeptember2010),attemptshavebeenmadeto use alternative ex vivo methods (e.g. theUssing chamber, everted sack and intestinalsackmethods).

The first aim of the present study wasto investigate possible effects of a prebioticfeed on epithelial histology and indigenousGI tract microbiota in the proximal intes-tine (PI) and distal intestine (DI) of Atlanticsalmon. Furthermore, the same effects,including morphological changes of epithelialcells after ex vivo exposure of the intestinaltracttoCarnobacteriumdivergens,aprobioticbacterium, are investigated by light micros-copyandelectronmicroscopy.The resultofCarnobacterium exposure is of high impor-tance to evaluate as translocation and celldamagearenegativecriteriawhenevaluatingtheuseofprobiotics inendothermicanimalsaswellasinfish.

Thesecondaimofthepresentstudywasto evaluate the bacterial community of thePIandDIofsalmonfedcontrolorprebioticdiets, before and after ex vivo exposure toprobioticbacteria,inordertoinvestigateiftheindigenous GI tract microbiota is modulatedbythedifferenttreatments.

Finally, we addressed the issue as towhethercarnobacteriaisolatedintheexvivostudieswereabletoinhibitinvitrogrowthofthe pathogenic bacteria Yersinia rückeri andAeromonas salmonicida ssp. salmonicida.

Fish husbandryTwohundredandfortyvaccinatedAtlantic

salmon (Salmo salar L.) were held at theEWOS Innovation AS Research Station,Dirdal, Norway. The average weight at thestartoftheexperimentwas350g.Twohun-dred and forty fish were distributed equally(i.e. 40 fish per tank) into six tankssuppliedwith500litresofseawaterandtwo diets were offered (i.e. triplicatetanks per diet). The control diet andprebiotic diet had the same ingredientcomposition(Table1)anddifferedonlyin the inclusion of 0.2 percent EWOSprebiosal®intheprebioticdiet.EWOSprebiosal®,isdescribedasamulti-com-ponentprebioticspecificallydesignedforsalmonidfish;moredetailedinformationaboutthecompositionofEWOSprebi-osal® is not available for commercialreasons.Feedingwasconductedtwicea

daywithdurationof2.5hourbetweeneachfeedingforaperiodof15weeks.Duringthefeeding period the water temperature andsalinityranged,withseason,from5.3-12.9°Cand26.7-30.9gl-1.

The samplings were carried out at twodifferentpoints:at thestart (week0)andattheendof the trial (week15).AnoverviewofthedifferenttreatmentsandgroupsislistedinTable2.

Probiotic bacteriaThe probiotic bacterium used in this

experiment was Carnobacteriumdivergensstrain Lab01 originally isolated from juvenileAtlantic salmon fed a commercial diet. Thebacteria were stored in glycerol-containingcryotubesat-80°Candinoculatedintotrypticsoybroth(Difco,USA)withglucose(10gl-1)

Table 2: Experimental treatments applied to Atlantic salmon intestine fed control and prebiotic diets

treatment grouop

type of treatment

type of feed

Week of feeding

1 Saline Control 0

2 C. divergens1 Control 0

3 Saline Control 15

4 C. divergens2 Control 15

5 Saline Prebiotic 15

6 C. divergens2 Prebiotic 15

table 1: Dietary formulation and chemical composition of the experimental diets

%

Fishmeal 31.25

north atlantic fish oil 13.50

Vegetable protein concentrates1 25.76

Vegetable oil 14.01

Carbohydrate-based binders2 13.00

Micro premixes3 2.48

Chemical composition (%)

Moisture 6.9

Protein4 44.2

Fat4 29.1

nFe4 1.6

ash4 8.41 Incudes soy protein concentrate, pea

protein concentrate, wheat gluten, sun flower meal.

2 Includes wheat and pea starch3 Includes vitamin, mineral, amino acid

and pigment premixes and 0.2% eWoS prebiosal® added to the prebiotic diet (at the expense of an equal volume of carbohydrate-based binders)

4 dry weight basis

8


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and NaCl (10 g l-1),viz. TSBgs medium.After approximately24 hours of pre-inoculation at roomtemperature with anagitationof190rpm,1percentofthepre-culture was trans-ferred to new TSBgsmedium and growth(same growth condi-tions as above) wasmeasured by opticaldensityforevaluationof the growth cycle(data not shown).Bacterial viability wasconfirmed by platingbacterial suspensionson tryptic soy agar(Difco)+glucose(15g l-1) and NaCl (15g l-1) (TSAgs) plates.The results obtainedfrom this studywereused to calculate thebacterial concentra-tion in the experi-mentalbacterialsolu-tions.

Table 3: Cultruable heterotrophic bacterial levels (log CFU g-1 wet weight) and identity (as determinedfrom phenotypic characteristics and 16S rRNA sequence analysus) obtained from different groups after the ex vivo assay

Proximal intestine Distal intestine

Group tVC (log CFU g-1) no Bacteria % tVC (log

CFU g-1) no Bacteria %

1 1.72 12Psychrobacter aquimaris - 16.7%

Psychrobacter glancincola - 16.7%Psychrobacter spp - 66.6%

1.73 11

Psychrobacter glancincola - 9.0%Psychrobacter spp - 36.3%

Pseudoalteromonas - 36.3%Brevibacterium sp. - 9.0%

Moraxella sp. - 9.0%

2 6.04 7 Carnobacterium divergens - 100% 5.56 7 Carnobacterium divergens - 100%

3 2.08 17

Carnobacterium divergens - 70.6%Pseudomonas fluva - 17.6%

Pantoea spp - 5.9%Gammaproteobacteria - 5.9%

2.69 15

Carnobacterium divergens - 33.3%Pseudomonas fluva - 6.6%Shewanella baltica - 6.6%Vibrio splendidus - 13.3%

Gammaproteobacteria - 40%

4 6.26 8 Carnobacterium divergens - 87.5%Pseudomonas spp - 12.5% 6.68 8 Carnobacterium divergens - 100%

5 2.34 47

Carnobacterium divergens - 29.8%Carnobacterium spp - 51%

Pseudomonas antartica - 2.1%Pseudomonas korensis - 2.1%Enterbacter hormaechi - 8.5

Gammaproteobacteria - 4.3%Uncultured bacterial clone CK20 - 2.1%

1.71 44

Carnobacterium divergens - 25%Carnobacterium spp - 52.3%

Pantoea spp. - 18.2%enterobacter spp. - 4.5%

6 6.63 25

Psychrobacter marincola - 4%Pseudomonas sp - 8%

Carnobacterium divergens - 20%Carnobacterium spp - 68%

6.7 17 acinetobacter sp. - 5.6%Carnobacterium divergens - 94.2%

*N = number of isolates identified


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Ex vivo exposure to bacteriaThree fish were randomly selected from

two of the tanks fed each diet and killedwithablowtothehead.Theentireintestine,from the last pyloric caeca to the anus,wasremoved aseptically and intestinal contentwasgentlysqueezedout,beforetheintestinewas flushed three times with sterile salinesolution(0.9%NaCl),inordertoremovetheallochthonousgutbacteria.Theposteriorendwas tightly tied with cotton thread beforefilling (ca. 1.5ml)with the appropriate assaysolution(Table2),tyingtheanteriorendandsuspendingthesealedintestinaltubeinsterilesalinesolution.Theintestinalsackswerethenincubatedat10ºCforonehour.

After incubation the intestine was cutopen, the contents discarded and flushedthreetimeswithsterilesalinesolution.

Post ex vivo bacterial assaysSamples for bacteriology from each seg-

mentfromthefirstsamplingpoint(groups1and2)werepreparedbyhomogenizing1gof

intestinaltissue(PIorDI)in1mlsterilesalineusingaStomacher(SeawardLaboratory,UK).Gutsamplesforbacteriologyfromthesecondsampling (groups 3-6) were prepared bygentlyscrapingoffmucuswithasterilescalpel.Thereafter,thesegmentswereweighed.Boththe homogenates and mucus were used tocreate serial ten-fold dilutions which werespreadplated (100μl) onTSAgs plates andincubatedat6ºCforupto1weektodeter-mineviablecountsofculturableheterotrophicbacteria.

After sub-culturing on TSAgs to achievepure cultures, phenotypicbacterial identifica-tion(Gramstain,colonymorphology,oxidase-andcatalasetestsandglucosefermentation)wascarriedoutonrandomcoloniesfromallplates containing between 10-300 colonies.Atotalof168bacterialstrainswere isolatedfromthetwosamplingpoints.

16S rRNA characterization of isolates

ThebacterialDNAwasisolatedfollowing

theprotocolfromacommercialkit(DNeasyBlood and Tissue, Qiagen, USA). Specifictreatment for Gram-positive and Gram-negative isolates was carried out accordingtothemanufacturer’s instructions.Template-DNA was diluted to a concentration ofapproximately 20-30 ng μl-1 using Milli-Qwater. The PCR mix constituted of 8 μl oftemplate-DNA, 36 μl Milli-Q water, 5 μl10x buffer F511, 0.25 μl dNTP, 0.25 μl27Fforwardprimer,0.25μl1492Rreverseprimerand0.25μlDNA-polymeraseyieldingatotalvolumeof50μl.PCRthermalcyclingconsistedof initial denaturation of 94 ºC, followed by35 cyclesof94 ºC for20 s, 53 ºC for20 sand72ºCfor90swithafinalextensionstepof72ºCfor7min.ToverifyPCRproducts,sampleswererunongelelectrophoresis.ThePCR-productsweredesaltedbymixing20μlof PCR-product with 40 μl of 100 percentethanoland2μlof3MNaOAc(pH5.3)andvortexedwell. Sampleswere then incubatedon ice for30min followedbycentrifugationfor20minutesat14,000gusinganEppendorfMicrocentrifuge Model 5417R. The superna-tantwas removedandpelletwashed in100μlof80percentethanolandcentrifuged foranother 5 minutes at 14,000 g. The super-natant was removed and the pellet dried atroomtemperaturefor60minutes.Thepelletwas then resuspended in 30 μl of Milli-Qwater.PurifiedPCRproductsweresequencedasdescribedelsewhere.

The resultant nucleotide sequences weresubmitted to a BLAST search in GenBank(http://blast.ncbi.nlm.nih.gov/Blast.cgi) toretrieve the closest known alignment identi-ties for the partial 16S rRNA sequences.Gene sequences that showed higher than95percentsimilaritytoagenusorspeciesinGenBankwerecategorizedaccordingly.

InvitrogrowthinhibitionofpathogensbyLABisolatedformtheexvivostudies

ElevenrandomlychosenLABisolatedfromthe intestinal tract after ex vivo exposureand one type strain, Carnobacterium inhibens(CCUG31728),weretestedforantagonisticeffects against two different fish pathogens.Thepathogenicbacteriaused in thepresentinvestigation were Yersinia rückeri (CCUG14190)andAeromonas salmonicida ssp. salmo-nicida(Ass4017).C. inhibens (CCUG31728)was used as a positive control as previousinvestigations have demonstrated that thisstrain has an inhibiting effect towards V. anguillarumandA. salmonicida.Invitrogrowthinhibition of the two fish pathogens by thetwelve LAB was tested using a microtitreplate assay described in detail by Ringø andco-authors. This method has been used intwo recent studies.Thepathogenic bacteriallevels at the start of assays were 106 cellsml-1. Positive in vitro growth inhibition wasdefinedwhennogrowth(turbidity<0.05atoptical density;OD600 nm)of the pathogen


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was detected. Sterilegrowth media and thepathogens were usedas controls. Growth (atOD600) of the patho-gens without additionof sterile supernatantofLAB was approximately0.6.Measurementswerecarried out each hourusinganautomaticplatereader (Bioscreen C,Labsystems,Finland).

HistologySamples for light

microscopy (LM) andtransmission electronmicroscopy(TEM)werecollected by excisingapproximately 5 mmfrom the posterior partofthePIandDI.Thesampleswereimmedi-ately fixed in McDowells fixative and storedat 4 ºC until processing. TEM and LM sam-pleswereprocessedasdescribedelsewhereMorphologicalobservationsweremadefrommultiplemicrographs(8) fromeach intestinalregion from two fish within each group.Thefollowingmorphologicalparameterswereobserved; detached microvilli, enterocytesdetached from the basalmembrane, disinte-gratedcelljunctions,presenceofgobletcells,presenceofabsorptivevacuolesandpresenceofintraepitheliallymphocytes.

ResultsBacteriallevelsafterexvivoexposureThe adherent bacterial levels, as deter-

minedbyusingastomacher(groups1and2)orbythecollectionofmucus(andsubsequentweighing of the segments the mucus wasremovedfrom)(groups3-6),didnotseemtodifferwhich indicates that the different sam-pling methods were similarly effective. Table3presentsanoverviewoftheautochthonousbacterial levels isolated from each segmentandeachgroupexposedtoeithersalineorC. divergens.Allvaluesareexpressedaslogcol-ony formingunits (CFU)g-1.Autochthonousbacteria isolated from intestines of fish fedthecontroldietattheexperimentalstartandexposed tosalinewasapproximately log1.7CFUg-1inbothPIandDI,whilethenumberof bacteria isolated from intestines of fishexposedtoC. divergenswaslog6.04CFUg-1inPIandlog5.56CFUg-1inDI.

After 15 weeks of feeding slightly highervalues were present in PI of fish fed theprebiotic diet post exposure to saline or C. divergens compared to fish fed the controldiet. Indeed, the bacterial level in the prebi-oticfedfishintestineexposedtoC. divergens(group6)was234percentgreaterthanthatofthecontrolfedfishintestineexposedtoC.

divergens (group 4). In both dietary groups,a similar bacterial level (~log 6.70 CFU g-1)was detected inDI exposed toC. divergens.However, a higher bacterial level (log 2.69CFUg-1)wasobserved in theDIof controlfedfishexposedtosalinethanthatofprebi-oticfedfish(log1.71CFUg-1).

Isolationandidentificationofbacteriaafterexvivoexposure

A total of 168 bacterial strains wereisolated from the two samplings. Amongthese,40isolateswereisolatedfromthefirstsamplingpointand128isolateswereisolatedfrom the second sampling point. All isolatesweretestedformorphologyandbiochemicalproperties(colonymorphology,Gram-testing,oxidase - and catalase tests and glucosefermentation).

One hundred and eleven isolates werefurtheridentifiedbypartialsequencingofthe16SrRNAgene.Isolatesnotidentifiedby16SrRNA gene sequencing but showing similarbiochemical and physiological properties tothose isolates identified by 16S rRNAgeneswere defined as ‘-like’. Table 3 provides anoverview of the different bacterial speciesisolatedineachexperimentalgroup.

Week 0 Microbiota of fish fed control diet and

intestines exposed to saline (group 1):Analysis of the adherent microbiota in thePI of fish fed the control diet and exposedto sterile saline (group 1) revealed that allisolatesbelongedtothegenusPsychrobacter.Of the 12 strains isolated from the PI ofthis group, two strains showed 96 percentsimilarity to Psychrobacter aquimaris, twostrains were identified as Psychrobacter gla-cincolawhileeight strainswere identifiedasPsychrobacter spp.-like.

TheDIoffishexposedtosalineatthefirstsamplingpointshowedamorediversecom-

munitywhichconsistedof4differentbacterialgenera.Ofthese,tenstrainswereindentifiedto genus level and one strain was identifiedto species level. The bacteria identified togenus level belonged to Pseudoalteromonas,Psychrobacter, Moraxella and Brevibacterium,while the last strains showed high similarity(98percent)toPsychrobacter glacincola.

Microbiota of fish fed control diet andintestines exposed toC. divergens (group2):All bacteria isolated from PI and DI of fishexposed toC. divergens at the first sampling(group2)wereidentifiedasC. divergens.ThisobservationindicatesthatC. divergensareableto adhere to the intestinal mucosa in bothsegments.

Week 15Microbiota of fish fed control diet and

intestinesexposedtosaline(group3):After15weeksof feedingonthecontroldiet,the iso-latedstrains(17)fromthePIexposedtosalineweredominatedbyC. divergens;70.6percentof the isolateswere identified asC. divergens,17.6 % were identified as Pseudomonas fulva,5.9%belongedtoPantoea spp.while5.9%oftheisolateswereidentifiedasmembersoftheclassGammaproteobacteria.Thebacteriaisolat-edfromtheDIwereidentifiedasC. divergens,two strains as Vibrio splendidus, one strain asShewanella baltica, one strain as Pseudomonas fulva and six other strains were identified asGammaproteobacteria.

Microbiota of fish fed control diet andintestinesexposedtoC. divergens(group4).Intheintestineoffishfedthecontroldietfor15weeksandexposedtoC. divergens,theidenti-fiedbacterialstrainsisolatedfrombothPIandDIweredominatedbyC. divergens.Onlyonestrain,identifiedasPseudomonas spp.,isolatedfrom the PI of 1 fish did not belong to thespeciesC. divergens.

Microbiota of fish fed prebiotic diet and

Table 4: Identification of LAB strains and pathogen antagonistic activity of extracellular products used in the in vitropathogen asays

Isolate code

Source group

Intestinal region

Closest known species Strain accession no Identity

(%) antagonism

33 Group 2 Proximal C. divergens lHICa_53_4 FJ656716.1 98 Y. ruckeri a. Salmon

40 Group 2 Distal C. divergens lHICa_53_4 FJ656716.1 98 + -

75 Group 3 Proximal C. divergens lHICa_53_4 FJ656716.1 99 + -

84 Group3 Distal C. divergens lHICa_53_4 FJ656716.1 100 + -

14 Group5 Proximal Carnobacterium sp H126a eF204312.1 86 + -

57 Group5 Proximal C. divergens lHICa_53_4 FJ656716.1 99 + +

17 Group 5 Distal Carnobacterium sp H126a eF204312.1 99 + -





*_originally isolated from the digestive tract of Atlantic salmon (salmo salar) [20]


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intestines exposed to saline (group 5): Theintestineexposedtosalineoffishfedtheprebi-oticdietfor15weeksshowedhigherdiversitycompared to the other groups exposed tosaline (groups 1 and 3). Of the 47 strainsisolated from thePI, 14were identified asC. divergens,oneasPseudomonasantarctica,oneasPseudomonas koreensis,fourasEnterobacter hor-maecheiandoneasunculturedbacterialcloneCK20. The remaining isolated strains wereidentified as members of the CarnobacteriumandAcinetobacter genera.Thedominantbacte-ria in thePIof thisgroupbelongedtocarno-bacteria(81%)and30percentoftotalisolateswereidentifiedasC. divergens.

The bacterial composition of the isolatesfromtheDIof fish fedtheprebioticdiet for15weekswererelatively low indiversity.Ofthetotalnumberofstrainsisolated(44)fromtheDI exposed to saline 34were identified

as Carnobacterium, eight strains showed highsimilarity(%)toPantoea spp.andtwostrainsbelongedtothegenusEnterobacter.

Microbiota of fish fed prebiotic diet andintestines exposed to C. divergens (group 6):In group 6, fish fed the prebiotic diets for15 weeks and exposed to C. divergens, theisolated strains in the PI were dominated byC. divergensandC. divergens-likestrains.Ofthe22 carnobacteria isolated, fivewere identifiedasC. divergensby16SrRNAsequencingwhile17 isolateswere identifiedasC. divergens-like.Threeother isolateswere identifiedasmem-bers of the genera Pseudomonas (2 strains)andPsychrobacter(onestrain).Ofthe17strainsisolatedandidesntifiedfromtheDIofgroup6,C. divergens andC. divergens-like strainsdomi-natedwithonlyoneisolate,whichshowedhighsimilarity(99percent)toAcinetobacter spp.,notbelongingtothisspecies.

Microscopical analysesLight microscopy (LM): All LM micro-

graphs,both fromPIandDIoftheprebioticgroups (5 and 6) showed no morphologicaldifferences compared to the control feedingregime (groups 1-4). All intestinal sectionsexamined appeared normal and healthy; nosignsofdetachedenterocytes,necroticente-rocytes, widened lamina propria or necrosiswereobservedandthenumberofgobletcellsweresimilarinbothtreatments(examplesaredisplayedinFigure1).

Transmission electron microscopy (TEM):Similar to the observations using LM, TEMrevealed no differences between treatmentsorexposuregroups;allmicrographsrevealedhealthyepithelialbrushborder,nodeteriationoftightjunctionswasobservedandmicrovilliappeared uniform. The presences of rodlet-likecells(asshowninFigure2)werepresent

inthePIandDIofallgroups.Thenumbersofrodletcellspresentinthe PI displayed great differencesbetween individual fish but werealways observed in the upperhalf of the epithelium, above theunderlying intraepithelial lym-phocytes.

In vitro growth inhibition oftwofishpathogensbyextracellularextracts of LAB isolated from exvivostudies

Identification by partialsequencingofthe16SrRNAgenesoftheelevenLABstrainsisolatedfromtheexvivoexperimentsandsubsequently used in the in vitropathogen antagonism assays aredisplayed in Table 4. The resultsshowthatgrowth inhibitionofY.rückeriwasobtained fromextra-cellularextractsfromallstrainsofcarnobacteria isolated from theex vivo experiment. However, invitro growth inhibition of A. sal-monicida ssp. salmonicida wasonlyobtained from the extracellularextractofCdivergens isolate57.The extracellular products fromthe positive control, C. inhibens CCUG31728,didnot inhibit thegrowth of A. salmonicida ssp. sal-monicida.

DiscussionThe ex vivo intestinal sack

methodhasbeenused in severalstudiestoevaluatepossiblehisto-logicalchangesinthefishintestineafter exposure to high levels ofLAB.TheresultofLABexposureto the intestine is of high impor-tance as translocation and celldamage have been proposed asimportantcriteriawhenevaluating


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theuseofprobiotics inendothermicanimalsaswellasinfish.Recently,theeffectofexvivoLAB exposure on the gut microbiota in fishwasdocumented,buttotheauthors’knowl-edge theeffectofprebiotic supplementationandexvivoLABexposureofthefishintestinehasnotbeeninvestigated.

The culturable bacterial levels recoveredon TSAgs plates from groups exposed tosaline were relatively low, ranging from log1.72 to 2.34 CFU g-1. These values are lowcomparedtoautochthonouslevelspreviouslyreportedinAtlanticsalmonandrainbowtroutOncorhynchus mykiss.Thisislikelyduetothethorough rinsing process, three times priorand three times post probiotic/saline expo-sure. Culturable adherent bacteria in the PIobserved in the prebiotic group exposed tosaline (group 5) was log 2.34 CFU g-1. Thefact that the value from this group is higherthan in control groups exposedtosalinewater(log1.72and2.08CFUg-1),mightbeduetoafeed-ing effect of the prebiotic diet.However, this hypothesis meritsfurtherinvestigations.

Investigationsofthequalitativeandquantitativebacterialcompo-sitionof the intestinalmicrobiotawerebasedon the studyof168pure cultured bacterial isolates.These isolates were biochemi-cally tested in order to obtain ageneral classification. From thisclassification 111 isolates wereselected by the lottery methodandidentifiedby16SrRNAgenesequencinganalysis.

The bacterial levels observedingroupsexposedtosalinevariedbetween segments and feedingregime. By comparing differentfeedinggroupsit isclearthattheindigenous microbiota of the PIwere affected by the diet, whiletheeffectofprebioticfeedingonthe microbiota was less clear intheDI.IntheintestineexposedtoC. divergens the average numberofbacteriawerehigher in thePIwhenthefishwerefedtheprebi-otic diet compared to fish fedthe control diet. Whether thesefindingscanberelatedtoahigherC. divergens colonization successinprebioticfedfishmeritsfurtherinvestigations.

The bacterial compositionfromthePIincontrolgroups,i.e.fishfedcontroldiet,andthereaf-terexposedtosalineweredomi-natedbymembersofafewgen-era(Psychrobacter,CarnobacteriumandPseudomonas).Psychrobacter,Carnobacterium andPseudomonas

spp.arecommonlyreportedintheGItractoffish,andthesebacteriahavepreviouslybeenisolatedandidentifiedfromtheGItractofsal-monids.Carnobacterium spp.haveoftenbeenreportedtobecomponentsofthegutmicro-biotaofsalmonids;indeed,C. maltaromaticum,C. mobile,C. divergensandCarnobacterium spp.have been identified from Atlantic salmon.Theconsistencyof isolationof these speciesindicates that these might be common corecomponentsoftheGImicrobiomeofAtlanticsalmonandarelikelytobeofimportancetothehost.

The bacterial composition isolated fromthe DI in the first control group exposedto saline (group 1) were dominated byPseudoalteromonasspp.andPsychrobacter spp.,while in the other control group (group 3)Acinetobacter spp. and C. divergens were thedominantbacteriaisolated.Alloftheselisted

bacteria have previously been isolated fromthe intestine of Atlantic salmon. The bacte-rial composition of the PI observed in theprebiotic fed fish exposed to saline (group5) showed greater bacterial diversity thanthatobserved fromcontrol fed fishexposedto saline (groups 1 and 3). The majority ofthe bacteria from the PI of group 5 wereC. divergens and Carnobacterium spp., whichtogetheraccountedfor81%ofalltheisolatedbacteria.Thetwootherbacterialspeciesthatwere isolated from this groupwerePantoea spp. and an unidentified member of theclassGammaproteobacteria.TheabundanceofculturableadherentCarnobacterium spp.(77%of isolates identified)washigher intheDIoftheprebioticfedfish(group5),comparedtocontrol groups (group 1= 0% and group 3=36%).Theseresultssuggestthattheprebi-oticsupplementationelevatesautochthonous


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Carnobacterium spp. levels, particularly in theDI. To the authors’ knowledge there is verylittleinformationregardingtheeffectofprebi-oticsoncarnobacteriawithin theGI tractoffish.However, somestudiessuggest that thecarnobacteriapopulationswithintheGItractof salmonids are effected by various dietaryfactorssuchaskrillmealandoxytetracyclineinAtlantic salmonanddietary carbohydrates inArctic charr (Salvelinus alpinusL.).However,itwasobservedthat thepresenceofdietaryinulin(aprebiotic-typecarbohydrate)tendedto lower culturable autochthonous carno-bacteria levels(byca.90%)inthehindgutofArcticcharrandalsoelevatedtheproportionof C. maltaromaticum at the expense of C. divergens.Thesefindingssuggestthatdifferentprebioticsmayinfluencedifferentcarnobacte-riastrainsindifferentfishspecies.

InallgroupsexposedtoC. divergensinthe

ex vivo studies the same C. divergens strainwas identified to dominate both the PI andDIafterexposure.C. divergens levelswereintherangeof104-106CFUg-1intestinewhichindicatesthatthebacteriaareabletopopulateand potentially colonize the intestinal mucusand out-compete other adherent bacteriaafteronlyonehourofexposure.TheseresultsareinaccordancewithcorrespondingstudiesinthatLABareabletocolonizetheintestineofAtlanticsalmonafteronehourexposure.

Despite theplethoraof informationavail-ableontheprebioticefficacyofelevatingpro-biotic colonization (i.e. synbiotics) in variousterrestrialspecies,littleinformationisavailablein fish. Further studies should focus on thistopicasthepresentstudydemonstratedthatthe presence of the dietary prebiotic, prebi-osal®, elevated theproportionof carnobac-teriafrom71-81percentintheDI(aswellas elevating total bacterial levels, effectivelyquadruplingthenumberofcarnobacteria)andfrom33%to77%inthePI(althoughthetotalbacterialpopulationwaslower).

Thehistologicaleffectofexposing theGItractofAtlantic salmon tohigh levelsof theC. divergenswasinvestigatedbylightandelec-tron microscopy. Furthermore, the intestinaleffectsof feedingaprebioticdiet toAtlanticsalmonwereevaluated.

Results from LM-investigations in thepresent study showed no apparent his-topathological changes of the epithelium inthe PI or DI, after exposure of C. divergens.In particular the micrographs demonstrated

that enterocytes showed no signs of junc-tionalrupturefromthebasementmembranewhichisincontrasttoobservationsofthePIof Atlantic salmon after exposure to VibrioanguillarumandA. salmonicida.

TEM observations confirmed the findingsobservedinLMregardingalackofhistologicalchanges. TEM revealed no observable dif-ferences between the groups in respect tothepresenceofcelldebris inthe lumen,theamountofmucus,thenumberbacteria–likeparticlesinthelumenandbetweenthemicro-villi, disorganized microvilli and disintegratedtight junctions. The enterocytes within allgroups displayed normal cell contacts withunaffectedtightjunctionsandzonaadherens.ThefactthatC. divergensdidnotinflictdam-agetotheintercellularunctionisgreatimpor-tantancesincethelooseningofthesejunctionscontributestoaparacellularportofentryfor

potentialpathogens.Rodlet cells were present in

large numbers in the PI of allgroups,whileintheDIthenumberobservedwerelower.SincegroupsexposedtoC. divergensdidnotdis-playanycleardifferencesinnumberofrodletcellscomparedtogroupsexposed to saline, their presence

inthosegroupsmaythereforenotberelatedto an immunological function towards theexposed bacteria. On the other hand, theroleofrodletcellsas immunecellsandtheirlarge number in the PI compared to the DImaybeadefensefunctiontowardspotentialinvading bacteria of the PI. Since the PI hasbeen confirmed as being an infection routeforpathogenicbacteriabyseveralstudies,theroleof rodletcellsas immunecells in thePIispossibleandwarrantsfurtherinvestigation.

TheantimicrobialeffectsofLABhavelongbeenutilizedinfoodpreservationbyfermenta-tion and several comprehensive reviewshavebeenpublishedon theabilityof LAB topro-duceproteinaceousantimicrobialsubstances.Infishstudies, theantagonisticeffectofLABhasbeencarriedoutonGram-negativefishpatho-genssuchasVanguillarumandAsalmonicida.In the present study strong growth inhibitionofY. rückeriwas recorded fromextracellularextracts from late exponential growth phasefromalloftheelevenCarnobacteriastrainsiso-latedfromtheexvivoexperiments.However,the ability of the isolated strains to inhibitgrowth of A. salmonicida ssp. salmonicida wasonly observed from one strain isolated fromthePI.Thefactthatonlyone(isolate57)ofthe11 strains displayed inhibitory effects towardsA. salmonicidassp.salmonicidais inaccordancewiththeresultsofRingøwhoobservedalackofantagonismwhenchallengingA. salmonicida ssp. salmonicida to extracellular extracts fromC. divergensstrainLab01.Theseresultsindicatethat the production of extracellular products

only, might not be sufficient for strains of C. divergens in lateexponentialgrowthphase, toinhibitgrowthofA. salmonicidassp.salmonicida.

The positive control bacteria, C. inhibens whichJöbornetal.reportedtodisplayantago-nistic effect against A. salmonicida, showed nosign of antagonism in the present study. ThisobservationthereforeindicatesthatantagonisiticactivityofC. inhibens isonlyeffectivewhencellsareactively incubatedtogetherorthatantago-nistic extracellular products areonly producedbyC. inhibens inthepresenceofA. salmonicida.

TheabilityofC. divergensasusefulprobiot-icswitheffectsagainstY.rückeriandA. salmoni-cidahavepreviouslybeenreportedinvivoandinvitro.KimandAustinobservedthatdietaryprovision of C. divergens strain B33, isolatedfrom the intestine of healthy rainbow trout,increased survival of rainbow trout againstA. salmonicida and Y. rückeri challenge by 60percentcomparedtothecontrolgroup.EventhoughstrainsofC. divergensshowantagonisticeffects against pathogens, the precise mecha-nism of action of antimicrobial compoundsisolatedfromfishremainsunclear,butsugges-tionsabouttheirabilityofpenetratingcellwallsbyformingporesandchannels,thusrenderingit more fragile and incapable of carrying outnormalmetabolismhasbeenproposed.

In order to confirm the in vitro probi-oticeffectofC. divergensagainstY.rückeriinAtlantic salmon, further investigations shouldtherefore include in vivo challenges studies.By further applying electronmicroscopy, thephysical interferencemechanismsbetweenC. divergensandY.rückeri intheGItractmightbeobserved.

AcknowledgementsThe authors thank the technical staff at

EWOSInnovationASforfeedmanufacture,analysis and running the feeding trial andthank Dr. Sigmund Sperstad and Dr. ChunLi, Norwegian College of Fishery Science,University of Tromsø for their inestimablehelpduring16SrRNAgenesequencingandin vitro growth inhibition. We also thankRandi Olsen and Helga Marie By at theEM department at University of Tromsø,and Anne Nyhaug at Molecular ImagingCentre, Institute forBiomedicine,Universityof Bergen for their inestimable help duringlight and electron microscopy analysis. Thisstudywaspartiallysupportedbygrantsfromthe Norwegian MABIT-program (projectnumberAF0038).

"The histological effect of exposing the

GI tract of Atlantic salmon to high levels

of the C. divergens was investigated

by light and electron microscopy"

Thisarticlewasoriginallypublishedon


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