expert topic 1305- salmon

7/29/2019 EXPERT TOPIC 1305- SALMON

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September | October 2013EXPERT 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 ofinformation published.Copyright 2013 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any formor by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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3/1636 | InnaIOnal AquAFeed | September-October 2013

EXPERTTPIC

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

SALMON

EXPERT TOPIC


4/16

World viewAccordingtostatisticsfromtheGlobalSalmon

Initiative (GSI) approximately 60 percent of

the worlds salmon is farmed. Figures show

that in 2011, wild-caught salmon reached

approximately930,000tonnes.Adropinthe

ocean compared to the 1,600,000 tonnes

producedbyaquaculture.

Salmon belongto a family offishknown

asSalmonidaeandbasedontheirdistribution,

are further classified into two main genera;

Atlantic dwellers (Salmo) and Pacific Ocean

basedspecies(Oncorhynchus).Thedistinguish-

ing factor in this classification is that unlike

the Salmo genus, species belonging to the

Oncorhynchusgenusdieafterspawning.

Duetocomplexproductionneeds,widewatertemperaturerangesandbiologicalconditions,farm-

ingofAtlanticsalmon-themostpopularspecies

ofSalmonidae-isdominatedbyjustahandfulof

countries.Currently,theEU,USAandJapanhave

thelargestsalmonaquaculturemarkets.

Atlantic salmon is pisciverous and there-

forerequiresadietrichinproteinandlipids.

Farmedfishareusuallyfedacombinationof

fishmealandfishoilandalthoughthewaste

producedfromfishprocessingcanbeusedin

certain components of fishmeal production,

theriskoftransferringdiseasemeansitcannot

beuseddirectlyinfishfeed.Thereison-going

research into supplementing fish feed with

plant or microbe-based products, though

currentlynosupplementhasbeenfoundfor

pisciverousspecies(FAO).

www.globalsalmoninitiative.org

www.fao.org/fishery/en

1

Iceland

The Ice land ic Ministry o f F ishe rie s andAgriculture states that salmon farming first

beganinIcelandatthebeginningofthe19th

century,withthefirstattemptstorearsalmon

fryoccurringin1961.

The first land-based salmon production

farmwasdevelopedin1978andbythelate

1980sbiggerfarmswerebeingconstructed.

Between1984and1987,salmoneggs

w er e i mp or te d f ro m N or wa y. A t t hi s

tim e, the re wer e lar ge inves tme nt s in the

production of salmon smolts for export.

Later,oceanranchingandcageandland-

based farming attracted interest among

investors.Oceanranchinginvolvesreleas-

ing young reared smolts into rivers and

streams.Theyoungsmoltsusethecoastal

environmenttomatureforaroundayear

beforereturningatwhichpointtheyare

harvested.

Thecountryowesmuchofitsfarming

capabilitiestoitsclimate.Withunpolluted

seas and an abund ance o f cle ar wat er ,

aquaculture condit ions are regarded as

among the best in the world in Iceland.

In 2007, 600 tonnes of Atlantic salmonwereexported.By2009,therewereabout

45 r egiste re d f ish far ms o n t he i sl and.

Figuresshowthatofthese,about30were

producing juveniles, mostly for Salmonid

on-rearing.Accordingto figuresproduced

September-October 2013 | InnaIOnal AquAFeed | 37

EXPERTTPIC

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34

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5

TasmaniaSalmon farming commenced

in Tasmania in the mid-1980s

after a report to theTasmanian

FisheriesDevelopmentAuthority

concluded that a salmon farm-

ingindustrycouldbesuccessfully

developedontheislandstate.

Asaresult,in1984fertilised

Atlantic salmon eggs were pur-

chased from the Gaden Trout

Hatchery,Jindabyne,NewSouth

Wales,Australiafromstockorigi-

nallyimportedinthe1960sfrom

NovaScotia,Canada.Aseafarm

was then established at Dover

inthe south ofTasmaniaand a

hatcherydevelopedatWayatinah

inthecentralhighlands.

The first 53 tonne commer-cial harvest of Atlantic salmon

occurred between 1986 -1987.

Nowadays,theTasmanianindus-

trynow producesalmost 40,000

tonnesperannum.

Nearly 93 percent of

Tasmanian salmonid production

wassoldinthedomesticmarket

in2006.(DPIW)

www.tsga.com.au

6NewZealandNewZealandKingSalmon'splans

formarinefarmsinMarlborough

Sound, New Zealand, may get

the go ahead after the High

Cou rt dis miss ed a n a ppeal

againstthem.

Thedecisionof theBoard

ofInquiry,reachedinFebruary

2 01 1, t o a pp ro ve f ou r n ew

s al mo n f ar mi ng s it es i n t he

M arlboro ug h So un ds w as

appealed by two part ies and

that ap pea l was he ard at the

H ig h C ou rt i n B len hei m i n

May.

T he n ew s h as be en w el -

c om ed b y t he g ov er nm en t,

Th e i mp ac ts o f t he se n ew

m ar ine far ms o n t he i mp or -

tant recre ation and cons erva-

tion values of the Marl borough

Soundsaresmall.Thisisabout

u se of o nl y s ix h ec ta res o f

more than 100,000 hectares

ofwaterspaceintheSounds,

said Conservation Minister Dr

NickSmith.

We are a BluegreenGovernmentthatwantsjobsand

development but also wants to

ensure we look after our envi-

ronment and greatkiwi lifestyle.

This decision confirms this bal-

ancedapproach.

Primaryindustriesarevitalfor

economicgrowthinourregions,

andaquacultureplaysanimpor-

tant role in the Marlborough

economy. I welcome the news

that extra jobs will be created

asaresultofthesenewfarms,

said Primary Industries Minister

NathanGuy.

Thisdecisionisanotherstep

forward for New Zealand King

Salmon in its plans to establish

four new farms, delivering an

additional $60 million a year in

exportincomeandproviding200

newjobs.

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7/16

Globalsalmon farmingindustry

joins forceswith newsustainabilityinitiativeby Alice Neal, Associate editor,International Aquafeed magazine

The Global Salmon Initiative (GSI)

unites 15 global farmed salmon

producers committed to greater

industrycooperationandtranspar-

ency,inordertoachievesignificantandcon-

tinuousprogressinindustrysustainability.

Together, these15 companies represent

70percenttheglobalsalmonindustry,mean-

ingtheinitiativecouldhavearealimpacton

salmonaquaculture.

Themajorsalmonproducingcountriesof

Chile,Norway,Scotland,theFaroeIslandsand

CanadaareallrepresentedintheGSI.

YgnveMyhre,CEO,SalMar,Norway and

GSImember,said,Whilewehavebeenmak-

ingattemptsatsustainability,salmonfarmingis

ayoungindustryandwerecognisethatmore

needstobedoneandwecandobetter.

Weknowitwilltaketimeandwillbea

continuousprocess,butthroughtheGSIwe

have committed to the significant improve-

ment that is needed. This initiative is about

significant improvement in sustainability. It is

notaboutsatisfactionwiththestatusquo.

The GSI wil l achieve its aim through

global collaborationandresearch,poolingof

resourcesandsharingknowledge.

Whatis different isthatas theGSI,the

companies have committed tohelping each

other towards improved sustainability. Its

about cooperation, not competition, saidMyhre.

AlfonsoMarquzdelaPlata,chairofthe

GSIstandardscommitteeandCEO,Empresas

AquaChile S.A., Chile, said, We cannot

choose betweena healthy environmentand

healthyfood,weneedboth.Thisinitiativeis

apracticalapproachtoachievingboth.While

meetingthestandardatagloballevelwillbe

asignificantchallenge,thisisamajorcommit-

ment from the salmon farming industry and

wehopethatthroughGSIcollaboration,we

cangettheretogether.

TheinitialimpetusfortheGSIcamefrom

a meeting in 2011 which was attended by

a number of CEOs. At that meeting, the

CEOsheardaboutsignificantprogressother

industrieshadmadeinsustainabilitybywork-

ingtogether.ThatgroupofCEOsdecidedto

meetagainandinviteotherCEOsandindue

courseitwasagreedtoformtheGSI.

Currently,theGSIisfocusingonbiosecu-

rity,feedandnutritionandmeeting industry

standards.

In terms of feed ingredients, the GSI is

keentofindsourcesthatdonotputfurther

stress onmarineresources.TheGSI iscon-

sidering utilizing by-products and isworking

closelywiththeFAOtoassessavailabilityof

theseresources.

The GSI has chosen the Aquaculture

StewarshipCouncil(ASC)asitsaccreditation

bodyandaimstohaveallitsmembersmeet

theASCSalmonStandardby2020.

Chris Ninnes, chief executive, ASC said,

GSIscommitmentto significantlyimproving

the sustainability of salmon farming mirrors

ASCsaimtotransformaquaculturetowards

environmentalsustainabilityandsocialrespon-

sibility.

A commitment at this scale presents

an unprecedented opportunity to realise a

meaningful reduction in the environmental

andsocial impact ofthe sector. Itis ahuge

statementofleadershipintenttotackletheseissues.

The initiative ties in with ASC plans to

launch a certified salmon to the market in

early2014.

I consider it extremely positive that a

major proportion of the salmon farming

industry is voluntarily seeking to become

environmentally responsible and to do this

ina transparentway sothatallcan see the

reductionofindustryimpact.

Transparency is one of the corner-

stones of ASC. The standards require an

unprecedentedamountofpublicdisclosure

offarm-leveldatafromcertifiedfarmsthat

arecurrentlynotpubliclyavailableinmost

cases. GSI members are aware of these

requirements.However, as an industry-led

initiativeandbyworkingtogethermembers

are wel l p laced to meet them as they

achievecertification.

Theinitiativehasbeenwarmlywelcomed

by the aquaculture industry. Mary Ellen

Walling ,executivedirector,BritishColumbia

Salmon Farmers Association, Canada said,

This initiative recognises that there is no

limit to how sustainable you can be. You

dontreachthehighestlevelandstop;there

is always room for improvement, always

more youcan learn. Thiscollaboration will

benefitthe industry inBC and around the

world.

GSI member companies include

AcuinovaChile;Bakkafrost;Blumar;Cermaq;

CompaaPesqueraCamanchaca;Empresas

AquaChile; Grieg Seafood; Lery Seafood

Group ; Los F iordos ; Mar ine Harvest ;

NorwayRoyalSalmon;SalMar;MultiexportFoodsSA; TheScottishSalmonCompany;

ScottishSeaFarms.

More InforMatIon:

www.globalsalmoninitiative.org

Ygnve Myhre, CEO, SalMar, Norwayand GSI member, While we have

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

we recognise that more needs to bedone and we can do better"

Chris Ninnes, chief executive, ASC,GSIs commitment to significantlyimproving the sustainability of salmonfarming mirrors ASCs aim to transformaquaculture towards environmentalsustainability and social responsibility"

40 | InnaIOnal AquAFeed | September-October 2013

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8/16September-October 2013 | InnaIOnal AquAFeed | 41

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9/16

Evaluationof prebiotic

and probioticeffects on theintestinal gutmicrobiota andhistology ofAtlantic salmonby Mads Kristiansen, Einar RingNorwegian College of Fishery, Facultyof Bioscience, Fisheries and Economics,University of Tomso, Norway-Aquamedical Contract Research, Vikan,Norway; Daniel Merrifield, Aquacultureand Fish Nutrition Research Group,School of Biomedical and BiologicalSciences, University of Plymouth, UK; JoseGonzalez Vecino, EWOS Innovation AD,Dirdal, Norway and Reidar Myklebust,Molecular Imaging Center, Instituteof Biomedicine, University of Bergen,

Norway

Today it is generally accepted that

thethreemajorroutesofinfection

infisharethrough:a)skin,b)gills

and c) the gastrointestinal (GI)

tract.TheGImicrobiota,includinglacticacid

bacteria (LAB), have beensuggested to be

importantinfishhealthandithasbeensug-

gestedthattheautochthonousgutbacterial

communitymaybe responsibleforcontrol-

lingthecolonizationofpotentialpathogensbyadhesioncompetitionandproductionof

antagonistic compounds. If the GI tract is

involvedasaninfectionroute,scientistshave

addresswhetherprobioticbacteriaareable

toadheretoandcolonisemucosalsurfaces

and outcompete endogenous bacteria and

pathogens.

Investigating these topics effectively in in

vivomodelscanbedifficultastheyaretime

consuming and costly. Furthermore, as the

EU has recommend reductions of in vivo

experimentsandthenumbersofanimalsused

inexperiments(RevisionoftheEUdirective

fortheprotectionofanimalsusedforscien-

tificpurposes[Directive86/609/EEC];8th of

September2010),attemptshavebeenmade

to use alternativeex vivomethods(e.g. the

Ussing chamber, everted sack and intestinal

sackmethods).

The first aim of the present study was

to investigate possible effects of a prebiotic

feed on epithelial histology and indigenous

GI tract microbiota in the proximal intes-

tine (PI) and distal intestine (DI) of Atlantic

salmon. Furthermore, the same effects,

including morphological changesof epithelial

cells after exvivo exposureof theintestinaltracttoCarnobacteriumdivergens,aprobiotic

bacterium, are investigated by light micros-

copyandelectronmicroscopy.Theresultof

Carnobacterium exposure is of high impor-

tance to evaluate as translocation and cell

damagearenegativecriteriawhenevaluating

theuse ofprobioticsin endothermicanimals

aswellasinfish.

Thesecondaimofthepresentstudywas

to evaluate the bacterial community of the

PIandDIofsalmonfedcontrolorprebiotic

diets, before and afterex vivo exposuretoprobioticbacteria,inordertoinvestigateifthe

indigenousGI tractmicrobiota ismodulated

bythedifferenttreatments.

Final ly , we addressed the issue as to

whethercarnobacteriaisolatedintheexvivo

studieswereabletoinhibitinvitrogrowthof

the pathogenic bacteria Yersinia rckeri and

Aeromonas salmonicida ssp. salmonicida.

Fish husbandryTwohundredandfortyvaccinatedAtlantic

salmon (Salmo salar L.) were held at the

EWOS Innovation AS Research Station,Dirdal, Norway. The average weight at the

startoftheexperimentwas350g.Twohun-

dredand forty fishwere distributed equally

(i.e. 40 fish per tank) into six tanks

suppliedwith500litresofseawaterand

two diets were offered (i.e. triplicate

tanks per diet). The control diet and

prebioticdiethad thesame ingredient

composition(Table1)anddifferedonly

in the inclusion of0.2 percent EWOS

prebiosalintheprebioticdiet.EWOS

prebiosal,isdescribedasamulti-com-ponentprebioticspecificallydesignedfor

salmonidfish;moredetailedinformation

aboutthecompositionofEWOSprebi-

osal is not available for commercial

reasons.Feedingwasconductedtwicea

daywithdurationof2.5hourbetweeneach

feedingforaperiodof15weeks.Duringthe

feeding period the water temperature andsalinityranged,withseason,from5.3-12.9C

and26.7-30.9gl-1.

The samplings were carried out at two

differentpoints:atthestart(week0)andat

theendof thetrial(week15).Anoverview

ofthedifferenttreatmentsandgroupsislisted

inTable2.

Probiotic bacteriaThe probiotic bacterium used in this

experiment was Carnobacteriumdivergens

strain Lab01originally isolated from juvenile

Atlantic salmon fed a commercial diet. Thebacteria were stored in glycerol-containing

cryotubesat-80Candinoculatedintotryptic

soybroth(Difco,USA)withglucose(10gl-1)

Table 2: Experimental treatments applied to Atlantic salmon

intestine fed control and prebiotic diets

reatmentgrouop

ype oftreatment

ype offeed

Week offeeding

1 Saline Control 0

2 C. divergens1 Control 03 Saline Control 15

4 C. divergens2 Control 15

5 Saline Prebiotic 15

6 C. divergens2 Prebiotic 15

able 1: Dietary formulation and chemicalcomposition of the experimental diets

%

Fishmeal 31.25

orth tlantic 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

F4 1.6

sh4 8.4

1 Incudes soy protein concentrate, peaprotein concentrate, wheat gluten, sunflower meal.

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

and pigment premixes and 0.2% WSprebiosal added to the prebiotic diet(at the expense of an equal volume ofcarbohydrate-based binders)

4 dry weight basis


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andNaCl (10 g l-1),

viz. TSBgs medium.

After approximately

24 hours of pre-

inoculation at room

temperature with an

agitationof190rpm,

1percentofthepre-

culture was trans-

ferred tonewTSBgs

medium and growth

(samegrowth condi-

tions as above) was

measured by optical

densityforevaluation

of the growth cycle

(data not shown).

Bacterial viabilitywas

confirmed by plating

bacterial suspensions

on tryptic soy agar

(Difco)+glucose(15

g l-1) and NaCl (15g l-1) (TSAgs) plates.

The results obtained

fromthisstudywere

usedtocalculatethe

bacterial 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 dierent groups after the ex vivo assay

Proximal iesie Disal iesie

GrouptVC (logCFU g-1)

no Baceria %tVC (logCFU g-1)

no Baceria %

1 1.72 12Psychrobacer aquimaris - 16.7%

Psychrobacer glacicola - 16.7%

Psychrobacer spp - 66.6%

1.73 11

Psychrobacer glacicola - 9.0%Psychrobacer spp - 36.3%

Pseudoalteromonas - 36.3%

Brevibacerium sp. - 9.0%Moraxella sp. - 9.0%

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

3 2.08 17

Carobacerium divergens - 70.6%Pseudomonas fluva - 17.6%

Pantoea spp - 5.9%Gammaproteobacteria - 5.9%

2.69 15

Carobacerium divergens - 33.3%Pseudomonas fluva - 6.6%Shewaella balica - 6.6%Vibrio spledidus - 13.3%

Gammaproteobacteria - 40%

4 6.26 8Carobacerium divergens - 87.5%

Pseudomonas spp - 12.5%6.68 8 Carobacerium divergens - 100%

5 2.34 47

Carobacerium divergens - 29.8%Carobacerium spp - 51%

Pseudomonas antartica - 2.1%

Pseudomonas korensis - 2.1%Enterbacter hormaechi - 8.5Gammaproteobacteria - 4.3%

Uncultured bacterial clone CK20 - 2.1%

1.71 44

Carobacerium divergens - 25%Carobacerium spp - 52.3%

Paoea spp. - 18.2%erobacer spp. - 4.5%

6 6.63 25

Psychrobacer maricola - 4%Pseudomonas sp - 8%

Carobacerium divergens - 20%Carobacerium spp - 68%

6.7 17cieobacer sp. - 5.6%

Carobacerium divergens - 94.2%

*N = number of isolates identified


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11/16

Ex vivo exposure to bacteriaThree fish were randomly selected from

two of the tanks fed each diet and killed

withablowtothehead.Theentireintestine,

fromthelastpyloriccaecatotheanus,was

removed aseptically and intestinal content

wasgentlysqueezedout,beforetheintestine

was flushed three times with sterile saline

solution(0.9%NaCl),inordertoremovethe

allochthonousgutbacteria.Theposteriorend

was tightly tied with cotton thread before

filling(ca.1.5ml)withtheappropriateassay

solution(Table2),tyingtheanteriorendand

suspendingthesealedintestinaltubeinsterile

salinesolution.Theintestinalsackswerethen

incubatedat10Cforonehour.

After incubation the intestine was cut

open, the contents discarded and flushed

threetimeswithsterilesalinesolution.

Post ex vivo bacterial assaysSamples for bacteriology from each seg-

mentfromthefirstsamplingpoint(groups1

and2)werepreparedbyhomogenizing1gof

intestinaltissue(PIorDI)in1mlsterilesaline

usingaStomacher(SeawardLaboratory,UK).

Gutsamplesforbacteriologyfromthesecond

sampling (groups 3-6) were prepared by

gentlyscrapingoffmucuswithasterilescalpel.

Thereafter,thesegmentswereweighed.Both

the homogenates and mucus were used to

create serial ten-fold dilutions which were

spreadplated(100l)onTSAgsplatesand

incubatedat6Cforupto1weektodeter-

mineviablecountsofculturableheterotrophic

bacteria.

After sub-culturing on TSAgs to achieve

purecultures,phenotypicbacterial identifica-

tion(Gramstain,colonymorphology,oxidase

-andcatalasetestsandglucosefermentation)

wascarriedoutonrandomcoloniesfromall

plates containing between 10-300 colonies.

Atotalof168bacterialstrainswereisolatedfromthetwosamplingpoints.

16S rRNA characterizationof isolatesThebacterialDNAwasisolatedfollowing

theprotocol froma commercialkit (DNeasy

Blood and Tissue, Qiagen, USA). Specific

treatment for Gram-pos itive and Gram-

negative isolates was carried out according

tothemanufacturers instructions.Template-

DNA was diluted to a concentration of

approximately 20-30 ng l-1 using Milli-Q

water. The PCRmix constitutedof 8 lof

template-DNA, 36 l Milli-Q water, 5 l

10x buffer F511, 0.25 ldNTP, 0.25 l27F

forwardprimer,0.25l1492Rreverseprimer

and0.25lDNA-polymeraseyieldingatotal

volumeof50l.PCRthermalcyclingconsisted

ofinitialdenaturationof94 C, followedby

35cyclesof94Cfor20s,53Cfor20s

and72Cfor90swithafinalextensionstep

of72Cfor7min.ToverifyPCRproducts,

sampleswererunongelelectrophoresis.The

PCR-productsweredesaltedbymixing20l

of PCR-productwith 40 l of 100 percent

ethanoland2lof3MNaOAc(pH5.3)and

vortexedwell.Sampleswerethenincubated

onicefor30minfollowedbycentrifugationfor20minutesat14,000gusinganEppendorf

MicrocentrifugeModel 5417R. The superna-

tantwas removedandpelletwashed in100

lof80percentethanolandcentrifugedfor

another 5minutes at 14,000 g. The super-

natantwasremoved and the pellet driedat

roomtemperaturefor60minutes.Thepellet

was then resuspended in 30 l of Milli-Q

water.PurifiedPCRproductsweresequenced

asdescribedelsewhere.

Theresultantnucleotidesequenceswere

submitted to a BLAST search in GenBank

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to

retrieve the closest knownalignment identi-

ties for the partial 16S rRNA sequences.

Gene sequences that showed higher than

95percentsimilaritytoagenusorspeciesin

GenBankwerecategorizedaccordingly.

Invitrogrowthinhibitionofpathogensby

LABisolatedformtheexvivostudies

ElevenrandomlychosenLABisolatedfrom

the intestinal tract after ex vivo exposure

andone type strain,Carnobacterium inhibens

(CCUG31728),weretestedforantagonistic

effects against two different fish pathogens.

Thepathogenicbacteriausedinthepresent

investigation were Yersinia rckeri (CCUG

14190)andAeromonas salmonicida ssp. salmo-

nicida(Ass4017).C. inhibens (CCUG31728)

was used as a positive control as previous

investigations have demonstrated that this

strain has an inhibiting effect towards V.

anguillarumandA. salmonicida.Invitrogrowth

inhibition of the two fish pathogens by the

twelve LAB was tested using a microtitre

plateassay described indetail byRing and

co-authors. This method has been used intwo recent studies.The pathogenic bacterial

levels at the start of assayswere 106 cells

ml-1. Positive in vitro growth inhibition was

definedwhennogrowth(turbidity


12/16

was detected. Steri le

growth media and the

pathogens were used

as controls. Growth (at

OD600) of the patho-

gens without addition

ofsterilesupernatantof

LABwas approximately

0.6.Measurementswere

carried out each hour

usinganautomaticplate

reader (Bioscreen C,

Labsystems,Finland).

HistologySamples for light

microscopy (LM) and

transmission electron

microscopy(TEM)were

collected by excising

approximately 5 mm

from the posteriorpart

ofthePIandDI.Thesampleswereimmedi-ately fixedin McDowells fixativeandstored

at4 Cuntilprocessing. TEM and LMsam-

pleswereprocessedasdescribedelsewhere

Morphologicalobservationsweremadefrom

multiplemicrographs(8)fromeachintestinal

region from two fish within each group.

Thefollowingmorphologicalparameterswere

observed; detached microvilli, enterocytes

detachedfrom thebasalmembrane,disinte-

gratedcelljunctions,presenceofgobletcells,

presenceofabsorptivevacuolesandpresence

ofintraepitheliallymphocytes.

ResultsBacteriallevelsafterexvivoexposure

The adherent bacterial levels, as deter-

minedbyusingastomacher(groups1and2)

orbythecollectionofmucus(andsubsequent

weighing of the segments the mucus was

removedfrom)(groups3-6),didnotseemto

differwhichindicatesthatthedifferentsam-

plingmethodswere similarlyeffective.Table

3presentsanoverviewoftheautochthonous

bacterial levels isolated from each segment

andeachgroupexposedtoeithersalineorC.

divergens.Allvaluesareexpressedaslogcol-

onyformingunits(CFU)g-1.Autochthonous

bacteria isolated from intestines of fish fed

thecontroldietattheexperimentalstartand

exposedtosalinewasapproximatelylog1.7

CFUg-1inbothPIandDI,whilethenumber

of bacteria isolated from intestines of fish

exposedtoC. divergenswaslog6.04CFUg-1

inPIandlog5.56CFUg -1inDI.

After15 weeksof feeding slightlyhigher

values were present in PI of fish fed the

prebiotic diet post exposure tosaline orC.divergens compared to fish fed the control

diet. Indeed, thebacteriallevel in theprebi-

oticfedfishintestineexposedtoC. divergens

(group6)was234percentgreaterthanthat

ofthecontrolfedfishintestineexposedto C.

divergens (group 4). In both dietary groups,a similarbacteriallevel (~log6.70CFU g-1)

was detectedinDIexposedtoC. divergens.

However, a higher bacterial level (log 2.69

CFUg-1)wasobservedintheDIofcontrol

fedfishexposedtosalinethanthatofprebi-

oticfedfish(log1.71CFUg -1).

Isolationandidentificationofbacteriaafter

exvivoexposure

A total of 168 bacterial strains were

isolated from the two samplings. Among

these,40 isolateswereisolated fromthe first

samplingpointand128isolateswereisolatedfromthesecond sampling point. All isolates

weretestedformorphologyandbiochemical

properties(colonymorphology,Gram-testing,

oxidase - and catalase tests and glucose

fermentation).

One hundred and eleven isolates were

furtheridentifiedbypartialsequencingofthe

16SrRNAgene.Isolatesnotidentifiedby16S

rRNA gene sequencing but showing similar

biochemical and physiological properties to

those isolates identifiedby 16S rRNAgenes

were defined as -like. Table3 provides an

overview of the different bacterial species

isolatedineachexperimentalgroup.

Week 0Microbiota of fish fed control diet and

intestines exposed to sal ine (group 1):

Analysis of the adherentmicrobiota in the

PIoffishfedthecontroldietandexposed

to sterile saline (group 1) revealed that all

isolatesbelongedtothegenusPsychrobacter.

Of the 12 strains isolated from the PI of

this group, two strains showed 96 percent

similarity to Psychrobacter aquimaris, twostrains were identified as Psychrobacter gla-

cincolawhileeightstrainswereidentifiedas

Psychrobacter spp.-like.

TheDIoffishexposedtosalineatthefirst

samplingpointshowedamorediversecom-

munitywhichconsistedof4differentbacterialgenera.Ofthese,tenstrainswereindentified

to genus level and one strain was identified

to species level. The bacteria identified to

genus level belonged to Pseudoalteromonas,

Psychrobacter,Moraxella and Brevibacterium,

while the last strains showed high similarity

(98percent)toPsychrobacter glacincola.

Microbiota of fish fed control diet and

intestinesexposedtoC. divergens(group2):

All bacteria isolated from PI and DI of fish

exposed toC. divergensatthefirstsampling

(group2)wereidentifiedas C. divergens.ThisobservationindicatesthatC. divergensareable

to adhere to the intestinal mucosa in both

segments.

Week 15Microbiota of fish fed control diet and

intestinesexposedtosaline(group3):After15

weeksoffeedingonthecontroldiet,theiso-

latedstrains(17)fromthePIexposedtosaline

weredominatedbyC. divergens;70.6percent

ofthe isolateswere identifiedasC. divergens,

17.6 %were identifiedas Pseudomonas fulva,

5.9%belongedtoPantoea spp.while5.9%of

theisolateswereidentifiedasmembersofthe

classGammaproteobacteria .Thebacteriaisolat-

edfromtheDIwereidentifiedasC. divergens,

two strainsas Vibrio splendidus, one strain as

Shewanella baltica, onestrain asPseudomonas

fulva and six other strains were identified as

Gammaproteobacteria .

Microbiota of fish fed control diet and

intestinesexposedtoC. divergens(group4).In

theintestineoffishfedthecontroldietfor15

weeksandexposedtoC. divergens,theidenti-

fiedbacterialstrainsisolatedfrombothPIandDIweredominatedbyC. divergens.Onlyone

strain,identifiedasPseudomonas spp.,isolated

fromthePIof1fishdidnotbelongtothe

speciesC. divergens.

Microbiota of fish fed prebiotic diet and

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

Isoltecode

Sourcegroup

Intestinlregion

Closest knownspecies

Strin accession noIdentity

(%)antgonism

33 Group 2 Proximl C. divergens HICa_53_4 FJ656716.1 98 Y. ruckeri a. Slmon

40 Group 2 Distl C. divergens HICa_53_4 FJ656716.1 98 + -

75 Group 3 Proximl C. divergens HICa_53_4 FJ656716.1 99 + -

84 Group3 Distl C. divergens HICa_53_4 FJ656716.1 100 + -14 Group5 Proximl Carnobacterium sp H126 F204312.1 86 + -

57 Group5 Proximl C. divergens HICa_53_4 FJ656716.1 99 + +

17 Group 5 Distl Carnobacterium sp H126 F204312.1 99 + -





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


EXPERTTPIC


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intestines exposed to saline (group 5): The

intestineexposedtosalineoffishfedtheprebi-

oticdietfor15weeksshowedhigherdiversity

compared to the other groups exposed to

saline (groups 1 and 3). Of the 47 strains

isolated from the PI,14were identified asC.

divergens,oneasPseudomonasantarctica ,oneas

Pseudomonas koreensis,fourasEnterobacter hor-

maecheiandoneasunculturedbacterialclone

CK20. The remaining isolated strains were

identified asmembers of the Carnobacterium

andAcinetobacter genera.Thedominantbacte-

riainthePIofthisgroupbelongedtocarno-

bacteria(81%)and30percentoftotalisolates

wereidentifiedasC. divergens.

Thebacterial compositionof the isolates

fromtheDIoffishfedtheprebioticdietfor

15weekswererelativelylowindiversity.Of

thetotalnumberofstrainsisolated(44)from

theDI exposed to saline 34were identified

asCarnobacterium, eight strains showed high

similarity(%)to Pantoea spp.andtwostrains

belongedtothegenusEnterobacter.

Microbiota of fish fed prebiotic diet and

intestines exposed toC. divergens (group6):

In group 6, fish fed the prebiotic diets for

15 weeks and exposed to C. divergens, the

isolated strains in thePI were dominatedby

C. divergensandC. divergens-likestrains.Ofthe

22carnobacteria isolated, fivewereidentified

asC. divergensby16SrRNAsequencingwhile

17isolateswereidentifiedas C. divergens-like.

Threeotherisolateswereidentifiedasmem-

bers of the genera Pseudomonas (2 strains)

andPsychrobacter(onestrain).Ofthe17strains

isolatedandidesntifiedfromtheDIofgroup6,

C. divergensandC. divergens-likestrainsdomi-

natedwithonlyoneisolate,whichshowedhigh

similarity(99percent)toAcinetobacter spp.,not

belongingtothisspecies.

Microscopical analysesLight microscopy (LM): All LM micro-

graphs,bothfromPIandDIoftheprebiotic

groups (5and6)showed nomorphological

differencescompared tothe control feeding

regime (groups 1-4). All intestinal sections

examinedappearednormal andhealthy; no

signsofdetachedenterocytes,necroticente-

rocytes,widened lamina propria or necrosis

wereobservedandthenumberofgobletcells

weresimilarinbothtreatments(examplesare

displayedinFigure1).

Transmission electronmicroscopy(TEM):

Similar to the observations using LM, TEM

revealedno differencesbetween treatments

orexposuregroups;allmicrographsrevealed

healthyepithelialbrushborder,nodeteriation

oftightjunctionswasobservedandmicrovilli

appeareduniform.Thepresencesof rodlet-

likecells(asshowninFigure2)werepresent

inthePIandDIofallgroups.The

numbersofrodletcellspresentin

the PI displayed great differencesbetween individual fish but were

always observed in the upper

half oftheepithelium,above the

underlying intraepithelial lym-

phocytes.

In vitro growth inhibition of

twofishpathogensbyextracellular

extractsof LAB isolated fromex

vivostudies

Identification by partial

sequencingofthe16SrRNAgenes

oftheelevenLABstrainsisolated

fromtheexvivoexperimentsand

subsequently used inthe invitro

pathogen antagonism assays are

displayed inTable4. The results

showthatgrowthinhibitionofY.

rckeriwasobtainedfromextra-

cellularextractsfromallstrainsof

carnobacteria isolated from the

exvivoexperiment.However, in

vitro growth inhibition ofA. sal-

monicida ssp. salmonicida wasonly

obtained from the extracellular

extractofCdivergensisolate57.

The extracellular products from

the positive control, C. inhibens

CCUG31728,didnotinhibitthe

growth ofA. salmonicidassp.sal-

monicida.

DiscussionThe ex vivo intestinal sack

methodhasbeenusedinseveral

studiestoevaluatepossiblehisto-

logicalchangesinthefishintestine

after exposure to high levels ofLAB.TheresultofLABexposure

to the intestine is of high impor-

tance as translocation and cell

damage have been proposed as

importantcriteriawhenevaluating


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Carnobacterium spp. levels,particularlyin the

DI.Totheauthorsknowledgethereisvery

littleinformationregardingtheeffectofprebi-

oticsoncarnobacteriawithintheGItractof

fish.However,somestudiessuggestthatthe

carnobacteriapopulationswithintheGItract

of salmonids areeffectedby various dietary

factorssuchaskrillmealandoxytetracyclinein

Atlanticsalmonanddietarycarbohydrates in

Arcticcharr(SalvelinusalpinusL.).However,

itwasobservedthatthepresenceofdietary

inulin(aprebiotic-typecarbohydrate)tended

to lower culturable autochthonous carno-

bacterialevels(byca.90%)inthehindgutof

Arcticcharrandalsoelevatedtheproportion

of C. maltaromaticum at the expense of C.

divergens.Thesefindingssuggestthatdifferent

prebioticsmayinfluencedifferentcarnobacte-

riastrainsindifferentfishspecies.

InallgroupsexposedtoC. divergensinthe

exvivo studies the sameC. divergens strain

was identified todominate both the PIand

DIafterexposure. C. divergenslevelswerein

therangeof104-106CFUg-1intestinewhich

indicatesthatthebacteriaareabletopopulateandpotentiallycolonizethe intestinalmucus

and out-compete other adherent bacteria

afteronlyonehourofexposure.Theseresults

areinaccordancewithcorrespondingstudies

inthatLABareabletocolonizetheintestine

ofAtlanticsalmonafteronehourexposure.

Despitetheplethoraofinformationavail-

ableontheprebioticefficacyofelevatingpro-

biotic colonization (i.e. synbiotics) in various

terrestrialspecies,littleinformationisavailable

in fish. Further studies should focus on this

topicasthepresentstudydemonstratedthat

the presenceof thedietaryprebiotic,prebi-osal,elevatedtheproportionofcarnobac-

teriafrom71- 81percent intheDI(aswell

as elevating total bacterial levels, effectively

quadruplingthenumberofcarnobacteria)and

from33%to77%inthePI(althoughthetotal

bacterialpopulationwaslower).

ThehistologicaleffectofexposingtheGI

tractofAtlanticsalmon tohigh levelsof the

C. divergenswasinvestigatedbylightandelec-

tronmicroscopy. Furthermore, the intestinal

effectsoffeedingaprebioticdiettoAtlantic

salmonwereevaluated.Results from LM-investigations in the

present study showed no apparent his-

topathological changes of the epithelium in

thePI orDI, after exposureofC. divergens.

In particular the micrographs demonstrated

that enterocytes showed no signs of junc-

tionalrupturefromthebasementmembrane

whichisincontrasttoobservationsofthePI

ofAtlantic salmon afterexposure toVibrio

anguillarumandA. salmonicida.

TEMobservations confirmed the findings

observedinLMregardingalackofhistological

changes. TEM revealed no observable dif-

ferences between the groups in respect to

thepresenceofcelldebris inthelumen, the

amountofmucus,thenumberbacterialike

particlesinthelumenandbetweenthemicro-

villi, disorganized microvilli and disintegrated

tight junctions. The enterocytes within all

groups displayed normal cell contacts with

unaffectedtightjunctionsandzonaadherens.

ThefactthatC. divergensdidnotinflictdam-

agetotheintercellularunctionisgreatimpor-

tantancesincethelooseningofthesejunctions

contributestoaparacellularportofentryfor

potentialpathogens.

Rodlet cells were present inlarge numbers in the PI of all

groups,whileintheDIthenumber

observedwerelower.Sincegroups

exposedtoC. divergensdidnotdis-

playanycleardifferencesinnumber

ofrodletcellscomparedtogroups

exposed to saline, their presence

inthosegroupsmaythereforenotberelated

to an immunological function towards the

exposed bacteria. On the other hand, the

roleofrodletcellsasimmunecellsandtheir

largenumberinthePIcomparedtotheDI

maybeadefensefunctiontowardspotential

invadingbacteria ofthePI. SincethePIhas

been confirmed as beingan infection route

forpathogenicbacteriabyseveralstudies,the

roleofrodletcellsasimmunecellsinthePI

ispossibleandwarrantsfurtherinvestigation.

TheantimicrobialeffectsofLABhavelong

beenutilizedinfoodpreservationbyfermenta-

tionand severalcomprehensivereviewshave

beenpublishedontheabilityofLABtopro-

duceproteinaceousantimicrobialsubstances.In

fishstudies,theantagonisticeffectofLABhas

beencarriedoutonGram-negativefishpatho-genssuchasVanguillarumandAsalmonicida.

Inthepresent studystrong growthinhibition

ofY. rckeriwasrecorded fromextracellular

extracts from late exponential growthphase

fromalloftheelevenCarnobacteriastrainsiso-

latedfromtheexvivoexperiments.However,

the ability of the isolated strains to inhibit

growth ofA. salmonicida ssp. salmonicida was

only observed fromone strain isolated from

thePI.Thefactthatonlyone(isolate57)ofthe

11 strains displayed inhibitoryeffects towards

A. salmonicidassp.salmonicidaisinaccordancewiththeresultsofRingwhoobservedalack

ofantagonismwhenchallengingA. salmonicida

ssp. salmonicida to extracellular extracts from

C. divergensstrainLab01.Theseresultsindicate

that theproductionof extracellular products

only,might not be sufficient for strains ofC.

divergensinlateexponentialgrowthphase,to

inhibitgrowthofA. salmonicidassp.salmonicida.

The positive control bacteria, C. inhibens

whichJbornetal.reportedtodisplayantago-

nisticeffect againstA. salmonicida, showed no

sign of antagonism in the present study. This

observationthereforeindicatesthatantagonisitic

activityofC. inhibens isonlyeffectivewhencells

areactivelyincubatedtogetherorthatantago-

nisticextracellularproductsare onlyproduced

byC. inhibens inthepresenceofA. salmonicida.

TheabilityofC. divergensasusefulprobiot-

icswitheffectsagainstY.rckeriandA. salmoni-

cidahavepreviouslybeenreportedinvivoand

invitro.KimandAustinobservedthatdietary

provision of C. divergens strain B33, isolated

from the intestine of healthy rainbow trout,

increased survival of rainbow trout against

A. salmonicida andY. rckeri challenge by60

percentcomparedtothecontrolgroup.Even

thoughstrainsofC. divergensshowantagonisticeffects against pathogens, theprecisemecha-

nism of action of antimicrobial compounds

isolatedfromfishremainsunclear,butsugges-

tionsabouttheirabilityofpenetratingcellwalls

byformingporesandchannels,thusrendering

itmore fragile and incapableof carryingout

normalmetabolismhasbeenproposed.

In order to confirm the in vitro probi-

oticeffectofC. divergensagainstY.rckeriin

Atlanticsalmon,furtherinvestigations should

therefore include in vivo challenges studies.

Byfurther applyingelectronmicroscopy, the

physicalinterferencemechanismsbetweenC.

divergensandY.rckeriintheGItractmight

beobserved.

AcknowledgementsThe authors thank the technical staff at

EWOSInnovationASforfeedmanufacture,

analysis and running the feeding trial and

thank Dr. Sigmund Sperstad and Dr. Chun

Li, Norwegian College of Fishery Science,

University of Troms for their inestimable

helpduring16SrRNAgenesequencingand

in vitro growth inhibition. We also thankRandi Olsen and Helga Marie By at the

EM department at University of Troms,

and Anne Nyhaug at Molecular Imaging

Centre,InstituteforBiomedicine,University

of Bergen fortheir inestimablehelp during

light andelectronmicroscopy analysis. This

studywaspartiallysupportedbygrantsfrom

the Norwegian MABIT-program (project

numberAF0038).

"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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