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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Welcome to Expert Topic. Each issue will take an in-depth lookat a particular species and how its feed is managed.
SALMON
EXPERT TOPIC
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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
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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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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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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
42 | InnaIOnal AquAFeed | September-October 2013
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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
September-October 2013 | InnaIOnal AquAFeed | 43
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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
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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 + -
154 Group 5 Distl C. divergens HICa_53_4 FJ656716.1 92 + -
173 Group 4 Proximl C. divergens HICa_53_4 FJ656716.1 99 + -
127 Group 4 Distl C. divergens HICa_53_4 FJ656716.1 99 + -
99 Group 8 Proximl C. divergens HICa_53_4 FJ656716.1 99 + -
*_originally isolated from the digestive tract of Atlantic salmon (salmo salar) [20]
September-October 2013 | InnaIOnal AquAFeed | 45
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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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