January | February 2013

Novel additives to reduce the economic impact of disease on shrimp production

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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16 | InternatIonal AquAFeed | January-February 2013

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Penaeidshrimpproductionisundercontinuous threat from bacte-rialandparticularlyviral infectionswhich have caused disastrous col-

lapses of the industry in all major shrimpproducing countries. Disease problemsin shrimp production are complex andoften still poorly understood. Regulations,consumerdemandsandsustainablemanage-mentstrategiesrestrictthenumberofdrugsavailable to treat pathogens. Vaccines arelikelytobeineffectiveincrustaceans,whichlackaspecificimmunesystemsimilartothatof vertebrates. Therefore, shrimp produc-ers must consider the seed stock quality,husbandryproceduresandhealthynutritionas the major tools to control disease.Thecurrent article reports on recent progressinthedevelopmentoffeedadditivescapableof reducing the impact of diseases on pro-ductivityandprofitabilityinshrimpfarming.

Diseases are number one threat The production of crustaceans has shown

an average annual growth rateof18percentover the period 1970-2008, which by farexceedsgrowthforallotheraquaculturespe-cies (FAO, 2010). World shrimp aquacultureisproducingnowwelloverfourmillionmtofshrimp(Valderrama,2011).Thisrapidincreasein crustacean production largelyreflectsthedramaticincreaseinwhitelegshrimpculture inChina,Thailand,VietnamandIndonesiasince2000.

Despite this apparent successstory in termsofproductionexpan-sion, shrimp production in manyregionscontinuestosufferimportanteconomic losses due to the impactofawidevarietyofdiseases.Recenteventsillustratetheimpactofdiseaseoutbreaks on shrimp production inmajorproducingcountries.

The white spot syndrome virus(WSSV), one of the main causesof stagnation in the shrimp indus-try in the nineties, is significantlyaffectingshrimpproductioninrecentyears in Mexico and Brazil. Early Mortality

Syndrome (AcuteHepatopancreaticNecrosis Syndrome,AHPNS),forwhichthecausativeagenthasnotbeen identified so far(Flegel,2012),isaffect-ing shrimp productionin China, Vietnam,Malaysia, and to alesserextent,Thailand.

Traditional approaches to boost shrimp health through the feed

A traditionalapproach to reducethe impact of shrimp

diseasesconsistsofincreasingthelevelofkeynutrientsaffectingthehealthandimmunologyof shrimp, including vitamin C and E, phos-pholipids, essential fatty acids, trace mineralsand carotenoids. These ‘booster feeds’ areoften supplemented with immunostimulants,

mostly derived from the cell envelope ofmicro-organisms, such as polysaccharides,lipoproteins,andlipopolysaccharides.

The continuous use of immunostimu-lants is generally discouraged due to therisks for over-stimulation of the immunedefense system. Alternating on/off regimesfor feedadditives isoften impractical in farmoperations. Encouraging results to improvediseaseresistancehavebeenobtainedbythecontinuous use of health enhancing boosterfeedsbasedontheselectionoftheappropri-ate immunostimulants in combinationwith abalanced nutritional supply of key nutrientstosupport theenhancementof the immunesystem(Table1).

However,theefficacyofvariouscommer-cially available immunostimulants to improvestress and/or disease resistance of fish andshrimp strongly depends on the type of theproductandonthesupplyofadjuvantnutri-

Figure 1 - Table: Effect of booster feed on production parameters in a farm in NE Brazil during episode of increased disease incidence due to a combination of intensive rains and increased incidence of infectious myonecrosis virus (IMNV) and necrotising hepatopancreatitis (NHP). Booster feed based on enhanced nutritional specifications and supplementation of an immunomodulator (AQUASTIM S, Nutriad) versus standard feed.

Control feed Boosted feed % change

total pond area (ha) 25 25

Culture period (days) 107 111

Survival (%) 77.1 80.7 +5%

Final weight (g) 12.77 14.01 +10%

Harvest yield (kg/ha) 1771 2034 +15%

FCr 1.86 1.85 -1%

avg weekly growth (g) 0.84 0.88 +6%

relative production cost 100% 100%

relative crop value 100% 119% +19%

Figure 2: The hepatopancreas is the main organ of the shrimp’s digestive system responsible for digestion, absorption and storage of nutrients. Esophagus (E), gastric mill (GM), hepatopancreas (HP), mid gut (MG), hind gut (HG), and anus (A).

Novel additives to reduce the economic impact of disease on shrimp productionby Peter Coutteau PhD, Business Unit Manager Aquaculture, and Tim Goossens PhD, R&D Engineer Gut Support, Nutriad International NV, Belgium

44 | InternatIonal AquAFeed | January-February 2013

FEATURE

January-February 2013 | InternatIonal AquAFeed | 45

64 | InternatIonal AquAFeed | January-February 2013

entsthatareessentialtosupportthebuildupoftheimmunesystem.

Novel approaches (1): boosting the nutritional status and lipid reserves of the hepatopancreas

Shrimps do not tolerate high levels ofdietary fat very well. A number of studiesshowreducedgrowthatlevelsabove10per-centofdietarylipid.Nevertheless,qualityandquantityofdietarylipidsplayaprimordialroleingrowthandhealthofshrimp.Shrimphavenoorverylimitedcapacitytobiosynthesizeanumberoflipidmoleculeswhichareessentialfornormalgrowth,includingcholesterol,high-lyunsaturatedfattyacidsandphospholipids.

Fishmeal and fish oil are often the mostimportant sources of cholesterol and HUFAin the diet. Increasing cost of these marineingredientshas forced formulators to reducedietaryspecificationsfortheseessentiallipids.Althoughthesenutrientlevelsmaynotshowsignificantdifferencesongrowthperformancein feeding trials under controlled conditions,they may become critical for maintaininghealth and immune defenses under diseasechallengesand fluctuatingambientconditionsencounteredinproduction.

Furthermore,theenergystatusofshrimpislargelydeterminedbyitslipidreservesdepos-ited in the hepatopancreas which functions

both as a digestive glandas well as a storage depotfor energy. Therefore,farmers routinely look atsquashpreparatestoevalu-atethenutritionalstatusofthe hepatopancreas, withample lipid reserves beinganindicatorofbetterresist-ance to stress and diseasechallenges.

Lipiddigestioninshrimpoccursforabigproportionintracellular in the hepato-pancreas epithelium fromwhere it is transported tothe target organs via thehaemolymphe under theformoflipoproteins(Fig.2).Theformationandabsorp-tion of lipid micelles fromthe lumen of the hepato-pancreas tubuli is therefore a limiting stepin the lipid digestive process. Digestibilityenhancers based on natural emulsifyingagents, selected for their compatibility withtheshrimp’sdigestivesystem,haveshowntobecapableofcomplementingtheprocessofemulsificationandabsorptionofdietaryfatsinthe hepatopancreas (Coutteau et al., 2012).Thisinturnimprovestheefficiencyofshrimp

to use fats as essential components and assource of energy for growth and survivingepisodes of stress or disease pressure. Theenhancement of the lipid reserves in thehepatopancreas of white shrimp Penaeus indicus as a resultof the supplementationofadigestibility-enhancingadditivewasdemon-stratedrecentlybyvandeBraaketal.(2012).Histological analyses showed a three-fold

Figure 3. Effect of the supplementation of a digestibility-enhancing additive (Aquagest®S, Nutriad) on the degree of lipid vacuolization in the hepatopancreas of shrimp fed the different feeds during 30 days (van de Braak et al., 2012).

44 | InternatIonal AquAFeed | January-February 2013 January-February 2013 | InternatIonal AquAFeed | 45

FEATURE

ASIAN GATEWAY TO AN AQUATIC WORLD OF WONDER

www.aquarama.com.sgFor more information, please contact: Iman Tam • aquarama-sg@ubm.com

co-located with

The 4th International Pet & Accessories Exhibition

couldbeeffectivewaysofprovidingthe‘researcharm’ forpartneringwith ‘largepharma’

• Technology Centers for marinebiotechnology, with specialised infra-structure might provide ‘one-stopshops’forchainconnection

Science,technologyandinfrastructures:• Marine biotech is not being well-

servedbylackofknowledgeamongsttechnology-transferoffices

• More fora for meeting of scientistsandindustrialplayerswouldgeneratebetter understanding and sharing ofneedsandpossibilities

• A global source of ‘soft’ fundingwould promote the transition fromproof of concept to demonstrationandcommercial-scaleforinnovations

• Specific incubatorprogrammes couldberecommended

• Discouragetheuseofnon-sustainablesourcesofMBtlibraries

Commercial context – investment, regu-lation,IP• Theneedforlong-termstabilitymeans

that VC and short-term investmentstrategies are not appropriate eitherat set-up or for longer survival ofnewbusinesses;encouragingbusinessangel groups and raising awarenessand knowledge-levels amongst thesewouldbefruitful

• Smaller players in innovation shouldconsidermorecollaborationsbetweenthem,andsellingskillsandknowledge,rather than pushing molecules atbig pharma or trying to go too fardownthevalue-chain;itisusuallytoodifficultforsmallcompaniestohandlethe cost and stresses of regulatoryprocesses

• Nevertheless, the existence of smallcompanies willing to take part inbiodiscovery de-risks this activityfor big companies and justifies theperceived need for entrepreneurialcompanies to supply into biggerpharma (and equivalent ‘big’ compa-nies – food/nutraceutical, cosmetics/cosmeceutical)

• Better and more efficient recogni-tion, development and transfer ofacademicIPinthisareaisneeded

• The attributes and benefits of MBtcould be better communicated. IntermsofgivingMBtadifferentimage,thehigherhitratecouldbeastartingpoint

Someof the points raised abovewerediscussed further in Think Tank 5 in thecontext of Marine Biotechnology and theEnvironment.

32 | InternatIonal AquAFeed | January-February 2013 January-February 2013 | InternatIonal AquAFeed | 33

FEATURE

Companies attending this Think Tank:AquaBioTechnologyASA-A-SparkGoodVentures-AlgalBioenergySpecialInterestGroup-Bioalvo-BioNova-BioTechNorth-

BretagneDeveloppementInnovation-CCMar-EMPA-EuropeanMarineBiologicalResourceCentre(EMBRC)-GovernmentofPortugal-GretteLaw-innoVactiv-InnovationNorway-JPIOceans-TheResearchCouncilofNorway-KielCenterformarinenaturalproducts-Marealis-MarineBiotechnologyProgrammeofIreland-MaxPlanckInstitute-NationalResearchCouncilofCanada-NautilusBiosciencesCanada-NovagraafTechnologies-NovusInternational-Soliance-MarineBio-TechnologiesCenterofInnovation-NationalUniversityofIreland,Galway-OceanGate,Inc.-Oceanomicsproject,Roscoff-Polaris-PolytechnicInstituteofLeiria-PwC-RoscoffMarineBiologicalStation-SaintMaloAgglomeration-ScandiDermaAS-UniveristyCollegeCork-UniversityofAveiro

AquaStar®Fast growth in improved environment!Probiotic strains support gut health. Biodegrading strains and enzymes stabilize water quality and pond bottom.

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increase of the percentage of shrimp witha high degree of lipid vacuolization in thehepatopancreasaftersupplementingthefeedadditiveduringonemonth(Figures3,4).Theresultsofaparallelpondstudyindicatedtwopercenthigheraveragebodyweight (ABW),four percent higher survival, and six percenthigher biomass for the treatment ponds.

However,removalofoutliersforsurvivalfromcontrolandtreatmentsetshowedeightper-centhigherABW,12percenthighersurvival,and23percenthigherbiomass.

Novel approaches (2): Quorum Sensing technology

QuorumSensing(QS)isaformofbacteri-alcommunication.Overthelastdecade,manybacterial species have been documented tobeable toproduceandsecrete small signal-ing molecules, such as acyl homoserine lac-tonesorcertainoligopeptides,whichcanbedetectedbyadjacentbacteriaofthesameorof distinct species. When population densityrises, thesemoleculeswill accumulate in theextracellular environment, thereby providingameansforbacteriatoquantitativelymonitorthepresenceofotherbacteria.Thesesignal-ing molecules will, upon reaching a certainthresholdconcentration, initiate intrabacterialsignaling that culminates in the activation ofspecificgenes.QScommunicationisthereforeusedbybacteriatosynchronizegeneexpres-sion alterations and coordinate biochemicalresponseswithintheentirepopulation.

In most pathogenic bacteria from whichthe QS system has been studied, QS hasbeen associated with pathogenicity, such asbiofilm formation and the production of

proteases,invasionfactorsorother virulence factors(Defoirdt, et al., 2011).In recent years, researchfocusing on ways to dis-turb QS signaling (alsocalledquorumquenching)is therefore gaining par-ticular interest (Figure 5).This is especially true inthe field of human medi-cine, where QS-inhibitors

are investigated as potential alternatives toantibiotics in tackling pathogenic bacterialinfections (Sintim et al., 2010). Interestingly,chances that bacteria build up resistanceagainstQSdisruptorsarepredictedtobelow,givingthattheselectivepressureagainstthesein se non-lethal molecules is limited. Thisstandsinstarkcontrastwithwhatisseenwith

conventionalantibiotics(Defoirdtetal.,2010).Initialstudiesofquorumsensinginaquacul-

tureorganismsarevery limitedbutpointoutexcitingresults.Halogenatedfuranonesisolatedfromredmarinealgae,forexample,havebeendemonstrated to reduce QS-regulated geneexpression in Vibrio and to protect fish andshrimpfromvibriosis(Raschetal.,2004;Defoirdtet al., 2006). At the Nutriad TechnologyCenter, QS technology is being applied ina novel generation of natural feed additivescapable of modulating gutmicro flora.Compoundsaretested for their capacity toinhibit QS-signaling using anarray of genetically modi-fied bacterial biosensors andQS-dependent infectionprotocols in simple modelorganisms.Usingthesesensi-tiveassays,potentQSmodu-lators, able to shut downQS signaling at concentra-tionsfarbelowtheminimuminhibitory concentration, arebeing identified. DifferentQS quenching activities areselected for agriculture andaquaculturespeciesbasedonscreening work using spe-

cific bacterial biosensors and model organ-isms. Synergistic blends of different naturalcompoundsresultedtobeextremelyefficientin QS quenching activity against signaling byVibrioharveyi,anpathogencausingvibriosisinpenaeidshrimp(Figure6).

Putting QS inhibition into practice: effect of optimising gut health on productivity and economics of semi-intensive shrimp farming

Shrimp are actively ‘grazing’ on the sub-strate present in the pond bottom andwater column, and therefore highly exposedto exchanges of microflora between theenvironment and the digestive system. Thisincreases the risk for the proliferation ofan unfavorable gut microflora or frequentdestabilization of the microflora, which canaffecttheoptimalfunctioningofthedigestivesystem.Furthermore, thedigestivesystemofshrimpisthemainentryportforbacterialandviral infections,whichremainamajorriskfortheprofitabilityofshrimpproduction.

Sustainable approaches to modulate thegutmicroflora in farmed animals include theuseof selectedbacteria to inoculate thegut(probiotics), specific nutrients promoting thedevelopment of selected bacterial strains(prebiotics), and specific natural compounds(mostlyderivedfromyeastandherbalextractscalled ‘phytobiotics’) capable of modulatingthemicroflora towardsa favorablecomposi-tion, favoring the development of beneficialbacteria and inhibiting potentially pathogenicmicro-organisms. The latter strategies havethe advantage of being easily applicable atthe feedmill on large volumes of feed andavoidingmajoradaptationsoftheproductionprotocolsatthefarm.

A synergistic blend of botanical extracts(Sanacore®GM,Nutriad)wasoriginallyselectedfor itsbacteriostaticandbactericidalpropertiesagainst pathogenic and potentially pathogenicbacteria in vitrousingthediskdiffusionmethod.

Figure 4: Histological determination of the degree of lipid vacuolization of the hepatopancreas in shrimp Penaeus indicus, showing a high (left picture) and low (right picture) level of lipid vacuolization (100x magnification; van de Braak et al., 2012)

Figure 5: Quorum Sensing (QS), an innovative mechanism to tackle pathogenicity

Feeding manually from a boat and harvesting shrimp at the CAMACO farm, Panama (with courtesy of Jorge Cuéllar-Anjel)

46 | InternatIonal AquAFeed | January-February 2013

FEATURE

January-February 2013 | InternatIonal AquAFeed | 47

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Furthermore, this synergistic blend has provento be a powerful interrupter of bacterial QSsignaling at concentrations well below minimalinhibitory concentrations, allowing it to effec-tively modulate the gut flora towards a morefavorable composition. The supplementationofSanacoreGMpromotedgrowthsignificantlyinhealthyshrimpgrowingundercontrolledlabconditions; showing a remarkable 20 percentincreaseofweeklyweightgainandfourpercentimprovementonfoodconversion(Coutteauetal., 2010). The effect of this botanical productshowing combined activities in QS inhibitionand bactericidal action against a wide rangeof pathogenic bacteria was verified in a semi-intensive shrimp farm in Panama.The secondproductionseasoninPanama,stockedbetweenAugust-September, ischaracterisedbyunstable

Figure 6: Dose – response of a synergistic blend of botanical compounds (SANACORE GM, Nutriad) on Quorum Sensing signaling activity of Vibrio harveyi. Graphs show signaling activity in QS biosensor system Vibrio harveyi BB170, relative to control, exposed to for different dilutions of the product extract (Nutriad Technology Center, in-house results).

Table 2: Production results for P. vannamei in Panama during the second production cycle for control ponds and treatment ponds receiving a phytobiotic supplement after 141 days of culture (average and standard deviation of eight replicate ponds of three ha per treatment).

treatment Survival Shrimp size Crop Yield (kg/ha)

Feed (kg/pond 3ha) FCr

Weekly Growth(g/wk)

average coefficient of variation for

parameters listed (CV%)

Sanacore® GM 55.5 ± 7.1 a 16.6 ± 1.5 a 735 ± 78 a 4,170 ± 338 a 1.91 ± 0.23 a 0.825 ± 0.075 a 10%

Control 44.6 ± 10.6 b 15.7± 2.9 a 543 ± 90 b 3,464 ± 396 b 2.17 ± 0.39 a 0.776 ± 0.137 a 18%

% change Sanacore vs Control +24.4% +5.8% +35.2% +20.4% -12.1% +6.3% -41%

P Value 0.0304 0.4395 0.0004 0.0018 0.7130 0.3876 ---

46 | InternatIonal AquAFeed | January-February 2013 January-February 2013 | InternatIonal AquAFeed | 47

FEATURE

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climatologicalconditions,resultinginstrongtem-peraturefluctuationswhichinturnaffectshrimpgrowthandincreasetheimpactofoutbreaksofwhite spot virus (WSSV).The first productioncycle, seeded between January-April, providesmore suitable growth conditions and generallyresultsinbettersurvivalandproductivity.

Two treatments were compared whichonlydifferedwithregardtothesupplementa-tion or not of the phytobiotic growth pro-moter(Sanacore®GM)tothestandardfeedusedatthefarm.Thesupplementationofthebotanical feed additive drastically improvedsurvival,amountingtoarelativeincreasewith24percentand18percentcomparedtothecontrol group for the secondand first cycle,respectively(Figure7).

NaturalWhite SpotDiseaseoutbreakswereobservedduringshrimpfarminginbothtreatmentsundersimilarfrequencyandseverity;WSSVvirus

wasconfirmedbyimmu-no-chromatography andnested-PCR tests. Thepresence of a synergis-tic blend of phytobiot-ics provided an array ofantimicrobial activities,including quorum sens-ing inhibition capabilities,in the shrimp’s digestivesystem. This offers addi-tional protection againstco-infectionswithoppor-tunistic bacteria such asvibriosis, often the majorcause of mortality inWSSV-infected shrimp(Phuocetal.,2009).

The evaluation inthe second cycle oneight replicates per

treatment allowed a good evaluation ofvariability amongponds for thedifferentproduction parameters. The addition ofthe phytobiotic reduced drastically thevariability of production results amongponds fed thesame feed(averagecoef-ficient of variation between ponds forthe six production parameters: control18 percent versus Sanacore group 10percent; Table 2). This further indicatedthe importance of increased control ofgut microflora on the reproducibility ofproduction in semi-intensive pond envi-ronments.

References available on request

More InforMatIon:Email: p.coutteau@nutriad.comWebsite: www.nutriad.com

About the authorsTim Goossens gota Masters degreein Biotechnology atGhent University,and subsequentlyworked as an aca-demic researcher inthe Laboratory of

Molecular Biotechnology at the Universityof Antwerp. After working for four yearson the characterisation of a gene familyinvolved inbonedevelopment, hemovedto the KU Leuven, where he took up adoctoral project on neurodevelopment intheLaboratoryofDevelopmentalGenetics.After obtaining his PhD. in BiomedicalSciences,he joinedNutriadtoworkasanR&D Engineer, focusing on the develop-ment and technical support of the GutSupportrangeoffeedadditives.

Peter Coutteau, cur-rently Business UnitManager Aquaculturefor Nutriad, obtainedin 1992 a PhD. inBiological Sciences atthe Laboratory of

Aquaculture & Artemia Reference Center,UniversityofGentonthefilterfeedingbiol-ogyofArtemiaandbivalves.Hecontinuedacademicresearchatpostdocleveltill1997onlipidnutritionofbivalves,fishandshrimp,publishingover40refereedpapersinscien-tificjournals.In1997,joinedtheINVEgroup,as head of research and product develop-ment in the aquaculture division. During2002-2008, he was responsible for globalproductdevelopmentandcustomerserviceforfeedconcentratesandadditivesasprod-uct manager farm nutrition for INVE’sBusiness Unit Aquaculture. FollowingrestructurationoftheINVEgroupinJanuary2009, the support team, research activitiesand product lines for aquaculture additiveswere reorganised under Nutriad’s BusinessUnitAquaculture.

Figure 7: Survival percentage at harvest for control ponds and treatment ponds receiving the phytobiotic supplement in two production cycles in semi-intensive production of white shrimp L. vannamei (average and standard deviation of eight and five replicate ponds of three ha per treatment, respectively; data from Vaca et al., 2010, 2011).

48 | InternatIonal AquAFeed | January-February 2013

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48 | InternatIonal AquAFeed | January-February 2013

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