Enterocytozoon Hepatopenaei (EHP) | Disease guide

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Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that was first characterized and named from the giant or black tiger shrimp Penaeus monodon ... WhatisEHP? Enterocytozoonhepatopenaei(EHP)isamicrosporidianparasitethatwasfirstcharacterizedandnamedfromthegiantorblacktigershrimpPenaeusmonodonfromThailandin2009(Tourtipetal.2009.J.Invertebr.Pathol.102:21-29). Itwasdiscoveredinslowgrowingshrimpbutwasnotstatisticallyassociatedwithslowgrowthatthattime. EHPisconfinedtotheshrimphepatopancreas(HP)andmorphologicallyresemblesanunnamedmicrosporidianpreviouslyreportedintheHPofPenaeusjaponicasfromAustraliain2001. Together,thesestudiessuggestthatEHPisnotanexoticpathogenbutthatitisendemictoAustralasia.Later,itwasfoundthatEHPcouldalsoinfectexoticPenaeusvannameiimportedforcultivationinAsiaandthatitcouldbetransmitteddirectlyfromshrimptoshrimpbytheoralroute(Tangprasittipapetal.2013.BMCVetRes.9:139). Thisdifferedfromthemostcommonmicrosporidianpreviouslyreportedfromcottonshrimp,wheretransmissionrequiredanintermediatefishhost,allowingdisruptionoftransmissionbyexclusionoffishfromtheproductionsystem. WhyisEHPimportant? AlthoughEHPdoesnotappeartocausemortality,informationfromshrimpfarmersindicatesthatitisassociatedwithseveregrowthretardationinP.vannamei.Thus,webegantowarnAsianfarmersandhatcheryoperatorsafter2009tomonitorP.vannameiandP.mondonforEHPinbroodstockandpostlarvae(PL). However,thewarningswerenotheededbecauseoftheoverwhelmingfocusonearlymortalitysyndrome(EMS)oracutehepatopancreaticnecrosisdisease(AHPND). ScientistsfearedthatlackofinterestinEHPwouldleadtoitsbuildupinproductionsystemsandthatitsspreadwouldbemaskedbyEMS/AHPNDbecauseitkillsshrimpbeforethenegativeeffectsofEHPongrowthareapparent.TheyalsofearedthatsolutionoftheEMS/AHPNDproblemwouldprobablyleadtosucceedingwidespreadproblemswithslowgrowth.Indeed,thisseemstohavehappenedinthepastyearorso. ScientistsnowhaveinformationindicatingthatEHPoutbreaksareoccurringwidelyinChina,Indonesia,Malaysia,VietnamandThailand.Veryrecently,theyhavealsoreceivedsamplesPCR-positiveforEHPfromslowgrowingshrimpinIndia.Thus,EHPisanemergingproblemthatisunderurgentneedofcontrol. HowtocontrolinternationalspreadofEHP AnestedPCRdetectionmethodandaLAMPmethodareavailabletocheckfecesofbroodstockandtocheckwholePLforthepresenceofEHP(Tangprasittipapetal.2013.BMCVetRes.9:139;Suebsingetal.2013.JApplMicrobiol114:1254-1263). Thepathogencanalsobedetectedbylightmicroscopyusinga100timesobjectivewithstainedHPtissuesectionsorHPsmears,butthisisbasedonfindingthecharacteristicsporesthatareextremelysmall(lessthanonemicroninlength)andaresometimesproducedonlyinsmallnumbers,eveninheavilyinfectedspecimens.Thus,thePCRdetectionmethodispreferred. ScientistshavedataindicatingthatmostSPFstocksofP.vannameiimportedtoThailandarenegativeforEHPbutthattheyoftenbecomecontaminatedinrecipientmaturationfacilitiesandhatcheriesbecauseofpoorbiosecurity. Oneseriousfaultinbiosecurityisthewidespreadpracticeofusingliveanimals(e.g.,polychaetes,clamsetc.)fromlocalsourcesorasimportstofeedbroodstockshrimp,despiteourconstantwarningsagainstthepractice. ScientistshavefirmdatathatsomelivepolychaetesfromlocalandimportedsourcesinAsiacangivepositivePCRtestresultsforbothAHPNDbacteriaandEHP.However,thereisalsoapossibilitythatsomeimportedstocksofP.vannameilabeledSPFmayalsobepositiveforEHP,sinceitisnotontheOIElistthatisusedbymanySPFsuppliersorquarantineagenciesresponsibleforconfirmingSPFstatus. ThisproblemcouldberectifiedbyaddingEHPtotheSPFlistofbothsuppliersandquarantineagencies.ThefecesofthebroodstockcanbetestedforthepresenceofEHPbynestedPCR. ThebestapproachformaturationandhatcheryfacilitiestoavoidEHPistoneveruseliveanimals(e.g.,livepolychaetes,clams,oysters,etc.)asfeedsforbroodstock.Ifthisadviceisignored,attheveryminimum,suchfeedsshouldbefrozenbeforeusesincethiswouldatleastkillAHPNDbacteriaandEHP. Betterwouldbepasteurization(heatingat70oCfor10minutes)sinceitwouldalsokillmajorshrimpviruses(whichfreezingwouldnot).Anotheralternativewouldbetousegammairradiationwithfrozenfeeds. HowtocontrolEHPinhatcheries EHPandAHPNDbacteriahavebothbeenfoundinbroodstockfromChina,VietNamandThailand.Bothhavealsobeenreportedfromlivingpolychaetesamplesusedtofeedbroodstockshrimp.EHPcanbesuspectedifpostlarvaefromanyhatcherygrowslowerthanwouldbeexpected. Therefore,thefirstissueistoensurethatbroodstockmaturationfacilitiesandhatcheryfacilitiesareCLEAN!Toachievethisgoal,allshrimpmustberemovedfromthehatcheryanditshouldbewashedfollowedbycleaningusing2.5percentsodiumhydroxidesolution(25gmsNaOH/Lfreshwater)withthesolutionleftonandwashedoffafterthreehourscontacttime. Thistreatmentshouldincludeallequipment,filters,reservoirsandpipes.AfterwashingtoremovetheNaOH,thehatcheryshouldbedriedforsevendays.Thenitshouldberinseddownwithacidifiedchlorine(200ppmchlorinesolutionatpH<4.5=""p="">. Thenextissueisthebroodstock.AsindicatedabovesomeSPFshrimpbroodstockgavepositivePCRtestresultsforEHPbutnoneforAHPNDbacteria.Thus,purportedSPFbroodstockshouldalsobecheckedforEHPwhileinquarantineandbeforebeingadmittedtoacleanedmaturationandhatcheryfacility. ThescientistsworkinThailandrevealedthatlocallypond-rearedbroodstockderivedfromimportedSPFstocksinitiallyfreeofEHPshowedveryhighlevelsofprevalenceforEHPinfection.Asstatedabove,broodstockfecesmaybecheckedforEHPbynestedPCRusingDNAextractsfromfecesasthetemplate.ConfirmationshouldbeconductedonHPtissueaftertheusefulnessofthebroodstockhasexpired. HowtocontrolEHPinfarms Forfarmers,therearetwomainissuestocontendwith.ThefirstissueistoinsurethatthePLusedtostockpondsarenotinfectedwithEHP.ThiscanbedonemosteasilybyPCRtesting.IfDNAhasalreadybeenextractedfromthePLtocheckforAHPNDbacteriabyPCR,aportionofthesameDNAextractcanbeusedtotestforEHP.AfarmershouldnotusebatchesofPLpositiveforeitherofthesepathogensforstockingponds. Thesecondissueforfarmerconcernsappropriatepreparationofpondsbetweencultivationcycles,especiallywhenacultivationpondhaspreviouslybeenaffectedbyEHP.ThesporesofEHPhavethickwallsandarenoteasytoinactivate.Evenhighlevelsofchlorinealonearenoteffective.Inaddition,potentialenvironmentalcarriersarecurrentlyunknown.Bothmayremaininapondafterharvestanditisimportantthatbothbeinactivatedbeforethenextcultivationcycle. TodisinfectearthenpondsofEHPspores,applyCaO(quicklime,burntlime,unslakedlimeorhotlime)at6ton/ha.PlowtheCaOintothedrypondsediment(10-12cm)andthenmoistenthesedimenttoactivatethelime.Thenleaveforoneweekbeforedryingorfilling.AfterapplicationofCaO,thesoilpHshouldriseto12ormoreforacoupleofdaysandthenfallbacktothenormalrangeasitabsorbscarbondioxideandbecomesCaCO3. AspecialwarningforMexico TherearerumorsthattheoutbreaksofAHPNDinMexicooriginatedfromcontaminatedbroodstockofP.vannameiillegallyimportedtoMexicofromAsiaforproductionofPLtostockrearingponds.Iftheserumorsaretrue,giventhehighprevalenceofEHPinAsia,itisquiteprobablethattheimportedshrimpwouldalsohavebeeninfectedwithEHP. Thus,itisurgentthattheMexicanquarantineauthoritieschecktheircurrentandarchivedDNAsamplesusedtomonitorforAHPNDbacteriabyPCRtoalsocheckforthepresenceofEHPtargetDNAbyPCR.Iftheyfindit,itwouldsupportthehypothesisthatAHPNDbacteriawereimportedfromAsia. Itisalsopossiblethattimelypreventativemeasuresorcontinuedsurveillanceofimported,livingshrimpstockscouldpreventtheunfortunateintroductionandestablishmentofwhatisprobablyanexoticparasitetoMexicoandtherestoftheAmericas. FurtherReading YoucanviewthefullreportontheNACAsiteandlistofauthorsbyclickinghere. 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