The effect of salinity on Enterocytozoon hepatopenaei ...

In advanced stages of the disease, EHP-infected shrimp typically display soft shells, lethargy, reduced feed intake, an empty midgut, and ... Skiptomaincontent Advertisement SearchallBMCarticles Search TheeffectofsalinityonEnterocytozoonhepatopenaeiinfectioninPenaeusvannameiunderexperimentalconditions DownloadPDF DownloadPDF Researcharticle OpenAccess Published:02February2021 TheeffectofsalinityonEnterocytozoonhepatopenaeiinfectioninPenaeusvannameiunderexperimentalconditions L.F.ArangurenCaro1,F.Alghamdi2,K.DeBelder1,J.Lin1,H.N.Mai1,J.Millabas1,Y.Alrehaili2,A.Alazwari2,S.Algetham2&…A.K.Dhar1 Showauthors BMCVeterinaryResearch volume 17,Article number: 65(2021) Citethisarticle 3651Accesses 5Citations Metricsdetails AbstractBackgroundEnterocytozoonhepatopenaei(EHP)isanentericpathogenthataffectsPenaeusvannameiandPenaeusmonodonshrimpinmanySEAsiancountries.Inthewesternhemisphere,EHPwasreportedforthefirsttimein2016infarmedP.vannameiinVenezuela.AnecdotalevidencesuggeststhatEHPismoreprevalentingrow-outpondswherethesalinityishigh(> 15partsperthousand(ppt))comparedtogrow-outpondswithlowsalinities( 90%)inimmersionchallengemethods.Incontrast,alowerdose(2.0 × 104CFU/ml)doesnotcausemortalitynorhistologicallesionsinthechallengedpopulation[9].Inthepresentstudy,theinoculumwithlowcopynumber(1.6 × 103copiesofEHP/ngoftotalfecalDNA)usedinchallenge#1causedmildinfectionsinthechallengedshrimp.However,severeinfections(GradesG3toG4)andahigherprevalenceoccurredinthechallenge#2whentheEHPcopynumberintheinoculumwashigher(1.1 × 106copiesofEHP/ngoftotalfecalDNA).Thehistologicallesionsinshrimpmaintainedat30 pptsalinityweremoresevere.Amoderate-severegradeofinfection(G3-G4)wasfoundin50%oftheshrimpaffected.Incontrast,only16%ofshrimprearedatsalinityof2 pptshowedgradeG3levelofinfectionand0%ofshrimprearedatsalinityof15 pptshowedgradeG2-G4levelofinfection,accordingtotheLightner’sscale[18].ThedifferenceintheseverityoftheEHPinfectionatthethreedifferentsalinitieswasprobablyduetothedifferentialeffectofsalinityonsporegermination.Oneofthecriticalphasesinthesporegerminationistheincreaseofintra-sporeosmoticpressure.Thedifferenceinsalinitiesledtoahypotonicenvironmentat2 pptand15 pptcomparedtohypertonicenvironmentat30 ppt.Itispossiblethatthehypertonicsolutionenhancesthegerminationofthesporebyincreasingthesporeactivationprocess.Heetal.[19]foundadifferenceofeversionofthepolartubeindifferentosmoticenvironmentsinEncephalitozoonintestinalis,anobligateintracellularmicrosporidiumthatcausesgastrointestinaldiseasesinimmunocompromisedandimmunocompetentpeople.Differencesinpolartubegerminationassociatedwithchangesinsalinityhavealsobeenreportedbyotherresearchers.DeGraffetal.showedanincreaseingerminationofNosemaapissporesat0.5 NNaClconcentration[20].Similarly,anincreaseofgerminationofNosemaalgeraesporesat0.1 MNaClconcentrationvs.0.05 MNaClwasreportedbyotherresearchers[21].Inourstudy,theNaClconcentrationwasabout0.5 M,0.2 M,and0.03 MNaClat30 ppt,15 ppt,and2 ppt,respectively.ThedifferenceinNaClconcentrationsperhapscouldexplain,inpart,aneffectonthegerminationofEHPsporesandresultingprevalencelevels.Hardnessisanothervariablethatwasdifferentinthethreesalinitiesusedinthisstudyandcouldhavebeenafactorthataffectedthesporegermination.Thehardnessinlowsalinity(2 ppt)wasabout240 mg/L(CityofTucsonhttps://www.tucsonaz.gov/water/water-quality-reports-and-publications),vs.themarinewaterartificiallypreparedat15 pptand30 pptthatwasaround787and1575 mg/Lrespectively(CrystalSea,Marinex).Ithasbeenreportedthatcalciumisanimportantsecondmessengerthatactivatesmanycelleventsandcalciuminfluxmightbe,inpart,responsiblefortheactivationofmicrosporidiansporedischargeathighersalinities[21].Ingrow-outpondsofsomeEHPendemicareasinAsia,thesalinityconditionsarefoundtovarywidely.Forexample,inIndiatherearesomeshrimpfarmingareasinhighandlowsalinity,andtheprevalenceofEHPseemstobeloweratlowersalinities(below5 ppt),asobservedduringashrimpdiseasesurveyinAndhraPradeshin2019(Arangurenetal.,Unpublisheddata).SimilarconditionswererecordedintwomajorshrimpfarmingareasinVenezuela,i.e.Maracaibolakewherethesalinityisaround4–6 pptandinFalconstatewherethesalinityvariesfrom36to40 ppt.InVenezuela,shrimpfarmingisnotfullyintegrated,andthemovementofnaupliiandpost-larvaebetweenFalconandMaracaibolakeareaisacommonpractice.ThissuggeststhatEHP-infectedPLand/orbroodstockhavebeenmovedbetweenthesetwozones.However,EHPhasonlybeendetectedintheFalconareawherethesalinitiesarehigh.IntheMaracaibo’slakewherethesalinitiesarelow,EHPhasnotbeenreported.OnepossibilitythathaslimitedthespreadofEHPcouldbethedifferenceinwatersalinity.ConclusionThisstudydemonstratedthatfecalstringsfromknownEHP-infectedshrimpcouldbeusedasareliablesourceofinoculumtoconductEHPexperimentalinfectionsviathefecal-oralroute.AnEHPinfectioncanoccuratalowsalinity(i.e.2 ppt)althoughtheprevalenceandtheseverityofinfectionishigheratasalinityof30 ppt.ThesefindingshaveimplicationsindiseasemanagementinEHP-endemicareas.MethodsShrimpSpecificPathogenFree(SPF)PenaeusvannameiwereobtainedfromacommercialvendorinFlorida,US.TheSPFpopulationhasbeenscreenedforthelast2 yearsattheUA-APLwithoutpresenceofanyoftheOIE-listedandnon-listeddiseasesincludingEHP.ThebioassayswerecarriedoutintheAquaculturePathologyLaboratory(UA-APL)ofTheUniversityofArizona.TheAmericanVeterinaryMedicalAssociation(AVMA)guidelineswerefollowedwhileprocessingshrimpforhistopathologyanalysisandforeuthanasiabysaltwater/iceslurryattheendoftheexperiment(https://www.avma.org/sites/default/files/2020-01/2020-Euthanasia-Final-1-17-20.pdf).EnterocytozoonhepatopenaeibioassayTheEHPisolateusedinthisstudywasobtainedfromaP.vannameipopulationoriginatinginThailand.TwoindependentEHPchallengeswereconducted.Inbothchallenges,shrimpweremaintainedatthreedifferentsalinitiesof2 ppt,15 ppt,and30 ppt.Foreachexperimentalchallenge,six90-Ltankswerefilledwithartificialseawater(CrystalSeaMarinex,Baltimore,Maryland)thatcorrespondedtothethreesalinitylevelswithtworeplicatesforeachsalinitytreatment.Temperaturewasadjustedat25 °C(±0.6)withmeasurementseachmorningwithaNISTthermometer.pHwasmeasuredwithapHindicatorstrips(Baxter®)onceaweekwitharangeof7.5–8.0.Salinitywasadjustedbychanging3partsofthesalinityeveryhourfrom25 ppt(initialsalinityoftheSPFP.vannameipopulation)downto5 ppt.Inordertoachieveasalinityof2 pptfrom5 ppt,thesalinitypartwaschangedevery2h.Oncesetup,salinitywasmeasuredwithasaltrefractometer(SperScientific®)onceaweekoverthelengthofthechallenges.Ten(10)SPFP.vannamei(weights:2.0–2.1 g)werestockedineachtankfortheexperimentalinfection,respectively.Three90-Lcontroltanksweresetupforeachsalinityasnegativecontroltreatment.InoculumpreparationOverthecourseofthetwochallenges,theEHP-infectedshrimpwerefeddailyat5%ofthetotalbiomass.FecalstringswerecollecteddailybysiphoningthefecalstringsfromtheEHP-infectedtanksintoatube1hafterfeeding.Inordertopreparetheinoculumforchallenge#1,theEHP-infectedfecalstringswerepooledfromthreetankswith5shrimppertank(meanweight12.5 g).Forchallenge#2,EHPinoculumwaspreparedbypoolingfecalstringsfromone1000 Ltankcontaining60EHP-infectedshrimp(meanweight9.0 g).ThesalinityoftheEHP-infectedtanksusedasinoculumsourceswereconstantforbothchallengesat25 ppt(±1.0).Aftersiphoningthefecalstringsfromthetanks,itwasweighedandaliquotedinsevenequalparts.Sixofthealiquotswereusedastheinoculumfortheexperimentalinfectionofeachtreatmenttank.Eachofthesixaliquotswereaddedtothetreatmenttanksbymixingthetubecontentsintothewaterofthetreatmenttanks.Theremainingaliquotwaspreservedin95%ethanolforEHPdetectionbyPCRfollowingapreviouslypublishedprotocol[4].Starting1 haftertheinoculationprocess,shrimpwerefedonceadayat2%ofthetotalbiomasswithacommercialpelletedfeed(Rangen35%,Buhl,Idaho).ThisprocessforcedtheshrimptoeatfirsttheinoculathatcontainedEHP-infectiousfecalstrings.Survivalwasrecordeddailyfromthestartofthechallenge.Attheendofthechallenge,shrimpsamplesfromthechallenge#1and#2fromeachtreatmenttankwerefixedinDavidson’s(AFA)fixative[22].DetailsofthenumberofsamplesforH&Erangedfrom4to6inthechallenge#1andfrom6to10inthechallenge#2.ThedetailsofthenumberofsamplesisdescribedinTable2.Pooledhepatopancreastissuefrom3to4shrimpwerecollectedfromeachtankinchallenge#1and#2foratotalof6pools.Sampleswerepreservedin95%ethanolforEHPdetectionbyPCR.Thedurationofthechallengeswas20 daysand26 daysforthechallenge#1and#2,respectively.HistopathologyTheDavidson’salcohol-formalin-aceticacid(AFA)-fixedsampleswereprocessed,embeddedinparaffin,andsectioned(5 μmthick)inaccordancewithstandardmethods[18,22].Afterstainingwithhematoxylinandeosin(H&E),thesectionswereanalyzedbylightmicroscopy.SeveritygradesoftheEHPinfection/lesionrangedfromG0-G4accordingtoLightner(1996)withG0beingabsenceofthediseaseandG4beingpresenceofseverelesionsandadvancedtissuedestruction.ConventionalandquantitativePCRindetectingandquantifyingEHPTwodifferentPCRmethodswereusedinthisstudy:ConventionalnestedPCRforEHPdetectionandreal-timePCRforquantitationofEHPload.EHPdetectionwascarriedoutbyaconventionalnestedPCRthattargetstheSporeWallProtein1(SWP1)gene.Theprimersforthefirststepare:SWP1F/1R(1F:5′-TTGCAGAGTGTTGTTAAGGGTTT-3,1R:5′-CACGATGTGTCTTTGCAATTTTC-3′).Theprimersgeneratea514 bpamplicon.Theprimersforthenestedstepare:SWP2F/2R(2F:5′-TTGGCGGCACAATTCTCAAACA-3′,2R:5′-GCTGTTTGTCTCCAACTGTATTTGA3′)whichgeneratesa148 bpamplicon.ThePCRprotocolforthefirstPCRreactionconsistedofa5-mininitialdenaturationat95 °Cfollowedby30 cyclesofdenaturationfor30 sat95 °C,annealingfor30 sat58 °Candextensionfor45 sat68 °Cwithafinal5-minextensionstepat68 °C.Forthesecond(nested)PCRstep,thetemplateconsistedof1 μlofthefinalreactionsolutionfromthefirstPCRstep.ThePCRprotocolforthesecond,nestedPCRreactionconsistedofaninitialdenaturationat95 °Cfor5 minfollowedby20 cyclesof30 sdenaturationat95 °C,30 sannealingat64 °Cand20 sextensionat68 °Cwithafinalextensionfor5-minat68 °C[23].ForEHPquantificationbyreal-timePCR,primersF:157(5′-AGTAAACTATGCCGACAA-3′)andR:157(5′-TTAAGCAGCACAATCC-3′),andaTaqManprobe(5-FAM-TCCTGGTAGTGTCCTTCCGT-TAMRA-3′)wereused.Thereal-timePCRprotocolconsistedofa20 sinitialdenaturationat95 °Cfollowedby40 cyclesofdenaturationfor1 sat95 °Candannealing/extensionfor20 sat60 °C,followingapreviouslypublishedmethodwithminormodifications[24].StatisticalanalysisTheEHPcopynumberobtainedfromthequantificationanalysiswastransformedtologbase10valuepriortocarryingoutstatisticalanalysisusingSPSSv16.0.Aone-wayANOVAwithTukey’smultiplecomparisonwereperformedandP-value