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Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy AUSTRALIAN AQUATIC VETERINARY EMERGENCY PLAN (AQUAVETPLAN) Whirling disease Version2,2016 AQUAVETPLAN–Disease Strategy AQUAVETPLANisaseriesofmanualsthatoutlineAustralia’sapproachtonational diseasepreparednessandproposethetechnicalresponseandcontrolstrategiestobe activatedinanationalaquaticanimaldiseaseemergency. NationalBiosecurityCommittee Whirling Disease 1 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Thisdiseasestrategyformspartof: AQUAVETPLAN Thisstrategywillbereviewedregularly.Suggestionsandrecommendationsfor amendmentsshouldbeforwardedto: AQUAVETPLANCoordinator AquaticPestandHealthPolicy AnimalHealthDivision AustralianGovernmentDepartmentofAgricultureandWaterResources GPOBox858,CanberraACT2601 Tel:(02)62725402;Fax:(02)62723150 email:[email protected] Approvedcitation:DepartmentofAgricultureandWaterResources(2016).Whirling Disease(Version2).In:AustralianAquaticVeterinaryEmergencyPlan (AQUAVETPLAN),AustralianGovernmentDepartmentofAgricultureandWater Resources,Canberra,ACT. Publicationrecord: Version2[August,2016] AQUAVETPLANisavailableontheinternetat: http://www.agriculture.gov.au/animal‐plant‐health/aquatic/aquavetplan ISBN978‐1‐76003‐119‐0 ©CommonwealthofAustralia2016 Ownershipofintellectualpropertyrights Unlessotherwisenoted,copyright(andanyotherintellectualpropertyrights,ifany)in thispublicationisownedbytheCommonwealthofAustralia(referredtoasthe Commonwealth). CreativeCommonslicence AllmaterialinthispublicationislicensedunderaCreativeCommonsAttribution3.0 AustraliaLicence,saveforcontentsuppliedbythirdparties,logosandthe CommonwealthCoatofArms. CreativeCommonsAttribution3.0AustraliaLicenceisastandardformlicence agreementthatallowsyoutocopy,distribute,transmitandadaptthispublication providedyouattributethework.Asummaryofthelicencetermsisavailablefrom creativecommons.org/licenses/by/3.0/au/deed.en.Thefulllicencetermsareavailable fromcreativecommons.org/licenses/by/3.0/au/legalcode. Thispublication(andanymaterialsourcedfromit)shouldbeattributedas: DepartmentofAgricultureandWaterResources2016,AQUAVETPLANDisease Strategy:WhirlingDisease.CCBY3.0 Whirling Disease 2 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Cataloguingdata DepartmentofAgricultureandWaterResources201,AQUAVETPLANDisease Strategy:WhirlingDisease,DepartmentofAgricultureandWaterResources,Canberra. Internet AQUAVETPLANDiseaseStrategy:WhirlingDiseaseisavailableatagriculture.gov.au Contact DepartmentofAgricultureandWaterResources Postaladdress:GPOBox858 CanberraACT2601 Australia Web:agriculture.gov.au Inquiriesregardingthelicenceandanyuseofthisdocumentshouldbesentto [email protected] TheAustralianGovernmentactingthroughtheDepartmentofAgricultureandWater Resourceshasexercisedduecareandskillinthepreparationandcompilationofthe informationanddatainthispublication.Notwithstanding,theDepartmentof AgricultureandWaterResources,itsemployeesandadvisersdisclaimallliability, includingliabilityfornegligence,foranyloss,damage,injury,expenseorcostincurred byanypersonasaresultofaccessing,usingorrelyinguponanyoftheinformationor datainthispublicationtothemaximumextentpermittedbylaw. Theinformationinthispublicationisforgeneralguidanceonlyanddoesnotoverride commonlaw,lawsoftheCommonwealthoranyAustralianStateorTerritory,orplace anylegalobligationofcomplianceoractionontheCommonwealth,aStateora Territory. Itistheresponsibilityoftheusersofthispublicationtoidentifyandensuretheyhave compliedwithalllegislativeorregulatoryrequirementsoftherelevantAustralian StateorTerritoryandtheCommonwealthpriortoundertakinganyoftheresponse optionssetoutwithinthispublication. Beingaguideonly,outbreaksorsuspectedoutbreaksmustbeassessedonacaseby casebasisandexpertadviceshouldbeobtainedtodeterminethemostappropriate managementplaninresponsetotherisk. DISEASEWATCHHOTLINE 1800675888 TheDiseaseWatchHotlineisatoll‐freetelephonenumberthatconnectscallerstothe relevantStateorTerritoryofficertoreportconcernsaboutanypotentialemergency animaldiseasesituation.Anyonesuspectinganemergencydiseaseoutbreakshould usethisnumbertogetimmediateadviceandassistance. Whirling Disease 3 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Preface Thisdiseasestrategyforthecontrolanderadicationofwhirlingdiseaseisanintegral partoftheAustralianAquaticVeterinaryEmergencyPlan,orAQUAVETPLAN. AQUAVETPLANdiseasestrategymanualsareresponsemanualsanddonotinclude informationaboutpreventingtheintroductionofdisease. TheDepartmentofAgricultureandWaterResourcesprovidesquarantineinspectionfor internationalpassengers,cargo,mail,animals,plantsandanimalorplantproducts arrivinginAustralia,andinspectionandcertificationforarangeofagriculturalproducts exportedfromAustralia.QuarantinecontrolsatAustralia’sbordersminimisetheriskof entryofexoticpestsanddiseases,therebyprotectingAustralia’sfavourablehuman, animalandplanthealthstatus.Informationoncurrentimportconditionscanbefound attheDepartmentofAgricultureandWaterResourcesBICONwebsite (agriculture.gov.au/import/online‐services/bicon). Thisstrategysetsoutthediseasecontrolprinciplesforuseinanaquaticveterinary emergencyincidentcausedbythesuspicionorconfirmationofwhirlingdiseasein Australia.ThestrategywasscientificallyreviewedbytheSub‐CommitteeonAquatic AnimalHealthoftheAnimalHealthCommittee,beforebeingendorsedby: theAnimalHealthCommitteeoftheNationalBiosecurityCommitteeinJanuary 2016,andtheNationalBiosecurityCommitteeinJune2016. WhirlingdiseaseislistedonAustralia’sNationalListofReportableDiseasesofAquatic Animal(DA2015a).Detailedinstructionsforthefieldimplementationof AQUAVETPLANarecontainedinthediseasestrategies,operationalproceduresmanuals andmanagementmanuals.Industry‐specificinformationisgivenintheenterprise manual.ThefulllistofAQUAVETPLANmanualsthatmayneedtobeaccessedinan emergencyisshownbelow: Diseasestrategies Enterprisemanual Individualstrategiesforeachdisease Includessectionson: –opensystems Operationalproceduresmanuals –semi‐opensystems Disposal –semi‐closedsystems Destruction Decontamination Managementmanual Controlcentresmanagement AquaticAnimalDiseasesSignificanttoAustralia:IdentificationFieldGuide(DA2012)is asourceofinformationabouttheaetiology,diagnosisandepidemiologyofinfection withwhirlingdiseaseandshouldbereadinconjunctionwiththisstrategy. Whirling Disease 4 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy ThefirsteditionofthismanualwaspreparedbyDrPaulHardy‐Smith,withthe assistanceofProfessorRonHedrick,DrCraigStephensandDrMarkCrane,in2005.This revisionwaspreparedbyDrBrendanCowledandDrAndyShinnin2016.Theauthors wereresponsiblefordraftingthestrategy,inconsultationwithawiderangeof stakeholdersfromaquaculture,recreationalfishingandgovernmentsectorsthroughout Australia.Thetext,however,wasamendedatvariousstagesoftheconsultationand endorsementprocess,andthepoliciesexpressedinthisversiondonotnecessarily reflecttheviewsoftheauthors.TherevisionauthorswouldliketothankDrMatt Longshaw.Contributionsmadebyothersnotmentionedherearealsogratefully acknowledged. TheformatofthismanualwasadaptedfromsimilarmanualsinAUSVETPLAN(the Australianveterinaryemergencyplanforterrestrialanimaldiseases)andfromthe AQUAVETPLANEnterpriseManual.Theformatandcontenthavebeenkeptassimilaras possibletothesedocuments,inordertoenableanimalhealthprofessionalstrainedin AUSVETPLANprocedurestoworkefficientlywiththisdocumentintheeventofan aquaticveterinaryemergency.TheworkoftheAUSVETPLANwritingteamsandthe permissiontousetheoriginalAUSVETPLANdocumentsaregratefullyacknowledged. Therevisedmanualhasbeenreviewedandapprovedbythefollowingrepresentatives ofgovernmentandindustry: Government CSIROAustralianAnimalHealthLaboratory DepartmentofPrimaryIndustries,NewSouthWales DepartmentofPrimaryIndustryandFisheries,NorthernTerritory DepartmentofAgricultureandFisheries,Queensland DepartmentofPrimaryIndustries,Parks,WaterandEnvironment,Tasmania DepartmentofFisheries,WesternAustralia DepartmentofEconomicDevelopment,Jobs,TransportandResources,Victoria DepartmentofPrimaryIndustriesandRegions,SouthAustralia BiosecurityAnimalDivision,DepartmentofAgricultureandWaterResources, AustralianGovernment DepartmentoftheEnvironment,AustralianGovernment Industry NationalAquaticAnimalHealthIndustryReferenceGroup ThecompleteseriesofAQUAVETPLANdocumentsisavailableontheinternet (http://www.agriculture.gov.au/animal‐plant‐health/aquatic/aquavetplan). Whirling Disease 5 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Contents Preface...............................................................................................................................................4 1 1.1 1.1.1 1.1.2 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.2.7 1.2.8 1.3 1.4 1.4.1 1.4.2 1.4.3 1.4.4 1.4.5 1.4.6 1.4.7 1.5 1.5.1 1.5.2 1.5.3 1.5.4 1.6 1.6.1 1.6.2 1.6.3 1.6.4 1.7 Natureofthedisease.......................................................................................................8 Aetiology...................................................................................................................................................8 Salmonidstage.....................................................................................................................................10 Tubifextubifexwormstage............................................................................................................11 Susceptiblespecies............................................................................................................................12 Susceptiblefishspecies...................................................................................................................12 Susceptibilitytoinfectionwithinsusceptiblefishspecies..............................................12 Within‐fishspeciessusceptibility...............................................................................................13 SusceptibilityofT.tubifexstrains...............................................................................................13 Susceptibilityofintroducedpopulationsofsalmonid......................................................13 Australiannativefishsusceptibility..........................................................................................14 Vectors.....................................................................................................................................................14 Notahumanpathogen.....................................................................................................................14 Worlddistribution.............................................................................................................................14 DiagnosisofinfectionwithM.cerebralis.................................................................................15 Presumptivediagnosis.....................................................................................................................15 Confirmatorydiagnosis...................................................................................................................15 Casedefinitions...................................................................................................................................16 Fieldmethods:clinicalsignsandgrosspathology..............................................................17 Laboratorymethods..........................................................................................................................17 Confirmationofinfection................................................................................................................19 Differentialdiagnosis........................................................................................................................20 Resistanceandimmunity................................................................................................................20 Resistancetoinfection.....................................................................................................................20 Innateimmunity..................................................................................................................................20 Adaptiveimmunity............................................................................................................................21 Vaccination............................................................................................................................................21 Epidemiology........................................................................................................................................21 Incubationperiod...............................................................................................................................22 Persistenceofthepathogen...........................................................................................................22 Modesoftransmission.....................................................................................................................22 Factorsinfluencingtransmissionandexpressionofdisease.........................................23 Impact......................................................................................................................................................23 2 2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 Principlesofcontrolanderadication.....................................................................25 Introduction..........................................................................................................................................25 Methodstopreventspreadandeliminatepathogens.......................................................27 Quarantineandmovementcontrols..........................................................................................27 Zoningandcompartmentalisation.............................................................................................30 Diseasemanagementinaquaticenvironments....................................................................31 Tracing.....................................................................................................................................................31 Surveillance...........................................................................................................................................32 Treatmentofhostproductsandby‐products.......................................................................33 Destructionofhosts..........................................................................................................................33 Whirling Disease 6 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 2.2.8 2.2.9 2.2.10 2.2.11 2.3 2.4 2.5 2.6 2.6.1 2.6.2 Disposalofhosts.................................................................................................................................35 Decontamination.................................................................................................................................35 Vaccination............................................................................................................................................37 Vectorcontrol.......................................................................................................................................37 Environmentalconsiderations.....................................................................................................37 Sentinelanimalsandrestockingmeasures............................................................................37 Publicawareness................................................................................................................................38 FeasibilityofcontroloreradicationofwhirlingdiseaseinAustralia........................38 ResponseOption1:Eradication..................................................................................................38 ResponseOption2:Containmentandcontrolviazoningand/or compartmentalisation......................................................................................................................40 2.6.3 ResponseOption3:Controlandmitigation...........................................................................42 2.7 Tradeandindustryconsiderations............................................................................................42 2.7.1 Exportmarkets....................................................................................................................................42 2.7.2 Domesticmarkets...............................................................................................................................43 3 3.1 3.2 3.2.1 3.2.2 3.2.3 3.3 3.4 3.5 PreferredAustralianresponseoptions.................................................................44 Overallpolicyforwhirlingdisease.............................................................................................44 Responseoptions................................................................................................................................45 Option1:Eradication........................................................................................................................47 Option2:Containmentandcontrolviazoningand/orcompartmentalisation.....48 Option3:Controlandmitigationofdisease...........................................................................49 Criteriaforproofoffreedom.........................................................................................................49 Fundingandcompensation............................................................................................................49 Exportmarkets....................................................................................................................................50 4 Appendix1OIEAquaticAnimalHealthCodeandManualofDiagnostic TestsforAquaticAnimals...........................................................................................51 OIEAquaticCode................................................................................................................................................51 OIEAquaticManual...........................................................................................................................................51 Furtherinformation..........................................................................................................................................51 5 Appendix2ApprovalofchemicalsforuseinAustralia....................................52 Registration...........................................................................................................................................................52 Minorusepermit................................................................................................................................................52 Emergencyusepermit.....................................................................................................................................52 Appendix3 ExtractionandPCRmethodforwhirlingdisease(Myxobolus cerebralis)—NewZealandMinistryforPrimaryIndustries..........................53 Whirlingdisease(Myxoboluscerebralis)—extractionandPCRmethod................................53 IANZaccreditedtest..........................................................................................................................................57 References.....................................................................................................................................59 Whirling Disease 7 Department of Agriculture and Water Resources 1 AQUAVETPLAN–Disease Strategy Nature of the disease Whirlingdiseaseisadiseaseoffreshwatersalmonidfishcausedbythemyxozoan parasiteMyxoboluscerebralis.TheparasitehasneverbeendetectedinAustralia,butis presentinNewZealandandareasofNorthAmerica,Europe(includingIceland),Russia, Africa(MoroccoandSouthAfrica),theMiddleEast(Lebanon)andAsia(Blaylock& Bullard2014;Hallett&Bartholomew2012). Importantly,theparasitehastwohosts—salmonidfishspeciesandafreshwater oligochaeteworm,Tubifextubifex(orspeciesassemblage)―hereaftercalledT.tubifex. Therearetwosporestages,onereleasedfromthesalmonidfish,whichisinfectiveforT. tubifex,andtheotherreleasedfromT.tubifexworms,whichisinfectiveforfish. Generally,theyoungerandsmallerfishareinfectedwhenfirstexposed(Ryceetal. 2005),andthehighertheparasiteexposuredose,thegreatertheseverityoftheclinical disease.Watertemperaturehasasignificantinfluenceonallstagesoftheparasitelife‐ cycle.Fishpopulationsaremostaffectedattemperaturesof10–15°C.Temperatures greaterthan20°Carenotconducivetoparasitedevelopmentorsurvival. Allsalmonidscanbeinfected,butrainbowtrout(Oncorhynchusmykiss)andcutthroat salmon(O.clarki)areverysusceptibletoclinicaldisease(Blaylock&Bullard2014).The parasitecausedseverehatcherylossesofrainbowtroutintheearlystagesofindustry developmentinEurope.Earlytroutcultureinearthenpondsprovidedhabitatfor T.tubifexworms.Changedculturetechniquesfortroutaquaculturehavegreatly reducedtheincidenceofthediseaseinaquaculture,particularlyrearingofyoungfishin concreteorplastic‐linedraceways.IntheUnitedStates,significantdeclinesinwildtrout populationshaveoccurredinsomeareas.Variabilityintheseverityofclinicaldiseaseis associatedwithecologicalconditionsanddifferentstrainsofrainbowtrout.Other salmonidscanbesubclinicallyinfected. Australiannativefreshwaterfisharenotsalmonids,anditisthereforeunlikelythatthey aresusceptibletothisdisease.Bothwildandfarmedsalmonidsusedinrestocking programswouldbesusceptible.InmanyareasofsouthernAustralia,troutpopulations aremaintainedorenhancedbyrestockingusinghatchery‐rearedfish.Thesestocksare mostatriskfrompotentialM.cerebralisinfection.Stockingwithtroutisnolonger consideredenvironmentallyresponsibleinsomeareas,butcontinuesinmanyareasof southernAustraliaforrecreationalfisheryenhancement.Hatcheriesalsosupplystock foraquaculture.Theinlandrecreationalfisheryforsalmonids(primarilybrownand rainbowtrout)inTasmaniaisworthover$40Mintourismrevenuealone,animportant contributiontolocaleconomies(IFS2008). WhirlingdiseaseisanationallyreportablediseaseinAustralia.Stateandterritory governments,therecreationalsalmonidfishingindustryandthesalmonidaquaculture industryneedtobeadequatelyeducatedandpreparedforthepossibleincursionofthis disease.Thiswillgreatlyminimisetheimpactofwhirlingdiseaseonsusceptible salmonidpopulationsiftheparasiteiseverintroducedintoAustralia. 1.1 Aetiology TheaetiologicalagentofwhirlingdiseaseistheparasiteMyxoboluscerebralis(formerly namedMyxosomacerebralis).Myxozoansareadiversegroupofmulticellularorganisms withthousandsofspecies,mostofwhichparasitisefishatsomestageoftheirlife‐cycle (Gilbert&Granath2003;Hallett&Bartholomew2012).Myxozoansarecharacterisedby sporestages. MyxoboluscerebralisbelongstothephylumMyxozoa,classMyxosporea,order Bivalvulida,suborderPlatysporinaandfamilyMyxobolidae. Whirling Disease 8 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Myxoboluscerebralisisthemoststudiedmyxozoanandisatinymetazoanparasitewith twomorphologicallydistinctspores(Fig1): anactinomyxonsporewhichisreleasedfromT.tubifexworms;the triactinomyxonhasthreefloatcellsformingatri‐radialanchorshape amyxosporewhichisreleasedfromfish(mostlyafterdeath)andisovalto lenticularinshapewithvalvecellsencapsulatingabinucleatesporoplasmand polarcapsules. Identificationanddiagnosiscanbedifficult,asseveralspeciesofmyxozoasharesimilar morphologyandcancausesimilarclinicalsigns(Bentzetal.2012;Hallett& Bartholomew2012;Hoggeetal.2008).Traditionaldiagnosishasbeenbasedontissue tropism(identificationofcharacteristicmyxosporesinthecartilageofsalmonids), speciesaffected(salmonids,especiallyrainbowtrout)andpresentingclinicalsigns.DNA diagnosticmethodsarenowusedtoconfirmidentification(Hoggeetal.2008). Figure 1 Life‐cycle of M. cerebralis (whirling disease) (courtesy of R. Hedrick, adapted from El‐Matbouli et al. (1992) Theimportantelementsofthelife‐cycleofM.cerebralisare: Two‐hostlife‐cycle Salmonids asexualreplicationonly susceptiblesalmonidsarepresentinsouthernAustralia. Freshwateroligochaeteworms,T.tubifex Whirling Disease sexualandasexualreplication T.tubifexwormsarepresentthroughoutAustralia. 9 Department of Agriculture and Water Resources Twoinfectivestages Myxospore formsinthecartilageofthefish infectivemyxosporesarepresentincartilageapproximately50–120 daysafterinitialinfection,dependingonwatertemperatureand salmonidspeciesinfected resistanttoenvironmentaldegradation,survivalmeasuredinmonths toyears. Actinosporetriactinomyxon(TAM) AQUAVETPLAN–Disease Strategy formsintheintestinalliningoftheT.tubifexworm infectiveTAMspresent100–170daysafterinitialexposure, dependingontemperatureandstrainofT.tubifexworm susceptibletoenvironmentaldegradation,survivalmeasuredindays. Alllife‐cyclestagesaresignificantlyaffectedbytemperature withinlimits(10–15°C),thehigherthetemperature,thefasterthelife‐cycle (seeGilbert&Granath[2003]andHallett&Bartholomew[2012]who summarisediverseliteratureontemperatureeffectsatseverallifestages). Significantvariabilityinhostsusceptibility bothT.tubifexandsalmonidfishdisplaystrainvariabilityinsusceptibilityto infection. 1.1.1 Salmonidstage BuoyantTAMsarereleasedfromtheT.tubifexhostsandpassivelyfloat.Whenafish comesincloseproximity,chemicalcues,mostlyinmucus,causepolarfilamentstofire andanchortheTAMtotheskin(Kallertetal.2011).Firingcanbestimulatedbymany fishspecies(Kallertetal.2009).Insalmonids,theamoeboidsporoplasmthenemerges frombetweenthevalvecellsoftheTAMandenterstheskinthroughthesecretory openingsinthemucouscells(El‐Matboulietal.1999a).Theparasitecanalsopenetrate acrossthegills,throughthefinsandthroughtheliningofthemouth. Thesporoplasmthenmigratesthroughtheepithelium,peripheralnervesandcentral nervoussystem(CNS)toreachcartilage(El‐Matboulietal.1999a). Within24hours,therecanbenumerousparasitecellsdeepintheskinofthefishand,by day4,parasitecellscanbefoundinnervoustissue.Atfirst,thecellsarefoundin peripheralnerves.ThecellsthenmovethroughgangliatotheCNS,replicatingasthey go.Asearlyas20daysafterexposure,parasitestagescanbefoundinthecartilageofthe susceptiblefish.Trophozoites(plasmodialforms)inthecartilagecausepathological changes.Thisisdependentonwatertemperature;optimaltemperaturefor developmentbeingaround10–15°C.Atthispoint,twoparasiticcellswilljointoinitiate sporogenesisortheformationofspores(‘myxospores’)inthecartilageofthefish. Cartilagethroughoutthebody,includingthecranium,spine,fins,vertebrae,ribsand operculumcanbeaffected(Antonioetal.1999). Myxosporescantake52–120daystodevelopinthefishat7–17°C(Halliday1973a).At 16–17°C,fullydevelopedmyxosporesappear52daysafterinfection.Thesesporesare elliptical,multicellular,havethickprotectiveshellsandareapproximately Whirling Disease 10 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 10micrometresindiameter.Myxosporesremaininthecartilageandmaybe‘trapped’ byboneasitformsaroundtheinfectedcartilage. OneTAMcanpotentiallyproduce2800–7800infectivemyxosporesinthecartilageofa fishheadandpossiblymoreincartilageelsewhereinthebody(Hedricketal.1999b; Kerans&Zale2002). Inlivefish,someviablemyxosporesescapefromdestroyedcartilageandarereleased directlyintotheenvironmentorpassoutinthefaeces(Nehringetal.2002).Themost significantreleaseofmyxosporesoccurswhenthefishdiesanddecomposes.The decompositionofonerainbowtroutcanreleasemorethanamillionmyxospores (Hedricketal.1999b). Oncereleased,myxosporesarehighlypersistentandareabletosurviveinthe environmentforlongperiods,possiblyyears(Hoffman1990). 1.1.2 Tubifextubifexwormstage TubifextubifexisthedefinitivehostforM.cerebralis.Itisfoundonlyinfreshwater, usuallyinareasofsedimentsrichinorganicmatter.ItistheonlyspeciesofTubifexin Australiaandiswidespreadacrossarangeofhabitatsbutisnotcommonlyencountered (Pinder&Brinkhurst2000).InAustralia,T.tubifexisknownfromallmainlandstates andTasmania,includingbeingrecordedaspresentatatrouthatchery(Pinder& Brinkhurst2000).ItisnotknownwhetheranyofthefivegeneraoftheAustralian TubificinaearesusceptibletoM.cerebralis,butothertubificids(e.g.Limnodrilussp.) overseasarerefractorytoinfection(Keransetal.2004). Forthepurposesofthismanual,susceptibilityofAustralianT.tubifexwormsto M.cerebralisshouldbeassumed,unlessotherwisedemonstrated. Whenaninfectedsalmonidfishdies,itsmyxosporessettleinsediment.Filterfeeding T.tubifexwormsmayingestmyxosporeswhilethesporesareinthewatercolumnorin suspendedsediment.WithinT.tubifex,theinfectivesporoplasmfromthemyxosporeis released,andmigratesintotheliningoftheintestinewhereitmultipliesandreplicates (Hedrick&El‐Matbouli2002).Replicationisbothasexualandsexual.Thisdefines T.tubifexasthedefinitivehostforM.cerebralis(El‐Matbouli&Hoffmann1998). TAMsarereleasedintothelumenoftheintestineasearlyas74days(at15°C)after ingestionofmyxosporesbytheT.tubifexworm(Gilbert&Granath2001).Tubifextubifex isthoughttoremainpersistentlyinfectedforlife(Gilbert&Granath2001). Experimentally,peakreleaseofTAMswasfoundtooccurat120–170daysafter exposure,butthisishighlytemperaturedependent(Markiw1986). TAMsaremorphologicallydistinctfromthemyxosporesreleasedfromfish.Theyare shapedlikeagrapplinghook,i.e.alongrod(approximately146micrometres)with threefloatcells,eachapproximately193micrometres.TAMsareproducedinveryhigh numbersfrominfectedT.tubifexworms.Theirmaximuminfectivedurationhasnot beendeterminedbutisatleast15days,dependingontemperature(thecoolerthe temperature,thelongerthesurvival)(El‐Matboulietal.1999b;Markiw1992). TherearedifferencesinthesusceptibilityofT.tubifextoM.cerebralis.Studies conductedwithintheUnitedStateshaveshownthatfourlineagesofT.tubifexexist, basedonmitochondrialDNA(lineagesI,III,VandVI)(Hallettetal.2009).Onlylineage IIIwormsreleaseTAMs,andonlypopulationsdominatedbythislineageamplifiedthe parasite.HigherproportionsofmoresusceptiblelineageswithinalocalT.tubifex populationappeartoincreasetheprevalenceofwhirlingdiseaseinlocalpopulationsof susceptiblesalmonids(Beauchampetal.2005;Zielinskietal.2011). Whirling Disease 11 Department of Agriculture and Water Resources 1.2 AQUAVETPLAN–Disease Strategy Susceptiblespecies 1.2.1 Susceptiblefishspecies SalmonidsaregenerallysusceptibletoinfectionbyM.cerebralis,butthereis considerablevariabilityamongsalmonidspeciesintheirsusceptibilitytoclinical disease. 1.2.2 Susceptibilitytoinfectionwithinsusceptiblefish species ItisthoughtthatM.cerebralisevolvedwithinEuropeanpopulationsofbrowntrout (Salmotrutta), asbrowntroutarerelativelyresistanttoclinicaldisease(Gilbert& Granath2003;Hallett&Bartholomew2012).Browntroutneedtobeexposedtovery highnumbersoftheinfectiveTAMstagesforanyclinicalsignstodevelop(Hedricketal. 1999b).Evenatveryhighdoses,nocharacteristictailchasingswimmingbehaviourhas beenobservedinbrowntrout. Incontrast,rainbowtrout,steelheadtroutandcutthroattrout(nativetoNorth America)areamongstthespeciesmostsusceptibletoclinicaldisease. Table1detailsthesusceptibilitiesofthedifferentspecies(afterHallett&Bartholomew (2012)wheretheoriginalreferencescanbefound). Table1Susceptibilityofdifferentsalmonidspeciestowhirlingdisease.Informationis basedonlaboratoryandobservationalstudiesonfishatvulnerablelifestages.Adapted fromHallett&Bartholomew(2012). Commonname Suscept.a Comp.suscept.b Oncorhynchus clarkic Cutthroattrout S‐hS 1 Oncorhynchus gilae Gila hS 1 Oncorhynchus gorbuscha Pinksalmon pR,U 3 Oncorhynchus keta Chumsalmon pR,U 3 Oncorhynchus kisutch Cohosalmon pR 3 Oncorhynchus masu Cherrysalmon pR,U 3 Oncorhynchus mykiss Rainbowtrout S‐hS 1 Oncorhynchus mykiss Steelheadtrout S‐hS 1 Oncorhynchus nerka Sockeyesalmon hS 1 Oncorhynchus tshawytscha Chinooksalmon S 2 Salvelinus confluentus Bulltrout pR 3 Salvelinus fontinalis Brooksalmon S 2 Salvelinus malma Dollyvarden pR,U 3 Salvelinus namaycush Laketrout R 4 Genus Whirling Disease Species 12 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Genus Species Commonname Suscept.a Comp.suscept.b Salmo salar Atlanticsalmon S,U 2 Salmo trutta Browntrout pR 3 Prosopium williamsoni Mountain whitefish S 2 Thymallus arcticus Arcticgrayling R‐pR 3 Thymallus thymallus Europeangrayling S,U 2 Hucho hucho Danubesalmon hS 1 Susc., susceptibility; Comp., comparative. a. S, susceptible (clinical disease common at high parasite doses, e.g. > 1000 TAMs per fish, or when very young, but greater resistance to disease at low doses, i.e. 100–200). hS, highly susceptible (clinical disease common). pR, partially resistant (clinical disease rare and develops only when exposed to very high parasite doses). U, susceptibility unclear (conflicting reports or insufficient data). R, resistant (no spores develop). b. 1 = highly susceptible, through to 4 = resistant to infection. c. including several subspecies of cutthroat trout. 1.2.3 Within‐fishspeciessusceptibility Considerablevariationinclinicaldiseasehasbeenshownwithinsusceptiblespecies, especiallyinrainbowtrout,anddependsonseveralfactors. Olderandlargerrainbowtroutaremoreresistanttoclinicaldisease,althoughthelevel ofskeletalossificationwasnotimportantinthedevelopmentofdisease(Ryceetal. 2005). SomeGermanrainbowtrouthatcherystrains(andtheircrosses)areresistanttoclinical disease,butsomeUnitedStatesstrainsarenot(Fethermanetal.2014).Thereisa geneticbasisforthisresistance(Baerwald2013).Naturalselectionforresistantfishis possibleinrelativelyfewgenerations(Miller&Vincent2008). 1.2.4 SusceptibilityofT.tubifexstrains DifferentstrainsofT.tubifexhavedifferentsusceptibilitytoinfectionwithM.cerebralis, ormayaffectseverityofclinicaldiseaseinlocalrainbowtrout(Baxaetal.2008; Beauchampetal.2005;Hallettetal.2009).Despitethis,theabilitytoreduceinfectionin salmonidsbymanipulatingT.tubifexstrainswillbedifficultbecausesomesusceptible strainswillalwaysremainandthesewillbeenoughtosustaintransmission(Elwelletal. 2006). 1.2.5 Susceptibilityofintroducedpopulationsofsalmonid Australiausesseveralexoticstrainsofsalmonidfishforstockingfisheriesor aquaculture,includingrainbowtrout,browntrout,brooktrout(Salvelinusfontinalis), redsalmon(O.nerka),Chinooksalmon(O.tshawytscha)andAtlanticsalmon(Salmo salar).Thesespeciesarehighlylikelytobesusceptibletoinfection,anditislikelythat rainbowtrout,brooktrout,redsalmonandChinooksalmonwouldbesusceptibleto clinicaldisease.Browntroutwouldbelesslikelytoshowclinicaldisease.Atlantic salmonmayresistclinicaldisease,butarevariableintheirresponsetoinfectionand thereisinsufficientevidencetobeconfidentoftheirresponse(Hallett&Bartholomew 2012). Ingeneral,clinicaldiseaseseverityincreaseswithincreasingparasitedose(MacConnell &Vincent2002).Alowdoseisconsideredtobe100–200TAMsperfish,andahighdose Whirling Disease 13 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy is1000–2000TAMsperfish(Hedricketal.1999a;Hedricketal.1999b).Veryhigh doses(2000–10000TAMsperfish)canevencausesevereclinicaldiseaseinless susceptiblespeciessuchasbrowntrout. Myxoboluscerebraliscannotcompleteitslife‐cycleinseawater,sofarmedsalmonids rearedinseacageswillnotbeexposed.Fishinfectedinfreshwatercanremaininfected insaltwater.Ifinfectedfishreturntofreshwater(e.g.broodstockreturningtospawn), myxosporesmaybereleasedandaninfectioncouldestablishinT.tubifexworm populations. 1.2.6 Australiannativefishsusceptibility TherearenoAustraliannativefishinthefamilySalmonidae.Thetwofamiliesof AustralianfishmostcloselyrelatedtothesalmonidswithintheorderSalmoniformes aretheGalaxiidaeandtheRetropinnidae(southerngraylings)(CSIRO2015). ArangeofgalaxiidspeciesarefoundinNewZealandandarenotsusceptibletowhirling disease(B.Jones,pers.comm.).Todate,allfishdeterminedtobesusceptibletowhirling diseaseareinthefamilySalmonidae(El‐Matboulietal.1999a;Gilbert&Granath2003; Nelson1994).Althoughexposuretrialshavenotbeenconducted,itisunlikelythat Australiannativefishwillbesusceptibletowhirlingdisease. 1.2.7 Vectors Myxosporesareresistanttodegradationinthealimentarytractofpiscivorousbirdsand fish,andremaininfectiousinfaecalmaterial.Viablemyxosporescanthereforebewidely dispersedbythemovementoftheseanimalvectors,potentiallytravellingsomedistance fromtheoriginalsiteofinfectionandacrosscatchmentboundaries(El‐Matbouli& Hoffmann1991;Koeletal.2010). 1.2.8 Notahumanpathogen Myxoboluscerebralisisnotknowntoinfectorcauseclinicaldiseaseinhumans.Infected fisharelikelytobesafetoeat,butclinicallydiseasedfishmaynotbemarketabledueto thepathologicalchangesassociatedwithdisease. 1.3 Worlddistribution MyxoboluscerebralishasneverbeendetectedinAustralia.OtherMyxobolusspecies, however,havebeenreportedinAustralia(Langdon1990),andanactinosporean identifiedasbelongingtothegenusSphaeractinomyxonhasbeenisolatedfromamarine oligochaeteinAustralianwaters(Hallettetal.1995).MyxobolusplectroplitesJohnston& Bancroft,1918wasdescribedfromthefreshwaterfishgoldenperch(Macquaria ambigua)(Borehametal.1998).Lom&Dykova(1994)reportedsixMyxobolusspecies fromestuarinefishesinNewSouthWales. MyxoboluscerebraliswasfirstdescribedinGermanyandwasspreadacrossEuropewith transportofliverainbowtrout.Myxoboluscerebralissubsequentlyspreadaroundthe world,generallywithmovementsofsalmonidssuchasrainbowtroutforstocking, acclimatisationandfarming.Additionally,widespreadtransportofT.tubifexwormsfor aquariumfishfoodandforjuvenilefishfood(includingtrout)mayalsohave contributedtothespreadofthisparasite.IthasbeenfoundinNewZealandandpartsof NorthAmerica(in25states),Europe(includingIceland,[Kristmundsson&Richter 2009]),Russia,Africa(MoroccoandSouthAfrica),theMiddleEast(Lebanon)andAsia (JapanandKorea). Whirlingdiseasewasoriginallythoughttobeaproblemconfinedtotrouthatcheries, butinrecentdecadeshasemergedasasignificantpathogenofwildsalmonidsinthe UnitedStates,butnotinNewZealand. Whirling Disease 14 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy SeeBartholomew&Reno(2002)forahistoricalperspectiveofdisseminationand Hallett&Bartholomew(2012)forareviewofdistribution. 1.4 DiagnosisofinfectionwithM.cerebralis 1.4.1 Presumptivediagnosis Apresumptivediagnosisofwhirlingdiseasereliesonhostcharacteristics,clinicalsigns andsporeextraction(MacConnell&Bartholomew2012). Hostcharacteristics Myxoboluscerebralismyxosporesinfectsalmonids,withclinicaldiseasepredominantly presentincertainspecies,particularlyrainbowtrout.Riskfactorsforclinicaldisease includeage(e.g.fingerlingsandyearlingsofsusceptiblespecies)anddevelopment(i.e. lessdeveloped).Suspectcasesofwhirlingdiseasearethereforemorelikelytooccurin youngfishofsusceptiblespecies(SeeSection1.2). Clinicalsigns Clinicalsignsincludewhirlingbehaviour(corkscrewswimming),black‐tail,skeletal deformities,stuntedgrowthanddeath(SeeSection1.4.1).Howeverclinicalsignsarenot pathognomonic(i.e.arenotspecificforwhirlingdisease)andsomespeciesandage classesofsusceptiblefishcanbesubclinicallyinfected. Sporeextraction Pepsin–trypsindigestandidentificationofsporescanbeusedasascreeningtestbut willnotdetectpre‐sporogonicstagesoftheparasite. 1.4.2 Confirmatorydiagnosis Confirmatorydiagnosisreliesonhistopathologicalexamination(sporesincartilage)and parasiteDNAamplificationusingspecificpolymerasechainreaction(PCR)‐basedtests. Histopathologicalconfirmation Histopathologicaldiagnosisreliesonobservationofpre‐sporogonicandsporeparasite stagesincartilagetissuesurroundedbybone.Thesensitivityofthemethodishighbut isnot100%.Forexample,myxosporescansimplybemissed(Kelleyetal.2006)or clinicalsignscanbedisplayedbeforecharacteristicsporeshaveformed. Polymerasechainreaction(PCR) DiagnosisusingaPCRassaydoesnotrelyonterminaldevelopmentalstagesofthe parasiteorontissuedamage,butinsteadontheamplificationofparasiteDNA(Kelleyet al.2006).Subclinicalinfectioncanthereforebedetectedbeforesporesform.The methodcanbeextremelysensitiveandspecific(Kelleyetal.2006).Purcelletal.(2011) notedthechallengesintransferringPCRforresearchintodiagnostictestssuitablefora regulatedframework.ThatreviewcentredontheuseofqPCR,andmanyparallelscan bedrawnfromitandconsideredintheapplicationofotherPCR‐basedmethodologies. CurrentrecommendationsaretouseqPCR(SeeSection1.4.2). Proceduresfordetectingsubclinicalinfectionsaresimilarandrelyonpepsin–trypsin digests,histopathologyandqPCR. AfullyvalidatedandstandardisedInternationalAccreditationNewZealand(IANZ)test isdetailedinAppendix3.FurtherdetailsondiagnosiscanbefoundintheFishHealth SectionBlueBookoftheAmericanFisheriesSociety(MacConnell&Bartholomew2012). Whirling Disease 15 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 1.4.3 Casedefinitions Suspectcasedefinition Asuspectcasedefinitionshouldbehighlysensitive(i.e.noorfewfalsenegatives)to avoidmissingcriticalnewoutbreaks,yetalsoquitespecific(i.e.fewfalsepositives).A casedefinitionshouldalsobeeasilyandrapidlyappliedinthefieldtoassistrapid outbreakmanagement.Asuitablesuspectcasedefinitioniscontextspecificbutwould includeoneormoreofthefollowingpoints: observanceoftypicalclinicalsignsinyoungsusceptiblesalmonidspecies(e.g. rainbowtroutfingerlingsoryearlings) asusceptiblepopulationofsalmonidsthathashadcontactorisincloseproximity toaknowncase salmonidsthatreturnapositiveresulttoascreeningtest(e.g.sporeextraction andvisualisation,orPCRtestinterpretedwithoutfurtherlaboratoryor epidemiologicalinvestigations) otherepidemiologicalevidencestronglysuggestiveofinfectionordisease(e.g. tracing). Allsuspectcasesshouldbefurtherinvestigatedtoconfirmwhetherinfectionispresent. Confirmedcasedefinition Aconfirmedcasedefinitionisrequiredtobehighlyspecificandhighlysensitive.The appropriatedefinitionmustdependonamixtureofevidence,suchashost characteristics,clinicalsigns,epidemiologicalevidenceandlaboratorytestresults. AconfirmedcasewastraditionallydiagnosedbydetectionofM.cerebralissporesinthe cartilagefromaknownsusceptiblesalmonidpopulationdisplayingclinicalsignsor typicalepidemiologicalpatternsofwhirlingdisease.ApositivePCRtest(amplification ofM.cerebralisDNAfromsamplematerial)iscurrentlyanalternativeto histopathologicalexamination,providedthetestisvalidatedandusedbyanappropriate laboratoryaccreditedtoISO/IEC17025andoperatingunderaQualityAssurance SystembasedontheISO/IEC17025standardandaccreditedbytheNational AssociationofTestingAuthorities(NATA)oritsequivalent.Thereisafullyvalidated andstandardisedtestavailablefromtheNewZealandMinistryforPrimaryIndustries (seeAppendix3).ApositivePCRtestmustbeconfirmedusingothermethodssuchas sequencingorhistopathologicalexamination. Asuitableconfirmedcasedefinitioniscontext‐specificbutwouldinclude: confirmedidentificationofsporesorpresporogonicstagesintissueofclinically diseasedfish OR positivePCRtest(seeAppendix3)followedbysequencingofPCRproduct. AllsuspectedexoticdiseasecasesmustbereferredtotheAustralianAnimalHealth Laboratory(AAHL)forconfirmatorydiagnostictesting. TherearenostandardsdetailedbytheOfficeInternationaldesÉpizooties(OIE[World OrganisationforAnimalHealth])orAustralianNewZealandStandardDiagnostic ProceduresfordiagnosingM.cerebralisinfection.AnaccreditedqPCRandStandard OperatingProcedureisshowninAppendix3.TheFishHealthSectionBlueBookofthe AmericanFisheriesSocietyalsodetailssuitabletests(MacConnell&Bartholomew 2012). Whirling Disease 16 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 1.4.4 Fieldmethods:clinicalsignsandgrosspathology Clinicalsigns Insusceptiblesalmonidsinfectedatanearlyage,clinicalsignsofwhirlingdisease includeerratictail‐chasing(‘whirling’),blackeningofthetailregion,andskeletal deformitiesincludingskulldepressionandspinalcurvatures.Infectedsalmonidscan alsoshownoclinicalsigns.Bartholomew&Reno(2002)notedthat‘sinceclinicalsigns arenotpathognomonic,andmaybesubtle,theymightnotbenotedexceptwhenthe diseasereachesepizooticlevels’. Theclinicalsignsofwhirlingdiseasewillvarydependingontheageoffishwhenfirst infected,theinfectivedoseandthespeciesandstrainofsalmonid. Whenrainbowtroutareexposedasfry: ‘whirling’signs(rapidcircular/corkscrewswimming)firstappearapproximately threetoeightweeksafterinfection.Fishmaydieduetoexhaustionand/orsevere malnutrition.Thisswimmingbehaviourisconsideredtobeduetolowerbrain stemandspinalcordcompressionandconstriction(Roseetal.2000) ‘blacktail’(caudalmelanosis)duetopressureoncaudalnervescontrolling pigmentation(Halliday1976).Thismaysubsideiffishareanaesthetisedorafter theydie. Ifinfectedfishsurvive,theyrarelyshowwhirlingbehaviourorblacktail,butmayhave: skeletaldeformities,e.g.skulldepression,misshapedjaws,shortenedoperculae (gillcovers),spinalcurvatures;thesesignscanvarysignificantlyinseverity,and lightinfectionscanbedifficulttodetect opercularcysts decreasedgrowthrateduringclinicaldiseasestage. Whenexposedatolderthannineweeks,thereareveryfewornoclinicalsigns. Othersalmonidspeciesshowsimilarsigns,dependingontheirsusceptibilityto M.cerebralis,butsignsmayvarywiththeageanddevelopmentalstageatwhich resistancetoclinicaldiseasedevelops. Pathology Insusceptiblefishexposedwhenyoungerthannineweeks,theremaybeobvious pathologicalchanges.Infishexposedwhenolderthannineweeks,pathologicalchanges (grossandhistological)maybeminimal.Theremaybenoobviousinternallesionsin fishsurvivinginfection. 1.4.5 Laboratorymethods Samplesubmission Samplesshouldfirstbesubmittedtotheappropriatestateorterritorylaboratory.These laboratorieswillprovideappropriateadviceonwhichspecimenstosubmit.Additional detailedinformationonsamplesubmissionisinAppendix3ortheBlueBookoftheFish HealthSectionoftheAmericanFisheriesSociety(MacConnell&Bartholomew2012). Fishwiththehighestrisk(i.e.susceptiblesalmonidspeciesandagegroups,andfish managedusingpracticeswhichmayincreasetheriskofinfection)shouldbeselectedfor submissionandsubmittedliveorfreshlykilled.High‐riskgroupsincluderainbowtrout fingerlingsoryearlings,andfishraisedinsoil‐linedpondsorracewayswithuntreated water. Whirling Disease 17 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Toincreasethelikelihoodthatatleastpartiallydevelopedsporesarevisibleon histopathologicalexamination,ensuresomefishhaveexperienced1800degree‐days; thisistheperiodoverwhichthesumofaveragedailytemperaturesis1800(e.g.ifmean dailytemperatureis10°C,the1800degree‐dayperiodwouldbe180days). Alternatively,ifrecordsarepoor,ensurethatfishare6monthsorolder.Samplingoffish shouldfocusonthosewithclinicalsignswhicharemorelikelytohavesporesinthe cartilage.Forfishwithpresporogonicstages,histopathologicalexaminationtoidentify thesestagesandM.cerebralisspecificPCRwouldbeadequatetoconfirmthediagnosis, althoughmaturesporesmaynotbereadilyobserved. Multiplefishshouldbesubmitted.Uptofivefishcanbepooledforscreeningwith pepsin–trypsindigestbuthistopathologicalexaminationrequiresindividualfish. Wholeheadsshouldbesubmittediffisharenottoolarge.Theseshouldbefromfreshly killedfishandshippedonice.Ifthefishisverylargeawedgeshapedsampleorcore samplecanbesubmittedinlieuofafreshhead.Forfish>15centimetres,atriangle‐ shapedwedgeiscutposteriortotheorbitatthedorsalsurfacealmosttotheventral edgeoftheopercula.Thetop(dorsal)portionofthewedgeshouldmeasure 1.5centimetres.Additionally,freshtissuesamplesfromtheheadshouldbesubmitted preservedin10%neutralbufferedformalin. Equipmentforcollectingsamples,reagentsforsamplepreparationandfacilitiesfor chilledorfrozenstorageandtransportofsampleswillberequired.Samplingequipment maybeavailableon‐site,ormaybeobtainedfromstateorterritoryauthorities.See AQUAVETPLANEnterpriseManualforcontactdetails(DA2015b). Microscopy Theparasiteinitiallyhasanaffinityfortheskinandsubsequentlyforthenervesand skeletalcartilageinthesalmonidhost.Thegranulomatousresponseseenincartilageis themostprominentsignofinfection. Inacutestages: thereislittlecellularresponseinthefirstfewdays macrophagesattackingresidualepithelialstagesmaybeseeninsub‐cutis nervoustissuecontainingparasiteappearsnormalwithnotissuereaction. Inlaterstages: lysisandphagocytosisofthecartilagebytrophozoitesinitiateanintense inflammatoryresponseinsusceptiblespecies(El‐Matboulietal.1995;Feist& Longshaw2006) lesionstypicallycontainremnantcartilage,developmentalandsporogonicstages oftheparasite,andfocaltodiffusegranulomatousinflammation.Granulomas consistpredominantlyofepithelioidandmononuclearcells,fibroblastsand multinucleategiantcells. Agradingsystemhasbeendevelopedtoassessthehistologicallesionsseeninwhirling disease(Baldwinetal.2000). Histopathologicalexamination Presumptivediagnosisdependsonvisualisingcharacteristicpre‐sporeorsporogonic stagesoftheparasiteincartilagesurroundedbybone,andconfirmatorydiagnosis requiresamplificationofM.cerebralisDNAusinganestedPCRassay(Andreeetal. Whirling Disease 18 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 1998).Characteristicpre‐sporeorsporogonicstagesoftheparasitearevisualisedwith histopathologicalexamination. Fishheads(wedgesorcoresamplesforlargeheads)areplacedinfixative(either10% neutralbufferedformalinorDavidson’sfixative).Smallsamples(fish<15centimetres) fixedinDavidson’sfixativecanbetransferredto70%ethanolafter24–48hours;larger samplesrequire48hoursandmayrequireadditionaldecalcification.Samplesfixedin 10%formalinshouldbedecalcifiedafter24–48hours.Decalcifiedsamplesarethen transferredtoethanol. Tissuesareembeddedandsectionedusingstandardmethods.Theyarethenstained withhaematoxylinandeosinorGiemsaandexaminedtoconfirmthatthemyxospores ordevelopmentalstagesoftheparasitearepresentincartilage(MacConnell& Bartholomew2012).Thisisessentialfordiagnosis,asthereareotherMyxobolus myxosporesthatcanbefoundassociatedwithotherheadtissuesoffish(Andreeetal. 1998). Failuretodetectsporesofthecorrectmorphologyinanytissueisnotsufficientto determinethatthesampleisnegativeforM.cerebralis.Detectionofcharacteristic sporesintissuesotherthancartilagecanbereportedasnegativeforM.cerebralis (MacConnell&Bartholomew2012). A‘SlideoftheQuarter’(Case#03‐3011:rainbowtrout(Oncorhynchusmykiss),whirling diseasecausedbythemyxosporeanprotozoan,Myxoboluscerebralis)wascirculated aroundgovernmentlaboratoriesinJuly–September2007.Materialwasprovidedbythe FishDiseasesLaboratory,CSIRO‐AAHL. Preparationforparasiteidentification TofurtheraididentificationofM.cerebralissporesintissue,purificationofmyxospores (e.g.withpepsin–trypsindigestion)canbeundertaken.Furtherstainingmethods(e.g. silvernitrateordirectfluorescentantibody[Wolf&Markiw1979])canbeusedtoaid identification.Anexperiencedparasitologistwillberequiredasidentificationof M.cerebralismyxosporescanbedifficult. Moleculartechniques AmplificationofM.cerebralisDNAallowsaconfirmatorydiagnosisofinfectionwith M.cerebralis. TissuesamplesforPCRanalysisshouldbefrozenorpreservedin80–95%analytical‐ gradeethanol.SeveralPCRtestshavebeenresearchedandcomparedwithtraditional methods(Andreeetal.1998;Cavenderetal.2004;Kelleyetal.2006;Kelleyetal. 2004b;Thompson2007).PCRmethodsgenerallyperformwell,arehighlysensitive,and allowdiagnosisearlierthanhistopathologicaldiagnosisandatlowconcentrationsofthe parasite.TheqPCRisarguablythemostusefultestbutchallengesremainwithusing qPCRintheregulatoryframeworkofaquaticanimalhealth(Purcelletal.2011).A validatedandstandardisedqPCRisusedinNewZealand.AnestedPCRiscurrentlythe testrecommendedbytheFishHealthSectionoftheAmericanFisheriesSociety(Andree etal.1998;MacConnell&Bartholomew2012). 1.4.6 Confirmationofinfection Forthepurposesofthismanual,confirmatorydiagnosisofwhirlingdiseaseorinfection dependsprincipallyonamplificationandsequencingofM.cerebralisDNA,forexample usinganestedPCRassay(Andreeetal.1998). Whirling Disease 19 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 1.4.7 Differentialdiagnosis Acutewhirlingdiseaseshouldbeonthedifferentialdiagnosislistwheneveryoung salmonidsinAustraliashowneurologicalsignssuchastail‐chasing,spinningor spirallinginthewatercolumn. Thedifferentialdiagnosesthatshouldbeconsideredwhentheclinicalsignsassociated withwhirlingdiseaseareseeninAustraliansalmonidsinclude: septicaemicconditionsthatcauseinflammatoryresponsesinthebrain(e.g. Yersiniaruckeri);theaetiologicalagentcausingtheassociatedinflammationmay bebacterial,viralorprotozoal nutritionaldisorders;some,e.g.vitaminCdeficiency,havebeenassociatedwith skeletaldeformitiesinsalmonids earlyinfection(e.g.salmonids<5grams)withFlexibacterspeciesresultingin shortenedoperculaeinfishthatsurvive highincubationtemperatures(e.g.inAtlanticsalmon,>8Cuntilfirstfeeding) andfluctuatingtemperaturesduringincubationcausingskeletaldeformities, rangingfromminorlesionsinsinglevertebrae,‘shorttails’and‘humpbacks’,to shortbodydwarfism,inwhichthevertebralcolumniscompressedandankylosed (GreteBaeverfjord,ResearchScientist,Akvaforsk,pers.comm.) electroshockinjuriescausingskeletaldeformitiesand/ormelanosis(Margoliset al.1996;Wolfetal.1981) injectiondamagecausingcaudalmelanosis,wherethecaudalveinhasbeenused astheinjectionsite. Soundjudgmentoffishhealthmustbeusedtodistinguishbetweenthemanyconditions onthelistofdifferentialdiagnoses. 1.5 Resistanceandimmunity 1.5.1 Resistancetoinfection InterspecificdifferencesinsusceptibilitytoinfectionwithM.cerebralisarewell documentedinfish.Onlysalmonidsareaffected,andthereisalsowidevariability betweensalmonidspeciesinsusceptibilitytobothsubclinicalinfectionandwhirling disease.SeeSection1.2(especiallyTable1). Thereisalsogoodevidenceofintraspecificvariationinresistancetoinfection. Considerablevariationinsusceptibilitytoclinicaldiseasehasbeenshownwithin susceptiblespecies,dependingonseveralfactors.Olderandlargerrainbowtroutare moreresistanttoclinicaldisease,althoughthelevelofskeletalossificationwasnot importantinthedevelopmentofclinicaldisease(Ryceetal.2005). Withinhighlysusceptiblespeciessuchasrainbowtrout,someGermanhatcherystrains (andtheircrosses)areresistanttoclinicaldisease,butotherstrainsfromtheUnited Statesarenot(Fethermanetal.2014).Thereisageneticbasisforthis(Baerwald2013); asingleQTLgenomicregionexplainsmuchofthephenotypicvariationinresistanceto M.cerebralis(Baerwaldetal.2011).Naturalselectionforresistantfishisalsopossible withinafewgenerations(Granath&Vincent2010;Miller&Vincent2008). 1.5.2 Innateimmunity Theinitialportalsofentryforthewaterborneinfectivestageoftheparasiteincludethe epidermis,respiratoryepitheliumandbuccalcavity.Non‐specific,innatedefence mechanismssuchaslysozymesarelocatedintheseareas(Gomezetal.2014)butlittleis Whirling Disease 20 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy knownaboutinteractionsbetweenmyxozoanparasitesandfishhosts(Kallertetal. 2009). Skinpenetrationbythesporoplasmisfacilitatedbytheenzymaticroleofproteases (Kelleyetal.2003;Kelleyetal.2004a).WhileM.cerebralisexhibitsnon‐specific invasionbehaviour(Kallertetal.2011;Kallertetal.2009),non‐salmonidspecies(e.g. carp)caneitherpreventinitialskinpenetrationofthesporoplasm(Kallertetal.2009), orpreventsubsequentdevelopmentalphasesthatwouldotherwiseresultinthe formationofsporesintissues(El‐Matboulietal.1999a;Kallertetal.2009). Myxoboluscerebralistargetsimmune‐privilegedhosttissuessuchasnervoustissue (avoidingthehostimmunesystem),depressestheimmunesystemofinfectedfish,and modifiesitsantigenexpressionatdifferentlife‐cyclestages(avoidinghostimmune responses)(Densmoreetal.2004;El‐Matboulietal.1995;Knaus&El‐Matbouli2005; Sitja‐Bobadilla2008a;2008b). 1.5.3 Adaptiveimmunity Myxoboluscerebralisinfectioninfishmaybecharacterisedbyarelativelyshort exposuretothehostimmunesystem(approximatelyfourdays),duringtheparasite’s migrationthroughtheepidermallayersandintothenervoustissue(El‐Matboulietal. 1992). Circulatingantibodiestotheparasitehavebeendetectedinrainbowtrout(Griffin& Davis1978)andthereisevidencethatserumwithanti‐M.cerebralissporeantibodies collectedfrominfectedtrout(atbothearlyandlatestagesofinfection)mayoffer incompletepassiveprotectionwhentransferredtoyoungrainbowtrout(Hedricketal. 1998). Cell‐mediatedimmunitymayalsobeimportantinthisdisease(Hedricketal.1998). Thereislittlecellularresponseseeninthefirstfewdaysafterinfection,but macrophagesareseensoonafter,attackingresidualepithelialstagesoftheparasite(El‐ Matboulietal.1999a).Thisresponseoccursduringtheactivefeedingdevelopmental phaseoftheparasite,andduringcartilagedestruction,butdoesnotoccuronce myxosporesareformed(Halliday1974). Presporogonicandsporogonicstagesassociatedwithcartilageinduceinflammationand agranulomatousresponse.Thisresponsemayeliminatesome,butnotall,ofthe parasites,andvarieswithspeciessusceptibility.Inlaterstages,akeycharacteristicof thediseaseistheformationofgranulomas.Thishasalsobeenshowntovarybetween species,fromanextensive,diffusegranulomatousresponse,toawell‐defined, encapsulatedgranulomacontainingfewintactparasites. 1.5.4 Vaccination Whilefishdohavethephysiologicalelementstomountanadaptiveimmuneresponse andacquireimmunity,andthishasbeenobservedforsomemyxozoanparasites,there arecurrentlynovaccinesforanymyxozoanparasite,includingM.cerebralis.UV‐ irradiatedM.cerebralismayinduceimmunityinrainbowtroutwhilenotcausingdisease (Hedricketal.2012).Intheabsenceoftreatmentandimmunoprophylacticagents, avoidanceofinfectionistheprimarymeansofcontrollingmyxozoandisease.SeeGomez etal.(2014)foranextensivereviewoffishimmunologicalresponsestomyxozoan infections. 1.6 Epidemiology ThemajorspeciesaffectedbyM.cerebralisincludethesalmonids,manyofwhichcanbe infectedandsomeofwhicharemoresusceptibletoclinicaldisease(e.g.rainbowtrout Whirling Disease 21 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy andcutthroattrout,seeSection1.2fordetails).TransmissionoccursfromT.tubifex wormstosalmonidsviaTAMsandfromsalmonidsbacktoT.tubifexviamyxozoan spores.TemperatureandecologicalfactorsaffectthedistributionandprevalenceofT. tubifexinfreshwateraquaticenvironments,includingtheamountofavailablesediment. ResistanceinsomestrainsofsusceptiblefishcanemergeandsomelineagesofT.tubifex arealsoresistanttoinfection.Susceptiblesalmonidspeciesaremoresusceptibleat youngandless‐developedstagesandwhenhigherinfectiousdosesarereceived. 1.6.1 Incubationperiod Salmonidscanbeinfectedwithoutshowingsignsofclinicaldisease.Whenclinical diseaseoccurs,clinicalsignsaregenerallyduetotheparasitecausinginflammationand damagetocartilage.Theparasitecanlocaliseincartilage20daysafterexposure,with maturesporesforming52–120daysafterexposureat7–17°C(Halliday1973b).The firstclinicalsignscanbeseenatthreetoeightweeksafterexposure(MacConnell& Vincent2002). 1.6.2 Persistenceofthepathogen ThetwoinfectivestagesofM.cerebralis,myxosporesandtriactinospores,caneach surviveforthelifespanoftheirhost(salmonidfishandT.tubifex,respectively)(Gilbert &Granath2001;Hedricketal.2008).Onceoutsidethehost,sporessurvivefora variabletimeandaredispersedinfreshwater. AfterreleasefromT.tubifex,TAMsremaininfectiveforsusceptiblefishforsixto15days ormoreat7–15°C(El‐Matboulietal.1999b;Markiw1992). Themyxosporescansurviveforconsiderableperiodsoftimeafterreleasefroman infectedfish—usuallyacarcass.Earlyresearchexaminedmyxosporesurvivability usingvitalstainingandwithoutknowledgeofthetwo‐hostlife‐cycle.Morerecent researchhasexaminedsurvivabilityofthemyxosporebasedonitsabilitytoinfect T.tubifex(Hedricketal.2008).MyxosporeswereabletoinfectT.tubifexforatleasttwo monthsat4,10and20°C,althoughlittletransmissionoccurredat20°C(Hedricketal. 2008).El‐Matbouli&Hoffmann(1991)showedthatsporescanretaininfectivityforup tofivemonthsinmudat13°C.Experimentalstudiessuggestthatmyxosporesremain infectivetosusceptiblelinesofT.tubifexforbetweensixand12monthsatlow temperatures(5–15°C)(Nehringetal.2015). PleaseseeSection2.2.6andtheAQUAVETPLANdecontaminationmanual(DAFF2008) forinformationonhowtoreducethepersistenceofsporesusingdisinfection. Vectorsformyxosporesincludepredatoryfishandbirdspecies(El‐Matbouli& Hoffmann1991;Hallett&Bartholomew2012;Koeletal.2010). 1.6.3 Modesoftransmission Transmissionisindirectandhorizontalwithnoknownverticaltransmissionin T.tubifexwormsorsalmonids. TransmissionfromsalmonidstoT.tubifexwormsisviamyxosporesinwater(see Section1.1). Infectedsalmonidsarethepredominantmeansofdispersalofmyxospores(Hallett& Bartholomew2012).Thereforelivesalmonids(e.g.straymigratinganadromousfishor fishfromrestockingprograms),salmonidproducts,andby‐productsthatcontainraw cartilage,cantransmitmyxosporestocleanwaterwhereuninfectedwormpopulations canbecomeinfected.Themovementofwaterorsedimentcontainingsinking myxosporescandisperseinfection.Fomitessuchasfishingoraquacultureequipment, waders,boots(Gatesetal.2008)andotherobjectscantransmitmyxospores. Whirling Disease 22 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Piscivorousbirds(El‐Matbouli&Hoffmann1991;Koeletal.2010)andfish(El‐Matbouli &Hoffmann1991)canalsotransmitmyxospores,asmyxosporescanretaintheir infectivityaftertransitionthroughthegut. Ofmostconcerninanoutbreakaremovementsoffish,fishproductsandfomitesby people(Gatesetal.2008),anddispersalbypredatorybirds,asthesemovementsmay transmitinfectiousorganismsmosteasilytonewcatchments(Koeletal.2010). TransmissionfromT.tubifexwormstosalmonidsisviaTAMs(seeSection1.1). TransportofT.tubifexwormsorwatercontainingTAMscanleadtothereleaseofTAMs intocleanwaterwayswiththesubsequenttransmissionofM.cerebralistosalmonids. Tubifextubifexwormsmaylivelongerthanthreeyearssoanyfallowperiodforfresh watersthathavebeendepopulatedofsalmonidswouldneedtoremainsoforatleast threeyears(Nehringetal.2015).Tubifextubifexworms,bothliveandimportedfreeze‐ driedproduct,aretradedwidelyintheaquariumindustryasfishfood. SalmonideggsfromM.cerebralis‐infectedbroodstockhavebeenshowntobefreeof whirlingdisease(O'Grodnick1975).However,thereisthepotentialformechanical transmissionofmyxosporesandTAMsinpackingmaterialandwaterusedforegg transport. 1.6.4 Factorsinfluencingtransmissionandexpressionof disease Severalriskfactorshavebeenidentifiedthatinfluenceclinicaldiseaseexpression, includingtemperature,ageandsizeofsusceptiblespecies,strainsofsusceptiblefishand husbandry. Individualfishofsusceptiblestrainsthataresmallerandyoungeraremorelikelyto showclinicaldiseasethanolderfish(Ryceetal.2004).Forexample,fingerlingsand yearlingsaremostsusceptible(MacConnell&Bartholomew2012).Thedevelopmentof resistancetoclinicaldiseaseinrainbowtroutisassociatedwithboththeageandsizeof thefishattimeofexposure.Rainbowtroutmustbebothnineweeksofageorolderand atleast40mminforklengthattimeofexposuretoexhibitincreasedresistanceto whirlingdisease(Ryceetal.2005). Theprimarydeterminantofclinicaldiseaseistemperature.Theoptimaltemperature fordevelopmentandmultiplicationofM.cerebraliswithinitshostsis10–15°C (Blaylock&Bullard2014).Temperaturesabove20°Carerefractorytodevelopmentof M.cerebralisineitherhost(inFeist&Longshaw2006). Susceptibilityofbothspeciesandstrainisalsoimportantfordevelopmentofclinicalor subclinicalinfection(seeSection1.2). Certainhusbandrypracticesfacilitateinfection.Forexample,earthenponds,raceways ortanksprovidehabitatforT.tubifexandthecompletionofthelife‐cycleoftheparasite. Theuseofconcreteorplastic‐linedpenswillreducetheavailabilityofsuitablehabitat, althoughT.tubifexcanliveinaccumulatedsediments.Therefore,facilitiesmustbekept cleantoreducetheincidenceofinfection. Likewise,parasitetransmissionisfacilitatedbyusinguntreatedwaterfrominfected waterways. 1.7 Impact Whirlingdiseasecausedlargeeconomiclossesintheformativeyearsofrainbowtrout aquacultureinEurope.Controlmeasuresledtoincreasedcosts,mortalityledtoreduced Whirling Disease 23 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy production,andsomefacilitiesrequiredde‐populationtopreventcontaminationof nearbywaterways. EcologicalimpactshavebeenobservedinNorthAmericawithlargeandsometimes persistentpopulationcrashesinendemicsalmonids(especiallycut‐throatandrainbow trout).Salmonidcommunityandpopulationstructureshavechangedasaresult.Despite this,recreationalfishinghasbeenrelativelyunharmedasashifttoresistantspecies occurred.SeeHallett&Bartholomew(2012)foradiscussionoftheimpacts. ThereisnoevidencethatAustraliannativefisharesusceptibletowhirlingdisease(see Section1.2).Australianimpactswilllikelybelimitedtofresh‐watersalmonid aquaculturefacilities,andrecreationalfisheriesthatrelyonsalmonidspecies.Large populationimpactswouldbepossibleininfectedareasinthesecircumstances.Some ruralareasbenefitgreatlyfromrecreationalfishing,sothisdiseasemayhavedirect economicimpactsontheimmediatelyaffectedtownshipsandneighboringrural communities. Whirling Disease 24 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 2 Principles of control and eradication 2.1 Introduction ThepresenceofM.cerebralisinAustraliawouldbelikelytocausesevereimpactsin infectedfreshwatersalmonidaquaculturefacilitiesandrecreationalfisheriesrelyingon salmonidspecies.Thissectionprovidesbackgroundinformationtoenablethemost appropriateresponsetodetectionofM.cerebralisinAustralia. Therearethreebroadresponseoptionsavailable.Theseoptionsare: 1. Eradication EradicationofM.cerebralisfromAustraliawouldinvolvethehighestlevelofcontroland wouldhavethehighestcost. 2. Containmentandcontrolviazoningand/orcompartmentalisation Thiswouldinvolvecontainmentoftheparasitetoareaswithendemicinfection, preventionoffurtherspreadandprotectionofuninfectedareas. 3. Controlandmitigation Implementationofmanagementpracticesthatdecreasetheincidenceandseverityof clinicaloutbreakswouldinvolvethelowestlevelofcontrolandcost. Thebasicprinciplesoferadicationandotherresponseoptionsaredescribedinthe AQUAVETPLANEnterpriseManual(DA2015b)andtheAQUAVETPLANControlCentres ManagementManual(DAFF2001).TheAQUAVETPLANEnterpriseManual,Appendix1, listsstateandterritorylegislationrelatingtodiseasecontrolanderadication. Thenatureofanoutbreakwillaffectwhichresponseoptionisconsideredmost appropriate.Outbreakswhicharewidelydisseminatedorinbothwildandaquaculture populationsmaymakeeradicationmoredifficult,orimpossible.Forexample,infection mayhavealreadybeentransmittedtowildpopulationsofT.tubifexworms,orthere mayhavebeenareleaseofmyxosporesacrossawideareaofnaturalhabitat.In contrast,anoutbreakatasingleaquaculturefacilitymayberelativelyeasytoeradicate. Thegeneralstrategiesthatshouldbeusedinthecontrolofthisdiseaseinclude: Rapidlydelineatingtheinfectionaccordingto: Rapidlypreventingfurtherdisseminationoftheinfectionbyimplementing: time(e.g.byageingoflesionstoestimateintroductiondate) fish(e.g.aquacultureversuswildsalmonidsorspecies) place(geographicalextentofepizootic). immediatezoningandmovementrestrictions(betweenandwithinfarms) ofstock,water,fomitesandvectors enhancedbiosecurity(reducefomitespread) educationofrelevantgroups. Reducingoreradicatinginfectionby: Whirling Disease 25 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy depopulation,disinfectionanddisposalofinfectedaquaculturepopulations (considerpracticalityforwildpopulations) breakinglife‐cycleofparasite usingwaterwithlowTAMsrisk(filtered,treatedorgroundwater) movingfishtosaltwater eliminatingT.tubifexwormhabitat(usingconcreteracewaysorbyhabitat restoration) implementingexcellentmanagementpracticesandhygienestandards. Therearelimitationsinourabilitytosatisfytheseprinciplesinthecontroland/orthe eradicationofM.cerebralis.Forexample,iftheoutbreakhasspreadtowildpopulations, itmaybedifficulttoaccuratelysurveydistributionandmanageinfection. Acknowledgingsuchlimitations,themostappropriatecontroloptionwilldependonthe probabilityofsuccessfuleradicationandresourcesavailabletoattemptmanagement. Theseprobabilitiesmaybeassessedbyexamining: thescaleandsizeofanoutbreak whetherinfectionisconfinedtoaquaculturefacilitiesorhasspreadtowild reservoirs(salmonidsorT.tubifexworms) thenatureofwildreservoirs: susceptibility(e.g.strainofwormorsalmonid) abilitytode‐stocklocalisedwildpopulations(e.g.viabilitywithout restocking,fishingpressureetc.) ecology accessibilityandtoolstodepopulate. theabilitytoidentifywildreservoirs(distributionandspecies) theavailabilityofalternativestockforrestockingofaquaculturefacilities expertiseandcapabilitiesoffishhealthmanagementandresponsepersonnel thelevelofriskacceptedforfuturespreadofinfection(e.g.associatedwithgrow‐ outofinfectedpopulations) cost–benefitanalyses. Susceptiblesalmonidpopulationswillgenerallybefoundintwoproductionphases,a hatcheryphaseandagrow‐outphase.Inthehatchery,eggscanbehatchedintankswith re‐circulatingorflow‐throughwatersystems,andthesecanthereforebeconsidered closedorsemi‐closedproductionsystems,respectively.Inthegrow‐outphasein Australia,salmonidsarerearedincages,generallyinseawaterbutsometimesinfresh water.Therearealsoafewraceway‐typesystemsinfreshwater.Thesecanbe consideredsemi‐opensystems.SeetheAQUAVETPLANEnterpriseManualforfurther information(DA2015b).Wildpopulationsareconsideredtomeetthedefinitionofan opensystem. Inclosedsystems(i.e.tanksthatusere‐circulatingwater),itmaybepossibletotreat inflowandoutflowwatertoreducetheriskofsporeentryorexitfromthetanks, meaningcontrolofsporesandpreventionoftransmissionispossible.Sourcing uncontaminatedwater(e.g.groundwater)isbestforgeneraldiseasemanagement. Insemi‐closedsystems(i.e.hatcherieswithflow‐throughwater),theabilitytocontrol themovementofsporesinwaterisreduced(myxosporesineffluent,TAMsininfluent). Thismakescontroloftransmissionmuchharder(eitherspreadofM.cerebralistoor fromcaptivepopulations).Althoughcontrolandtreatmentofinputwateris theoreticallypossiblefortheremovalofviableTAMs(e.g.sandfiltration,ozonationand Whirling Disease 26 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy UVlight(Arndt&Wagner2004;Arndtetal.2006;Wagneretal.2003)),underthe currentmanagementpracticesofmanyfarmssuchtreatmentwouldnotbefeasible.A betteroptionwouldbeeliminatingpotentialhabitatforT.tubifexworms(e.g.lining earthenpondswithconcreteorsyntheticlinersandkeepingthemclean). Treatmenttoensureinactivationofmyxosporesineffluentwaterisalsoconsideredless feasible.ThiscouldpossiblybedonewithUVlighttreatment(Hedricketal.2008; Hedricketal.2007)butmaynotbepracticalinmanyfacilities.Dailyremovalofdead fishwillreducemyxosporereleasefromthedecomposingcarcasses.Daily‘mort’ (mortality)removalispossibleinsemi‐closedsystems,andshouldbestandardpractice inanyaquaculturesystemasfarasispractical. Semi‐opensystemsforgrow‐outhavenocontrolofwatermovementintooroutofpens, butmostgrow‐outoccursinsaltorestuarywaterwithhighsalinity.Fishinitiallyreared infreshwaterandexposedtoTAMscouldremaininfectedwhentransferredtothe marineenvironment,butmyxosporesreleaseddirectlyfromfishintothemarine environmentareunlikelytoencounterasusceptiblewormhost,asTubifexspp.donot toleratesalinitiesabove10partsperthousand(Pinder&Brinkhurst2000).Therefore, grow‐outinsemi‐openseapensisaviableoptiontocontroltransmissionriskandallow productiontocontinue.Thereisalowtheoreticalriskthatfishcontainedincages mooredinestuariesorshelteredareasoftheseacouldescapeintothewildandmigrate tofreshwater.Iffisharegrownoutinsemi‐opencagesorracewaysinfreshwaterthere isahighriskoftransmission. Controlofinfectioninopensystems(i.e.wildpopulations)willbeverydifficult(e.g. depopulationanddisinfectionmaybeimpossible).Despitethis,eradicationmaybe possibleinsomesmallisolatedcatchmentsofwildsalmonidsifinfectionwaslimitedto theseareasandsalmonidpopulationswerenon‐viablewithoutrestocking.Sustained heavyfishingpressuremayreducetheabundanceoffishtoaverylowlevel,and preventionofrestockingmayfurtherreducepopulationstonegligiblenumbers.Habitat modificationtoremoveT.tubifex(insediments)mayreducetheotherhostreservoir. Sentinelfishcouldthenbeusedafterseveralyearstogaugesuccessoferadication. Cautionisrequiredbecausethisisatheoreticaldiscussionanderadicationfromwild populationshasnotbeenachievedoverseas(wheresalmonidpopulationsareendemic andhencewidelydispersedandabundant).InAustralia,itmaybeaviableoptionin somecircumstances,particularlywhereaffectedpopulationsareentirelydependenton stocking.Afallowperiodofthreeyearsbeforerestockingisrecommendtoallow infectedT.tubifexwormstodieoutnaturally(Nehringetal.2015). 2.2 Methodstopreventspreadandeliminate pathogens 2.2.1 Quarantineandmovementcontrols Thefollowingquarantineandmovementrestrictionsshouldbeimplemented immediatelyonsuspicionofM.cerebralis. Establishmentofquarantineareas Establishmentofspecifiedareas(Figure2)(seeAQUAVETPLANEnterpriseManual SectionAformoredetails)(DA2015b),including: declaredarea—infected,restrictedandcontrolareas: Whirling Disease infectedareaorpremises—thepremises(e.g.farm)orareawherethe infectionispresent,andtheimmediatevicinity 27 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy restrictedarea—areaaroundandcontaininginfectedpremisesor area controlarea—abufferbetweentherestrictedareaandfreeareas. freearea—uninfectedarea;notconsideredadeclaredareaandmayincludelarge areasofAustraliainwhichthepresenceofM.cerebralishasnotbeendetected throughsurveillanceactivities. Figure 2 Establishment of specified areas to control M. cerebralis Inthedeclarationofquarantineareas,thefollowingfactorsneedtobetakeninto account: Epidemiologicalandsurveillanceinformation Epidemiologicalandsurveillancedataoninfectiondistributionandsusceptible populationsisthebestmeansofestablishingzones,butthisisunlikelytobe comprehensivelyavailable,especiallyearlyinanoutbreak.Considerationofthe followinginformationmayallowpredictionoflikelytransmissionanddispersaland earlyestablishmentofzonesintheabsenceofgoodsurveillancedata. Factorsaffectingtransmission Naturalfactorsthatcouldfacilitateorhindertransmissioninclude: contiguousdistributionofnaturalisedsalmonidpopulations vectormovements(e.g.movementsofpiscivorousspecies) movementsofsalmonidcarriers(e.g.adultsorlesssusceptiblesalmonidspecies) watermovementsthatcandispersespores naturalcatchmentdivisionsthatmaycontrolinfectionbecausewaterorwildlife doesnotmovebetweencatchments. Anthropogenicfactors(fromtheaquacultureindustry)thatcouldfacilitateorhinder transmissioninclude: connectivitybetweenaquaculturefacilities(e.g.companystructureand movementofstock,equipmentorfomites) Whirling Disease 28 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy proximityofotheraquaculturefacilitieswithsusceptiblespecies facilitationofbusinesscontinuity(e.g.whenpossible,declaredareasshould facilitatemovementofessentialequipment,personnelandproductbetweenfarms ortoprocessingplants). Anthropogenicfactors(fromrecreationalfishing)thatcouldfacilitateorhinder transmissioninclude: establishmentofareasbasedonlikelyhistoricalmovementoffishersthatmay havetransmittedinfection structuringofareastominimisedisruptiontorecreationalfishing(where possible) structuringofareassothatmovementbanscanbelegallyandpractically enforced. Thefollowingpracticesmustbeconsideredwhenimplementingresponseoptions: transportoffish,fomitesandproducts transportofstockandequipmentbetweenfarms transporttoandbetweenharvestingandprocessing transportofconsumerproducts movementsofrecreationalfishers movementsofotherriverusers dischargeofeffluentfromprocessorsandfarms recreationalfishing movementofpotentialvectors disposalofdeadfishandproducts decontamination. Movementcontrols Movementcontrolsinclude: banningmovementswithinandoutofrestrictedareas,includinginfectedareas: bansonthemovementofliveanddeadsalmonids,T.tubifexworms(or oligochaetewormsifidentificationisdifficult),water,aquaticplants (especiallyifmudandpotentiallyT.tubifexwormsareattached)and equipmentshouldbeconsidered,includingmovementswithinrestricted (includinginfectedareas)areas,andmovementsfromrestrictedareasto controlordisease‐freeareas duetothefreshwaterlife‐cycleofthewormhost,movementoffishfroma restrictedareaintosaltwatermaybepermitted,anditmaybepermissible tomovefishdirectlytoslaughterfacilitiesiffisharecarefullyprocessedand theresultantwasteistreatedtokillmyxospores(orotherwisedisposedof safely) somemovementscouldbepermittedforlow‐riskfomitesafter decontamination permittingmovementfromcontrolareastofreeareas: permitsrequiredbeforemovementofliveanddeadsalmonids,T.tubifex(or oligochaetewormsgenerallyifidentificationisdifficult),water, decontaminatedequipmentorotherfomitesandplantsfromcontrolareas tofreeareas Whirling Disease 29 Department of Agriculture and Water Resources permittingmovementfromthecontrolareatoarestricted(includinginfected) area: AQUAVETPLAN–Disease Strategy permitsrequiredbeforemovementofliveanddeadsalmonids,T.tubifex(or oligochaetewormsgenerallyifidentificationdifficult),water,any equipmentorotherfomitesandplantsfromcontrolareastorestricted (includinginfected)areas unrestrictedmovementsinthefreearea: implementationofmovementrestrictionscanassistsignificantlyinthe earlystagesofadiseaseresponsebypreventingfurtherdisseminationof infection,andcanalsobuytimewhiletheextentoftheinfectionisassessed implementationofmovementbansandrestrictionswillbeadynamic process,determinedbythelocationandextentofthediseaseoutbreakand whethertheaimistoeradicatethediseaseagentortocontrolitsspread; somerestrictionsmaybeimpracticalorunnecessarybutotherswillbe criticallyimportanttoeradicationorcontrol thefeasibilityofmovementrestrictionsandbansandtheextenttowhich theycanbeenforcedwilldependonthelocationofinfection,thelocation andtypeofenterprisesaffectedandthecontrolresponseoptionchosen. 2.2.2 Zoningandcompartmentalisation Itissometimespossibletomaintainasub‐populationofsalmonidswithdistinctaquatic animalhealthstatus(e.g.infectedorfreeofwhirlingdisease).Thiscanbedoneona geographicalbasis(referredtoaszoning)oronthebasisofcommonbiosecurityfactors, e.g.managementpractices(referredtoascompartmentalisation).Theseprocessesare discussedbelow. Zoning IfM.cerebralisweretobecomeendemicinspecificregionsofAustralia,azoningpolicy specificforM.cerebralismaybenecessarytoprotectnon‐infectedareasandtoprevent furtherspreadofinfection.ZoneswouldbebasedonthedistributionofM.cerebralis andofanyvectorspeciespresent(ifappropriate),thegeographicalandhydrological characteristicsofwaterbodiesandlandforms,andpredictionsofthemostlikelymethod ofspreadofinfection.Zoningmayrelyontheidentificationofbiogeographicalbarriers. AcorrespondingsurveillanceandmonitoringprogramforM.cerebraliswouldbe requiredtosupportthezoningpolicy.Principlesofzoningforinfectedandnon‐infected zonesinAustraliaareoutlinedintheAQUAPLANZoningPolicyGuidelines(DAFF2000) andintheOIEAquaticAnimalHealthCode(OIE2015a). Establishingdefinitiveinfectedanduninfectedzonesforwhirlingdiseasemaybe challenginginAustralia.DifficultiesinidentifyingbothT.tubifexandinfectedfish,and excludinginfectionfromdisease‐freeareas(e.g.fomitessuchasfishingwaderscan transmitspores)maymakeitdifficulttomaintainorconfirmzones.Thepresenceof geographicalbarrierswouldassistzoning,forexampleTasmaniansalmonidsmaybe protectedfromanoutbreakinmainlandAustralia. Compartmentalisation Acompartmentinthiscontextisdefinedasoneormoreaquacultureestablishments underacommonbiosecuritymanagementsystem,containinganaquaticanimal populationwithadistincthealthstatusrelatingtoaspecificdiseaseordiseases,for whichrequiredsurveillanceandcontrolmeasuresareappliedandbasicbiosecurity Whirling Disease 30 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy conditionsaremet.Suchcompartmentsmustbeclearlydocumentedbythe‘competent authority’(theveterinaryauthorityofthejurisdiction)ifusedforinternationaltrade. Acompartmentdoesnothavetoincludecontiguousfacilities—itcanapplytoaseriesof farmsoveralargearea,includingoverseveraljurisdictions.Thekeyisthatitmusthave inplaceabiosecuritymanagementsystemthatmeetstheguidelinesdetailedin Chapters4.1and4.2oftheOIEAquaticAnimalHealthCode,andthatthesesystemshave beendocumentedbythecompetentauthority. 2.2.3 Diseasemanagementinaquaticenvironments TheestablishmentofDiseaseManagementArea(DMA)boundariesduringanoutbreak ofwhirlingdiseasepresentsparticulardifficultiesrequiringdetailedconsideration beyondthatnormallyrequiredforterrestrialanimaldiseasecontrol.Watermovement throughandaroundfarmsandwithinstreamsorriversrepresentsasubstantialriskfor spreadofM.cerebralisthroughtransferofsporesinthewatercolumn,movementof infectedmaterial(particularlydeadfishpartsandsedimentcontainingspores)andany infectedwildsalmonids. Forexample,althoughaninfectedareamaybeestablishedaroundanindividualland‐ basedhatcheryorfarm,waterbodiesadjacenttotheinfectedareaandinthesame catchmentshouldbeconsideredformonitoringandcontrolmeasures.The establishmentofDMAboundariesaroundwildsalmonidfisheriesmayneedtobe comparativelylarge,andtakeintoconsiderationlocalflows,naturalbarriersandthe rangeofsusceptiblewildsalmonids. EstablishmentoftherelevantDMAboundariesmustalsotakeintoaccountthedispersal ofsporesinwaterdischargedfromanyinfectedsemi‐closedaquaculturesystems(e.g. hatcheries)orpotentiallyinfectedprocessingfacilities,andhowthisentersadjacent water.Potentialspreadofinfectionbyotherspeciesalsoneedstobeconsidered,as piscivorousfishandbirdscanactasvectors,withviablesporespassedinfaecesthatcan infectT.tubifex(El‐Matbouli&Hoffmann1991;Koeletal.2010). Therefore,ratherthanpropertyboundaries,thegeography,waterflow,distributionof susceptiblespecies,distancebetweenfarmingareasandtherangeofsusceptiblespecies willdefinewhereDMAboundariesareplaced. EstablishmentofDMAboundariesandtheirclassificationmustalsotakeintoaccount potentialmechanismsbywhichM.cerebralismaymovebeyondtheseboundaries(e.g. recreationalfishers,movementoflivefishassociatedwithrestocking,vector movementsoraquacultureactivities).Inmostcircumstances,itisadvisableto overestimatethesizeofDMAsandreducetheirareaastheresponsetakeseffect.In mostcases,intheinitialresponse,theDMAboundarieswillneedtoincludethewhole catchmentareainfreshwatersystems. 2.2.4 Tracing Tracingadiseaseoutbreakistheprocessofretrospectivelydeterminingthemethodand patternofdiseasespread.Tracinginvestigationsarecrucialindeterminingall confirmedandpotentiallocationsofthedisease,aswellasdefiningrestrictedand controlareas.Theinformationgatheredfromtracingwillhelpdeterminethemost appropriateresponseaction.Theimmediatestepsrequiredaretotracebackallcontacts withinfectedsalmonidsandT.tubifex,premisesandsites(toestablishtheoriginofthe outbreak),andtotraceforwardallcontactswithinfectedsalmonidsandT.tubifex, premisesandsites(toestablishthecurrentlocationandpotentialspreadofinfection). AquaticsurveillanceinformationisavailablefromtheDepartmentofAgricultureand WaterResourcesaquaticdiseasesurveillanceguidelines(Cameron2004). Whirling Disease 31 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Thefollowingitemsmustbetraced: fishmovements(includinglivefish,deadfishandfishproducts) effluentandwaterfromthefacility(potentiallyincludinghydrologicalmodelling) personnel vehicles equipment naturalmovementsofvectors,wildsalmonidsandwater(includingmodelling theoreticalmovementswhentracingactualmovementsoftheseentitiesis impractical) distributionofT.tubifexassemblages. Fishfarmsonthesamewatercourseorinthesamewatershedmayalreadybeinfected. Somefreshwateroperationsslaughterandprocessfishonsite.Anywastefromsuch processingcouldbeasourceofmyxosporesifitreachesfreshwater.Infectioncouldbe establisheddownstreamifsusceptibleT.tubifexwormsarepresent.Mapswiththe locationofneighbouringfishfarms,waterwaysandhydrographicdataarenecessaryto monitorthepotentialspreadofthepathogen.Thelocationofsusceptiblefishspecies shouldalsobenotedbothupstreamanddownstreamoftheinfectedsite.Further sourcesofinfectionmaybeidentifiedifanumberoffacilitiesshareacommonwater source.BothmyxosporesandTAMs(whichareneutrallybuoyant)cantravel considerabledistancesinwater. Predatorssuchasbirdsandfishcanalsocarryinfectedfishtoneighbouringwaterways. Myxosporescansurvivepassagethroughthegutofsomebirdsandfish. Forinformationonthelocationoffarmingestablishmentsandwildfishpopulationsat riskofinfection,contacttherelevantstateorterritoryauthority(seeAQUAVETPLAN EnterpriseManualforcontactdetails)(DA2015b). 2.2.5 Surveillance Surveillance,byscreeningforclinicalsignsandbylaboratorytestingofsamplesfor subclinicalinfection,isnecessaryto: definetheextentoftheinfectionandestablishrestrictedandcontrolareasto whichquarantineandmovementrestrictionsareapplied detectnewoutbreaks establishinfectedandnon‐infectedareasforanM.cerebraliszoningand/or compartmentalisationprogram monitortheprogressandsuccessofaneradicationstrategy demonstratefreedomfrominfectionafteranoutbreak. Asurveillanceprogramneedstobedesignedtomeettherequirementsofthe investigation―whetheritisfordelimitationofinfectedareaforcontrolpurposes,orfor longertermmanagementincludingcertificationfortranslocationorexportpurposes. Duringsurveillanceatapremises,laboratorysamplesrequiredforsubmissioninclude fishheadsfromseveralfishpreservedinchilledand10%neutralbufferedformalinor Davidson’sfixative(seeSection1.4).Theseshouldbefromsusceptiblesalmonidspecies ofappropriateages(e.g.youngrainbowtrout),preferablyshowingclinicalsigns.Other salmonidspeciesorolderrainbowtroutcanbesampledtodetectsubclinicalcarriers. ThesesampleswillbeusedforPCRtests,microscopicandhistopathological examination,andpossiblyforsporeextractionandvisualisation. Whirling Disease 32 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Subclinicallyinfectedstockmustbeidentifiedbeforeeradicationcanoccur.Diagnosisof subclinicalinfectionismorecomplexthandiagnosisofclinicaldisease,becausethere willbelittleevidenceofwhichindividualfishtosampleandfewersporestovisualiseon histopathologicalexamination.Todiagnosesubclinicallyinfectedstockrequires epidemiologicalinvestigation(e.g.age,speciesandcontacts)and,importantly, applicationofdiagnostictests,especiallyawellvalidatedandcorrectlyinterpretedPCR test.Giventhereducedsensitivityofhistopathologicalexaminationinthediagnosisof subclinicalinfection,PCRistherecommendedtestforsurveillancepurposes. Conversely,thepresenceofaknownoutbreakofwhirlingdiseaseinaregion(i.e.where anindexcasehasalreadybeendiagnosed)andclinicalsignsofdiseaseinsalmonidsof knownsusceptibility(e.g.youngrainbowtrout)willbeenoughevidenceto presumptivelydiagnosesuspectwhirlingdiseasecaseswithoutusingdiagnostictests. Despitethis,applicationofaPCRtestorhistopathologicalexaminationisrequiredfor confirmatorydiagnosis. 2.2.6 Treatmentofhostproductsandby‐products Thereisnotreatmentavailabletoeliminatetheparasitefromliveinfectedfish,although sometreatmentshavereducedboththeprevalenceofdiseaseandthenumberof generatedspores.Foramorecompletediscussionoftheliteraturerelatingtothis,see Hallett&Bartholomew(2012). Traderegulations,marketrequirements,foodsafetystandardsandpotentialspreadof thepathogenmustbeconsideredwhendeterminingthetreatment,processingand destinationoffishproductsandby‐products. Untreatedproductfrominfectedfishcantransmitinfectionasmyxosporeswillbe presentincartilage.Myxoboluscerebraliscansurvivewellindeadfish,evenwhen frozenat–20Cforuptothreemonths.Brinedfishalsoretainviablespores,although hot‐smokingat66Cfor40minutesinactivatesspores(Wolf&Markiw1982). Thespeciesofsalmonidaffectedwillinfluencethepotentialnumberofmyxosporesper fish.Infectedrainbowtroutcanhavehighnumbersofmyxosporesincartilage,and affectedfishproductsandby‐productswillpotentiallyhavehighermyxosporeburdens comparedtolesssusceptiblespecies,suchasbrowntrout. Eggs Myxoboluscerebralisisnottransmittedvertically.MyxosporesandTAMscouldbe mechanicallytransmittedduringtransportinpackingmaterialandfluidssurrounding fisheggsifcontaminatedwaterhasbeenusedinspawning.TheOIEAquaticAnimal HealthCodehasachapterondisinfectiontechniquesforsalmonideggs.Thosemethods werespecificallydevelopedtolimitvirustransmission,buttheywouldreduceriskof transmissionofalldiseases(seehttp://www.oie.int/en/international‐standard‐ setting/aquatic‐code/access‐online/). Humanhealth Myxoboluscerebralisisnotknowntoinfectorcauseclinicaldiseaseinhumans.Infected fisharelikelytobesafetoeat,butclinicallydiseasedfishmaynotbemarketabledueto thegrosspathologicalchangesassociatedwithdisease. 2.2.7 Destructionofhosts Destructionoffish Whirling Disease 33 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Slaughtermustbebothhygienicandhumane.Itisunlikelythatthechosendestruction methodwillinfluencemyxosporesheddingatslaughter,asmostmyxosporesare releasedfromcartilageondeathanddecompositionoffish. Therearemanydifferentmethodstoanaesthetiseand/orslaughterfish,allofwhich havelimitations.Acommonprocedureinvolvesloweringthewaterlevelinatankoffish andusingawater‐solubleanaestheticagenttosedatetheanimalsbeforeslaughter(e.g. isoeugenol[Aqui‐S®],whichhasnowithholdingperiod).Thedoseofanaestheticcanbe varieddependingonwhetherthefisharetobesubsequentlyharvestedorimmediately euthanased. Stunning(oftenfollowedbyslaughterandbleeding)maybemechanised(percussive stunning)ormanual(e.g.withaclub). Pithing(e.g.usingtheikejimemethod)israpidandeffective,althoughitislabour‐ intensive. Fishmayalsobekilledbyremovingwaterwhichasphyxiatesandcrushesfish. Themostappropriatemethodofslaughterdependsonthefollowingfactors: sizeandnumberoffish deadlineforslaughter(whichdependsonthemortalityratesandtheabilityto containthedisease) destination(forhumanconsumptionordisposal) slaughterfacilities(site,equipmentandmethodsavailable) welfareconsiderations. TheNationalAquacultureCouncil’sAquaticAnimalWelfareGuidelines(Johnston& Jungalwallan.d.),OIEAquaticAnimalHealthCode(chapter7.3and7.4)(OIE2015a)and theAQUAVETPLANDestructionManual(DAFF2009a)provideusefulguidelinesand informationrelatingtosalmonidwelfareduringdestruction.Ingeneral,fishshouldbe killedrapidly(forexamplewithpithing);orshouldbestunned(e.g.usingamechanical percussionstunningdevice)orsedated(e.g.withAqui‐S)beforebeingkilled(Johnston &Jungalwallan.d.).Othermethodscanachieveasimilareffect.Insomeinstancesduring anemergency,lesshumanemethodsmaybenecessarytomaximisebiosecurity outcomes. Experienceandavailabilityofpersonnel Experiencedstaffshouldconductdestructiontoensureitisdoneappropriatelyand safely.Theavailabilityofstaffmayinfluencewhichmethodofdestructionisbest,as somearemoreintensiveandrequiremoreexperiencedstaff. AnychemicalsusedmustbeapprovedforthatusebytheAustralianPesticideand VeterinaryMedicinesAuthority(APVMA)(seeAppendix2). Inaddition,anychemicalthatisuseddirectlyorindirectlyforthecontrolofananimal diseaseisgovernedinitsusebyrelevant‘controlofuse’legislationineachstateand territory.Therelevantstateorterritoryauthorityshouldbeconsultedforadvicebefore useofthechemical. SeetheAQUAVETPLANDestructionManualforfurtherinformation(DAFF2009a). Eliminationofoligochaeteworms Physicalalterationofnaturalhabitats(generallyhabitatalterationtoremove sediments)hasbeenattemptedinothercountriestoreducethedensityofT.tubifex Whirling Disease 34 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy withtheaimtoreducetheprevalenceofwhirlingdiseaseinnaturalsettings.Significant engineeringandcostscanbeassociatedwithsuchmeasures.Thesestudieshave producedvariableresults;severaldemonstratedreducedwhirlingdiseaseprevalence afterthehabitatmodification(Granath2014;Hansen&Budy2011)butothersshowed nosignificantchangeinwhirlingdiseaseprevalencewithhabitatmodification(Pierceet al.2014;Thompson2011).Itislikelythatthesuccessofhabitatalterationiscontext‐ specificandpredictionoftheoutcomeiscomplex.Significanthabitatalterationis unlikelytobesociallyacceptableevenifitwerepracticalorcost‐effective. Inaquaculturefacilities,thebestmeansofremovingthewormhostistoeliminate wormhabitat,eitherthroughtheuseofconcreteorlinedraceways,orwithregular cleaningofpondstoremovesediments.Ifitisnotviabletoreplaceearthenfacilities, destockinganddryingracewaysisaneffectivemeansofreducingwormnumbersand disinfectingsedimentofviablemyxospores(Bartholomewetal.2007). Generally,disinfectionofpondswithcalciumcyanide,calciumcanamideorchlorinewill rendersporestagesnon‐viableandkilltheinvertebratehost.Amolluscicide,Bayluscide (5,2’‐dichloro‐4’‐nitrosalicylanilide),reducedwormdensitiesby73–82%(Kowalski& Bergersen2003).Thiscompoundistoxictofishatthedosesusedtokillworms.Anyuse ofunregisteredproductswouldrequireanemergency‐usepermitfromtheAPVMA(see Appendix2),andappropriatepermissionsfromlocalenvironmentalauthoritiesfor disposalofwastetosafeguardtheenvironment. 2.2.8 Disposalofhosts Correctdisposalofcarcassesiscriticalbecauseofthehighlyresistantnatureof myxospores.Rapidremovalofcarcasses(‘morts’)frompondsorracewaysisalso essentialtobothminimisemyxosporereleaseandpreventexposureofsusceptible T.tubifexworms.Disposalsitesmustbecarefullychosentoensurethereisnocontact withwaterwaysorbirdsandotheranimalsthatcouldtransmitthemyxosporesto T.tubifexhabitats. Myxosporesarenotdirectlyinfectioustothefish,soinfectedfisharenotadirectsource ofinfectiontouninfectedfish. SeetheAQUAVETPLANOperationalProceduresManual(Disposal)fordetailsof destructionanddisposaloffishcarcasses(DAFF2009b). 2.2.9 Decontamination Duetodifferencesinfarmingenterprises,disinfectionprotocolsmayneedtobe determinedonanindividualbasisandinvolvethefarmmanager,thestateorterritory ChiefVeterinaryOfficer(CVO)and/ortheDirectorofFisheries.Theprotocolshould takeintoconsiderationthefactorsoutlinedinSection2oftheAQUAVETPLAN DecontaminationManual,inparticular: thesourceandlocationofinfection thetypeofenterprise(e.g.farm,processingplant,hatchery,grow‐outponds, watersourceetc.) theconstructionmaterialsofthebuildingsandstructuresonthesite thedesignofthesiteanditsproximitytootherwaterwaysorbuildings currentdisinfectionprotocols workplacesafetyconcerns environmentalimpactofthedisinfectantprotocol legislativerequirements(occupationalhealthandsafety,environmental protection,chemicaluse) availabilityofapproved,appropriateandeffectivedisinfectants. Whirling Disease 35 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy SeetheAQUAVETPLANOperationalProceduresManual–Decontaminationfordetailsof decontaminationmethodsandtheirindicators(DAFF2008),inwhich,aswellasthe generalinformationdetailedabove,therearesomespecificdetailsforM.cerebralis. Myxospores Duetotheresistantnatureofmyxospores,effectivedecontaminationofequipment, materials,personnel,tanksandbuildingsmustinactivatethisstageoftheparasite,or ensurethatviablemyxosporesdonotenterfreshwaterwaterwayscontaining susceptibleT.tubifexworms.Decontaminationrequiresthoroughcleaningbefore disinfection.Thewaterusedforthecleaningwhichthenentersafreshwaterhabitat maycontainviablemyxosporesifitisnotdisinfectedbeforerelease. Practicesandcompoundseffectiveinkillingmyxospores(reviewedinWagner[2002] unlessotherwisestated)include: calciumhydroxideat>0.5%for24hours calciumoxide(quicklime)orpotassiumhydroxideat>0.25%for24hours chlorineat1600partsperthousandfor24hours,orat5000partsperthousand for10minutes;500partsperthousandfor15minutesdisinfectedmost myxospores(Hedricketal.2008) heatingfor10minutesat90°C,and20°Cfortwomonths(Hedricketal.2008) dryingofpondsmaybeeffective;contaminatedmuddriedfor13–19monthsdid notinduceinfectionwhenthepondwasrestocked UVirradiationat40MJ/cm2(Hedricketal.2008) freezingat–20°and–80°Cinactivatesmyxosporesaftersevenand56days, respectively(Hedricketal.2008) alkyldimethylbenzylammoniumchloride(ADBAC)at1500mg/Lfor10minutes chitinaseinactivatesmyxosporesfromotherspeciesofmyxozoans(Liuetal. 2011)butisnotcurrentlyaviableproductforcommercialuse. TAMs TAMsarelessresistantthanmyxospores,e.g.dryingorfreezingforatleastanhourwill inactivateTAMs(Wagneretal.2003).OthermethodsofTAMinactivationinclude: temperatures>75°Cforatleastfiveminutes(at7°C,TAMscansurvivesevento eightdays)(Wagneretal.2003) chlorineat130partspermillionfor10minutesiseffectiveattemperatures rangingfromice‐watertoroomtemperature,andtotalhardnessrangingfrom10 to500mg/L hydrogenperoxideat10%for10minutes(Wagneretal.2003) povidone‐iodineat5000partspermillionactiveiodine,for60minutes(Wagner etal.2003) UVirradiationof40MJ/cm2willinactivateTAMs(Hedricketal.2007) sandfiltersaresuccessfulatremovingmostTAMSfromhatcherywater,thereby markedlyreducingtheinfectionprevalenceinfish(Arndt&Wagner2004;Arndt etal.2006). Stringentdecontaminationofsaltwaterfacilitiesfromwhichfishneedtoberemoved (i.e.fishinfectedinfreshwater,thentransferredtosaltwater)willnotberequired,asit isunlikelythatmyxosporesreleasedintoasaltwaterenvironmentwouldinfecta freshwaterT.tubifexworm.Likewise,processingplantsdischargingintosaltwaterare unlikelytoneedstringentdecontaminationprotocolsunlessthereispotentialtraffic Whirling Disease 36 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy betweenthefacilityandfreshwater,orthepotentialforbirdsorotheranimalstocarry carcassesoroffaltofreshwaterhabitats. 2.2.10 Vaccination Generally,fishcanmountanadaptiveimmuneresponseandacquireimmunity. Immunologicalmemoryspecificforsomemyxozoanparasiteshasbeenobserved. Additionally,UV‐irradiatedM.cerebralishaveinducedimmunityinrainbowtroutwhile notcausingdisease(Hedricketal.2012).Currently,however,therearenovaccinesfor anymyxozoanparasite,includingM.cerebralis.SeeGomezetal.(2014)foranextensive reviewofcurrentresearch. 2.2.11 Vectorcontrol Myxosporescansurvivepassagethroughthegutofpiscivorousbirdsorfish(El‐ Matbouli&Hoffmann1991;Koeletal.2010).OftheninebirdspeciestestedbyKoelet al.(2010),allspecieshadM.cerebralisDNAdetectedinthefaecalmaterialofbirdsfed infectedrainbowtrout.However,onlyonespecies(thegreatblueheron)wasshownto beadefinitivevector,asmeasuredbytheproductionofviableTAMsinlaboratory T.tubifexwormsexposedtomyxosporesofinfectedbirdfaeces.TheabilityofAustralian birdsandfishtoactasvectorsisunknown. Openairtanks,pondsandespeciallyprocessingfacilitiesandareaswherecarcassesare disposedofmayattractbirdsandmustbecovered(e.g.usingnetsortankroofs). Predatoryorscavengingfishthatmayactasvectors(orbecomeinfected)mustbe excludedfromaquacultureandprocessingfacilities. Waterratscouldalsoactasmechanicalvectorsofmyxosporesandwilleasilymove betweennaturalandaquaculturehabitatsunlessadequatefencingisinplace. 2.3 Environmentalconsiderations Environmentalconsiderationsinthecontrolofwhirlingdiseaseinclude: 2.4 Dischargeofinfectedorpotentiallyinfectedeffluentintofreshwatercatchment areasornaturalwaterwaysmayleadtofurtherspreadofinfection,andcouldlead totheestablishmentofreservoirsofinfectioninT.tubifex,wildfishpopulations andwaterways. Theuseofdisinfectantscouldhaveanimpactontheenvironment,especiallyif usedinlargerquantitiesorconcentrationsthanusual.Thelocalenvironmental protectionagencywillneedtobeconsulted(seetheAQUAVETPLANEnterprise Manual[DA2015b]). Environmentalimpactsrelatedtothedestructionanddisposalofinfected carcassesandmaterialmustbeminimisedwhileensuringthatthereisno disseminationofinfection. Sentinelanimalsandrestockingmeasures Removingallsalmonidsfromanareaistheonlywaytoensuretherearenosusceptible fishhostsinwhichtheparasitecancompleteitslife‐cycle.IfT.tubifexisremovedfroma facility,andthereisnopossibilityofTAMsenteringthefacility,thenrestockingcanstart immediately. Foreradicationinfarmingfacilities,restockedfishmustbefreeofsubclinicalinfection orclinicaldisease.IfareasaredeclaredfreeofM.cerebralis,fishintroducedintothose areasmustalsobefreefrominfection.Declaringanareafreeoftheparasitemaybevery difficult. Whirling Disease 37 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Rainbowtroutareconsideredthemostsusceptiblefishtoinfection.Youngrainbow troutmaybestockedassentinelfishtodeterminethepresenceorabsenceofthe parasite.Forexamples,seeBartholomewetal.(2007)andKelleyetal.(2006). Atlanticsalmonandbrowntroutcanbeinfectedwiththeparasiteandmyxosporescan formintheircartilage,butclinicaldiseaseisrareandmorbiditylow.Hencerestocking withlesssusceptiblespecies(andwhenfishareoldere.g.at>nineweeks)maybean optioninsomeareastomaintainproductioniferadicationisnotfeasible.Restocking withfishstrainsthatareresistanttoclinicaldiseasemayalsobeanoption. 2.5 Publicawareness Acommunityengagementprogramemphasisingeducation,surveillanceand cooperationfromindustryandthecommunityisessential.Thepublicshouldbe informedthat: 2.6 whirlingdiseasedoesnotinfectpeople thetwo‐hostlife‐cycleiscomplex goodbiosecurityisrequiredtopreventfurtherspread surveillanceisessentialtodeterminethescaleoftheepizootic cooperationisessential. Feasibilityofcontroloreradicationof whirlingdiseaseinAustralia Thefeasibilityofcontrollinganoutbreakofwhirlingdisease(orinfectionwith M.cerebralis)dependsonthenatureandlocationoftheoutbreak.AsoutlinedinSection 2.1,therearethreeresponseoptions:eradication;containmentandcontrolviazoning and/orcompartmentalisation;andcontrolandmitigation. 2.6.1 ResponseOption1:Eradication Eradicationreliesonrapidlyimposedmovementrestrictions,surveillanceandtracing, destructionanddisposalofinfectedfish,anddecontamination.Ifthetechnical capabilitiestoimplementeachofthesesteps,orresourcesrequiredarelimited, attemptederadicationislesslikelytosucceed. Ifwildpopulationsareaffected,itisgenerallyacceptedthatM.cerebraliscannotbe eliminatedonceitisestablished.Failederadicationeffortsfromareaswherethe parasiteisnowendemic(e.g.manystatesintheUnitedStates)havebeendocumented intheliterature,includingdrasticmeasuressuchaschlorinatinganentirestream.As Wagner(2002)states,‘Thebestmanagementistoavoidinfectingnegativewaters’. Thesefailedattempts,however,havebeeninhigh‐density,widelydispersedandwell‐ establishednativesalmonidpopulations.Incontrast,theremaybethepotentialfor eradicationinsomeareasofAustraliawhereanepizooticislimitedtosmallisolated populationsofnon‐endemicsalmonidsthatarenotsustainablewithoutrestocking.This isatheoreticalpossibilityandhasnotbeenrigorouslyinvestigated. Eradicationisunlikelytobesuccessfulorfeasibleifepidemiologicalinvestigations determinethatinfectioniswidespread,hasnodetectablepointsource,isunabletobe containedandispresentorpotentiallywidelyestablishedinwildfishspecies,ornatural waterbodies.Thisisdueto: theabilityofM.cerebralistospreadrapidlyandestablishreservoirsofinfectionin wildfishpopulationsthatmaybeimpossibletoeradicate theabilityofM.cerebralistoproducesubclinicalinfectionsthataredifficultto detect Whirling Disease 38 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy theabilityofinfectedwildfishandvectorstotransmitandestablishinfectionin riversandotherfreshwaterhabitats closecontactbetween,andrelativelackofcontrolover,farmedandwildfish populations,andwaterinAustraliansalmonidfarmingoperations(bothsemi‐ openandsemi‐closedsystems) experienceinaffectedcountrieswhereeradicationwasunsuccessfulonce reservoirsofinfectionbecameestablishedinwildfishpopulationsandthenatural environment. Incertaincircumstances,itmaybepossibletoeradicateM.cerebralisfromafish farmingfacility.Thishasoccurredoverseas(Anderson1993).Theprinciplesareto ensurethat: sourcewateriseitherfreeofTAMsorthereisthepotentialtoadequatelytreat incomingwater therearenopotentialhabitatsforT.tubifexwormswithinthefacility. Theoptionchosenmustensurethatthereisnofurther: exposureofunexposedfishpopulationstoTAMs spreadofinfectionviathereleaseofmyxosporesintotheenvironment. Unexposedsalmonids Inaneradicationresponse,unexposedsalmonidsindisease‐freeareaswouldnotbe subjecttodestructionnotices,andcommercialfarmscouldcontinuenormaloperations, providedthatfutureexposuretoinfectioncanbeprevented.Young(pre‐marketsized) unexposedfishindeclaredcontrolareasmaybeallowedtogrow‐out,alsounderthe provisothatfutureexposuretoinfectioncanbeprevented,whileolderunexposedfish maybeemergency‐harvestedandslaughteredforhumanconsumption.Alternatively, fishmaybetransferredtosaltwatertonegatethepossibilityofexposuretoTAMs.This isonlyaviableoptionwhen: fishwilltoleratesuchsalinitychanges saltwaterfacilitiesareavailableandsuitable. Emergencydestockingofunexposedfishpopulationslocatedwithindeclaredcontrol areascouldbeconsideredaspartofaneradicationresponse(withtheaimbeingto increasethesizeofthebufferzonebetweeninfectedanduninfectedareas,andto increaseconfidenceintheirputativediseasestatus).However,giventhenatureofthe parasiteandthelikelyhighcommercialvalueoftheaffectedfish,thisactionmaybe consideredexcessive,evenwithintheconservativeapproachtypicalofadisease eradicationresponse.Myxoboluscerebraliscannotbetransmitteddirectlyfromfishto fish,andthereisasignificanttemporallagfordevelopmentoftheparasitewithinthe T.tubifexwormhostbeforeinfectiveTAMsarereleased(>90days,dependingon temperature). Amoremeasuredapproach,involvingconcurrentandrigoroussurveillanceof unexposedfishpopulationsincontrolanddisease‐freeareas,maythereforebe preferabletoemergencydestockingincontrolareas.DetectionofM.cerebralisinfection incontrolordisease‐freeareaswouldindicatethatcontainmentoftheoutbreakhad failed.Adecisionwouldneedtobemadeaboutwhetherdiseaseeradicationremained feasible,orwhethertheresponsewouldneedtotransitiontoacontainmentandcontrol phase(Section2.6.2). Whirling Disease 39 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Exposedorpotentiallyexposedfish Alllivesalmonidfishwithinaninfectedorrestrictedareaareassumedtobeexposed. Immediatedestructionanddisposalofthesefishcouldbeanoptionduringan eradicationresponse.However,thiswillnoteradicatetheparasiteifthereisthe possibilitythateffluentwaterorescapedinfectedfishfromtheaffectedfacilityhave spreadmyxosporestodownstreamwormhabitats.Thereisnoneedforimmediate destructionifthefisharelocatedinsaltwater. Normalorcontrolledgrow‐outofexposed,orpotentiallyexposed,clinicallynormal farmedsalmonidpopulationscouldalsobeconsidered,providedanymyxospores potentiallyreleasedduringthegrow‐outperiodarepreventedfromcomingincontact withT.tubifexworms.Grow‐outinsaltwatermayachievethiswherefishcantolerate suchsalinities(e.g.smoltedAtlanticsalmon),andwherethefacilitiesexist.Othergrow‐ outoptionsincludefacilitiesthathavetheabilitytosendeffluentwatertoground,or wherethereisnopossibilityofcontaminatedwaterenteringareaswithsuitableworm habitat. Treatmentofexposedorpotentiallyexposedfishisnotanoptionduringaneradication response,astherearenoeffectivetreatmentsthatdestroyallmyxosporesinthefish host.Emergencyharvestingofexposedfishcanbeconsidered,andisunlikelytoresult infurthertransferofinfectionprovidingthereisnopossibilityofuntreatedprocessing waste,especiallyskeletalelements,beingreleasedintofreshwaterhabitats.Infectedfish aresafeforhumanconsumption. Thestrictcontrolmeasuresnecessarytopreventfurtherspreadofinfectioninclude: disinfectionofallequipment/personnelinvolvedinharvesting,slaughterand processingtoeliminatetheriskoftransferringmyxosporesoffsite. quarantinerestrictionsandproceduresapplyingtotheinfectedsite,including personnel,equipmentandvehicles;quarantineisaimedatmyxospores,asitis unlikelythatTAMswouldbetransferredoffsitebythisroute processing,possiblyonsiteoroffsite,providedwasteandfishcarcassescannot comeincontactwithfreshwaterT.tubifexhabitats holding,treatmentandsafedisposalofslaughterandprocessingeffluent (includingholdingwaterandwasteoffal) ensuringthatthefinalproductwillnotresultinthespreadofinfection disinfectionofeffluentwater. Infectedfish Ifclinicallydiseasedanddeadfisharelocatedinfreshwater,andthereisnopossibility ofdisinfectingorredirectingeffluentwaterfromthefacility,theirremoval,destruction anddisposalisessentialtopreventmyxosporestransmittinginfection.Ifclinically infectedordeadfisharetoberemoved,burialsitesshouldbechosencarefullytoensure thereisnocontactwithwaterways,birdsoranimals. Tubifextubifexwormsmayalsobeinfected,andwillremaininfectedforthelifeofthe worm,whichcanbeuptothreeyears. 2.6.2 ResponseOption2:Containmentandcontrolvia zoningand/orcompartmentalisation Thepurposeofthisresponseoptionistocontaintheparasitetoareaswithendemic infectionbypreventingfurtherspreadandprotectinguninfectedareas.Itmaybe feasiblewhereanepizooticislimitedtoawellcircumscribedgeographicalarea(e.g.an Whirling Disease 40 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy islandsuchasTasmaniaoraremotewildpopulationinawellcircumscribedcatchment) orwheretheepizooticislimitedtoasingleindustrycompartment. Therearechallengestoimplementingthisresponse.Forexample,itmaybedifficultto effectivelycontaintheinfection;fishersandwildvectorsmaytransmitinfection betweencatchments;andspores(bothTAMsandmyxospores)canbetransmittedlong distancesinwater.Additionally,thesubclinicalnatureoftheparasiteinsomesalmonid speciesandolderfish,thelongincubationperiodanddifficultyinidentifyingT.tubifex maymakesurveillanceanddelineatinginfectedareasdifficult. Insummary,ifanepizooticwaswidelydispersedeitheracrosswidegeographicareasor acrossseveralsectorsofcommercialindustry,andifsignificantresourceswerenot availableforsurveillanceandenforcementofbiosecuritymeasures,containmentand controlwouldbeverydifficult.Incontrast,iftheoutbreakwasdetectedearly,before widedissemination,wasconfinedtowelldelineatedgeographicorindustry compartments,andpublicandindustrycooperationwashigh,thencontainmentand controlmaybepossible. Unexposedfish Managementoptionsforunexposedfisharethesameasthoseoutlinedforeradication. Theimplementationofazoningand/orcompartmentalisationprogram,andassociated controlmeasures,tomaintainuninfectedareaswouldbenecessary.Forzoning,see Section2.2.2. Exposedorpotentiallyexposedfish Exposedorpotentiallyexposedfishwithinaninfectedzoneorcompartmentare assumedtobeinfected.Immediatedestructionofthesefishisanoptionfora containmentandcontrolresponse,asitcanhelpdecreasetheinfectiousloadonasite andminimisethespreadofinfection.IfsusceptibleT.tubifexhostsarealsoinfected, thendestructionoffishmustbecarefullyconsideredagainsttheoverallbenefitof removingonehostbutnottheother.Removingbothhostsbydestructionand eliminationofT.tubifexhabitat,ifthisispossible(e.g.cleaninganddryingponds,or conversiontoconcreteraceways),willbeamoreeffectiveoption. Inaninfectedzoneorcompartment,normalorcontrolledgrow‐outandslaughtermay befeasiblewithoutfurtherspreadofinfection.Topreventthespreadofinfection, however,finalproductsmustbeprocessedtopreventspreadtouninfectedareas. Therearenoeffectivetreatmentsthatdestroyallmyxosporesinthefishhost,and treatmentisnotconsideredaviableoptionaspartofacontainmentandcontrol responseinAustralia. Asuccessfulcontainmentandcontrolresponsewillrelyheavilyontheimplementation ofmovementrestrictionsforexposedorpotentiallyexposedfishtopreventspreadof M.cerebralistouninfectedzonesorcompartments.Thezoningand/or compartmentalisationprogrammustalsotakeintoaccountwatermovementand possiblespreadoftheparasite’stwosporestagesbythismeans.Thefeasibilityofthese restrictionswilldependonfarmmanagementpractices,theextenttowhichinfection hasalreadyspreadandthelocationofreservoirsofinfection.Feasibilitycanonlybe assessedatthetimeoftheoutbreak,takingintoaccountrequiredmovement restrictionsonfish,people,vehiclesandwatercraft,andalsomarketaccessforthefish productsandby‐products.Eventhen,assessmentmaybelimited. Whirling Disease 41 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Infectedfish Clinicallydiseasedanddeadfish,alongwithinfectiouswastes,areconsideredtobethe mainsourceofmyxosporesintheenvironment.Unmanaged,theyconstitutean unacceptableriskforspreadingtheinfectiontouninfectedzonesduetotheresistant natureofmyxospores. Theonlyrealoptionforclinicallydiseasedfish,ifeffluentwatercannotbecontainedor disinfected,isimmediatedestructionortransporttosaltwaterforgrow‐out.Itis unlikelythatmyxosporeswouldcontactasusceptibleT.tubifexhostinthis environment.Escaped,infectedfishmaybeariskiftheyreturntofreshwater. 2.6.3 ResponseOption3:Controlandmitigation Inacontrolandmitigationprogram,theaimmaysimplybetoreducetheprevalence andseverityofclinicaldiseasetobiologicallyand/oreconomicallyacceptablelevels. Critically,theremaybealevelofdiseaseinthepopulationbelowwhichthecostof furtherexpenditureoncontrolwouldbegreaterthanthelikelybenefits. Theprinciplesofcontrolandmitigationaretoreducetheimpactofdisease.Therefore, theuseofanyoptions(orpartsofoptions)listedinSection2.2couldbeconsidered.In general,theresourcesusedtoimplementavailablecontrolandmitigationmeasures, andtheextentoftheirimposition,willoftenbelessthaniferadicationorcontainment andcontrolwerepursued. Considerableresearchintocontrolandmitigationoptionsinwildsalmonidpopulations hasoccurredinrecentyears,andhabitatrestorationisonemeansthatissometimes successful(seeSection2.2.6).Restockingprogramsthatconcentrateonreleasingolder fishthatarelesssusceptibletoclinicaldiseasecanreducetheimpactsofwhirling disease.Inrecentyears,restockingwithresistantstrainsofrainbowtrouthasbeen practisedintheUnitedStateswithevidencethatresistancespreadsrapidlyinthewild population(Fethermanetal.2014). 2.7 Tradeandindustryconsiderations Incountrieswherewhirlingdiseaseisendemic,affectedindustriesincludesalmonid farmingandrecreationalfishing.Itisunlikelythatotheraquaticfarmingindustriesin Australiawouldbeaffectedbythisdisease. Traderegulations,marketrequirementsandfoodsafetystandardsmustbeconsidered aspartofacontrolstrategy.Permitsmayberequiredfromtherelevantauthoritiesto allowproductsderivedfromdiseasecontrolprogramstobereleasedandsoldfor humanconsumption. 2.7.1 Exportmarkets WhirlingdiseaseisendemicthroughoutmanypartsoftheUnitedStates,Europe,North andSouthAfrica,NorthEastAsiaandtheeastcoastofthesouthislandofNewZealand. ItisnotlistedasanotifiablediseasebytheOIE.Despitethis,therearesomecountries thatrequireimportstobecertifiedfreefromwhirlingdisease.Somecountriesalsohave regionalrequirementsthatdifferwithinthecountry;forexamplesomestatesofthe UnitedStates. TheDepartmentofAgricultureandWaterResourcesisresponsibleforthehealth certificationofallexportsandshouldbecontactedforfurtherinformation ([email protected]). Whirling Disease 42 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 2.7.2 Domesticmarkets Acautiousapproachisrequiredforthesalvageofexposedorpotentiallyexposed productforthedomesticmarket.Themyxosporeishighlyresistantandcansurvivefor longperiods.Decisionsonthereleaseofsalmonidsorsalmonidproductstothe domesticmarketwilldependonthecontrolstrategyimplemented. Eradication Iferadicationisconsidered,decisionsrelatingtothereleaseofproductforthedomestic marketmustensurethereisnopotentialforthespreadofM.cerebralis. Containmentandcontrolviazoningand/orcompartmentalisation Thereleaseofexposedorpotentiallyexposedsalmonidproducttothedomesticmarket mustbecarefullycontrolledtoensurethereisnopotentialspreadofviableM.cerebralis touninfectedareas. Controlandmitigation Requirementsforthereleaseofexposedorpotentiallyexposedsalmonidproducttothe domesticmarketwouldbelessstringentifwhirlingdiseasebecameendemicin Australia. Whirling Disease 43 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 3 Preferred Australian response options 3.1 Overallpolicyforwhirlingdisease Whirlingdiseasehasthepotentialtocausesignificantmortalityandmorbidityin farmedandwildsalmonidpopulationsinAustralia.Rainbowtroutareparticularly susceptibletoclinicaldisease,especiallyifinfectedwhenlessthansevenweeksold. Ittakesthreetoeightweeksafterinfectionforclinicalsignstodevelopinsusceptible fish;manyinfectedfishmaynevershowclinicalsigns.Fishcanonlybeinfectedwith sporesreleasedfromtheT.tubifexwormhost. Therearethreepotentialresponseoptions: Option1:Eradicationoftheparasite Option2:Containmentandcontrolviazoningand/orcompartmentalisation (restrictionoftheparasitetoareaswithendemicinfection,preventionoffurther spreadandprotectionofuninfectedareas) Option3:Controlandmitigationbyimplementingmanagementpracticesthat decreasetheincidenceandseverityofthedisease. Thediseaseresponseoptionchosenwilldependonthenatureoftheoutbreakand/or isolationoftheparasite.TheDirectorofFisheriesand/ortheCVOofthestateor territoryinwhichtheparasiteisisolatedwilldecidethecontroloption(s). Itisimportantthattheselectedresponseisdynamic,toallowevolutionofthestrategy withthechangingsituation,e.g.choosingcontainmentandcontrolviazoningand/or compartmentalisationintheshorttermdoesnotprecludeadoptingeradicationasa long‐termpolicy. Strategieswhichmaybeusedundertheseoptionsinclude: quarantineandmovementcontrolsonfish,fishproducts,T.tubifexwormsand fomitesindeclaredareastopreventspreadofinfection destructionanddisposalofclinicallydiseasedanddeadfishtopreventfurther myxosporereleaseintotheenvironment decontaminationoffacilitiestoinactivatetheresistantmyxosporestageofthe parasiteoninfectedpremisesandtopreventspreadtoT.tubifexworms surveillanceandtracingtodeterminetheextentofpossibleinfectedwormand fishhosts,andtoprovideproofoffreedomfromtheparasite zoningand/orcompartmentalisationtodefineandmaintaininfectedand parasite‐freezones restockingwitholder,lesssusceptiblefish,lesssusceptiblespeciesorresistant strainsoffishunlikelytodevelopclinicaldisease alterationofthehabitattoeliminatetheT.tubifexwormhost,e.g.changing earthenpondstoconcreteracewaysornaturalhabitatrestoration preventionofpredators(e.g.birdsandfish)gainingaccesstoinfectedfish educationofthepublic,aquaculturistsandgovernment. TheDirectorofFisheriesand/ortheCVOinthestateorterritoryinwhichtheoutbreak occurswillberesponsiblefordevelopinganemergencyanimaldisease(EAD)response plan.ThisplanwillbesubmittedtotheAquaticConsultativeCommitteeonEmergency Whirling Disease 44 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy AnimalDiseases(AqCCEAD),whowillprovideadviceonthetechnicalsoundnessofthe plananditsconsistencywithAQUAVETPLAN. DirectorsofFisheriesand/orCVOsintheaffectedstatesorterritorieswillimplement thediseasecontrolmeasuresasagreedintheEADresponseplanandinaccordance withrelevantlegislation.Theywillmakeongoingdecisionsonfollow‐updisease‐ responsemeasuresinconsultationwithAqCCEAD.Thedetailedresponsemeasures adoptedwillbedeterminedusingtheprinciplesofcontrolanderadication(seeSection 2),epidemiologicalinformationabouttheoutbreak,andthefinancialfeasibilityofthe option. Forinformationontheresponsibilitiesoftheotherstateorterritorydiseasecontrol headquartersandlocaldiseasecontrolcentres,seetheAQUAVETPLANControlCentres ManagementManual(DAFF2001). 3.2 Responseoptions Thecircumstancessurroundinganoutbreakofwhirlingdisease(orinfectionwith M.cerebralis)willgreatlyinfluenceselectionofthemostsuitableresponseoption. Figure3detailstheactionsthatshouldoccuroninitialsuspicionofwhirlingdisease. Figure4isaflowcharttoassistselectionoftheappropriateresponseoption. Whirling Disease 45 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Figure 3 Actions to be undertaken by key response agencies during the initial phase of a suspected outbreak of whirling disease Whirling Disease 46 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Figure 4 Selection of most appropriate response to a whirling disease outbreak 3.2.1 Option1:Eradication Eradicationofwhirlingdiseasehasthehighestshort‐termeconomiccosts,butif successful,thelong‐termeconomicbenefitswilllikelyoutweightheseshort‐termcosts. Eradicationislikelytobesuccessfulinaclosedsystemorwheretheoutbreakisan obviouspointsourceepizooticwithminimalspread.Inopen,semi‐openandpossibly semi‐closedsystemswheremyxosporesmayhavewidelycontaminatedthe surroundingarea,successofaneradicationstrategywoulddependonwhetherthe: infectionhadspreadtowildsalmonidpopulations Whirling Disease 47 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy resourcesforsurveyinganddestockingwildsalmonidsintheimmediatearea weresufficient infectionwaswidelydispersedacrossmultipleaquaculturefacilities. Aneradicationplanmustincludethefollowingactivities: Quarantineandmovementcontrolsmustbedeclaredimmediatelyandstringently enforcedonsalmonids,salmonidproducts,water,T.tubifexandanyvectorsor fomiteslocatedindeclaredareas.Restrictionsmustapplytomovementoutofthe infectedareaofanythingcapableoftransmittingM.cerebralisfrominfectedto uninfectedsalmonids,andtoaquaculturefacilitiesorprocessingplants. Movementcontrolsshouldbemaintaineduntiltheagentiseithereradicatedor declaredendemic.However,movementtoprocessingplantswithgood biosecurityproceduresandadequateeffluenttreatmentcouldbepermitted, providingthatadequatebiosecurityisobservedonroutetotheplant.Giventhe resistantnatureofspores,anyeffluentwouldneedtreatmentsufficienttokill spores,safedisposal(e.g.seepagepitsawayfromnaturalwaters),ordisposalin areaswhereT.tubifexcannotlive(e.g.seawater).Proposalstomovepotentially infectedfishforprocessingwouldneedindividualconsideration. Surveillanceandtracingshouldberapidlyimplementedtoallowdetectionofall infectedpremisesandpopulationssothatcontainment,implementationof responsesandappropriatedecision‐makingcanoccur. Destructionordepopulationofallexposedfishandwormsshouldoccur.Insome instances,flexibilitycanbeallowedinhowthisisachieved(e.g.fishcanbegrown outinsaltwaterfirst). Allexposeddeadandculledfishandproductsshouldbedisposedofappropriately topreventescapeofmyxosporesintofreshwater. Allcontaminatedareas,equipmentandmaterialshouldbedecontaminatedand effluenttreated. Restockingofsusceptiblesentinelfish(e.g.youngrainbowtrout)canoccurafter destocking,thoroughdecontaminationandremovalofthesourceofTAMs.This willallowdetectionofresidualinfection(e.g.remnantinfectedworm populations). Australiannativefisharelikelytoberesistantandcanberestocked,ascanless susceptiblesalmonidspecies(e.g.browntrout). 3.2.2 Option2:Containmentandcontrolviazoningand/or compartmentalisation Iferadicationisdiscounted,thencontainmentandcontrolisthepreferredresponse optioninordertoprotectandmaintainuninfectedareas. Measurestobetakenunderthisresponseoptioninclude: Zoningand/orcompartmentalisationprogram:caremustbeexercisedinthe developmentofsuchaprogramforwhirlingdisease.Factorssuchaslaboratory testperformance,casedefinitionsandbiasinsamplingproceduresmustbe consideredbeforeimplementationtoensuresuchaprogramwouldachievethe desiredobjectives.Principlesofzoningandcompartmentalisationforinfectedand non‐infectedzonesinAustraliaareoutlinedintheAQUAPLANZoningPolicy Guidelines(DAFF2000). Managementoptionstoreducetheseverityandincidenceofinfection:managersof farmsintheaffectedareaswouldneedtoconsideroptionstoreduceexposureto M.cerebralis(seeChapter2). Whirling Disease 48 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Restockingwithlesssusceptiblefish:farmsincontrolareasmayelecttorestock withlesssusceptiblefishspecies(e.g.Australiannativesorhybridsalmonids)if thisisdeemedeconomicallyviable.Alternatively,preventingexposureofyoung salmonids(<sevenweeks)toTAMswillsignificantlyreducetheincidenceof clinicaldisease,althoughsuchfishmaystillbeinfected. 3.2.3 Option3:Controlandmitigationofdisease Ifthiscontroloptionischosen,measurestakenwillbeaimedatmanagingthediseasein affectedareas.SuchmeasuresaredetailedinSection2,andwhichmeasurestouseare context‐specific. 3.3 Criteriaforproofoffreedom Duetotheeffectivelyglobaldistributionofwhirlingdisease,exportofAustralian salmonidfishproductsisnotlikelytobeseverelyaffected.TheOIEdoesnotlist whirlingdiseaseandhasnoguidelinesforproofoffreedom,butindividualcountries maystillhaverestrictionsontheimportoffishproducts. Despitethis,proofoffreedomtoolsareavailableandcanbeusedtodesignan appropriatesurveillanceprograminAustralia.Toolsincludesuitablediagnostictests andsurveillancetechniques.Therearesuitablediagnostictestsforidentifyingboth T.tubifex(Eszterbaueretal.2006;Hallettetal.2005;Lodhetal.2012;Lodhetal.2011) andM.cerebralis(Kelleyetal.2006;Kelleyetal.2004b;MacConnell&Bartholomew 2012);andsuitablesurveillancetechniquesincludepassiveandactivesurveillance. Passivesurveillanceusesexistingsystemsandprocessesfordiseaseidentificationand notification.Ifasuitablepassivesurveillancesystemexists,theabsenceofdisease reportsfromputativelydisease‐freeareas(e.g.zonesoreradicatedareas)canbeused asevidencethatsuchareasareindeeddisease‐free. Activesurveillancerelatestocollectionofnewinformation(e.g.samplesoffishor worms)todetectdiseaseifitispresent.Therearepracticalactivesamplingstrategiesto demonstratefreedomfromdisease(Cameron1999;2002;2004;Cameron&Baldock 1998a;1998b).Forexample,asurveillanceprogramcanbeundertakenusingasample sizethatissufficienttodetectinfectionifitwaspresentataspecifieddesignprevalence value(oftensetat2%forthepurposesofdemonstratingdiseasefreedom;seeOIE 2015a).Theabsenceofinfectionfromthesurveillanceprogramcanthenbeusedto showthatwhirlingdiseaseiseffectivelyabsent. Approacheshavealsobeendevelopedthatallowaholisticanalysisofdisparate surveillancesources(e.g.activeandpassivesources)todeterminetheprobabilitythat diseasefreedomhasbeenachievedinafarm,regionorcountry(Cameron2012;Martin etal.2007). Thesetechniquesarewidelyacceptedinternationallyandareoftenconsideredrequired informationduringinternationaltradenegotiations.Theycouldbeappliedtowhirling diseasetoshowfreedomfromdisease. 3.4 Fundingandcompensation Therearecurrentlynonationalcost‐sharingagreementsinplaceforemergency responsestoaquaticanimaldiseases,includingwhirlingdisease.Itistheresponsibility oftheusersofthispublicationtoseekadviceinrelationtoanyrelevantfundingor compensationarrangementswithintherelevantjurisdictions. Whirling Disease 49 Department of Agriculture and Water Resources 3.5 AQUAVETPLAN–Disease Strategy Exportmarkets Somecountriesmayhaveimportconditionsinplacerelatedtowhirlingdisease,suchas requiringimportstobecertifiedfreeofwhirlingdisease.TheDepartmentofAgriculture andWaterResourcesisresponsibleforthehealthcertificationofallexportsandshould becontactedforfurtherinformation([email protected]). Whirling Disease 50 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 4 Appendix 1 OIE Aquatic Animal Health Code and Manual of Diagnostic Tests for Aquatic Animals Note:currentversionsoftheAquaticCodeandAquaticManualareavailableontheOIE website(http://www.oie.int/en/international‐standard‐setting/overview/).These standardsareupdatedannually. OIEAquaticCode TheobjectiveoftheOIEAquaticAnimalHealthCode(OIE2015a)istopreventthe spreadofaquaticanimaldiseases,whilefacilitatinginternationaltradeinaquatic animalsandaquaticanimalproducts.Thisannuallyupdatedvolumeisforuseby veterinarydepartments,importandexportservices,epidemiologistsandpeople involvedintheinternationaltradeofaquaticanimalsandtheirproducts. ThecurrenteditionoftheOIEAquaticCode(18thedition)waspublishedin2015andis availableontheOIEwebsite(OIE2015a). OIEAquaticManual ThepurposeoftheOIEManualofDiagnosticTestsforAquaticAnimals(OIE2015b)isto contributetotheinternationalharmonisationofmethodsforthesurveillanceand controlofthemostimportantaquaticanimaldiseases.Standardsaredescribedfor laboratorydiagnostictestsandtheproductionandcontrolofbiologicalproducts (principallyvaccines)forveterinaryuseglobally. ThecurrenteditionoftheOIEAquaticManual(7thEdition)waspublishedin2015andis availableontheOIEwebsite(OIE2015b). Furtherinformation FurtherinformationabouttheOIEAquaticCodeandAquaticManualisavailableonthe OIEwebsiteat: http://www.oie.int/international‐standard‐setting/overview/ (Accessed22/07/2016) Whirling Disease 51 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy 5 Appendix 2 Approval of chemicals for use in Australia TheAustralianPesticidesandVeterinaryMedicinesAuthority(APVMA)evaluates, registersandregulatesagriculturalandveterinarychemicals.Beforeaveterinary chemical(e.g.anantibioticorvaccine)canentertheAustralianmarket,itmustgo throughtheAPVMA’srigorousassessmentprocesstoensurethatitmeetshigh standardsofsafetyandeffectiveness.Inaddition,animportpermitisrequiredfromthe DepartmentofAgricultureandWaterResourcesifaproductcontainingbiological materialistobesourcedfromoverseas. Detaileddataabouttheproductanditsproposedusepatternmustbesubmittedtothe APVMAwiththeapplicationforregistrationorpermits.Becausetheassessmentprocess issodetailed,theevaluationmaytakesometimetocomplete. Registration RegistrationisthedefaultmethodfortheAPVMAtoallowtheuseofaveterinary chemicalinAustralia.Registrationistimeconsumingandexpensiveanditmaybe necessarytoapplyforaminororemergencyusepermitduringanemergency. Minorusepermit Theminorusepermit(MUP)systemisatemporaryapprovalsystemfortheuseofdrugs andchemicals.ThesystemwasdevisedbytheAPVMAforAustralia,andallowsthe restricteduseofalimitedamountofadrugorchemicalinaspecifiedspecieswhen inadequatedataareavailabletosatisfyAPVMArequirementsforregistration. Conditionsareappliedtothepermit,whichoftenincludethecollectionofdatarelated totheuseoftheproduct.TheMUPsystemaimstoenablerestricteduseofadrugor chemicaluntilsufficientdataareavailabletoenablefullregistration. Forexample,theAPVMAmaysetatemporarywithholdingperiodwithawidemarginof safetyforaMUP.Thiswithholdingperiodmayhavebeenextrapolatedfromdata relatingtotheuseoftheproductinotherspecies.Insuchcases,aconditionoftheMUP willbethecollectionofresiduedatafortesting.Resultsfromthedataareassessedby theAPVMA(usuallyafter12months,whichisthedurationofmostpermits)andusedto moreaccuratelysetawithholdingperiodfortheproduct. Emergencyusepermit TheAPVMAhasapermitsystemfortheemergencyuseofaproductthatiseither unregisteredinAustraliaorregisteredforuseinadifferentspeciesorforadifferent use.TheAPVMAwillverifywiththeappropriatestateandterritorycoordinatorsthat theemergencyisgenuine. Forfurtherdetailsorpermitapplicationforms,visittheAPVMAwebsite(APVMA2015). Whirling Disease 52 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Appendix 3 Extraction and PCR method for whirling disease (Myxobolus cerebralis) — New Zealand Ministry for Primary Industries SuppliedcourtesyoftheMPI,WallacevilleLaboratories,NewZealand,withsomeminor modifications(includingtheremovalofreferencestoNZ‐specificforms).Thetest, currentasatJuly2015,isaccreditedtoISO17025byInternationalAccreditationNew Zealand.Assistancewithsourcingpositivecontrolmaterialcanbeobtainedfrom: NationalCentreforBiosecurityandInfectiousDisease MinistryforPrimaryIndustries,ManatūAhuMatua,InvestigationandDiagnosticCentre 66WardSt,POBox40742,UpperHutt5018,NewZealand Whirlingdisease(Myxoboluscerebralis)—extractionandPCRmethod TM‐111 Safetystatement Careshouldbetakenwhenhandlingsamplesthatmaycontainzoonoticagents;all biologicalsamplesshouldbetreatedaspotentiallyharmful.Usetheassignedprotective clothingandequipmentatalltimes.Checkthattheworkareaissafebylocatingfire extinguishers,firstaidkitandanysafetyequipmentthatmayberequired.CheckMSD sheetsforanychemicalsorreagentsthatmaybeusedandhaveaclearunderstandingof thedangersassociatedwiththem. Whirlingdisease(Myxoboluscerebralis)—extractionand PCRmethod 1. Purpose ToextractDNAfromthecartilageofjuvenilesalmonheadsforusewithaPCRassaytotestfor Myxoboluscerebralis. 2. Scope Examinationoffarmedorwildsalmonidpopulationsforsporesofthechondrophilicprotozoan parasiteM.cerebralis. 3. LimitationsofMethod Thismethodisusedforfishlongerthan120mm(i.e.headslongerthan26mm,noseto caudalmarginofoperculum). TherateofM.cerebralissporedevelopmentisdependentontemperature.Salmonids canbetestedbythismethodiftheperiodbetweenexposuretoM.cerebralisand samplingisgreaterthanfourmonths. Whirling Disease 53 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy M.cerebralisinfestedcartilageisnotroutinelyavailableforcontrolpurposes. EscherichiacolitransformantswithM.cerebralisDNAinsertsarestoredonbeadsin– 70°Cfreezer: DNAisextractedfromthisandusedasapositivecontrolwitheachPCR. 4. SampleRequirements Sixtyfisharesampledfromeach‘lot’*atthelocation,givingastatisticalconfidencelevel of95%thata100%sensitivetestwilldetectatleastoneindividualinapopulationwith a5%orgreaterprevalenceofM.cerebralis.Anyfishshowingclinicalsignsofwhirling diseasearesampledfirstandthesamplenumbermadeupwithrandomlyselected individuals. Wholefishorheadsarekeptchilledduringtransporttothelaboratory. *A 'lot' is a group of fish of the same species and age which have shared the same holding facilities and water supply. 5. QualityControlMaterial PositivecontrolDNA: ClonedMyxoboluscerebralisusedataconcentrationof0.1pg/µL or DNAfrominfectedtissueofMyxoboluscerebralisusedataconcentrationof1 ng/µL ThisDNAisrunwitheachassay. 6. QualityControlProcedures Asperlaboratoryrequirements. 7. 8. Equipment Waterbath:45°C(±5°C),referSOPEQ‐46 Scissors Sterileforceps Largebeaker Sterilebladessize22 Plasticbottles Sterilepetridishes Ultra‐turraxhomogeniser:typeT25,IKA‐Labortechnik Heatingblocks‐56°Cand80°C,1.5mLmicrofugetubes Thermocyclers Pipettors, Benchcentrifuge, Freezer–20°C ReagentsandSolutions 96%Ethanol Distilledwater Instagenematrix 10%bufferedformalin Instagenelysisbuffer ZymoGenomicDNACleanandConcentratorTMkit Whirling Disease 54 Department of Agriculture and Water Resources PCRreagents 9. Procedure 9.1 Processingofsalmonheads Fishheadsshouldarrivedatthelaboratoryinpoolsof5—ifnot,poolintolotsof 5andlabel. Sagittallyhalvetheheads,andplacehalfin10%neutralbufferedformalin.Keep theseformalinfixedtissuesfor3monthsforconfirmationifneeded. Softenremainingheads,aspoolsinseparatebags,bywarmingtheheadsinalarge beakerofwarmtapwater,whichisimmersedinawater‐bathat45°C.Theheads arereadytobeprocessedwhentheeyeshaveturnedopaque(5–10minutesfor smallheadslessthan40mmlong,and20–30minutesforlargeheadsgreaterthan 40mm).Occasionallystirtheheadstomaintainaneventemperature.Removethe headsfromthebeaker. Usingforcepsremoveanddiscardtheeyes,brain,lowerjaw,skinandothersoft tissue,retainingallthecartilage/bonefromeachpoolinseparatepetridishes. Fishshowingclinicalsignsaretobepooledtogether. Processeachbatchseparatelyasfollows: 9.2 AQUAVETPLAN–Disease Strategy cutremainingcranialelementswithscissorsorscalpelbladeandplaceinto asmallplasticcontainer.Furtherdispersethischoppedmaterialfor30 secondsinupto30mLofdistilledwater(5–10mLwaterispreferable)in anUltra‐turraxhomogeniser.Rinseallsolidmaterialofftheprobeintoa1% solutionofsodiumhypochloriterinse,thensterilisetheprobebyflaming with96%ethanolbetweeneachpool. UsehomogenatefortheDNAextraction,Storetheremaininghomogenateat–20°C for3months. Brushcleanandsterilizeallequipmentina1%solutionofsodiumhypochlorite solutionfor4hours.Donotleavemetallicequipmentinsodiumhypochloritefor longerthanthis. Fleshscrapingsfromthefishheadsanddisposablepipettesshouldbeplacedin thebiohazardbinsfordisposal. Cleanthewaterbathbyraisingthewatertemperatureto90°Cforaminimumof onehour.Aftercoolingthecontentscanbedisposedofdownthesinkandthebath washedwithdetergent. DNAextraction—ModifiedInstagenematrixmethod Add~750µLhomogenatetoamicrofugetubeusingatransferpipette. Pelletthishomogenatebycentrifugingat10,000xgfor1minute.Remove supernatant. Weighpelletandrepeatifnecessarytoobtainbetween150mgand250mgof material. Add200µLlysisbuffertopellet.Vortexandincubateat56°Cforatleast30 minutes. Centrifugeat10,000xgfor1minute.Removesupernatant. Add200µLInstagenematrixtothepelletandincubateat80°Cforatleast30 minutes.Vortex. Incubateat100°Cfor8minutes. Centrifugeat10,000xgfor3minutes.Removesupernatantandplaceintoa microfugetubeforcleanup Whirling Disease 55 Department of Agriculture and Water Resources CleanupextractedDNAusingtheZymoGenomicDNACleanandConcentratorTM kit—followmanufacturersprotocolwiththeinclusionofthefollowingsteps: extendthefinalwashspinby2minutestoensurecompleteremovalofthe washbuffer elutein15µLusingDNAelutionbufferwarmedat60–70°C. 9.3 10. AQUAVETPLAN–Disease Strategy ConventionalPCR Interpretation—quantifytheextractedcleanedDNAandverifytheDNAis amplifiablebyrunningthroughaninternalcontrolPCRe.g.18SrRNA. RunsamplesthroughthewhirlingdiseaseconventionalPCR. NotetheDNAconcentrationonthetubes,andstoreinthestorageboxesinthe samplepreplaboratory–20°Cfreezer. NotestoragelocationinthefishpathDNAstorageworkbooklocatedinthesample preplab. Allsampleswillbestoredforthreemonthsbeforebeingdiscarded. Recordallinformationontheappropriatemolecularworksheets InterpretationandRecordingofResults Expectedproductsizeis507bp. Positivecontrol: Noproduct=runnotvalid,repeat 507bpproduct=validrun Samples: Noproductornon‐specificbanding=negative 507bpproduct=notnegative Notnegativesamples: 11. PCRtoberepeated ifanotnegativeresultisproduced,theproductshouldbesequencedtoconfirm theidentificationofM.cerebralis.ThePCRprimers(Tr5‐16/Tr3‐17)areusedto sequencepartofthe18srRNAgene. Sagitallysectionedheadsfixedinformalinshouldbeprocessedhistologically, stainedwithhaematoxylinandeosin,andexaminedmicroscopicallyforevidence ofMyxosporeanorganismsinthecartilage. Calculations Notapplicable. 12. ReportingofResults AtypedreportisenteredinLIMSorappropriatelabrecordingsystemwhichdetailsthe particularsofthefishsampled,thetestmethodandresult.Thisreportissenttothe person/swhorequestedthetestandcopiesareheldbothelectronicallyandwiththe casepapers. 13. ReferenceDocuments Thoesen,JC,editor1994,‘Suggestedproceduresforthedetectionandidentificationof certainfishandshellfishpathogens’,4thed.,Version1,FishHealthSection,American Whirling Disease 56 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy FisheriesSociety,SOSPublications,43DeNormandieAvenue,FairHaven,NewJersey 07704‐3303. Andree,KB,MacConnell,EM,&Hedrick,RP1998,‘Anestedpolymerasechainreaction forthedetectionofgenomicDNAofMyxoboluscerebralisinrainbowtrout Onchorhynchusmykiss’,DiseasesofAquaticOrganisms,vol.34,145‐154. Epp,JK,&WoodJS1998,‘Quantitativedeterminationofthesensitivityofasingleround PCRprocedurefordetectingMyxoboluscerebralis’.ProceedingsoftheWhirlingDisease Symposium:ResearchinProgress,19‐21February1998,FortCollins,CO.TheWhirling DiseaseFoundation,Bozeman,MT,pp.173‐175. IANZaccreditedtest WhirlingDisease(Myxoboluscerebralis)ConventionalPCR Nucleicacidextraction–DNA Primers Name Sequence(5’‐3’) Size Position Target* Forward Tr5‐16 GCATTGGTTTACGCTGATGTAGCGA 25 211 18srRNA EF370480 Reverse Tr3‐17 GGCACACTACTCCAACACTGAATTTG 26 693 18srRNA EF370480 * Please include a Genbank accession number for the target wherever possible. PCR kit: Kapa 2G Fast Hotstart Readymix Reaction volume: 25 µL Reagentmix Volume(µL) Kapa2Greadymix 12.5 Nucleasefreewater 10.7 Fprimer(10µM) 0.4 Rprimer(10µM) 0.4 DNAtemplate(1–100ng) 1* *DNAvolumemayvarydependingonconcentrationofDNA. PCRcontrols Description Positive DNAextractedfromMyxoboluscerebraliscloneusedat0.1pg/uLorDNA extractedfrominfectedtissueusedat1ng/uL Reagent Nuclease‐freewater Cyclingparameters Temp(°C) Time(sec) No.cycles Hold 95 120 1 Denature 95 15 35 Whirling Disease 57 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Anneal 67 15 35 Extension 72 1 35 Electrophoresis Description Sizeofamplicon(bp) Agarosegel 1.5% 507bp MWmarker 100bp References Andree,KB,MacConnell,EM,&Hedrick,RP1998,‘Anestedpolymerasechainreaction forthedetectionofgenomic‘DNAofMyxoboluscerebralisinrainbowtrout Onchorhynchusmykiss’,DiseasesofAquaticOrganisms,vol.34,145‐154. Epp,JK,&WoodJS1998,‘Quantitativedeterminationofthesensitivityofasingleround PCRprocedurefordetectingMyxoboluscerebralis’.ProceedingsoftheWhirlingDisease Symposium:ResearchinProgress,19‐21February1998,FortCollins,CO.TheWhirling DiseaseFoundation,Bozeman,MT,pp.173‐175. 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