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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
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DepartmentofAgricultureandWaterResources201,AQUAVETPLANDisease
Strategy:WhirlingDisease,DepartmentofAgricultureandWaterResources,Canberra.
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AQUAVETPLANDiseaseStrategy:WhirlingDiseaseisavailableatagriculture.gov.au
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informationanddatainthispublication.Notwithstanding,theDepartmentof
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Theinformationinthispublicationisforgeneralguidanceonlyanddoesnotoverride
commonlaw,lawsoftheCommonwealthoranyAustralianStateorTerritory,orplace
anylegalobligationofcomplianceoractionontheCommonwealth,aStateora
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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,>8Cuntilfirstfeeding)
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–20Cforuptothreemonths.Brinedfishalsoretainviablespores,although
hot‐smokingat66Cfor40minutesinactivatesspores(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
UVirradiationat40MJ/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)
UVirradiationof40MJ/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.
Schisler,GJ,Bergersen,EP,Walker,PG,Wood,J,&Epp,JK2001,‘Comparisonofsingle‐
roundpolymerasechainreaction(PCR)andpepsin‐trypsindigest(PTD)methodsfor
detectionofMyxoboluscerebralis’,DiseasesofAquaticOrganisms,vol.45,pp.109‐114.
Whirling Disease 58 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy References Anderson,CD1993,‘EpidemiologicalaspectsofMyxoboluscerebralis,theagentof
salmonidwhirlingdisease’,Surveillance,vol.20,no.2,pp.19–24.
Andree,KB,MacConnell,E&Hedrick,RP1998,‘Anestedpolymerasechainreactionfor
thedetectionofgenomicDNAofMyxoboluscerebralisinrainbowtroutOncorhynchus
mykiss’,DiseasesofAquaticOrganisms,vol.34,no.2,pp.145–54.
Antonio,DB,El‐Matbouli,M&Hedrick,RP1999,‘Detectionofearlydevelopmental
stagesofMyxoboluscerebralisinfishandtubificidoligochaetehostsbyinsitu
hybridization’,ParasitologyResearch,vol.85,no.11,pp.942–4.
APVMA2015,‘RegistrationsandPermits’,AustralianPesticidesandVeterinary
MedicinesAuthority,fromhttp://apvma.gov.au/node/1060,accessed05/05/2016.
Arndt,RE&Wagner,EJ2004,‘Rapidandslowsandfiltrationtechniquesandtheir
efficacyatfilteringtriactinomyxonsofMyxoboluscerebralisfromcontaminatedwater’,
NorthAmericanJournalofAquaculture,vol.66,no.4,pp.261–70.
Arndt,RE,Wagner,EJ,Bobo,C&StJohn,T2006,‘Laboratoryandhatchery‐scale
evaluationofsandfiltersandtheirefficacyatcontrollingwhirlingdiseaseinfection’,
JournalofAquaticAnimalHealth,vol.18,no.4,pp.215–22.
Baerwald,MR2013,‘Temporalexpressionpatternsofrainbowtroutimmune‐related
genesinresponsetoMyxoboluscerebralisexposure’,Fish&ShellfishImmunology,vol.
35,no.3,pp.965–71.
Baerwald,MR,Petersen,JL,Hedrick,RP,Schisler,GJ&May,B2011,‘Amajoreffect
quantitativetraitlocusforwhirlingdiseaseresistanceidentifiedinrainbowtrout
(Oncorhynchusmykiss)’,Heredity,vol.106,no.6,pp.920–6.
Baldwin,TJ,Vincent,ER,Silflow,RM&Stanek,D2000,‘Myxoboluscerebralisinfectionin
rainbowtrout(Oncorhynchusmykiss)andbrowntrout(Salmotrutta)exposedunder
naturalstreamconditions’,JournalofVeterinaryDiagnosticInvestigation,vol.12,no.4,
pp.312–21.
Bartholomew,JL,Lorz,HV,Atkinson,SD,Hallett,SL,Stevens,DG,Holt,RA,Lujan,K&
Amandi,A2007,‘Evaluationofamanagementstrategytocontrolthespreadof
MyxoboluscerebralisinaLowerColumbiarivertributary’,NorthAmericanJournalof
FisheriesManagement,vol.27,no.2,pp.542–50.
Bartholomew,JL&Reno,PW2002,‘Review:Thehistoryanddisseminationofwhirling
disease’,AmericanFisheriesSocietySymposium,vol.29,no.3,pp.3–24.
Baxa,DV,Kelley,GO,Mukkatira,KS,Beauchamp,KA,Rasmussen,C&Hedrick,RP2008,
‘Arresteddevelopmentofthemyxozoanparasite,Myxoboluscerebralis,incertain
populationsofmitochondrial16SlineageIIITubifextubifex’,ParasitologyResearch,vol.
102,no.2,pp.219–28.
Beauchamp,KA,Kelley,GO,Nehring,RB&Hedrick,RP2005,‘Theseverityofwhirling
diseaseamongwildtroutcorrespondstothedifferencesingeneticcompositionof
TubifextubifexpopulationsincentralColorado’,JournalofParasitology,vol.91,no.1,pp.
53–60.
Bentz,CR,Hogge,CI,Meyers,TR&Ferguson,JA2012,‘MyxobolusneurotropusInfecting
RainbowTroutinAlaska,aNewGeographicRecord’,JournalofAquaticAnimalHealth,
vol.24,no.4,pp.225–31.
Whirling Disease 59 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Blaylock,RB&Bullard,SA2014,‘Counter‐insurgentsofthebluerevolution?Parasites
anddiseasesaffectingaquacultureandscience’,JournalofParasitology,vol.100,no.6,
pp.743–55.
Boreham,RE,Hendrick,S,O’Donoghue,PJ&Stenzel,DJ1998,‘Incidentalfindingof
Myxobolusspores(Protozoa:Myxozoa)instoolsamplesfrompatientswith
gastrointestinalsymptoms’,JournalofClinicalMicrobiology,vol.36,no.12,pp.3728–30.
Cameron,AR1999,Surveytoolboxforlivestockdiseases:apracticalmanualandsoftware
package,AustralianCentreforInternationalAgriculturalResearch,Canberra,ACT,
Australia,fromhttp://aciar.gov.au/files/node/2268/mn94.pdf.
——2002,Surveytoolforaquaticanimaldiseases:apracticalmanualandsoftware
package,AustralianCentreforInternationalAgriculturalResearch,Canberra,ACT,
Australia.
——2004,PrinciplesfortheDesignandConductofSurveystohowPresenceorAbsenceof
InfectiousDiseasesinAquaticAnimals,DepartmentofAgriculture,Canberra,ACT,
Australia,fromhttp://www.agriculture.gov.au/animal/aquatic/guidelines‐and‐
resources.
——2012,‘Theconsequencesofrisk‐basedsurveillance:Developingoutput‐based
standardsforsurveillancetodemonstratefreedomfromdisease’,PreventiveVeterinary
Medicine,vol.105,no.4,pp.280–6.
Cameron,AR&Baldock,FC1998a,‘Anewprobabilityformulaforsurveysto
substantiatefreedomfromdisease’,PreventiveVeterinaryMedicine,vol.34,no.1,pp.1–
17.
——1998b,‘Two‐stagesamplinginsurveystosubstantiatefreedomfromdisease’,
PreventiveVeterinaryMedicine,vol.34,no.1,pp.19–30.
Cavender,WP,Wood,JS,Powell,MS,Overturf,K&Cain,KD2004,‘Real‐time
quantitativepolymerasechainreaction(QPCR)toidentifyMyxoboluscerebralisin
rainbowtroutOncorhynchusmykiss’,DiseasesofAquaticOrganisms,vol.60,no.3,pp.
205–13.
CSIRO2015,‘AtlasofLivingAustralia’,CommonwealthScientificandIndustrial
ResearchOrganisation,fromhttp://bie.ala.org.au/species/Tubifex+tubifex,accessed
24/02/2015.
DA2012,AquaticAnimalDiseasesSignificanttoAustralia:IdentificationFieldGuide(4th
Edition),DepartmentofAgricultureandWaterResources,ACT,Canberra,Australia.
——2015a,Australia’snationallistofreportablediseasesofaquaticanimals,
DepartmentofAgriculture,Canberra,ACT,Australia,from
http://www.agriculture.gov.au/animal/aquatic/reporting/reportable‐diseases.
——2015b,Enterprisemanual(version2.0),DepartmentofAgriculture,Canberra,ACT,
Australia,fromhttp://www.agriculture.gov.au/animal/aquatic/aquavetplan.
DAFF2000,ZoningPolicyGuidelines(AQUAPLAN),DepartmentofAgricultureFisheries
andForestry,Canberra,ACT,Australia,from
http://www.agriculture.gov.au/animal/aquatic/guidelines‐and‐resources.
——2001,Controlcentresmanagementmanual(version1.0),DepartmentofAgriculture
FisheriesandForestry,Canberra,ACT,Australia,from
http://www.agriculture.gov.au/animal/aquatic/aquavetplan.
Whirling Disease 60 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy ——2008,Operationalproceduresmanual—Decontamination,Departmentof
AgricultureFisheriesandForestry,Canberra,ACT,Australia,from
http://www.agriculture.gov.au/animal/aquatic/aquavetplan.
——2009a,Operationalproceduresmanual—Destruction,DepartmentofAgriculture
FisheriesandForestry,Canberra,ACT,Australia,from
http://www.agriculture.gov.au/animal/aquatic/aquavetplan.
——2009b,Operationalproceduresmanual—Disposal,DepartmentofAgriculture
FisheriesandForestry,Canberra,ACT,Australia,from
http://www.agriculture.gov.au/animal/aquatic/aquavetplan.
Densmore,CL,Ottinger,CA,Blazer,VS&Iwanowicz,LR2004,‘Immunomodulationand
diseaseresistanceinpost‐yearlingrainbowtroutinfectedwithMyxoboluscerebralis,the
causativeagentofWhirlingdisease’,JournalofAquaticAnimalHealth,vol.16,no.2,pp.
73–82.
El‐Matbouli,M,Fischer‐Scherl,T&Hoffmann,RW1992,‘Presentknowledgeonthelife
cycle,taxonomy,pathology,andtherapyofsomeMyxosporeaspp.importantfor
freshwaterfish’,AnnualReviewofFishDiseases,vol.2,pp.367–402.
El‐Matbouli,M&Hoffmann,R1991,‘Effectsoffreezing,aging,andpassagethroughthe
alimentarycanalofpredatoryanimalsontheviabilityofMyxoboluscerebralisspores’,
JournalofAquaticAnimalHealth,vol.3,no.4,pp.260–2.
El‐Matbouli,M&Hoffmann,RW1998,‘Lightandelectronmicroscopicstudiesonthe
chronologicaldevelopmentofMyxoboluscerebralistotheactinosporeanstageinTubifex
tubifex’,InternationalJournalforParasitology,vol.28,no.1,pp.195–217.
El‐Matbouli,M,Hoffmann,RW&Mandok,C1995,‘Lightandelectronmicroscopic
observationsontherouteofthetriactinomyxonsporoplasmofMyxoboluscerebralis
fromepidermisintorainbowtroutcartilage’,JournalofFishBiology,vol.46,no.6,pp.
919–35.
El‐Matbouli,M,Hoffmann,RW,Schoel,H,McDowelll,TS&Hedrick,RP1999a,‘Whirling
disease:hostspecificityandinteractionbetweentheactinosporeanstageofMyxobolus
cerebralisandrainbowtroutOncorhynchusmykiss’,DiseasesofAquaticOrganisms,vol.
35,no.1,pp.1–12.
El‐Matbouli,M,McDowell,TS,Antonio,DB,Andree,KB&Hedrick,RP1999b,‘Effectof
watertemperatureonthedevelopment,releaseandsurvivalofthetriactinomyxon
stageofMyxoboluscerebralisinitsoligochaetehost’,InternationalJournalfor
Parasitology,vol.29,no.4,pp.627–41.
Elwell,LCS,Kerans,BL,Rasmussen,C&Winton,JR2006,‘Interactionsamongtwo
strainsofTubifextubifex(Oligochaeta:Tubificidae)andMyxoboluscerebralis
(Myxozoa)’,DiseasesofAquaticOrganisms,vol.68,no.2,pp.131–9.
Eszterbauer,E,Marton,S,Racz,OZ,Letenyei,M&Molnar,K2006,‘Morphologicaland
geneticdifferencesamongactinosporeanstagesoffish‐parasiticmyxosporeans
(Myxozoa):difficultiesofspeciesidentification’,SystematicParasitology,vol.65,no.2,
pp.97–114.
Feist,SW&Longshaw,M2006,PhylumMyxozoa,inFishdiseasesanddisorders.Volume
1:ProtozoanandMetazoaninfections,WooPTK(eds),CABIPublishing,Wallingford,vol.
1,pp.230–96.
Whirling Disease 61 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Fetherman,ER,Winkelman,DL,Baerwald,MR&Schisler,GJ2014,‘Survivaland
ReproductionofMyxoboluscerebralis‐ResistantRainbowTroutIntroducedtothe
ColoradoRiverandIncreasedResistanceofAge‐0Progeny’,PlosOne,vol.9,no.5.
Gates,KK,Guy,CS,Zale,AV&Horton,TB2008,‘AdherenceofMyxoboluscerebralis
MyxosporestoWaders:ImplicationsforDiseaseDissemination’,NorthAmericanJournal
ofFisheriesManagement,vol.28,no.5,pp.1453–8.
Gilbert,MA&Granath,WO2001,‘PersistentinfectionofMyxoboluscerebralis,the
causativeagentofsalmonidwhirlingdisease,inTubifextubifex’,JournalofParasitology,
vol.87,no.1,pp.101–7.
——2003,‘Whirlingdiseaseofsalmonidfish:Lifecycle,biology,anddisease’,Journalof
Parasitology,vol.89,no.4,pp.658–67.
Gomez,D,Bartholomew,J&Sunyer,JO2014,‘Biologyandmucosalimmunityto
myxozoans’,DevelopmentalandComparativeImmunology,vol.43,no.2,pp.243–56.
Granath,WO2014,‘EffectsofhabitatalterationontheepizootiologyofMyxobolus
cerebralis,thecausativeagentofsalmonidwhirlingdisease’,JournalofParasitology,vol.
100,no.2,pp.157–65.
Granath,WO&Vincent,ER2010,‘EpizootiologyofMyxoboluscerebralis,thecausative
agentofsalmonidwhirlingdiseaseintherockcreekdrainageofwest‐centralMontana:
2004–2008‘,JournalofParasitology,vol.96,no.2,pp.252–7.
Griffin,BR&Davis,EM1978,‘Myxosomacerebralis—detectionofcirculatingantibodies
ininfectedrainbow‐trout(Salmogairdneri)’,JournaloftheFisheriesResearchBoardof
Canada,vol.35,no.9,pp.1186–90.
Hallett,SL,Atkinson,SD&Bartholomew,JL2005,‘Counteringmorphological
ambiguities:developmentofaPCRassaytoassisttheidentificationofTubifextubifex
oligochaetes’,Hydrobiologia,vol.543,pp.305–9.
Hallett,SL&Bartholomew,J2012,MyxoboluscerebralisandCeratomyxashasta,inFish
Parasites:PathobiologyandProtection,WooPTK&BuchmannK(eds),CABIPublishing,
Wallingford,OX108DE,Oxford,England,pp.1–383.
Hallett,SL,Erseus,C&Lester,RJG1995,‘AnactinosporeanfromanAustralianmarine
oligochaete’,BulletinoftheEuropeanAssociationofFishPathologists,vol.15,no.5,pp.
168–9.
Hallett,SL,Lorz,HV,Atkinson,SD,Rasmussen,C,Xue,L&Bartholomew,JL2009,
‘PropagationofthemyxozoanparasiteMyxoboluscerebralisbydifferentgeographicand
geneticpopulationsofTubifextubifex:AnOregonperspective’,JournalofInvertebrate
Pathology,vol.102,no.1,pp.57–68.
Halliday,MM1973a,‘StudiesonMyxosomacerebralis,aparasiteofsalmonids.1.
Diagnosisofinfection’,NordiskVeterinaerMedicin,vol.25,no.7–8,pp.345–8.
——1973b,‘StudiesonMyxosomacerebralis,aparasiteofsalmonids.2.Development
andpathologyofMyxosomacerebralisinexperimentallyinfectedrainbow‐trout(Salmo
gairdneri)fryrearedatdifferentwatertemperatures’,NordiskVeterinaerMedicin,vol.
25,no.7–8,pp.349–58.
——1974,‘StudiesonMyxosomacerebralis,aparasiteofsalmonids.4.Preliminary
immunofluorescentinvestigationofsporesofMyxosomacerebralis’,NordiskVeterinaer
Medicin,vol.26,no.3–4,pp.173–9.
Whirling Disease 62 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy ——1976,‘BiologyofMyxosomacerebralis—causativeorganismofwhirlingdiseaseof
salmonids’,JournalofFishBiology,vol.9,no.4,pp.339–57.
Hansen,ES&Budy,P2011,‘Thepotentialofpassivestreamrestorationtoimprove
streamhabitatandminimizetheimpactoffishdisease:ashort‐termassessment’,
JournaloftheNorthAmericanBenthologicalSociety,vol.30,no.2,pp.573–88.
Hedrick,RP&El‐Matbouli,M2002,Recentadvanceswithtaxonomy,lifecycle,and
developmentofMyxoboluscerebralisinthefishandoligochaetehosts,inWhirling
Disease:ReviewsandCurrentTopics,BartholomewJL&WilsonJC(eds),vol.29,pp.45–
53.
Hedrick,RP,El‐Matbouli,M,Adkison,MA&MacConnell,E1998,‘Whirlingdisease:re‐
emergenceamongwildtrout’,ImmunologicalReviews,vol.166,pp.365–76.
Hedrick,RP,McDowell,TS,Adkison,MA,Myklebust,KA,Mardones,FO&Petri,B2012,
‘Invasionandinitialreplicationofultravioletirradiatedwaterborneinfectivestagesof
Myxoboluscerebralisresultsinimmunitytowhirlingdiseaseinrainbowtrout’,
InternationalJournalforParasitology,vol.42,no.7,pp.657–66.
Hedrick,RP,McDowell,TS,Gay,M,Marty,GD,Georgiadis,MP&MacConnell,E1999a,
‘ComparativesusceptibilityofrainbowtroutOncorhynchusmykissandbrowntrout
SalmotruttatoMyxoboluscerebralis,thecauseofsalmonidwhirlingdisease’,Diseasesof
AquaticOrganisms,vol.37,no.3,pp.173–83.
Hedrick,RP,McDowell,TS,Mukkatira,K,Georgiadis,MP&MacConnell,E1999b,
‘Susceptibilityofselectedinlandsalmonidstoexperimentallyinducedinfectionswith
Myxoboluscerebralis,thecausativeagentofwhirlingdisease’,JournalofAquaticAnimal
Health,vol.11,no.4,pp.330–9.
Hedrick,RP,McDowell,TS,Mukkatira,K,Macconnell,E&Petri,B2008,‘Effectsof
freezing,drying,ultravioletirradiation,chlorine,andquaternaryammoniumtreatments
ontheinfectivityofmyxosporesofMyxoboluscerebralisforTubifextubifex’,Journalof
AquaticAnimalHealth,vol.20,no.2,pp.116–25.
Hedrick,RP,Petri,B,McDowell,TS,Mukkatira,K&Sealey,LJ2007,‘Evaluationofa
rangeofdosesofultravioletirradiationtoinactivatewaterborneactinosporestagesof
Myxoboluscerebralis’,DiseasesofAquaticOrganisms,vol.74,no.2,pp.113–8.
Hoffman,GL1990,‘Myxoboluscerebralis,aWorldwideCauseofSalmonidWhirling
Disease’,JournalofAquaticAnimalHealth,vol.2,no.1,pp.30–7.
Hogge,CI,Campbell,MR&Johnson,KA2008,‘Redescriptionandmolecular
characterizationofMyxoboluskisutchiYasutake&Wood,1957’,JournalofFishDiseases,
vol.31,no.9,pp.707–12.
IFS2008,TasmanianInlandRecreationalFisheryManagementPlan2008–2018,Inland
FisheriesService,NewNorfolk,Tasmania,NewNorfolk,from
http://www.ifs.tas.gov.au/publications/tasmanian‐inland‐recreational‐fishery‐
management‐plan‐2008‐2018.
Johnston,C&Jungalwalla,Pn.d.,AquaticAnimalWelfareGuidelines:Guidelineson
welfareoffishandcrustaceansinaquacultureand/orinliveholdingsystemsforhuman
consumption,NationalAquacultureCouncilInc.,Canberra,ACT,Australia,from
http://www.australiananimalwelfare.com.au/content/aquatic‐animals/aquatic‐animal‐
welfare‐guidelines.
Whirling Disease 63 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Kallert,DM,Bauer,W,Haas,W&El‐Matbouli,M2011,‘Noshotinthedark:Myxozoans
chemicallydetectfreshfish’,InternationalJournalforParasitology,vol.41,no.3–4,pp.
271–6.
Kallert,DM,Eszterbauer,E,Grabner,D&El‐Matbouli,M2009,‘Invivoexposureof
susceptibleandnon‐susceptiblefishspeciestoMyxoboluscerebralisactinospores
revealsnon‐specificinvasionbehaviour’,DiseasesofAquaticOrganisms,vol.84,no.2,pp.
123–30.
Kelley,GO,Adkison,MA,Leutenegger,CM&Hedrick,RP2003,‘Myxoboluscerebralis:
identificationofacathepsinZ‐likeproteasegene(MyxCP‐1)expressedduringparasite
developmentinrainbowtrout,Oncorhynchusmykiss’,ExperimentalParasitology,vol.
105,no.3–4,pp.201–10.
Kelley,GO,Adkison,MA,Zagmuff‐Vergara,FJ,Leutenegger,CM,Bethel,JW,Myklebust,
KA,McDowell,TS&Hedrick,RP2006,‘Evaluationofquantitativereal‐timePCRfor
rapidassessmentsoftheexposureofsentinelfishtoMyxoboluscerebralis’,Parasitology
Research,vol.99,no.4,pp.328–35.
Kelley,GO,Zagmutt‐Vergara,FJ,Leutenegger,CM,Adkison,MA,Baxa,DV&Hedrick,RP
2004a,‘IdentificationofaserineproteasegeneexpressedbyMyxoboluscerebralis
duringdevelopmentinrainbowtroutOncorhynchusmykiss’,DiseasesofAquatic
Organisms,vol.59,no.3,pp.235–48.
Kelley,GO,Zagmutt‐Vergara,FJ,Leutenegger,CM,Myklebust,KA,Adkison,MA,
McDowell,TS,Marty,GD,Kahler,AL,Bush,AL,Gardner,IA&Hedrick,RP2004b,
‘EvaluationoffivediagnosticmethodsforthedetectionandquantificationofMyxobolus
cerebralis’,JournalofVeterinaryDiagnosticInvestigation,vol.16,no.3,pp.202–11.
Kerans,BL,Rasmussen,C,Stevens,R,Colwell,AEL&Winton,JR2004,‘Differential
propagationofthemetazoanparasiteMyxoboluscerebralisbyLimnodrilushoffmeisteri,
Ilyodrilustempletoni,andgeneticallydistinctstrainsofTubifextubifex’,Journalof
Parasitology,vol.90,no.6,pp.1366–73.
Kerans,BL&Zale,AV2002,TheecologyofMyxoboluscerebralis,inWhirlingDisease:
ReviewsandCurrentTopics,BartholomewJL&WilsonJC(eds),AmericanFisheries
SocietySymposium,SaltLakeCity,Utah,vol.29,pp.145–66.
Knaus,M&El‐Matbouli,M2005,‘Characterisationofcarbohydrate‐bindingsitesin
developmentalstagesofMyxoboluscerebralis’,ParasitologyResearch,vol.97,no.6,pp.
505–14.
Koel,TM,Kerans,BL,Barras,SC,Hanson,KC&Wood,JS2010,‘Avianpiscivoresas
vectorsforMyxoboluscerebralisinthegreaterYellowstoneecosystem’,Transactionsof
theAmericanFisheriesSociety,vol.139,no.4,pp.976–88.
Kowalski,DA&Bergersen,EP2003,‘ThetoxicityofBayluscideandTFMtoTubifex
tubifex:implicationsforchemicalcontroloftheoligochaetehostofMyxoboluscerebralis,
thecausativeagentofwhirlingdisease’,NorthAmericanJournalofAquaculture,vol.65,
no.3,pp.171–8.
Kristmundsson,A&Richter,SH2009,‘Parasitesofresidentarcticcharr,Salvelinus
alpinus,andbrowntrout,Salmotrutta,intwolakesinIceland’,IcelandicAgricultural
Sciences,vol.22,pp.5–18.
Langdon,JS1990,MajorprotozoanandmetazoanparasiticdiseasesofAustralian
finfish,inFinfishdiseases:refreshercourseforveterinarians,PostgraduateCommitteein
VeterinaryScience,UniversityofSydney,Sydney,NSW,Australia,pp.233–55.
Whirling Disease 64 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Liu,YC,Zhou,ZG,Miao,W,Zhang,YT,Cao,YA,He,SX,Bai,DQ&Yao,B2011,‘Achitinase
fromAeromonasveroniiCD3withthepotentialtocontrolmyxozoandisease’,PlosOne,
vol.6,no.12.
Lodh,N,Kerans,BL&Stevens,L2012,‘Theparasitethatcauseswhirlingdisease,
Myxoboluscerebralis,isgeneticallyvariablewithinandacrossspatialscales’,Journalof
EukaryoticMicrobiology,vol.59,no.1,pp.80–7.
Lodh,N,Stevens,L&Kerans,B2011,‘PrevalenceofMyxoboluscerebralisinfections
amonggeneticlineagesofTubifextubifexatthreelocationsintheMadisonRiver,
Montana’,JournalofParasitology,vol.97,no.3,pp.531–4.
Lom,J&Dykova,I1994,‘StudiesonprotozoanparasitesofAustralianfishes.3.Species
ofthegenusMyxobolusbutschli,1882’,EuropeanJournalofProtistology,vol.30,no.4,
pp.431–9.
MacConnell,E&Bartholomew,J2012,‘Whirlingdiseaseofsalmonids’,inFHSbluebook:
suggestedproceduresforthedetectionandidentificationofcertainfinfishandshellfish
pathogens(2014edition),AmericanFisheriesSociety—FishHealthSection.
MacConnell,E&Vincent,ER2002,‘TheeffectsofMyxoboluscerebralisonthesalmonid
host’,inWhirlingDisease:ReviewsandCurrentTopics,BartholomewJL&WilsonJC
(eds),AmericanFisheriesSocietySymposium,SaltLakeCity,Utah,vol.29,pp.95–107.
Margolis,L,Kent,ML&Bustos,P1996,‘Diseasesofsalmonidsresemblingmyxosporean
whirlingdisease,andtheabsenceofMyxoboluscerebralis,inSouthAmerica’,Diseasesof
AquaticOrganisms,vol.25,pp.33–7.
Markiw,ME1986,‘Salmonidwhirlingdisease—dynamicsofexperimentalproductionof
theinfectivestage—thetriactinomyxonspore’,CanadianJournalofFisheriesandAquatic
Sciences,vol.43,no.3,pp.521–6.
——1992,‘ExperimentallyinducedwhirlingdiseaseII.Determinationoflongevityof
theinfectivetriactinomyxonstageofMyxoboluscerebralisbyvitalstaining’,Journalof
AquaticAnimalHealth,vol.4,no.1,pp.44–7.
Martin,RAJ,Cameron,AR&Greiner,M2007,‘Demonstratingfreedomfromdisease
usingmultiplecomplexdatasources1:anewmethodologybasedonscenariotrees’,
PreventiveVeterinaryMedicine,vol.79,no.2–4,pp.71–97.
Miller,MP&Vincent,ER2008,‘Rapidnaturalselectionforresistancetoanintroduced
parasiteofrainbowtrout’,EvolutionaryApplications,vol.1,no.2,pp.336–41.
Nehring,RB,Schisler,G,Chiaramonte,L,Horton,A&Poole,B2015,‘Assessmentofthe
long‐termviabilityofthemyxosporesofMyxoboluscerebralisasdeterminedby
productionoftheactinosporesbyTubifextubifex’,JournalofAquaticAnimalHealth,vol.
27,no.1,pp.50–6.
Nehring,RB,Thompson,KG,Taurman,KA&Shuler,DL2002,‘Laboratorystudies
indicatingthatlivingbrowntroutSalmotruttaexpelviableMyxoboluscerebralis
myxospores’,inWhirlingDisease:ReviewsandCurrentTopics,BartholomewJL&Wilson
JC(eds),vol.29,pp.125–34.
Nelson,JS1994,Fishesoftheworld(3rdedition),JohnWileyandSonsInc,NewYork.
O’Grodnick,J1975,‘Eggtransmissionofwhirlingdisease’,ProgressiveFish‐Culturist,vol.
37,no.3,pp.153–4.
Whirling Disease 65 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy OIE2015a,AquaticAnimalHealthCode2015,WorldOrganisationforAnimalHealth
(OIE),Paris,France,fromhttp://www.oie.int/international‐standard‐setting/aquatic‐
code/access‐online/.
——2015b,ManualofDiagnosticTestsforAquaticAnimals2015,WorldOrganisation
forAnimalHealth(OIE),Paris,France,fromhttp://www.oie.int/international‐standard‐
setting/aquatic‐manual/access‐online/.
Pierce,R,Podner,C,Marczak,L&Jones,L2014,‘Instreamhabitatrestorationand
streamtemperaturereductioninawhirlingdisease‐positivespringcreekinthe
BlackfootRiverbasin,Montana’,TransactionsoftheAmericanFisheriesSociety,vol.143,
no.5,pp.1188–98.
Pinder,AM&Brinkhurst,RO2000,‘AreviewoftheTubificidae(Annelida:Oligochaeta)
fromAustralianinlandwaters’,MemoirsofMuseumVictoria,vol.58,no.1,pp.39–75.
Purcell,MK,Getchell,RG,McClure,CA&Garver,KA2011,‘Quantitativepolymerase
chainreaction(PCR)fordetectionofaquaticanimalpathogensinadiagnostic
laboratorysetting’,JournalofAquaticAnimalHealth,vol.23,no.3,pp.148–61.
Rose,JD,Marrs,GS,Lewis,C&Schisler,G2000,‘Whirlingdiseasebehaviorandits
relationtopathologyofbrainstemandspinalcordinrainbowtrout’,JournalofAquatic
AnimalHealth,vol.12,no.2,pp.107–18.
Ryce,EKN,Zale,AV&MacConnell,E2004,‘Effectsoffishageanddevelopmentof
whirlingparasitedoseonthediseaseinrainbowtrout’,DiseasesofAquaticOrganisms,
vol.59,no.3,pp.225–33.
Ryce,EKN,Zale,AV,MacConnell,E&Nelson,M2005,‘Effectsoffishageversussizeon
thedevelopmentofwhirlingdiseaseinrainbowtrout’,DiseasesofAquaticOrganisms,
vol.63,no.1,pp.69–76.
Sitja‐Bobadilla,A2008a,‘FishimmuneresponsetoMyxozoanparasites’,Parasite‐
JournalDeLaSocieteFrancaiseDeParasitologie,vol.15,no.3,pp.420–5.
——2008b,‘Livingoffafish:Atrade‐offbetweenparasitesandtheimmunesystem’,
Fish&ShellfishImmunology,vol.25,no.4,pp.358–72.
Thompson,KG2007,‘UseofsiteoccupancymodelstoestimateprevalenceofMyxobolus
cerebralisinfectionintrout’,JournalofAquaticAnimalHealth,vol.19,no.1,pp.8–13.
——2011,‘Evaluationofsmall‐scalehabitatmanipulationtoreducetheimpactofthe
whirlingdiseaseparasiteinstreams’,AquaticEcosystemHealth&Management,vol.14,
no.3,pp.305–17.
Wagner,EJ2002,‘Whirlingdiseaseprevention,control,andmanagement:areview’,in
WhirlingDisease:ReviewsandCurrentTopics,BartholomewJL&WilsonJC(eds),
AmericanFisheriesSocietySymposium,SaltLakeCity,Utah,vol.29,pp.217–25.
Wagner,EJ,Smith,M,Arndt,R&Roberts,DW2003,‘Physicalandchemicaleffectson
viabilityoftheMyxoboluscerebralistriactinomyxon’,DiseasesofAquaticOrganisms,vol.
53,no.2,pp.133–42.
Wolf,K&Markiw,ME1979,‘Myxosomacerebralis—methodforstainingsporesand
otherstageswithsilver‐nitrate’,JournaloftheFisheriesResearchBoardofCanada,vol.
36,no.1,pp.88–9.
——1982,‘Myxosomacerebralis—inactivationofsporesbyhotsmokingofinfected
trout’,CanadianJournalofFisheriesandAquaticSciences,vol.39,no.6,pp.926–28.
Whirling Disease 66 Department of Agriculture and Water Resources AQUAVETPLAN–Disease Strategy Wolf,K,Markiw,ME,Cruz,JM,Galhano,MH,Eiras,J&Herman,RL1981,‘Non‐
myxosporidanblacktailofsalmonids’,JournalofFishDiseases,vol.4,no.4,pp.355–7.
Zielinski,CM,Lorz,HV,Hallett,SL,Xue,L&Bartholomew,JL2011,‘Comparative
susceptibilityofDeschutesRiver,Oregon,TubifextubifexpopulationstoMyxobolus
cerebralis’,JournalofAquaticAnimalHealth,vol.23,no.1,pp.1–8.
Whirling Disease 67