Seasonal recurrence of cowpox virus outbreaks in captive cheetahs

15 Pages·2017·7.63 MB·English

RESEARCHARTICLE
Seasonal recurrence of cowpox virus
outbreaks in captive cheetahs (Acinonyx
jubatus)
JuliaStagegaard1,AndreasKurth2,3,DanielStern2,PiotrWojciechDabrowski4,
AnnPocknell5,AndreasNitsche2,3*,LiviaSchrick2,3
1 ReePark–Safari,Ebeltoft,Denmark,2 CentreforBiologicalThreatsandSpecialPathogens,RobertKoch
Institute,Berlin,Germany,3 GermanConsultantLaboratoryforPoxviruses,RobertKochInstitute,Berlin,
Germany,4 MethodologyandResearchInfrastructure,RobertKochInstitute,Berlin,Germany,5 Finn
Pathologists,Norfolk,England
a1111111111
a1111111111 *[email protected]
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a1111111111 Abstract
Cowpoxvirusinfectionsincaptivecheetahs(Acinonyxjubatus)withhighmorbidityandmortality
havealreadybeenreportedintheUKandRussiainthe1970s.However,mostofthereported
caseshavebeensingularevents.Here,wereportatotaloffivecowpoxvirusoutbreaksinchee-
OPENACCESS
tahsinthesamesafariparkinDenmarkbetween2010and2014.Ninecheetahsshowedvary-
Citation:StagegaardJ,KurthA,SternD,
ingseverityofclinicaldisease;twoofthemdied(22%).AllepisodesoccurredbetweenAugust
DabrowskiPW,PocknellA,NitscheA,etal.(2017)
andOctoberoftherespectiveyear.Noothercarnivoreskeptatthesameinstitutionnorthe
Seasonalrecurrenceofcowpoxvirusoutbreaksin
captivecheetahs(Acinonyxjubatus).PLoSONE12 keeperstakingcareoftheanimalswereclinicallyaffected.Theclinicalpictureofcowpoxwas
(11):e0187089.https://doi.org/10.1371/journal. confirmedbyextensivelaboratoryinvestigationsincludinghistopathologicalandmolecularanal-
pone.0187089
ysesaswellascellcultureisolationofacowpoxvirus.Highanti-orthopoxvirusantibodytiters
Editor:MartinBeer,Friedrich-Loeffler-Institut, weredetectedinall9diseasedcheetahscomparedtosevencontactcheetahswithoutclinical
GERMANY
signsand13cheetahsnotindirectcontact.Additionally,wholegenomesequencingfromone
Received:February17,2017 sampleofeachclusterwithsubsequentphylogeneticanalysisshowedthatthevirusesfromdif-
Accepted:October15,2017 ferentoutbreakshaveindividualsequencesbutclearlyformacladedistinctfromothercowpox
viruses.However,theintra-cladedistancesarestilllargerthanthoseusuallyobservedwithin
Published:November9,2017
cladesofoneevent.Thesefindingsindicatemultipleandseparateintroductionsofcowpox
Copyright:©2017Stagegaardetal.Thisisan
virus,probablyfromwildrodentpopulations,wheretheviruskeepscirculatingnaturallyandis
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which onlysporadicallyintroducedintothecheetahs.Sero-positivityofvoles(Arvicolaamphibious)
permitsunrestricteduse,distribution,and caughtinzoogroundsstrengthensthishypothesis.Asaconsequence,recommendationsare
reproductioninanymedium,providedtheoriginal
givenformedicalandphysicalmanagementofdiseasedcheetahs,forhygienicmeasuresas
authorandsourcearecredited.
wellasforpre-shipmentisolationbeforecheetahexportfromzoogrounds.
DataAvailabilityStatement:Genomesequences
havebeendepositedinGenBankwithaccession
numbersKY569018-KY569022.TheGenBank
accessionnumberscanalsobefoundinthe
SupportingInformationtable.
Introduction
Funding:Theauthorsreceivednospecificfunding
forthiswork. Cowpoxviruses(CPXV)belongtothegenusOrthopoxvirus(OPV)inthefamilyPoxviridae
[1].CPXVareendemicinpartsofEuropeandWesternAsia[2].Theprimaryreservoirhost
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. seemstoberodents[3],asserologicalsurveyshaveshownthatthevirusiswidespreadamong
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Cowpoxvirusincheetahs
Europeanwildrodents[4].Inthewildrodentpopulations,thevirusdoesnotinduceobvious
signsofdiseasenordoesitseemtoaffectsurvival[5].Despiteitsname,CPXVhasnotbeen
isolatedfromcattleduringthelastthreedecades[6];instead,ithasbeendiagnosedmostfre-
quentlyindomesticcatsandzooanimals[7].Afterelephants,exoticfelidsarethesecondmost
commonlyinfectedgroupofexoticanimals[8].Casesincaptivecheetahs,withhighmorbidity
andmortality,havealreadybeenreportedintheUKandRussiainthe1970s[9,10].Indomes-
ticandlargecats,multipleskinlesions(primarilyseenonthehead,neck,forelimborpaws,
and/orintheoralcavity),conjunctivitis,and/orpurulentoculardischargemaydevelopupon
infection.Occasionally,systemicinfectionsoccurthatmaybefatalifinternalorganssuchas
thelungsareinvolved(e.g.necrotizingpneumonia)andincasesofco-infectionsorimmuno-
deficiency[11].Therouteandsiteofinfection,thedoseofvirus,aswellastheCPXVstrain
seemtoinfluencetheoutcomeoftheinfection[12].Cowpoxincidencesincatsandhumans
arereportedtobehighestinlatesummerandearlyautumn[1],whichcoincideswithnumbers
ofseroconvertingbankvoles(Clethrionomysglareolus)andwoodmice(Apodemussylvaticus)
thatpeakinthisperiod[3].
ReePark–Safariisa70-hasafariparklocatedonaruralsiteintheeastofJutland,Denmark.
Since2001,theinstitutionhasbeenkeepingcheetahs.Thetotalnumberofcheetahskeptinthe
groundsvaries;however,usuallythezoohouses2–3adultmales,3–4adultfemales,andtheir
offspringuntiltheyareapproximatelytwoyearsold.Therearetwoseparatecheetahholding
facilities(Fig1),oneinthesafaripark(Fig1A)withfourpensandoneatafarm(Fig1B)with
sixpenslocated1kmawayfromthepark.Allenclosureshavenaturalvegetationwithhigh
grassandsmallbushes.Mostoftheenclosuresconnecttoastablevaryinginsizebetween4
and9m2.Thereisnodirectcontactbetweentheenclosuresasallfencesarecoveredtoavoid
directvisibility.Thesameanimalkeeperstakecareofbothlocations.
BeginningwiththefirstoutbreakinOctober2010,atotaloffivesevereclinicalpoxvirus
infectionswerediagnosedincheetahsintheyears2010,2011,2012,and2014.Resultsof
histologicalandmoleculardiagnostics,phylogeneticanalysisofgenomesequences,anti-
bodytitersofcheetahsandotheranimals,aswellasmanagementstrategiesarepresented
anddiscussed.
Materialsandmethods
Animalspecimens
Allorgansamplesweretakenfromdiseasedanimalsinthecontextofclinicalinvestigations
exclusivelyfordiagnosticpurposesorfromanimalsthatnaturallyhaddiedfromdisease.
Thereforenoethicscommitteehasbeeninvolved.
Noneoftheanimalsmentionedinthisarticlewaseuthanized.Allbloodsampleswere
drawnduringanesthesia(inexoticcarnivoresatReeParkalwaysacombinationofketamine
andmedetomidine,oftenplusmidazolambyremoteinjection)foreitherpre-shipmenthealth
screeningordiseasediagnostics.Itisstateoftheartamongzoosalwaystofreezeextrablood
samplesdown,eitherforforensicorforretrospectiveinvestigationalpurposes.
Histopathology
Samplestakenfromthelipandthenasalplanum,askinnodulefromtheindexpatientinclus-
ter1,andthetoewoundfromtheindexpatientincluster2werefixedin10%neutralbuffered
formalinandthenembeddedinparaffin.Sectionsofthesampleswereroutinelyprocessed,
sectionedat5μm,andstainedwithhematoxylinandeosinforhistologicalexamination.
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Cowpoxvirusincheetahs
Fig1.OverviewofcheetahenclosuresatReePark–Safari,Denmark.Enclosures‘A’areinsidethesafaripark,enclosures‘B’atafarmlocated
1kmawayfromthepark.
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Moleculardiagnostics
DNAwasextractedfrommultiplespecimensbyusingtheQIAGENQIAampDNAMiniKit.
TheDNAwasanalyzedbyOPV-specificreal-timePCR(rpo18)[13],CPXV-specificreal-time
PCR[14],andreal-timePCRdetectingthecellularc-mycgeneforrelativequantification[15].
Additionally,conventionalPCRwasperformed,amplifyingtheopenreadingframe(ORF)of
thehemagglutinin(HA)genefollowedbysangersequencing[15].
Viralgenomesequencingandphylogeneticanalysis
Fullviralgenomesequencesweregainedfromonespecimenofeachclusterbyusingeither
DNAfromcellcultureisolates(ifavailable)orDNAdirectlyisolatedfromcrustorskintissue.
Samplesfromclusters1–4weresubjectedtoIonTorrentPGMsequencing(IonXpressPlusFrag-
mentPreparationKit,IonPGM™Sequencing200Kit,Ion318Chip).Forthesamplefromclus-
ter5Illuminasequencingwasutilized(Nexteralibrary,TruSeqRapidPEClusterKitv2,HiSeq
RapidSBSKitv2,250+250basessequencingonaHiSeq1500instrumentinrapidmode).
Assemblyofthesequencedrepresentativespecimens’genomeswasperformedinthreesteps.
First,readsoriginatingfromthehostwereseparatedfromthoseoriginatingfromthevirusby
usingRAMBO-K[16].Thengenomeswereassembledbyusingacombinationofvelvet[17]
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Cowpoxvirusincheetahs
andSOAPdenovo2[18],yieldingtheentireviralgenomeinasinglecontigofover200kbin
lengthinallcases(seeS1Table).Finally,errorcorrectionwasperformedbymappingallreads
fromeachsampletotherespectiveassembledgenomebyusingbowtie2[19],anddiscrepancies
werevisuallyinspectedand,wherenecessary,correctedmanuallybyusingGeneiousR9[20].
Forphylogeneticanalysis,analignmentoftheassembledgenomeswithallCPXVfull
genomesandasinglerepresentativesequenceofeveryotherOPVspeciesavailableinGen-
Bankwasperformedbyusingmafft[21].Inordertoremovespuriousphylogeneticsignals,
low-qualityalignmentregionswerestrippedfromthealignmentbyusingGBlocks[22],yield-
ingastrippedalignmentof136,924gap-freepositions.Aphylogenetictreewasthencalculated
fromthisstrippedalignmentbyusingPhyML[23](GTR+ɣ,4substitutionratecategories,
aLRTbranchsupports).
Virusisolation
Clinicalspecimenswerehomogenizedinphosphate-bufferedsalineandaddedatdifferent
dilutionstoconfluentlayersofVerocells(ECACC,Cat.No85020206)cultivatedwithmedia
with2%fetalcalfserumsupplementedwithpenicillin/streptomycinin24-wellcellculture
plates.Oncecytopathiceffectswereevidentunderthemicroscope,passagingwasinitiated.
Serology
Serumsamplesweretestedforantibodiesbyimmunofluorescentassay(IFA)byusingCPXV-
infectedHEp-2cellsandaFITC-conjugatedgoat-anti-humanIgG(H+L)asdescribedprevi-
ously[24]orbyindirectELISAbyusingvacciniavirus-infectedHEp-2celllysateasantigen.
Tothisaim,UV-inactivatedcelllysatewaspreparedbyinfectingHEp-2cells(ATCC,CCL-
23™)withvacciniavirusstrainNewYorkCityBoardofHealth(ATCC,VR-1536™)byusing
RIPAbuffersupplementedwithproteaseinhibitorcocktail(ThermoFisherScientific)as
describedbefore[25].
ForELISA,PolySorpmicrowellplates(Nunc)werecoatedwith100μLoflysateofinfected
ornon-infectedHEp-2cellsat4μg/mLin0.1Mcarbonatebuffer(pH9.6)overnightat4˚C.
Betweeneachstep,plateswerewashedwith4×300μLofwashingbuffer(100mMTris,pH
7.5,150mMNaCl,0.05%Tween20).Plateswereblockedatroomtemperaturefor1hour
with200μLperwellof3%bovineserumalbumin(BSA,CarlRoth)inwashingbuffer.Subse-
quently,100μLperwellofnon-inactivatedserawereincubatedata1:100dilutioninwashing
buffersupplementedwith0.25%BSAfor1hourbeforedetectionwitheithergoatanti-catIgG
(H+L)HRP,goatanti-dogIgG(H+L)HRP,orgoatanti-mouseIgG(H+L)(Dianova,usedata
1:5000dilution).Finally,signalsweredevelopedfor15minutesbyusing100μLofSeramun-
SlowTMBsubstrate(Diavita)perwellbeforethereactionwasstoppedbyadditionof100μL
of0.25MH SO .Theresultingabsorptionwasreadat450nmreferencedto620nmatan
2 4
ELISAreader(Tecan).Eachserumwastestedintworeplicatemeasurementsonbothinfected
andnon-infectedHEp-2celllysate.Bindingsignalsagainstnon-infectedHEp-2celllysate
weresubtractedfromsignalsagainstvacciniavirus-infectedcells,andserawithadifferential
bindingsignalabove0.05wereconsideredpositive.
Results
Clinicalcases
Anoverviewofgroupsofcheetahsaffectedinfiveoutbreaksbetween2010and2014isgiven
inTable1,whileexamplesofclinicalpresentationsfortypicalandatypicallesionsobserved
duringtheoutbreaksareshowninFig2.
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Cowpoxvirusincheetahs
Table1. SummaryofclinicalsymptomsofthecheetahsaffectedbyfiveCPXVoutbreaksatReePark,Denmark.
Cluster ID Age Enclosure Dateoffirstclinicalsigns Clinicalsigns Daysonsetofclinicalsignstoimprovement/death
1 Sheppard§ 18months A–1 Oct4,2010 Severefaciallesions 12
Tosha 8years Oct15,2010 Ulcersontongue Unknown
Grey 18months Oct17,2010 Lung 12†
Izzy 18months Oct18,2010 Fewfaciallesions Unknown
Split 18months -
2 TopCut§ 7months B–1 Aug31,2011 Generalized 19†
Clyde 7months Sep13,2011 Ulcersontongue Unknown
Aduke 6years -
Bonnie 7months -
3 Nuru§ 7years B–2 Sep7,2012 Severeskinlesions 20
4 Hurley§ 30months A–1 Oct5,2012 Faciallesions 13
Jack 30months -
Sawyer 30months -
5 Nova§ 30months A–1 Sep11,2014 Severeskinlesions 11
Heidi 30months -
Novi 30months -
Listedaretheaffectedanimalsaswellasthecontactanimalslivinginthesameenclosurebutnevershowingclinicalsigns.Enclosures:A—situatedinside
thesafari-park,B–situatedatafarm1kmawayfromthepark.
§Indexcase
†death,Ageatthetimeoftheoutbreak.
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Cluster1–2010(Sheppard). ThefirstclinicalcaseoccurredintheparkinOctober2010
(Fig1,enclosureA1),whereamother(Tosha)washousedtogetherwithoneson(Sheppard)
andthreedaughters(Grey,Izzy,Split).Theindexpatientwasthe1.5-year-oldsonSheppard.
Hepresentedwithulcerativeskinlesionsonthelowerlipandnostril.Theaffectedareaswere
biopsiedandtheanimalreceivedlong-actingbroad-spectrumantibiotic(8mg/kgcefovecin
oncei.m.).Theanimalwasisolatedinthestabletendaysafterinitialsymptomswhenhisto-
pathologicalresultsofthesubmittedsamplesindicatedpresumptivepoxvirusinfection.
Elevendaysafterthefirstlesionshadbeennoticedinthemale,afissurewasobservedon
thetongueofthemother(Tosha),andonthefollowingdayoneofitssisters(Izzy)hadalesion
ontheupperlip.Thenextday,respiratorydistressbecameobviousinanothersister(Grey).At
thattime,theentiregroupwasisolatedandputinsidethestable.Cefovecinwasgivenby
remoteinjectiontoalloftheminordertopreventsecondarybacterialinfections.
ThefemaleGreywithrespiratorysymptomsdied12daysafteronsetofsymptomsandwas
foundpartlyeatenbytheothers.Theotheranimalsrecovereduneventfully.Onlyoneofthe
fiveanimals(Split)didnotshowanysymptoms.
Cluster2–2011(TopCut). Afterthefirstoutbreakinthepark,thesecondoneoccurred
atthefarm(Fig1,enclosureB1)inAugust2011,inafamilygroupconsistingofabreeding
female(Aduke)andtwomale(TopCut,Clyde)andonefemaleoffspring(Bonnie).Theindex
patient,a7-month-oldmale(TopCut),presentedwithapoorlyhealingwoundonthefront
paw.Uponclinicalexaminationofthecheetahundergeneralanesthesia,multiplepapularder-
malnoduleswerenoticedontheentirebody,andpoxvirusinfectionwassuspected.Despite
antibioticandanti-inflammatorytreatment(initially8mg/kgcefovecini.m.once,fromday2
clindamycin11mg/kgBIDp.o.,andmeloxicam0.05mg/kgSIDp.o.),theanimaldied19days
afterthediseasewasfirstnoticed.
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Cowpoxvirusincheetahs
Fig2. Exemplaryclinicalpresentationsfortypical(A)andnon-typical(B)poxviruslesionsobservedduringtheoutbreaks.A.
ProgressionoftheinfectionobservedforNova(cluster5)fromd2whentypicaldermalnoduleswereclearlyvisible,d8atthepeakofclinical
diseasewithmultipleclassicalskinwounds,andd52withremainingpoxscars.Lesionswerepredominantlylocatedinthefaceoftheanimalbut
werealsodistributedoverthebodyandlegs.B.AtypicalulcerativeskinlesionsobservedonSheppard(cluster1).Thelesiononthelowerlipis
shownintheinsetontherightforbettervisibility.(d=daysafterfirstclinicalsigns).
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Onlyoneofitstwosiblings(Clyde)developedanulceronthetongueonday14,theother
sibling(Bonnie)andthedamdidnotshowanysignsofinfectionatall.Theanimalsremained
togetherintheirenclosurethroughoutthediseaseprocess.
Cluster3–2012a(Nuru). InSeptember2012,a7-year-oldmale(Nuru),housedaloneat
thefarmintheneighboringenclosuretocluster2(Fig1,enclosureB2),developedseverepox-
likelesionsintheface.Withindays,theentirebodywascoveredbyvisiblepustules.Toavoid
secondaryinfectionsandtoreducescratching,theanimalwaskeptunderoralantibiotics(5
mg/kgenrofloxacinSIDp.o.)andnon-steroidalanti-inflammatories(0.05mg/kgmeloxicam
SIDp.o.)untilobviousimprovementonday20.Herecovereduneventfullyandwasnotiso-
latedduringtheperiod.
Cluster4–2012b(Hurley). Onemonthlater,inOctober2012,a2.5-year-oldmale(Hur-
ley),housedatthepark(Fig1,enclosureA1)togetherwithhistwobrothers(Jack,Sawyer),
hadtypicalpoxlesionsonlyintheface.Herecoveredafter13dayswithoutanytreatment.No
lesionswereseeninthesiblings.
Cluster5–2014(Nova). Startingin2013,allcheetahswerevaccinatedwithModifiedVac-
ciniaAnkara(MVA)smallpoxvaccine,followingthevaccinationregimeasrecommendedby
theproducer.However,anotheroutbreakoccurredinSeptember2014inthepark(enclosure
A1)wherethreefemalesiblingswereliving(Nova,Novi,Heidi).The2.5-year-oldNova
showedsevereskinlesionsconsistentwithcowpoxintheface,onherbody,andherlegs.She
receiveddailyoralantibiotics(5mg/kgenrofloxacinSIDp.o.)andimprovedafter11days.Her
twosistersdidnotshowanylesions.
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Cowpoxvirusincheetahs
Histopathology
SectionsfromthelipandnasalplanumofSheppard(cluster1)showeddiffuseulcerativeexuda-
tivenecroticcheilitisandnasaldermatitis,respectively–theulceronthenasalplanumwassuper-
ficiallycolonizedbymixedbacteria.Sectionsfromtheskinnoduleshowedsimilarulceration,
exudation,andnecrosis,butalsoshowedsomeareaswithintactepidermisandhairfollicles.
Keratinocytesintheepidermisandinsomehairfolliclewallsshowedextensivedisruptionand
hydropicdegeneration,oftenaccompaniedbyonetoseverallarge,angular,eosinophilic,cyto-
plasmicinclusionbodies.Thecombinationofthenecroticandulcerativeinflammationandthe
cytoplasmicepithelialinclusionbodiesledtoapresumptivediagnosisofcutaneouspoxviral
infection,withsuspicionofCPXVinfection.Sectionsoftheskinatthesiteofthetoewound
fromTopCut(cluster2)showedverysimilarhistologicallesions,comprisingulcerativenecrotic
dermatitis,folliculitis,andperifolliculitis,againwithsimilarcytoplasmiceosinophilicinclusion
bodiesinkeratinocytes,supportingthepresumptivediagnosisabove.
Moleculardiagnostics
Samplesfromallindexcases(Sheppard,TopCut,Nuru,Hurley,Nova)andthedeadfemaleof
cluster1(Grey)weresubmittedtotheGermanConsultantLaboratoryofPoxvirusesatthe
RobertKochInstitutefordiagnosticsandfurtherclassificationoftheviruses.PCRanalysis
showedhighOPV-DNAloadswhennormalizedtocellularDNA(c-myc)inmultiplespeci-
mensfromseveralaffectedanimals(Table2).Viralloadwasespeciallyhighinthedeceased
Table2. Real-timePCRresultsofdifferentspecimensfromsymptomaticcheetahsatReePark.
Cluster ID Timefromfirstsymptomstosamplingind Material Cq Cq ΔCq
OPV c-myc (OPV—
c-myc)
1 Sheppard§ 3 Skinnodule(lip,formalin-fixed) 28.2 34.2 -6.0
Grey† 12 Lungtissue*# 9.8 20.7 -10.9
12 Blood 19.9& - -
12 Vulvarswab 29.9 21.6 8.3
12 Oralswab 24.0 23.1 0.9
12 Rectalswab 29.4 22.3 7.1
2 TopCut§† 19 Skintissue* 15.0 21.3 -6.3
Oralswab 24.3 26.2 -1.9
Rectalswab 19.8 23.7 -3.9
Preputialswab 15.7 22.6 -6.9
Bodyfluidsabdominalcavity 25.0& - -
Lungtissue 23.4 24.7 -1.3
Blood 17.8& - -
3 Nuru§ 11 Crust*# 13.1 20.5 -7.4
4 Hurley§ 7 Crust*# 20.7 20.5 0.2
5 Nova§ 18 Crust* 17.8 20.5 -2.7
SymptomaticanimalsfromeachclusterweresampledforPCRanalysis.MultiplespecimenswerecollectedfromthedeceasedanimalsGreyandTopCut.
ForeachsampleOPVDNA(locatedintherpo18gene)wasquantifiedinrelationtocellularc-mycDNA.LowervaluesforΔCqindicatehighervirusloadsin
arespectivetissue.Cq,quantificationcycle;OPV,orthopoxvirus
§indexcase
†death
&Cqvalueper5μlofDNA
#materialusedtoobtaincellcultureisolate
*materialappliedtoCPXV-specificreal-timePCR,HA-sequencing,andnext-generationsequencing.
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Cowpoxvirusincheetahs
Fig3.PhylogeneticplacementofCPXVfromoutbreakclustersincheetahsamongallknownCPXVgenomesandonerepresentativesequence
fromeveryotherOPVspecies.Maximumlikelihoodtreebasedonstrippedwhole-genomealignment.TherepresentativestrainsoftheCPXVclustersfrom
cheetaharehighlightedinred.GroupsofCPXVstrainsbelongingtoknownoutbreaksarecoloredinblue,samesuperscriptsdenotethesameoutbreak.The
clusteringbetweenthesequencesfromtheknownoutbreaks(blue)ismuchtighterthanbetweenthesequencesfromtheoutbreaksincheetah(red).All
branchsupports(aLRT[26])areabove0.99.
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animals(GreyandTopCut)withextraordinarilyhighviralloadsdetectedintheblood,indi-
catingasystemicinfection.
Foreachcluster,selectedsampleswereanalyzedfurtherasindicatedinTable2.ACPXV-
specificreal-timePCRandHAsequencingconfirmedtheinfectionwithOPVandfurtherclas-
sifiedthecausativeagentasCPXV.Viruswasisolatedfromcellcultureforsamplesindicated
inTable2.
Viralgenomesequencingandphylogeneticanalysis. Furthermore,awholegenome
sequencewasreconstructedfromonesampleofeachcluster(forselectionofmaterialsee
Table2,forcontiglengthandaccessionsseeS1Table),andphylogeneticanalysiswasper-
formed.Intheresultingtree(seeFig3),thefiverepresentativeoutbreaksequencesfromRee
Parkclearlyformadistinctclade,withintra-cladedistancesmuchlowerthanthedistanceof
anyoftheoutbreaksequencestoanyotherknownCPXV.However,theintra-cladedistances
arestilllargerthanthoseobservedwithincladesformedbyotherCPXVknowntocomefrom
distinctoutbreakevents.
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Serology. Allserologicalresultsofbloodsamplesfromcheetahsobtainedduringor
shortlyaftertheoutbreaksaswellasresultsfromfollow-upsandcheetahswithintheinstitu-
tionthathavenevershownanysymptomsofapoxvirusinfectionarecollectedinTable3.All
symptomaticanimalsdevelopedhighOPV-antibodytiterswithinthefirstmonthaftersymp-
tomonsetwithIgM(cid:21)1:320andIgG(cid:21)1:20,480.TheIgMtiterwasalreadyclearlyelevatedin
thetwoseratakenfromSheppardandTopCut3daysaftersymptomonsetanddecreased
Table3. Anti-OPVantibodytitersofcheetahsatReePark.
Cluster ID Dateofbloodsampling Timefromfirstsymptomstosampling IgM IgG
1 Sheppard§ Oct7,2010 3days (cid:21)1:5,120 1:20,480
Grey† Oct29,2010 12days 1:320 1:81,920
Izzy Dec5,2010 2months 1:80 1:81,920
Split Dec6,2010 1:80 1:20,480
2 TopCut§ Sep2,2011 2days 1:5,120 1:20,480
TopCut§† Sep19,2011 19days 1:1,280 1:20,480
Bonnie Jun17,2015 1:20 1:1,280
3 Nuru§ Jun29,2014 22months 1:80 1:20,480
4 Hurley§ Oct12,2012 7days 1:1,280 1:81,920
Hurley§ Oct7,2013 12months 1:20 1:5,120
Sawyer& Jul15,2014 1:20 1:1,280
Jack& Jul15,2014 1:80 1:1,280
5 Nova§& Sep29,2014 18days 1:320 1:20,480
Nova§& Dec10,2014 3months 1:20 1:20,480
Novi& Dec10,2014 <1:20 1:1,280
Heidi& Dec10,2014 1:20 1:1,280
controlanimals YellowEye Jul2,2007 <1:20 1:80
Desert Sep9,2011 <1:20 1:20
Cimber Mar7,2012 <1:20 1:320
Sterling Mar7,2012 <1:20 1:20
Suna Jul10,2012 1:20 1:80
Duma May22,2013 <1:20 1:80
Hollaender& Jun28,2014 1:80 1:320
Abayomi& Jan28,2015 <1:20 1:320
Maya& Nov2,2015 <1:20 1:320
Sarah& Nov2,2015 <1:20 1:320
controlanimals* Tosha* Sep14,2010 <1:20 1:80
Jack* Feb21,2011 <1:20 1:80
Sawyer* Feb21,2011 1:20 1:320
Kate* Sep5,2012 <1:20 1:80
Anti-OPVantibodytitersweredeterminedbyImmunofluorescenceassay(IFA).Intheupperpartofthetable(clusters1–5)resultsfromCPXV-infected
individualsareshownwithindicationoftimespanfromsymptomonsettosampling.TheseaffectedanimalsdevelopedhighOPVantibodytiters.
Furthermore,titersfromanimalsthatsharedthesameenclosurebutremainedsymptomfreeareshownintheupperpartofthetable.Theseanimalsshow
onlymoderateantibodyresponses.Animalsthathadneverbeenindirectcontactwithanaffectedanimalandthusareconsideredascontrolanimals
developednooronlylowantibodyresponses.Theseresultsareshowninthelowerpart.Inourexperience,onlyIgMtiters(cid:21)80andIgGtiters>320are
consideredmeaningful.
§Indexcase
†death
*samplesfromanimalsinvolvedinclusters1–5takenbeforetheoutbreak
&previouslyvaccinatedwithMVA.
https://doi.org/10.1371/journal.pone.0187089.t003
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Cowpoxvirusincheetahs
Table4. OPVseropositivityofotheranimalskeptattheinstitutionorcaughtinzoogroundsbetween2011and2015.
Keptattheinstitution(Inst)/Wildcaught(Wild) Species Noofindividualstested Noofseropositiveanimals
Inst Lion(Pantheraleo) 13 3
Inst Sumatrantiger(Pantheratigrissumatrae) 1 1
Inst Leopard(Pantherapardus) 1 0
Inst Sandcat(Felismargarita) 2 0
Wild Domesticcat(Feliscatus) 1 1
Wild Redfox(Vulpesvulpes) 1 1
Wild Watervole(Arvicolaamphibius) 21 14
SerostatuswasdeterminedbyIFAand/orELISA,whereasanIgG(cid:21)1:320ordifferentialELISA>0.05isconsideredpositive.Anti-OPVantibodieswere
detectedinmultipleanimalsfromdifferentspecies.
https://doi.org/10.1371/journal.pone.0187089.t004
withinthecourseofinfection.Ontheotherhand,animalsthatwerekepttogetherinthestable
orenclosurewiththesymptomaticanimalsbutremainedsymptom-freedevelopedonlymod-
erateantibodytiters,indicatingasymptomaticandmildinfections.Theanimalsthathadnever
beenincontactwithsymptomaticanimalsshowednooronlylowOPV-antibodyresponses
(IgM(cid:20)80andIgG(cid:20)1:320),whereasinourexperienceonlyIgMtiters(cid:21)80andIgGtiters>
320areconsideredmeaningful.
Additionally,otheranimalskeptorcaughtinthezoowereanalyzedforOPVantibodysta-
tus.Therefore,anewELISAwasestablishedwhichshowedexcellentagreementwithIFAdata
(seeS1Fig).Anoverviewofseroreactivityinfelinesotherthancheetahskeptinthezoo,as
wellasvolesandotheranimalscaughtinzoogrounds,isgiveninTable4.Althoughnoneof
theseanimalsshowedsymptomscompatiblewithCPXVinfection,OPVseropositivitywas
shownformultipleanimalsfromdifferentspecies.Incontrasttothestrongseroreactivity
observedinsymptomaticcheetahs,heretheoverallantibodyreactivityagainstOPVastested
byIFAandELISAwaslow(maximumIFAtitersof1:1,280).Additionally,serafromfiveani-
malkeeperstakingcareofthecheetahswereanalyzedforOPV-antibodieswithinmonthsof
theoutbreaks(datanotshown).Noneofthekeepersdevelopedsymptomsandtheyshowed
negativeoronlyweaklypositiveOPV-antibodytitersbyIFA,indicatingnosevereacute
infection.
Discussion
CheetahsseemtobemorepronetoCPXVinfectionsthanotherexoticcatspecies[9,10,27].
DuringanoutbreakatWhipsnadeParkintheUKin1977,twooutofthreesymptomaticchee-
tahsdied;anothersixcontactanimalsdidnothaveanyclinicalinfection[9].Incontrast,dur-
ingtheoutbreakatMoscowZoo,Russia,in1973[10],manydifferentfelinespecies,including
twocheetahs,allhousedinthesamebuilding,fellillandthemajoritydied.Inarecentout-
breakatChesterZoo,UK,in2012,afamilygroupoffivecheetahsgotsickandtwo4-month-
oldcubsdied,butfourothercheetahsinthesameinstitutionwerenotaffected[27].Even
thoughothercarnivoreswerealsokeptatWhipsnadeandChester,onlycheetahsdeveloped
clinicalpoxvirusinfection.Inbothevents,onlycontactanimalsbecamesick,indicatinghigh-
qualityhygienestandardspreventingtransmissionthroughstaff.Theseobservationsarein
accordancewiththeoutbreaksatReePark:althoughmanyotherfelinespecies(seeTable4)
arekeptundersimilarconditions,incloseproximitytothecheetahs,andarelookedafterby
thesamekeepers,noneoftheanimalsorkeepershasevershownanysymptoms.Thereasonof
cumulatedCPXVinfectionsofcheetahscomparedtootheranimalspeciesmightbeareduc-
tionofgeneticdiversity,correlatingwithanincreasedvulnerabilitytoinfectiousdiseases[28],
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