Endemic Circulation of Europeanbat Lyssavirus Type1 in Serotine

Endemic Circulation of EuropeanBat Lyssavirus Type1 in Serotine

Bats, Spain

Sonia Vázquez-Morón,*† Javier Juste,‡ Carlos Ibáñez,‡ Eduardo Ruiz-Villamor,§ Ana Avellón,* Manuel Vera,*

and Juan E. Echevarría*†

To determinethepresenceof Europeanbatlyssavirus type1insouthernSpain,westudied19coloniesof sero- tinebats(Eptesicusisabellinus),itsmainreservoir,during

1998–2003. Viral genomeandantibodies weredetectedin healthybats, whichsuggestssubclinicalinfection. Thedif- ferenttemporalpatternsofcirculationfoundineachcolony indicateindependentendemiccirculation.

he serotine bat (Eptesicus serotinus) is considered themainreservoirforEuropeanbatlyssavirustype1 (EBLV1).Recently,southernIberianpopulationsofthis specieshavebeenclassifiedasE.isabellinus(1),abatspe-

ciespreviouslyknown toexistonlyinNorth Africa.

In1989,5EBLV1-infected serotinebatswerefound deadduringasurveyofnaturalcoloniesinHuelva(An- dalusia).TheprevalenceofEBLV1antibodieswasupto

20%amongcompletelyhealthybatsthatwererecaptured1 yearlater,providingthefirstdirectevidenceofthesurvival ofserotinebatsafterEBLV1infection(2).Furthermore, highseroprevalenceandpresenceofviralRNAintheoral cavityandbloodstreamofdifferentlyssavirusspecieshave beenreportedinhealthybatscapturedinnaturalcolonies inwhichthenumbersofdeathshavenotincreased (3–5), showingdirectevidenceofsubclinical orasymptomatic diseaseafterviralinfection. EvenviralRNAandantigens havebeendetectedinthebrainsofhealthycaptivebats(6). However,reportsofexperimental lyssavirustransmission havedrawn discrepantconclusions(7–9).

*InstitutodeSaludCarlosIII, Majadahonda,Madrid,Spain;†Cen- tro deInvestigaciónBiomédicaenReddeEpidemiologíaySalud Pública(CIBERESP),Spain;‡EstaciónBiológicadeDoñanaCSIC, Sevilla.Spain;and§LaboratorioCentraldeVeterinaria deSanta Fe,Granada, Spain

The Study

During1998–2003,atotalof1,030E.isabellinusfrom

19colonieslocatedintheprovincesofHuelva,Seville,and Granada(Andalusia),insouthernSpain,weresampled.We focusedonthisregionbecauseallEBLV1casesreportedin SpainwerefromsouthernSpain.Distances betweencolo- niesvariedfrom0.5 to317 km.

Batswerecapturedmainlyinso-calledmaternitycolo- niesmadeupofadultfemalesandyoungofbothsexes. Eachanimal wasbanded andassessedforsex,age,size, andweight.Atotalof150(14.5%)wererecaptured. The frequencyofrepeatedcapturesreached22.7%inindividual batsfrom3coloniesthatweresampledinall6yearsofthe study.Allrecaptured batswerealwaysfoundinthesame colonyinwhichtheywerefirstmarked.

ViralRNAwasdetectedin34(2.8%)of1,226oropha- ryngealswabspecimens from33batsof8colonies(1bat wasresampledaftera 1-weekinterval).Onebatthattested positivewasrecaptured andtestednegativeinafollowup samplingeffort.Positiveresultswere identifiedasEBLV1 bydirectsequencing aspreviouslydescribed(4,10).Sam- plesforreversetranscription–PCRwerestoredinabuf- ferthatwasdesignedforRNApreservationbutwasnot suitableforkeepingthevirusviableforisolationoncell culture.

Atotalof626plasmasamplesweretestedforEBLV1- specificantibodiesbyusingamodification oftherapidflu- orescentfocusinhibitiontest(11),but77(12.3%)ofthem werefoundtobetoxictocells.EBLV1antibodieswere found in51 (9.3%) of theremaining549 from13 colonies. Only2of22samplesshowingviralRNAintheoralcavity wereantibodypositive(Table).Thirteenantibody-positive batswererecapturedinahealthyconditioninthefollowing campaigns.

Thetemporalpatternofcirculation wascompletely differentin eachcolony(onlineAppendixTable,available from www.cdc.gov/EID/content/14/8/12653-appT.htm). Only1colonyshowedcontinuouscirculationfrom1998 through2002.

Brainswereobtainedfrom20batsofthe samecolony thathadbeenstudiedpreviously(4).Differences inbody conditionbetweennoninfected bats,batswithpositive oropharyngeal swabsonly,andbatswithpositiveoropha- ryngealandbrainspecimensweretestedbyananalysis ofvariance; indexofbodycondition wasthedependent variable.Individualbodyconditionwasexpressedasthe residualsfromananalysisofcovariance withanopti- mizeddesign,includingbodymassasdependentvariable; forearmlengthasacovariate;andsex,age,andyearas fixedfactors.Wefoundasignificant negativeassociation (Figure1) betweenRNA presenceandbody condition (F=11.78;degreesoffreedom=2,281;p<0.001,n=292). Posthoctestsindicatedthatonlybatswhosebrainstested

Table.Europeanbatlyssavirustype1inEptesicusisabellinus

bats,Spain,1999–2003*

TypeoftestingRT-PCRRFFIT Totalcaptures1,226 626

No.colonies 19 13

Totalno.(%)positive 34(2.8) 51(9.3)

1998

No.captures164151

No.(%)positive4(2.4)10(6.6)

1999

No.(%)captures16190

No.positive4(2.5)5(5.6)

2000

No.captures204128

No.(%)positive16(7.8)12(9.4)

2001

No.captures20996

No.positive04(4.2%)

2002

No.captures287100

No.(%)positive10(3.4)5(5.0)

2003

No.captures20154

No.positive00

*Resultsobtainedduringactive surveillance.RT-PCR,reverse transcription–PCR, RFFIT,rapidfluorescentfocus inhibitiontest.

positivehadasignificantly worsebodycondition(Tukey honestlysignificantdifferences[HSD]test–2.33,p<0.001, andHSD–2.27,p<0.001,respectively). Differencesin body condition between oropharyngeal swab–negative and oropharyngealswab–positivebats werenot significant (TukeyHSD 0.57, p =0.99).

Brain,cerebellum, andspinalcordfrom1batcarcass withnaturalEBLV1infection werestudiedbyhistopatho- logicandhistochemicaltechniques.Thebatwascaptured whileflyingbutdiedduringmanipulation,whichisanex- tremelyrareevent.Thebrain andoropharyngealswabwere positiveforEBLV1byreversetranscription–PCR,andthe brainsmear wasalso positivebyimmunofluorescence.His- topathologicstudiesshowedmoderateneuronaldegeneration characterizedby neuronalhyperchromatosis,chromatolysis, andsatellitosis(Figure2,panel A).TheSellersstain results werenegative, provingtheabsenceofNegri bodies (Figure

2,panelB).Theoccasionalpresenceofbasophilicintracyto- plasmicstructures inneuronslocatedinthecerebralcortex thatunderwentnecrobiosiswasinterpretedasaneurophagic processthatwastakingplaceintheglialcellsbecausethese inclusionswereshowntobepositivebytheFeulgenreaction (Figure2,panelC),whereastheNegribodieshaveastrong- lyacidophilicnature. Moderategliosis withoutperivascular infiltrationwasobserved(Figure2,panel2D).

Conclusions

Despitethefactthatserotinebats(E.serotinusand

E.isabellinus)arethemostfrequently involvedspeciesin

casesofbatlyssavirusexposureinhumansinEurope,most publishedsurveysofEBLV1 infectioninnatural batcolo- nieshavefocused onotherbatspecies (5,12).Datafrom serotinebatshavebeenobtained byeitherserologic ordi- rectdetectiontechniquesbutnotfromboth(2,4),asin this report.

Ourrecapture datashowanabsenceofswitching amongcolonies,evenbetweencolonieslocatedonlya few kilometers apart.Thisresultindicateshighlyphilopatric behavioramongfemalebats,ashasbeenconfirmedina recentmtDNA-basedstudyofthesecolonies (J.Juste,un- pub.data).Thiscouldbethecauseofthedifferenttem- poraldistribution ofthepositiveresultsobservedineach colony,whichsuggestsapatternofindependent endemic viralcirculation differentfromthemodel,basedonperi- odicepidemic wavesoffastviralspreading proposedfor themouse-earedbat(Myotis myotis) (12).

Theanalysisofbodyconditionindexasameasure of physiologic condition gives additional evidence for mildorsubclinicalinfectioninthepreviouslydescribed long-termsurvivalofEBLV1RNAorantibody-positive bats (2,4,5,12). Similarbody conditionvaluesbetween batswithoropharyngeal swabsthatwerepositiveforthe virusandthosethatwerenegativecouldbeinterpreted asarecentvirusinfectionforwhichnosymptomshave

Figure1. Relationshipbetweenbodyconditionindex(mean± standarderror)anddiagnosisofEuropeanbatlyssavirustype1 byreversetranscription PCR.Malesandfemalesarerepresented asfilledandopencircles,respectively.1, onlynegativeinoro- pharyngealswabandbrainspecimens(z n=49;cn=225);

2,positiveinoropharyngealswabandbrainspecimens(zn=1;

cn=4);3,positiveinoropharyngealswabbutnegativeinbrain specimen (zn =3;cn =4).

European BatLyssavirus, Spain

Figure2.PathologicimagesobtainedfromacarcassofEptesicusisabellinus.Thebatwascapturedwhileflyingbutdiedduring

handling.BrainspecimenwaspositiveforlyssavirusantigensbyimmunofluorescenceandforEuropeanbatlyssavirus1RNAbyreverse transcription–PCR.A)Neuraldegenerationinbrainbyhematoxylinandeosinstain(H&E);magnification×400.B)NegativeSellerstain inspinalcordindicatingtheabsenceof Negribodies;magnification×400.C)PositiveFeulgenreactioninbrain,glialcellneurophagia; magnification ×200. D) Focal proliferation ofglial cells by H&E;magnification ×100.

developed.However,1lyssavirus-positive batshowedno viralRNA3years later. Onebateven showedRNAinthe oralcavityinconsecutivesamplesseparatedby1week. Ourfindingssuggestthat asymptomaticEBLV1RNAcar- riagemaybecommon inserotinebats(2–5,9,13,14).In onlyasmallsubsetofthemdoessymptomatic neurologic infectionprogresstomoreseverebodycondition.How- ever,mostofthesebatswerecapturedwhiletheywerefly- ing,andonly1ofthoseinvolved inanepisodeofhuman exposurehadlosttheabilitytofly.Thisbathadthepoorest bodycondition.TheabsenceofNegribodiesandthemod- eratepercentage ofnervecellsaffectedfoundinthebat, capturedwhileflying,suggestasubclinicalprocess,ashas beenproposedforEBLV1inRousettusaegyptiacus(6,14). Wecannotpredictwhethersevereencephalitiswasaboutto developinthesebats.Thelackofpathogenicitycouldhave arisenthroughlong-standingcoevolutionbetweenbatsand viruses,asthephylogeneticdatasuggest(15).Neverthe- less,additional workisnecessary toestablishwhetherthe presence ofEBLV1RNAintheoralcavityisassociated withtheexcretionof livevirus.

InasimilarstudyofM.daubentoniiintheUnited

Kingdom,onlyEBLV2antibodies,butnotviralRNAin

theoropharyngealcavity,werereported(3).Anotherstudy oftheexperimentaltransmissionofAravan,Khujand,and IrkutvirusestoE.fuscusfoundoralexcretion onlyinbats whoseconditions wereprogressingtoneurologic infec- tionanddeath(8).Thesefactssuggestdifferencesbetween modelsof pathogenesisof lyssavirus infectionsinbats.

Insummary,ourfindings indicatethatnaturalmater- nitycoloniesofE.isabellinusbehaveasclosecommunities inwhichEBLV1independentlycirculates,whichsuggests anendemicpatternassociatedwithasymptomatic ormild disease.Furtherstudiesareneededto1)investigate the circumstances underwhichneurologicmanifestations de- velopinbats(whichleadtotheabnormalbehaviorusually associated withhumanexposures)and2)toestablishthe epidemiologicimplicationsforpublichealthofasymptom- aticoralRNAcarriage.Theanthropophilicbehaviorofthis batspeciesmakesactivesurveillancehighlyrecommended topredictriskfactorsthatallowtakingpolitical decisions relatingtopublichealth.

Acknowledgments

WegratefullyacknowledgeJuanL.García-Mudarra,José

A. Garrido, Elena Migens, Joaquín Muñoz, Juan Quetglas,

JesúsNogueras,andCarlosRuizforprovidingtechnicalassistance asbathandlers.WealsothanktheGenomicUnitoftheNational CenterofMicrobiologyforanalyzingthegenomicsequences.We alsogratefullyacknowledgeThomas Müllerwhokindlydonated thespecificmonoclonalantibodyW239used inthiswork.

Thisprojectwasfinanciallysupportedbygrantsfromthe Instituto deSaludCarlosIII(MPY1364/99, MPY1271/01, and EXP03/0020),byanagreement betweenthePublicHealthDe- partmentoftheSpanish Ministry ofHealthandtheInstitutode SaludCarlosIIIforthedevelopmentof“Improvementrabiessur- veillanceinSpainthroughthedevelopmentofmoleculartools andtheestablishment ofanewsystemforactiverabiessurveil- lance,”bytheSpanishprogramofResearchinBiomedicine(FIS

98/0945),bytheWP5oftheEuropeanMed-Vet-Netnetworkof excellence, bytheprojectSAF2006-12784-C02-01/oftheGen- eralResearchProgramoftheSpanishMinistryofScienceand Education,andbytheEnvironmentalDelegationofSeville.This researchhasbeenapprovedbytheappropriate animalreview boardof theSpanishSocietyfor ConservationandStudyof Bats.

MsVázquez-Morónisadoctoralcandidateinscienceatthe ComplutenseUniversityofMadridandamicrobiologist whois developing anewsystemforactiverabiessurveillanceattheIn- stitutodeSaludCarlosIII, Madrid.Her researchinterestsinclude emergentviruses inbatsandtheirpublichealthimplications.

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Addressforcorrespondence: SoniaVázquez-Morón, Diagnostic Microbiology Service,NationalCenterforMicrobiology, Institutode SaludCarlosIII,CarreteradeMajadahonda-Pozuelokm2Majadahonda

28220, Madrid,Spain;email:

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