Diseases and causes of death in European bats: dynamics in disease susceptibility and infection rates


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Diseases and causes of death in European bats: dynamics in disease susceptibility and infection rates
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PLOS ONE
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Mühldorfer, Kristin
Speck, Stephanie
Kurth, Andreas
Lesnik, René
Freuling, Conrad
Müller, Thomas
Kramer-Schadt, Stephanie
Wibbelt, Gudrun
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Bats -- Diseases ( lcsh )
Bats -- Mortality ( lcsh )
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Europe -- Germany

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Bats receive increasing attention in infectious disease studies, because of their well recognized status as reservoir species for various infectious agents. This is even more important, as bats with their capability of long distance dispersal and complex social structures are unique in the way microbes could be spread by these mammalian species. Nevertheless, infection studies in bats are predominantly limited to the identification of specific pathogens presenting a potential health threat to humans. But the impact of infectious agents on the individual host and their importance on bat mortality is largely unknown and has been neglected in most studies published to date. Between 2002 and 2009, 486 deceased bats of 19 European species (family Vespertilionidae) were collected in different geographic regions in Germany. Most animals represented individual cases that have been incidentally found close to roosting sites or near human habitation in urban and urban-like environments. The bat carcasses were subjected to a post-mortem examination and investigated histo-pathologically, bacteriologically and virologically. Trauma and disease represented the most important causes of death in these bats. Comparative analysis of pathological findings and microbiological results show that microbial agents indeed have an impact on bats succumbing to infectious diseases, with fatal bacterial, viral and parasitic infections found in at least 12% of the bats investigated. Our data demonstrate the importance of diseases and infectious agents as cause of death in European bat species. The clear seasonal and individual variations in disease prevalence and infection rates indicate that maternity colonies are more susceptible to infectious agents, underlining the possible important role of host physiology, immunity and roosting behavior as risk factors for infection of bats.

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DiseasesandCausesofDeathinEuropeanBats: DynamicsinDiseaseSusceptibilityandInfectionRatesKristinMu ¨ hldorfer1* ,StephanieSpeck2,AndreasKurth3,Rene ´ Lesnik3,ConradFreuling4,Thomas Mu ¨ ller4,StephanieKramer-Schadt1,GudrunWibbelt11 LeibnizInstituteforZooandWildlifeResearch,Berlin,Germany, 2 BundeswehrInstituteofMicrobiology,Munich,Germany, 3 RobertKochInstitute,Berlin,Germany, 4 Friedrich-Loeffler-Institute,Wusterhausen,GermanyAbstractBackground:Batsreceiveincreasingattentionininfectiousdiseasestudies,becauseoftheirwellrecognizedstatusas reservoirspeciesforvariousinfectiousagents.Thisisevenmoreimportant,asbatswiththeircapabilityoflongdistance dispersalandcomplexsocialstructuresareuniqueinthewaymicrobescouldbespreadbythesemammalianspecies. Nevertheless,infectionstudiesinbatsarepredominantlylimitedtotheidentificationofspecificpathogenspresentinga potentialhealththreattohumans.Buttheimpactofinfectiousagentsontheindividualhostandtheirimportanceonbat mortalityislargelyunknownandhasbeenneglectedinmoststudiespublishedtodate.Methodology/PrincipalFindings:Between2002and2009,486deceasedbatsof19Europeanspecies(family Vespertilionidae )werecollectedindifferentgeographicregionsinGermany.Mostanimalsrepresentedindividualcases thathavebeenincidentallyfoundclosetoroostingsitesornearhumanhabitationinurbanandurban-likeenvironments. Thebatcarcassesweresubjectedtoapost-mortemexaminationandinvestigatedhisto-pathologically,bacteriologicallyand virologically.Traumaanddiseaserepresentedthemostimportantcausesofdeathinthesebats.Comparativeanalysisof pathologicalfindingsandmicrobiologicalresultsshowthatmicrobialagentsindeedhaveanimpactonbatssuccumbingto infectiousdiseases,withfatalbacterial,viralandparasiticinfectionsfoundinatleast12%ofthebatsinvestigated.Conclusions/Significance:Ourdatademonstratetheimportanceofdiseasesandinfectiousagentsascauseofdeathin Europeanbatspecies.Theclearseasonalandindividualvariationsindiseaseprevalenceandinfectionratesindicatethat maternitycoloniesaremoresusceptibletoinfectiousagents,underliningthepossibleimportantroleofhostphysiology, immunityandroostingbehaviorasriskfactorsforinfectionofbats.Citation: Mu ¨ hldorferK,SpeckS,KurthA,LesnikR,FreulingC,etal.(2011)DiseasesandCausesofDeathinEuropeanBats:DynamicsinDiseaseSusceptibilityand InfectionRates.PLoSONE6(12):e29773.doi:10.1371/journal.pone.0029773 Editor: AnthonyR.Fooks,VeterinaryLaboratoriesAgency,UnitedKingdom Received August11,2011; Accepted December4,2011; Published December28,2011 Copyright: 2011Mu ¨ hldorferetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisstudywassupportedbytheAdolfandHildegardIsler-Stiftung,theFAZIT-Stiftung(fellowshiptoKM)andtheKlaraSamariter-Stiftung.Thefun ders hadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:muehldorfer@izw-berlin.deIntroductionBatsareamongthemostsuccessfulanddiversemammalson earth.Approximately1230chiropteranspeciesarefoundonevery continentexceptAntarcticaandinhabitamultitudeofdiverse ecologicalniches[1].Batsplayessentialrolesinmaintaining healthyecosystems,astheyactasplantpollinators,seeddispersers, andpredatorsofpopulationsofinsectsincludingharmfulforest andagriculturalpests[2].MostbatspeciesarelistedintheIUCN Redlistofendangeredspeciesandalmosthalfoftheseare consideredthreatenedornear-threatened[3].Toestimateand preventfurtherpopulationdeclines,researchhasbeenprimarily focusedonbatbiology,ecologyandbehavior,whiledisease aspectswerelargelyneglected[4]. Inthelasttwodecades,theimportanceofchiropteranspeciesas potentialvectorsofsignificantviraldiseasesespeciallyinregardto zoonoseshasreceivedgrowingattention.Besidesbatrabiesthat hasbeenstudiedformorethanhalfacentury,extensiveresearch effortsidentifiedalargenumberofmicrobialagents[5]including importantemergingzoonoticvirusesdetectedinbatsacrossthe world[6–12].However,moststudiesarelimitedtothe identificationofmicroorganismsdetectedandinvestigations regardinginfectiousdiseasesandcausesofdeathinbatsare sparse[13–16]. InEurope,researchispredominantlyfocusedonEuropeanbat lyssaviruses[17,18]andcoronaviruses[19,20],butfirstindications ofbat-pathogenicbacteria[13,14,21–23]andnovelviruses[24,25] isolatedfromdeceasedbatsinGermanyandGreatBritainwere found.Inthisstudy,weprovidenewdataoninfectiousdiseasesin Europeanbatspecies,consideringfactorslikelytoaffectthe susceptibilityofbatstoinfectiousagentsincludingeffectsof seasonality,individualandspecies-specificheterogeneities,and possibleintra-andinter-speciestransmissiondynamics.MaterialsandMethodsAllbatspeciesinEuropearestrictlyprotectedundertheFloraFauna-HabitatGuidelinesoftheEur opeanUnion(http://ec.europa. eu/environment/nature/legislation /habitatsdirective/index_en.htm) (92/43/EEC)andtheAgreementontheConservationofPopulations PLoSONE|www.plosone.org1December2011|Volume6|Issue12|e29773

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ofEuropeanBats(www.eurobats.org)thatprohibitinvasivesampling ofbatsforresearchpurposes.Fortheanimalsinvestigatedinthis study,carcassesofdeceasedbatsfoundinGermanywerekindly providedbybatresearchersandbatrehabilitationcentersofdifferent federalstates.StudymaterialBetween2002and2009,atotalof486deceasedbatsof19 Europeanvespertilionidspecies(i.e.,FamilyV espertilionidae )were investigated(Fig.1A,[26]).Thebatcarcassesoriginatedfrom6 differentgeographicregionsinGermany,i.e.Berlingreater metropolitanarea(n=223),Bavaria(n=165),Brandenburg (n=38),LowerSaxony(n=36),Thuringia(n=21),andBadenWuerttemberg(n=3),andwerecollectedbybatresearchersand batrehabilitationcenters.Mostanimalsrepresentedindividual casesthatwerefounddead,injuredormoribundnearhuman habitation.Thus,thespeciescompositioninthisstudypredominatelyreflectedtheurbanandsuburbanbatfauna,whichis characterizedbyadisproportionateabundanceofafewbat species(Fig.1A,[27,28]).Twogroupsof2and21adultnoctules ( Nyctalusnoctula ),respectively,werecollectedfromtreehibernacula destroyedduringwoodlogging.Afurthergroupof25deceased adult N.noctula originatedfromacolonythatwastrappedinarain pipeinDecember.Ninedeadjuvenile Pipistrelluspipistrellus were collectedfromanurseryroost. Ifbatsdiedincareorhadtobeeuthanizedforanimalwelfare reasons,thecarcasseswereimmediatelystoredat 2 20 u Candwere shippedtotheLeibnizInstituteforZooandWildlifeResearch, Berlin,Germany,fordiagnosticinvestigations.Ofallcarcasses examinedhisto-pathologically,about90%weresuitablefor bacteriologicalinvestigation.Alesserextend(43%)wasalso examinedforselectedviralagentsattheRobertKochInstitute, Berlin,Germany.Inaddition,abrainsampleofeachanimalwas submittedtotheFriedrich-Loeffler-Institute,Wusterhausen,Germany,forrabiesdiagnosis.PathologicalinvestigationAfullnecropsywasperformedoneachbatandallmacroscopic findingsincludingectoparasiteinfestationwererecorded.For histo-pathologicalexamination,smallslicesofmultipleorgan tissues(i.e.,lung,liver,heart,kidney,adrenalgland,spleen, intestine,pancreas,brain,tongue,larynx,salivaryglandand pectoralmuscle)andtissuesconspicuousforpathologicalchanges werefixedinbuffered4%formalin,processedusingstandard methodsandembeddedinliquidparaffin.Sectionswerecutat2– 5mmandroutinelystainedwithhematoxylin-eosin(HE).In addition,specialhistologicalstainingmethodswereuseddependingonmicroscopicfindings,i.e.forthedetectionofbacteria (GramorGiemsastaining),fungi(periodicacidSchifforGrocott’s Gomorimethenaminesilvernitratestaining),iron(Prussianblue stain),mineralization(vonKossastaining),connectiveand collagentissue(trichromestaining).Detailsonpathologicalresults arepublishedelsewhere[26]. Thecausesofmortalitywererigorouslystandardizedwiththe primarycauseofdeathidentifiedforeachbatasthemostserious injury,diseaseoreventsubsequentlyfataltotheanimal.Toensure independenceofprimaryandcontributingcausesofdeath,the categorizationwasbasedontheseverityofpathologicalfindings.BacteriologicalinvestigationSamplesoflung,liver,heartandkidney,andtissuesconspicuous forpathologicalchanges(e.g.enlargedspleen)of430batswere platedontoColumbia(5%sheepblood),Chocolate,Gassner,and MacConkeyagar(Oxoid,Germany)andwereincubatedat37 u C (Chocolateagar5%CO2)for24–48h.Specificculturemediaand conditionsfortheisolationof Yersinia , Salmonella andanaerobic bacteriawereusedifappropriate.Primaryidentificationof bacterialstrainswasbasedoncolonymorphology,hemolysis, Gram-staining,indolproduction,catalaseandoxidasereaction. Bacterialspeciesidentificationwascarriedoutusingtherelevant commercialApitestsystem(bioMe ´rieux,Germany).Additional conventionalbiochemicaltests[29,30]wereappliedtoconfirm Apitestresultswherenecessary.Incaseofambiguousbiochemical testresults,16SrDNAgeneanalysiswasperformedforfinal identification[23]. Salmonella isolateswerecharacterizedatthe NationalReferenceLaboratoryfortheAnalysisandTestingof Zoonoses( Salmonella )attheFederalInstituteforRiskAssessment, Berlin,Germany.Identificationandcharacterizationof Yersinia and Pasteurella specieshavebeenreportedearlier[22,23].VirologicalinvestigationHomogenizedorgantissueoflung,liver,heart,kidney,spleen, brainandsalivaryglandof210batswerepooledforeach individualandusedforRNA/DNAextractionandfurther molecularanalysisbygenericPCRassaysdetectingflavi-[31], hanta-[32],corona-[33],andinfluenzaA-viruses[34].Also,PCR assaysspecificfor8previouslydescribedherpesviruses[24]from Europeanvespertilionidbatswereused.Forthispurpose,RNA/ DNAwasisolatedusingtheNucleoSpin H RNAIIKit(MachereyNagel,Germany)andtheNucleoSpin H TissueKit(MachereyNagel),respectively,accordingtothemanufacturer’sinstructions. Becauseoflimitationsinsamplevolume,for180outofthe210 batsPCRassayscouldonlybeappliedfor4differentbat herpesviruses.InternalcontrolswereusedforallPCRassaysto testforinhibition.Forconfirmation,allretrievedfragmentsofbat herpesvirus-specificPCRassayswerecheckedforsequence identitytopreviouslypublishedisolates[24]. Fordetectionoflyssavirusantigeninbraintissuethefluorescent antibodytest(FAT)usingapolyclonalantirabiesconjugate(Sifin, Germany)wasused[35].FAT-positivebraintissuesweresubject ofvirusisolationinmurineneuroblastomacellculture(Na42/13) usingtheRabiesTissueCultureInfectionTest(RTCIT)as describedelsewhere[36].Lyssavirusesisolatedincellculturewere characterizedusingbothapanelof10anti-nucleocapsid monoclonalantibodies(MAb)[37]andpartialsequencingofa fragmentofthenucleoproteingeneafterRNAextractionusing Trizol(Invitrogen,Germany)essentiallyasdescribed[18].GeneticidentificationofbatspeciesGenomicDNAwasextractedfromorganhomogenatesusing theNucleoSpin H TissueKit(Macherey-Nagel)accordingto manufacturer’srecommendations.Geneticidentificationofthe batspecieswasperformedbyamplificationandsequencingofa 241bpfragmentofthecytochromeB( cytB )gene[38]using primersFMup(5 9 -CCCCHCCHCAYATYAARCCMG ARTGATA-3 9 )andFMdown(5 9 -TCRACDGGNTGYCCT CCDATTCATGTTA-3 9 ).Inaddition,fordifferentiationof the2distinct Pipistrellus species, P.pipistrellus and P.pygmaeus ,a rapidmultiplexPCRassaywasperformedasdescribedbyKan uch etal.[39]usingprimersPIP-F(5 9 -CTCATTCATTGAYCT ACCAGC-3 9 ),PIP-R(5 9 -CAGCRAATAGTAAAATAA CTCC-3 9 )andPpip-F(5 9 -CATCTGTTTGGGACTACA GATCC-3 9 ).StatisticalanalysisThebatdatawerecategorizedinregardtodifferentexplanatory numericandfactorvariables,e.g.batspecies,sexandageclass. Thevariable‘ageclass’rankedbetween1and4withincreasingDiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org2December2011|Volume6|Issue12|e29773

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DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org3December2011|Volume6|Issue12|e29773

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age(i.e.neonates,juveniles,subadults,andadults)andwasusedas numericvariable.Forendoparasiticanalysis,wedefineda3level variable‘batsize’accordingtothebodysizeofacertainbat speciestoreducethedegreesoffreedomofthefullmodel,i.e.large species( N.noctula , Eptesicusserotinus ,and Vespertiliomurinus ), medium-sizedspecies( E.nilssonii , Plecotusauritus , Myotisdaubentonii , M.nattereri ,and P.nathusii )andsmallspecies( P.pipistrellus ,and M. mystacinus ).Todetecteffectsofseasonality,4differentactivity periodswerespecifiedaccordingtothedateofsampling,i.e. hibernationperiod(NovembertoMarch),post-hibernationperiod (April/May),maternityperiod(JunetoAugust),andswarming period(September/October).Asdependentbinaryvariableforthe respectivemodelsweeitherclassifiedthemortalitycausebeing diseaseornot(i.e.trauma),orthepresence-absenceofbacterial, ecto-andendoparasiticinfections. Weformulated4differenthypothesestotestforindividualand species-specificdifferencesindiseasesusceptibilityandinfection rates:(A)Disease-relatedmortalityinbatsisinfluencedbysex,age andspecies-specificdifferences,anddegreeofendoparasitic infection.(B)Bacterialinfectioninbatsisinfluencedbysex,age andspecies-specificdifferences,occurrenceoftraumaticinjuries andcatpredation.(C)Ecto-or(D)endoparasiticinfectioninbats isaffectedbyage,sexandspecies-specificdifferences.Seasonal effectswerenotanalyzedbecauseoftoomanymissingdatapoints. Becausethelong-termdatasetwashighlybiasedtowardssampling procedure,preservationofbatcarcassesandfollowingdiagnostic investigations,wesplitandfilteredthefulldataintoseveralsubsets reflectingthedifferentanalyses(Table1). AllstatisticalanalyseswereperformedusingtheRsoftwareV. 2.13.1(RDevelopmentCoreTeam2011,Vienna,Austria).We usedthechi-squaretestforgivenprobabilitiestoevaluate significantdifferencesinthesexratioamongbatsofdifferent species.ForhypothesesAandB,weusedageneralizedlinear mixedmodelingapproach(binomialGLMMusingfunctionlmer inlibrarylme4)withbatspeciesincludedasrandomeffect.This variablehadnotbeensignificantasfixedeffect(resultsnotshown), butfromotherstudieswecanassumethattherearespeciesspecificdifferencesinsusceptibilityofbatstocertaininfectious agentsandthereforeincludeditasrandomeffect.Wefurtherused generalizedlinearmodels(GLMwithlogitlinkandbinomialerror structure;fordatasetswithbatspecies . 10individuals)totestfor individualandspecies-specificdifferencesinparasiteinfection rates(hypothesesCandD). Wecreatedafullmodelforeachhypothesis(A–D)toexamine multipleandinteractioneffectsofthespecifiedvariables.Toselect thefinalmodelvariables,weusedastepwisebackwardalgorithm (functionstepAICinlibraryMASS)basedonAkaike’sinformation criterion(AIC)[40].The D AICofthefinalmodelwascalculated relativetoarandominterceptmodeltodemonstratetheeffectsize oftheselectedvariables.ResultsResultsofthediagnosticanalysesfollowthefulldatasplitting intoseveralsubsets(seesection‘Statisticalanalysis’inMaterialand Methods;Table1).Fulldataset: BatsamplesAllsampledbatsbelongedto7differentgenera(i.e. Pipistrellus , Nyctalus , Myotis , Eptesicus , Plecotus , Vespertilio ,and Barbastella )and19 Europeanvespertilionidspecies(Fig.1A).Threebatspecies,the commonpipistrelle( P.pipistrellus ,n=138),thenoctulebat( N. noctula ,n=92),andtheserotinebat( E.serotinus ,n=53)constituted about60%ofallbatcarcassesinvestigatedinthisstudy,whereas P. pygmaeus , Nyctalusleisleri , Myotisbrandtii , M.bechsteinii , M.dasycneme , Plecotusaustriacus and Barbastellabarbastellus wererepresentedinsmall numbersof1to4animals.Theoverallsexratiowas1.5malesto1 femalewithsignificantspecies-specificdifferences(Fig.1B).Animals intheirfirstyearoflife(neonates,juveniles,andsubadults) representedonethird(32.5%,n=158)ofbatsamples(Fig.1C). Table1. Descriptionofthedatasetsusedfordifferentanalyses.Analysis Dataset (totaln) Sex (%males) Age (%adults) Batspecies (totaln) FulldatasetBatsamples48655.667.519 Subset1Causesofdeath433a55.065.419 GLMM:disease-vs.trauma-relatedmortality(A)289a55.065.717 Subset2Bacteriologicalresults43058.465.318 GLMM:bacterialinfectionvs.noinfection(B)377a58.162.618 Subset3Virologicalresults210b56.764.316 Subset4Parasitologicalresults433a55.065.419 GLM:parasiticinfectionvs.noinfection(C,D)402a54.765.210 GLMM,generalizedlinearmixedmodelswithbatspeciesincludedasrandomeffect. GLM,generalizedlinearmodelsfordatasetswithbatspecies . 10individuals. A–D:referstothemodelsanalyzedonthedifferentdatasets(seechapter‘Statisticalanalyses’).aToavoidoverrepresentationofbatsamplesthatwerecollectedatthesametimeandlocation,arandomlyselectedindividualofeachgroupwasinclude dinthefinal dataset.bFordetectionoflyssavirusantigen,braintissueofall486batswastested. doi:10.1371/journal.pone.0029773.t001 Figure1.DetailsonbatsfromGermany. (A)Batspeciesdistributionamongthestudysample(n=486).(B)Male-to-femaleratio(batspecies . 10 individuals).Footnotes:1)Chi-squaretest, x2=11.1,df=1,p=0.0009,2) x2=8.8,df=1,p=0.003,3) x2=4.0,df=1,p=0.05,4) x2=3.5,df=1,p=0.06. Abbreviations:Ppip, Pipistrelluspipistrellus ;Pnath, Pipistrellusnathusii ;Nnoc, Nyctalusnoctula ;Mmyst, Myotismystacinus ;Mdaub, Myotisdaubentonii ; Mnatt, Myotisnattereri ;Eser, Eptesicusserotinus ;Enils, Eptesicusnilssonii ;Paur, Plecotusauritus ;Vmur, Vespertiliomurinus .(C)Age-sexdistribution amongthestudysample(n=486). doi:10.1371/journal.pone.0029773.g001 DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org4December2011|Volume6|Issue12|e29773

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Subset1: CausesofdeathOverall,wewereabletoassignacauseofdeathto70% (n=304)ofbatsinvestigatedinthisstudy.Twothirdsofmortality wereduetotrauma(n=145)ordisease(n=144),whilealmost4% ofbatshaddiedofothernon-infectiouscauseslikepulmonary edema,dehydrationandhypoglycemia(Table2).In30%(n=129) nosignificantpathologicalfindingscouldbefound. Amongthe145traumatizedbats,additionalmild(n=42), moderate(n=28)andsevere(n=4)inflammatoryorganchanges werenotedinonehalf(50.9%)ofindividuals,and23%ofthebats revealedbacterial(n=19)and/orparasiticinfections(n=15) (Table3).Ofthe144batsconsideredasdyingofdisease,fatal bacterial(n=54),viral(n=5)andparasiticinfections(n=2)were observedin42%.Besides,amnioticfluidaspirationwasnotedina neonatenoctulebat( N.noctula ),andajuvenilecommonpipistrelle ( P.pipistrellus )waseuthanizedbecauseofsevereforearmbone deformation.Theremaining81bats(56.3%)revealedmoderateto severepathologicalchangesofunknownetiologyorunconfirmed bacterialorviralcause(Table2). BasedontheGLMManalysis,significantage-andsexdependentdifferences( D AIC=23.13)weredetectedbetweenthe generalcausesofmortality,diseaseandtrauma(Table4).The diseasepresenceinbatsamplesdecreasedcontinuouslywith increasingage.Neonatesandjuvenilesofbothsexeswere significantlymoreaffectedbydiseasethanolderageclasses (Table4;Fig.2A).Wealsofoundasignificanttrendindiseaseassociatedmortalitybetweenthesexes,withadultfemales displayinghigherdiseaseprevalence(52.5%)thanmales(36.4%) (Table4).Nosignificantassociationwasobservedbetweena certaincauseofmortality(i.e.diseaseortrauma)andseverityof endoparasiticinfection( D AIC=0.75,resultnotshown).The seasonaldistributionofdisease-relatedmortalitycases(Fig.2B) describedatrimodalpattern,withpeaksinspring(April),summer (JunetoAugust)andwinter(December).Theproportionof traumatizedindividualsalsoincreasedobviouslyduringthe summermonthsuptoandincludingtheswarmingperiod,but waslowduringtherestoftheyear. Table2. CausesofmortalityofbatsfromGermany.AgeclassSexclass Causeofdeathn%Euthanasia , 1YearAdultFemaleMalen.d. Trauma 14533.5544110455873 Unknowntraumacause7116.529195233363 Catpredation6615.32319471847Roostdestructiona20.5--22-Trappedinrainpipea10.2--1-1Trappedinwindow10.2--1-1Trappedinlamp10.2-1--1Trappedinflystrip10.2-1-1-Barbedwireinjury10.211-1-Smokepoisoning10.21-1-1Disease 14433.37588664728 Unknownetiology8118.73354635388 Bacterialinfection5412.5220342727Viralinfectionb51.211414Parasiticinfection20.5--2-2Aspirationpneumonia10.2-1-1-Bonedeformation10.211--1Others 153.4-6969Pulmonaryedema92.1-3618Dehydration20.5--211Anemiac10.2--11-Hyperthermiad10.2-1-1-Hypothermia10.2-1-1-Hypoglycemia10.2-1-1-Nosignificantfindings 12929.814584337026 Total 4331006215028315823837 n.d.,notdetermined.aArandomlyselectedindividualof3differentgroupsofadult Nyctalusnoctula .bAdenovirus(batAdV-2)[25]andEuropeanbatlyssavirus(EBLV-1)infection.cDuetoseveretickinfestation.dArandomlyselectedindividualofagroupofjuvenile Pipistrelluspipistrellus . doi:10.1371/journal.pone.0029773.t002 DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org5December2011|Volume6|Issue12|e29773

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Subset2: BacteriologicalresultsAbout90%(n=430)ofbatsampleswereexaminedbacteriologically.Amongthese,42differentbacterialgenerawithmore than53bacterialspecieswereidentified(TableS1).Predominant bacteriaisolatedwere Enterococcusfaecalis (14.7%,n=63), Hafnia alvei (11.2%,n=48), Serratialiquefaciens (10%,n=43),and Pasteurellamultocida (7.7%,n=29).In37%(n=157)ofbatsno bacterialgrowthwasobservedatall. Comparativebacteriologicandhisto-pathologicanalysisidentified22differentbacterialspeciesthatwereclearlyassociatedwith pathologicallesionsand/orsystemicinfection,foundin17% (n=73)ofbatsinvestigatedbacteriologically(Table5).Members ofthefamilies Pasteurellaceae (aboveall P.multocida )(41.1%,n=30), Enterobacteriaceae (variousbacterialspecies)(28.8%,n=21),and Streptococcaceae (aboveall Enterococcus spp.)(21.9%,n=16)were predominantbacteriaassociatedwithdisease.Morethanhalf (54.8%,n=40)ofbacterialinfectionswereobservedinbatswith traumaticinjuries.TheGLMManalysisrevealedlowsex-and age-dependentdifferencesinbacterialinfection( D AIC=1.97, resultnotshown).Femalebats(21.9%)andadults(21.6%)showed marginallyhigherprevalenceofbacterialdiseasecomparedto males(18.3%)andtootherageclasses(15.6%),respectively. However,wefoundastronginfluenceofcatpredation ( D AIC=16)associatedwithbacterialinfectioninbats(Table4).Subset3: VirologicalresultsTestingforhuman-pathogeniczoonoticviruses,noexamined batsample(0/210)waspositiveforinfluenzaAvirus,corona-, hanta-andflaviviruses,respectively.NoinhibitionofthePCR assayswasnotified.Outof486batstestedforrabiesvirus infection,2serotinebats( E.serotinus )werepositiveforlyssavirusby FATandRTCIT.TheviruseswereidentifiedasEuropeanbat lyssavirustype1(EBLV-1)sublineagea,bothusingMAbsand sequencing. Applyingbatherpesvirus-specificPCRassays,63outof210 batsprovedtobeinfectedwith7ofthepreviouslydescribed8bat herpesviruses(Table6).Thehighestprevalenceof65%(24/37) wasobservedforbatgammaherpesvirus6(BatGHV6)incommon pipistrellebats( P.pipistrellus ),followedbybatgammaherpesvirus5 (BatGHV5,42.1%)innathusius’pipistrellebats( P.nathusii )and batgammaherpesvirus4(BatGHV4,33.8%)innoctulebats( N. noctula ).Co-infectionwithdifferentbatherpesviruseswere recognizedin4 N.noctula (7.4%)infectedwithBatGHV3and BatGHV4,andinone N.noctula (1.5%)infectedwithBatGHV4 andBatGHV5.BatGHV5wasnotonlydetectedinitsinitially specifichost P.nathusii ,butalsoin3otherbatspecies,i.e. N.noctula , Myotismyotis and M.mystacinus .Althoughtheprevalenceof BatGHV3(13.0%)andBatGHV4(33.8%)differedsignificantly withinitsmigratinghost N.noctula ,nodifferencewasobserved betweenthesexes.Twojuvenile N.noctula werefoundtobe infectedwithBatGHV4.Interestingly,forthesedentarybat species P.pipistrellus beinginfectedwithBatGHV6,aconsiderably higherprevalencewasobservedin22juvenilebats(72.7%) resultinginanoverallprevalenceof65%alsowithoutdifference betweenadultmaleandfemalebats.Subset4: ParasitologicalresultsEctoparasites(mites,fleas,andticks)werenotedin14%(n=62) ofbats,butapotentialbiasinectoparasitenumberscollectedfrom deadanimalsincomparisontoectoparasiteabundanceonlive animalshastobetakeninaccount.Femalebats(17.1%)were slightlymoreinfestedbyectoparasitesthanmales(14.7%),whereas indifferentageclassesectoparasiteprevalencewasalmost balanced.TheGLManalysisrevealedsignificantspecies-specific differencesinectoparasiteinfestation( D AIC=14.58,Table4). Mostbatspeciesrevealedlowectoparasiteprevalence(range5.3– 11.8%),whilealmost43%(n=20)of N.noctula wereinfestedwith mitesand/orfleas(Fig.3A). Microscopicexaminationoforgantissuesrevealedendoparasitic infectionin29%(n=124)ofinvestigatedbats,involvingdifferent protozoan(families Eimeriidae and Sarcocystidae )andhelminth parasites(trematodes,cestodes,andnematodes).Helmintheswere predominantlyfoundinthegastro-intestinaltractofthebats,while insomeanimals,granulomatousorganlesionswereassociated withlarvalmigrationofnematodespecies.BasedontheGLM analysis,clearage-andspecies-specificdifferences( D AIC=24.95) wereobservedbetweeninfectedandnon-infectedbats(Table4). Theprevalenceofendoparasiticinfectioninbatsamplesincreased significantlywithincreasingage,whereastheincreasein prevalencewasmorerapidbetweenjuvenilesandsubadults (8.5%)comparedtotheolderageclasses(4.5%).Marginal differenceswerealsoobservedbetweenthesexes,withfemale batsshowingslightlyhigher(30.4%)endoparasiteprevalencethan males(24.4%).Regardingspecies-specificdifferences,largebats like N.noctula , E.serotinus and V.murinus revealedhigher endoparasiteprevalencecomparedtoindividualsofmedium-sized orsmallvespertilionidspecies(Table4;Fig.3B).Discussion CausesofdeathanddiseasedynamicsinbatsThisstudywasbasedonapassivesurveillancesamplingstrategy thatinherentlyinfluencesthecompositionofbatssampledfor diagnosticinvestigations[27]andmightalsoeffectthedata presentedoncausesofdeathbyecologicalandanthropogenic impactsofurbanlandscapes[41].Traumaanddiseaserepresented themostimportantcausesofmortalityindeceasedbatsfrom Germany,whichdifferfromresultsofpreviousstudies[13–15] wheredisease-relatedmortalityoftenplayedasubordinaterole. Youngbatsandadultfemalesweresignificantlymoreaffectedby disease,indicatingthatsex-andage-relateddiseaseprevalencein Table3. Pathologicalfindingsandbacterial,viraland parasiticinfectionsspecifiedforthegeneralcausesof mortality,traumaanddisease.TraumaDisease n%n% Totalnumberofbats14533.514433.3 PathologicalfindingsaInjuries13693.83725.7 Inflammatorylesions7451.012486.1 Non-inflammatorylesions10.72013.9 Spleenactivation8155.98256.9 Circulatorychanges5336.34128.5 Metabolicdisorders106.8128.3 Bacterialinfection 1913.05437.5 Viralinfectionb--53.5 Parasiticinfectionc1510.3149.7aDetailsonpathologicalfindingsdescribedelsewhere[26].bAdenovirus(batAdV-2)[25]andEuropeanbatlyssavirus(EBLV-1)infection.cSevereintestinaltrematodeinfection,disseminatednematodeinfection,renal orintestinalcoccidiosis[26]. doi:10.1371/journal.pone.0029773.t003 DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org6December2011|Volume6|Issue12|e29773

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batsarestronglycorrelatedwiththematernalseason.This assumptionisfurthersupportedbythedistinctincreaseofdiseaserelatedmortalityfromJunetoAugust,whichcorrespondstothe maternityperiodofCentralEuropeanbatspecies.Similarseasonal prevalencepatternsinbatshavealsobeendescribedforparasitic (e.g.[42–45])andviralinfections(e.g.[19,46,47]).Incontrast,the increaseoftrauma-associatedmortalitycasesfromJulytoOctober resembles4majorbehavioralactivitypatternsofEuropeanbat species(i.e.weaning,mating,pre-hibernalfatstorage,and migration)[48]andcouldthereforepredisposebatstotrauma. However,bothseasonalpeaksalsocoincidewithtimeand locationswheresick,injuredordeadbatsaremorelikelytobe discoveredaswellaswiththeseasonalroostingbehaviorofbats adaptedonurbanhabitats[27].Theadditionalseasonalpeaksof disease-associatedmortalitycorrespondedtothepost-hibernal andtheearlyhibernalperiodoftemperatezonebats.Currently, thereisalackofknowledgeofbatimmunology.Itisknownfor othermammalianspeciesthathibernationreducestheinnate andadaptiveimmuneresponse;likewiseanincreasingriskof infectioncouldbeassumedforhibernatingbats[49].Withthe startofthehibernationseason,largeaggregationsofbats originatingfromvariouscoloniesmightenhancetheriskof spreadinginfectiousagentssimilartomaternitycolonies.Equally, thepost-hibernalincreaseofdisease-relatedmortalityissuggestiveforreducedimmunityinassociationwithprolongedfasting duringhibernation.BacterialdiseasesandcatpredationBacterialdiseaseshaverarelybeendocumentedinbats. Pasteurella spp.,hereidentifiedin7%ofbats,werethepredominant bacterialpathogensreportedinEuropeanbatsandinfection appearstobestronglycorrelatedwithcatpredation[13,14,23,26]. Inourstudy,bacterialinfectionswereconfirmedin17%ofbats investigatedbacteriologically.Mostofthesebacterialisolates representedopportunisticpathogensthatusuallydonotharmthe hostunlesstheimmunesystemisweakened[50]orpreceding injurytonaturalhostbarriers(e.g.skinabrasion).Primary bacterialpathogenslike Samonellaenterica serovarTyphimurium, S. Enteritidisand Yersiniapseudotuberculosis [22]wereidentifiedin almost12%ofaffectedbats.Someofthebacterialspecies(e.g. Burkholderia sp., Cedeceadavisae and Clostridiumsordellii )arenewly describedinbats.Nevertheless,bacteriologicanalysescan markedlybeinfluencedbypost-mortembacterialinvaders, freezingandstorageofbatcarcassesandtheinabilitytodetect certainbacteriabyroutineculturemethods,resultinginsome bacterialspeciesthatmighthaveescapeddetection. Wefoundastrongassociationbetweencatpredationand bacterialinfectioninbatsasalmostonehalfofbats(44%)caught bycatswereaffectedbybacterialdisease.Variousbacteriacanbe transmittedviacatbites[51];hencebatsattackedbycatsarelikely tosuccumbtobacterialinfectionevenifnon-fatalinjurieswere present.Thisrelationhasbeenprovenfor P.multocida infectionsin Europeanbatspecies[13,14,23,26].Ontheotherhand,bats alreadydebilitatedbydiseasemighteasierfallpreytopredators likecats.Consequently,batsmayalsoactasvectorsforzoonotic pathogens,asdomesticcatscouldpasstheseinfectiousagentsonto humans.Suchcross-speciestransmissioneventsfrombatsto domesticanimalsarewelldocumented[9,52].VirologicalinvestigationsForalltestedhuman-pathogeniczoonoticvirusesnoinfected batcouldbedetectedinthisstudyexceptlyssaviruses.Batrabies istheonlybattransmittedzoonosisinEuropethatisknownto haveresultedinhumancases[53].Unlikeinothermammals Table4. Resultofthefinalmodelvariablescorrespondingto4differentanalyses:(A)disease-vs.trauma-relatedmortality,and presence-absenceof(B)bacterial,(C)ecto-and(D)endoparasiticinfection.Analysis D AIC*VariableFactorlevelEstimateSEz-valuep-value (A)GLMM23.13Ageclass 2 0.560.18 2 3.090.002 Sex(male) 2 0.620.28 2 2.190.03 (B)GLMM16.00Catpredation1.200.284.32 , 0.0001 (C)GLM14.58Batspecies Nyctalusnoctula 2 0.300.30 2 1.020.3 Myotisdaubentonii 2 1.100.52 2 2.130.03 Vespertiliomurinus 2 1.560.55 2 2.830.005 Eptesicusnilssonii 2 2.010.75 2 2.680.007 Pipistrelluspipistrellus 2 2.040.27 2 7.42 , 0.0001 Eptesicusserotinus 2 2.060.43 2 4.75 , 0.0001 Plecotusauritus 2 2.400.74 2 3.250.001 Pipistrellusnathusii 2 2.740.73 2 3.760.0002 Myotisnattereri 2 2.771.03 2 2.690.007 Myotismystacinus 2 2.900.73 2 3.98 , 0.0001 (D)GLM24.95Ageclass0.430.152.880.004 BatsizeLargespecies 2 0.180.18 2 0.990.3 Medium-sizedspecies 2 1.300.23 2 5.64 , 0.0001 Smallspecies 2 1.290.19 2 6.86 , 0.0001 GLMM,generalizedlinearmixedmodelswithbatspeciesincludedasrandomeffect. GLM,generalizedlinearmodelsfordatasetswithbatspecies . 10individuals. AIC,Akaike’sinformationcriterion. * D AICofthefinalmodelrelativetoarandominterceptmodel. doi:10.1371/journal.pone.0029773.t004 DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org7December2011|Volume6|Issue12|e29773

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wherelyssavirusesultimatelycauselethalrabies,inbatsnonlethal lyssavirusinfectionsmayalsoleadtothedevelopmentof immunity[47].WiththedetectionofEBLV-1weconfirmthat thislyssaviruscirculatesamong E.serotinus aspreviousstudies showed[18].InGermany,batrabiesisalsocausedbyEBLV-2 andBokelohbatlyssavirus(BBLV)[54,55],butwhilethelatter wasrecentlyisolatedfrom M.nattereri ,EBLV-2isassociatedwith M.daubentonii and M.dasycneme [56].Theapparentabsenceof EBLV-2andBBLVinthesampledbatsislikelyduetothefact thatlyssavirusinfectionshaveaverylowincidenceinbat populations[18]andthatthesamplesizewastoolimited, especiallyconcerningtherelevantspecies. Thereisahighprevalenceforherpesvirusesindifferent insectivorousbatspeciesinGermany(thisstudy,[24]).Mostof thepreviouslydescribedbatherpesviruseshavebeendetectedin lownumbersinmorethanonebatspecies[24].Here,we observedahighspecies-specificprevalenceamongherpesvirusinfectedbats,indicatingthatacertaintypeofEuropeanbat herpesvirusisprimarilyassociatedwithasinglebatspecies.This issupportedbyBatGHV6andBatGHV7thatwereagainonly identifiedintheirinitialhosts P.pipistrellus and P.auritus (both sedentary),respectively,underliningthetypicalstrongspeciesspecificityofmammalianherpesviruses.However,speciesÂ’range overlapandcloseinter-speciescontactsinbatroostsmayresultin cross-speciestransmissionandcouldexplaintheobserved overcomingofthespeciesbarrier(thisstudyBatGHV5,[24]). Interspeciestransmissionhavealsobeendiscussedforother mammalianherpesviruses,i.e.bovineandequineherpesviruses (e.g.[57,58]).Furthermore,forRNAviruses(i.e.rabiesvirus) phylogeneticdistancebetweendifferenthostspeciesandoverlap ingeographicrangehaverecentlybeendemonstratedasstrong predictorsofhostshiftsandcross-speciestransmissioninbats [59].Someofthebatspecies(i.e. N.noctula , P.pipistrellus ,and P. nathusii )inthisstudyappeartobemoresusceptibletoherpesvirus infection.In N.noctula ,3differentgammaherpesviruses (BatGHV3,4,5)withsignificantprevalencedifferenceswere recognized.Suchtype-specificdifferencesinprevalencebetween thephylogeneticallydistantvirusesBatGHV3(13.0%)and BatGHV4(33.8%)withinonebatspeciesindicatesco-evolutionaryvirus-regulatedmechanisms. Figure2.Age-dependentdifferencesandseasonalvariationsamongthegeneralcausesofmortality,diseaseandtrauma. (A)Agespecificprevalence.(B)Seasonaldistributionoftrauma-anddisease-relatedmortalitycases. doi:10.1371/journal.pone.0029773.g002 DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org8December2011|Volume6|Issue12|e29773

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DifferencesinparasiteprevalenceParasiteinfestationinwildlifeoftenoccurswithoutclinical effects,butsevereinfectioncanreducehostfitnesseitherinterms ofsurvivalorreproductivesuccess[60].Mostdataoninfection dynamicsinbatscamefromparasitestudiesfocusingonindividual andseasonalvariationsinectoparasiteprevalence(e.g.[43– 45,61]).Hostdensity,roostpreferenceandmovementpattern seemtobeimportantfactorsexplainingindividualandspeciesspecificparasiteinfestationratesinbats[43–45].InEuropean vespertilionidspecies,female-biasedparasiteloadsaremostlikely associatedwithhostphysiologyanddifferencesinroosting behavior[42,44].Wealsofoundspecies-specificseasonalvariationsinectoparasiticinfestation,with N.noctula and M.daubentonii showinghigherectoparasiteprevalenceinspringandautumn comparedtothebreedingseason(datanotshown),whichisin accordancewithZahnandRupp[43]. Additionalfindingsofourparasiteanalysesaredistinct variationsinecto-andendoparasiteprevalenceinrelationtobat species.Batsprimarilyroostingintreesornestboxesweremore frequentlyinfestedwithectoparasiteslike N.noctula (43%)and M. daubentonii (25%)comparedtootherspecies(range5–12%) investigatedinthisstudy.Highectoparasiteloadshavegenerally beendescribedinbatspreferringenclosedroostslikeburrowsand cavities[61,62],suggestingthatstructuralcharacteristicsandthe microclimateofroostinghabitatsinfluenceectoparasitesurvival andre-infectionofbathosts.Thisassumptionisinaccordance Table5. BacteriaassociatedwithdiseaseinbatsfromGermany.BacteriaBatsClinicalstatus*Pasteurellamultocida 28Septicemia;pneumonia;pleuritis;peri-/epicarditis;myocarditis;nephritis;liver/spleennecroses; woundinfection;abscess Pasteurellamultocida,Pasteurella speciesB1Septicemia;glossitis(bitewoundinfection);livernecrosis Pasteurellapneumotropica,Vibrio spp.1Septicemia Serratialiquefaciens 5Systemicinfection;pneumonia;woundinfection Serratiamarcescens 1Systemicinfection;pneumonia Enterobactercancerogenus 2Systemicinfection;pneumonia Enterobactercancerogenus,Hafniaalvei 1Peritonitis;pneumonia Hafniaalvei 1Systemicinfection Klebsiellaoxytoca 3Systemicinfection;pneumonia Klebsiellamobilis 1Systemicinfection;pneumonia Escherichiacoli 2Systemicinfection;pneumonia;nephritis;cystitis Salmonellaenterica serotypeTyphimurium2Systemicinfection;pneumonia;meningitis Salmonellaenterica serotypeEnteritidis1Systemicinfection;pneumonia,woundinfection Yersiniapseudotuberculosis 1Systemicinfection;pneumonia;liver/spleennecroses Cedeceadavisae 1Pneumonia Burkholderia sp.1Systemicinfection Enterococcusfaecalis 9Septicemia;pneumonia;endocarditis;abscess Enterococcusfaecium 3Septicemia;pneumonia Enterococcusfaecalis,Enterococcusfaecium 2Septicemia;pneumonia;myocarditis;woundinfection Staphylococcusaureus 3Septicemia Staphylococcusaureus,Enterococcusfaecalis 1Septicemia;dermatitis Aerococcusviridans 1Systemicinfection;pneumonia Bacillus sp.1Pneumonia Clostridiumsordellii 1Hemorrhagicenteritis *Histo-pathologicalfindingsdescribedinmoredetailselsewhere[26]. doi:10.1371/journal.pone.0029773.t005 Table6. BatherpesvirusinfectioninbatsfromGermany.VirusBatspeciesTotalPositive(%) Batherpesviruses16species21063(30.0) BatGHV1aEptesicusserotinus 91(11.1) BatGHV3aNyctalusnoctulac547(13.0) BatGHV4bNyctalusnoctulac6522(33.8) BatGHV5bPipistrellusnathusii 198(42.1) Nyctalusnoctulac651(1.5) Myotismyotis 21n.d. Myotismystacinus 211(4.8) BatGHV6aPipistrelluspipistrellus 3724(64.9) BatGHV7bPlecotusauritus 122(16.7) BatBHV1bMyotisnattereri 21n.d. BatGHV,Batgammaherpesvirus. BatBHV,Batbetaherpesvirus.aTestedbatsfromasamplesetcontaining180animals.bTestedbatsfromasamplesetcontaining210animals.cCo-infectionofdifferentherpesvirusesrecognized. n.d.,notdeterminedduetoinsufficientsamplenumbers. doi:10.1371/journal.pone.0029773.t006 DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org9December2011|Volume6|Issue12|e29773

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withresultsofPearceandOÂ’Shea[63]whofounddifferencesin ectoparasiteprevalenceandintensityin Eptesicusfuscus inrelation toenvironmentalfactors(i.e.temperatureandhumidity)of differentroostsites.Incontrasttotheseresults,theendoparasite prevalenceinEuropeanvespertilionidbatsseemstobecorrelated withthebodysizeofthebatspecies[26].Species-specific variationsindietandpreyselectioncouldpossiblyeffect endoparasiteprevalenceininsectivorousbats[64],aslargerbats feedoninsectsofawidersizerangeincludinghard-bodiedprey [65,66].Thisassumptionissupportedbytheclearprevalence increaseinsubadultandadultbatscomparedtolowendoparasite infectionratesinjuvenilesprimarilyfeedingonmilk.ConclusionAmultitudeofpublicationsisrestrictedtopathogenpresenceor absenceindifferentchiropteranspecies;herewedemonstratethe impactofdiseasesandinfectiousagentsonbatsthemselves. Alongsidetotrauma-associatedmortalityandundefinedmortality cases,diseaseaspectsrepresentedonethirdofmortalitycausesin 486investigatedbatsof19Europeanvespertilionidspecies.By comparingpathologyandbacteriologyresults,wewereableto detect22differentbacterialspeciesthatwereclearlyassociated withdiseaseinbats.Atleast12%ofallbatshaddieddueto bacterial,viralandparasiticinfections.Finally,wefoundclear seasonalandindividualvariationsindiseaseprevalenceand infectionrates,indicatinganincreasedsusceptibilitytoinfectious agentsinfemalebatsandjuvenilesduringthematernityseason. Ourdataemphasizeandprovidethebasisfordiseaserelated studiesinbatspeciesonpopulationleveltoelucidatethe complexityoftheecologyofinfectiousagentsandhostspecies likewise.SupportingInformationTableS1BacteriaisolatedfrombatsfoundinGermany. (DOC) Figure3.Species-specificparasiteinfectionrates. (A)Ecto-and(B)endoparasiteprevalenceindifferentEuropeanvespertilionidbatspecies. Errorbarsrepresent95%binomialconfidenceintervals.Abbreviations:Nnoc, Nyctalusnoctula ;Mdaub, Myotisdaubentonii ;Vmur, Vespertiliomurinus ; Enils, Eptesicusnilssonii ;Ppip, Pipistrelluspipistrellus ;Eser, Eptesicusserotinus ;Paur, Plecotusauritus ;Pnath, Pipistrellusnathusii ;Mnatt, Myotisnattereri ; Mmyst, Myotismystacinus . doi:10.1371/journal.pone.0029773.g003 DiseasesandCausesofDeathinEuropeanBats PLoSONE|www.plosone.org10December2011|Volume6|Issue12|e29773

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AcknowledgmentsTheauthorswouldliketothankBerlinerArtenschutzTeam–BAT-e.V.,F. Brandes,I.Frey-Mann,H.Geiger,J.Haensel,J.Harder,L.Ittermann,M. Kistler,M.Kredler,S.Morgenroth,E.Mu ¨hlbach,K.Mu ¨ller,R.Pfeiffer, W.Rietschel,S.Rosenau,R.Straub,G.Strauß,andW.andH.Zoelsfor providingthebatcarcasses,toN.Jahn,C.Kohl,J.Kliemt,D.Krumnow, D.Lieckfeldt,P.MachnowskaandM.Sonntagfortheirexcellentlabwork, andM.GrobbelandG.-A.Czirja ´kfortheirassistanceandhelpful discussions.Wearealsogratefulto2anonymousreviewersfortheirhelpful suggestionsandcomments.AuthorContributionsConceivedanddesignedtheexperiments:SSAKTMGW.Performedthe experiments:KMSSAKRLCFTMGW.Analyzedthedata:KMAK SKS.Contributedreagents/materials/analysistools:KMSSAKRLCF TMSKSGW.Wrotethepaper:KM.References1.SchipperJ,ChansonJS,ChiozzaF,CoxNA,HoffmannM,etal.(2008)The statusoftheworld’slandandmarinemammals:diversity,threatandknowledge. 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