Aserological survey ofcommon felinepathogens infree-livingEuropeanwildcats (Felissilvestris) incentral Spain
Javier Millán& Alejandro Rodríguez
Abstract Twenty-five serum samples of 22 free-living
Europeanwildcats(Felissilvestris)capturedfrom1991to
1993incentralSpainweretestedforevidence ofexposure toseven felinepathogens.Allthewildcatsbutone(95.4%) presentedevidenceofcontactwithatleastoneof theagents (mean=2.2). Contactwith felineleukemiavirus (FeLV) wasdetectedin81%ofthewildcats(antibodies, 77%; antigenp27,15%).Antibodiestofelinecalicivirus(FCV,
80%),felineherpesvirus (FHV,20%),felineparvovirus (FPV,18%),andChlamydophila sp.(27%)werealso detected.Analyses werenegativeforfelineimmunodefi- ciencyvirusandfelinecoronavirus. Theprobability of havingantibodies toFPVwasinverselyrelated withthe concentrationofserumcholesterolandwithamorphomet- ricindexofbodycondition.Similarityinthecomposition ofantibodiesagainstdiseaseagents(numberandidentityof detectedandundetectedantibodies) wassignificantly higherinpairsoffemalewildcatsthaninpairsofmales orheterosexualpairs,suggestingthatfemaleshadamore homogeneousexposuretopathogens.Seroprevalencefor FHVwashigherinmalesthaninfemales.Antibodiesto FHVandChlamydophilasp.weremorefrequentinwinter thaninotherseasons.Inaddition,themeansimilarityofthe pathogencommunitybetweenpairsofserumsampleswas
C ommunicatedbyC.Gortázar
J.Millán(*) FundacióNaturaParc,
07142SantaEugènia(BalearicIslands),Spain e-mail:
A.Rodríguez
DepartmentofConservationBiology,EstaciónBiológicade
Doñana(CSIC),
Avda.MaríaLuisas/n,
41013Sevilla,Spain
higher ifbothwildcatswerecaughtduringthesameseason thaniftheywerenot.Meansimilaritywaslowestwhen serumsamplesobtainedinwinterwerecompared with thosefromspringorsummer.The resultssuggestthat some agentsprobablyhadareservoir indomesticcatsandmay causesomeundetectedmorbidity/mortalityinthestudied wildcatpopulation,whereasothers,suchasFeLVandFCV, maybeenzootic.
Keywords Felid.Felineleukemia.
Felinerespiratorydisease.Seasonality.Serology
Introduction
TheEuropeanwildcat(Felissilvestris Schreber1777)is cataloguedasvulnerable (IUCN2007).Thewildcathasa scattereddistributioninEurope,whereithasdisappeared frommuchofitsoriginaldistribution area,resultingin severefragmentationofitspopulations.TheMediterranean areathatincludestheIberianPeninsula representsonehalf ofthedistributionrangeofthespeciesinEurope(Lozanoet al.2003).AccordingtoGarcía-Perea(2002),thewildcat populationinSpaincouldhavedeclinedandexperienced considerablefragmentation. Acknowledgedthreatsare persecution,habitatloss,andhybridizationwithdomestic cats.However,theimportanceofotherfactorsofdecline hasbeensofarneglected.Inparticular, anincreasing concernexistsabouttheroleofinfectious diseasesasa causeofdeclineofpopulationsofsomewildspecies(Smith etal.2006).Domesticcatsarecommoninthedominant culturallandscapesofMediterranean Europe,allowingfor theirinteractionwithwildlifeandincreasingthepotential fordiseasetransmission betweendomesticcatsand wildcats.Infact,wildcatsfromanumberoflocationsin
northern Europehostvirusescommonlyfoundindomestic cats(McOrist etal.1991;ArtoisandRemond1994; Danielsetal.1999;Leuteneggeretal.1999;Fromontet al.2000).Contact withthepathogenicFelineleukemia virus(FeLV)wasdetectedinallthesestudies.Fromont et al.(2000)alsodetectedinfectionswiththeFelineimmu- nodeficiency virus(FIV).Incontrast,Račniketal.(2008) failedtodetectevidenceofcontactwithFeLVandFIVin wildcatsinSlovenia.
Exceptinga few parasitologicalstudies(Torreset al.
1989;RodríguezandCarbonell1998),noinformationis availableaboutthediseaseagentsinfectingtheEuropean wildcatintheIberian Peninsulaorotherareasofthe Mediterraneanregion.FIV,FeLV,andotherfelinepatho- gensareknowntoinfectdomesticcatsinSpain(Arjonaet al.2000;Solano-Gallegoetal.2006).Thesediseaseagents mightnotonlydecreasethepersistence oflocalwildcat populationsthroughincreasedmortalitybutalsoinmore subtleways,e.g.,alteringthebehaviororreducingthebody conditionandfitnessofinfectedindividuals(Scott1988). Furthermore, sincethemechanisms oftransmissionbe- tweenspecies ofsuchdiseaseagentsarepoorlyknownin the wild,wildcatscouldbeinvolvedinthe epidemiologyof infectious diseasesthatmayposeariskforsympatricrare endangeredspecies,specially theIberianlynx(Lynx pardinus),whichhavelittlecontactwithanthropicenviron- ments(Delibesetal.2000).
Theaimsofthepresent studywere(1)toassess the seroprevalenceagainstfelinediseaseagentsinfree-living wildcatsand(2)toexaminewhetherprevalence,numberof detectedagents,andsimilarity betweenwildcatsinthe compositionofthepathogenstheywereexposedtowere relatedwithsex,season,andbodycondition.
Materials andmethods
Westudied awildcat population ina110-km2areaof easternToledoMountains,southcentralSpain(39°18′N,3°
45′W).Thestudyareaconsistsofamosaic ofMediterra- neanshrubland, cereal fields,andtoalesser extent, pastureland.Major land uses other than agricultureare gameandsheepandgoat husbandry.Therearenotownsor otherformsofurbanuse,buttheareacontains scattered farms(0.1farm/km2),mostofthempermanentlyinhabited, wherefree-roaming domesticcatsanddogsarefairly common.Around30buildingsaggregateina60-haspot neartheeasternendofthestudyarea;afewareusedas holidayhouses;theotherarevirtually abandoned.
Inthecourseof ecologicalstudies,double-entrancebox trapsandpaddedno.2Victorcoil-spring traps(Wood- stream,Lititz,Pennsylvania)wereplacedduringperiods of2–16 weeksacrossthestudyarea.Trappingtookplace
fromwinter(January–March)tosummer(July–Septem- ber),between1991and1993.Twenty-two wildcatswere caughtandimmobilizedeitherwithketaminehydrochlo- ride(Ketolar©,Parke-Davis, Spain)mixedwithxylazine hydrochloride(Rompún©,Bayer,Spain),or withtiletamine- zolazepam(Zoletil©, Virbac,Spain).Wildcatswere measured,weighedandclassifiedasadultsorjuveniles (<1yearold),onthebasisoftoothwear,aswellassigns ofpregnancyorcurrent(milk)orpastlactation(hairless areasaroundnipples).Coatpatterns, size,weight, and spatialbehaviorindicated theseanimals werenotdomes- ticorferal cats.Oursampleconsisted offiveadultmales,
15adultfemales, andtwojuvenilefemales. Wholeblood wasobtainedfromvenipuncture ofthecephalicveins. Sampleswerecollected inserumseparator tubesand allowedtoclot.Then,theywerecentrifugedat2,000rpm for15min,andtheserumwasremoved.Afractionwas subjectedtobiochemicalanalysis,andtherestwaskept frozenat−20°Cuntilexamined forthepresenceof antibodies todiseaseagents.Sincethreeindividuals were caughttwice,25serumsampleswereobtained.Foreach wildcat,bodyconditionwasestimatedintwoways:(1) theresidualofaregression lineof(log)bodyweight(g) against (log) headplusbodylength(mm), and(2)the concentrationoftotalcholesterol(mg/dl)intheserum.In mammals, itiswellknownthattheintakeofanimal proteinsincreasesplasmatotalcholesterol(Forsythe etal.
1980;SuganoandRoba1993).Thus,wehypothesized thata higherconcentrationof serumcholesterolindicatesa higherproteinintakeandabettercondition.Morpholog- icalandbiochemicalestimatesofconditionwerestatisti- callyindependent(r=0.373,p=0.106).
Serumsamplesweretestedusingenzyme-linkedimmu- noassay(ELISA)usingcommercial kitsprovidedby Ingenasa(Madrid,Spain)andEVL-Eurovet (Madrid, Spain)tothefollowing diseaseagents:FIV(Ingezim FIV©),FeLV(IngezimFeLVgp-70©), Felinecoronavirus (FCoV;IngezimFCoV©),Felinecalicivirus(FCV;F1008- AB02-Feline calicivirusantibodyELISA©),Felineherpes- virus-1(FHV;F107-AB02-Feline herpesvirusantibody ELISA©),andChlamydophilasp.(F1009-AB01-Chlamyd- iaantibodyELISA©).Presenceofantibodiesagainstfeline parvovirus(FPV)weredeterminedusingatestforcanine parvovirus(IngezimCPV©)usinganti-catIgGasconju- gate,followingtherecommendationsofthemanufacturer. Finally,thepresenceofFeLVp27wasdetermined with ELISAforantigendetection(IngezimFeLV-Das©).Each procedureincorporatedpositiveandnegativecontrolsera accordingtothemanufacturer’sinstructions.
Weexploredtheunivariateeffectsofsex,season,and bodycondition onthepresence ofantibodies againsteach considered pathogen with logistic regression. Then we fitted generalized linear models with Poisson errors to
examinetheeffectofthesamepredictorsonthenumberof diseaseagentsperindividual. Finally,weexaminedhow similarthecompositionofpathogenswasineachsampleas afunction ofsexandseason. Foragivenpairofserum samples AandB,wecomputedthesimplematching coefficient(Krebs1998),SSM¼ðaþdÞ=ðaþbþcþdÞ, whereaisthenumberofpathogens thatoccurbothin samplesAandB,b isthenumberofantibodiesinsampleB but not in sampleA, c is the numberof antibodiesin sample AbutnotinsampleB,anddisthenumberof antibodiesabsentinbothsamples.Wecalculatedthisindex ofsimilarityintheidentityofantibodiesforallpairsof samplesandcomparedthemeansofthesimplematching coefficient betweencombinationsoflevelsofsexand seasonwithanalysisofvariance.
Results
Allthewildcatsbutone(95.4%) presented evidences of contact with at least one of the pathogens (mean=2.2 pathogens/individual).Four wildcats revealed antibodies foroneagent(18.2%),11fortwoagents (50.0%),threefor threeagents(13.6%), twoforfouragents(9.1%),andone for five agents (4.5%). Seroprevalencesare shown in Table1.ExposuretoFeLV andFCVwasdetected, respectively,in81%and80%ofindividuals.Antibodies againstFPV,FHV,andChlamydophilasp.werefoundin
20–30% ofindividuals. Therewasnoevidenceofcontact withFIVorFCoV(Table1).
AntibodiesagainstFeLVweredetectedin17wildcats.
Seraoftwootherindividuals yieldedanabsorbancevalue betweenthepositiveandnegativecutoffs.Inaddition,three wildcatsresultedpositivefor antigen detection.One of thempresentedbothantigenandantibodies. Asecond individual wassampledtwiceandpresentedantigenand antibodiesonly inthesecondsample,1yearlater.Thethird
wildcatwasalsosampled twice,andinthefirstsampling, bothtestswerenegative,but2weekslater, itpresented antigenemiabutnotantibodies(seeTable2fortheseand othercasesofseroconversion).
Theprevalence ofFHV inmaleswasfourtimeshigher thaninfemales, andthesesexualdifferencesweremargin- allysignificant(Table3).Prevalencewassimilarinboth sexesforotheragents,exceptChamydophila sp.whose proportion inmaleswasmorethantwiceashighasin females,thoughdifferences werenotsignificant. The seroprevalenceofFHVinwildcatscaughtinwinterwas significantly higherthaninsamplesobtainedinother seasons(Table3).TherewasalsoatendencyforChamy- dophilasp.todecreaseitsprevalence fromwinterto summer,whereaswefoundlowervariation inseasonal prevalenceforotheragents.Ingeneral,theprobabilityof havingcontactedadiseaseagentshowedaweakassocia- tionwithindicesofbodycondition.However,theproba- bilityofhavingantibodiesagainstFPVhadasignificant inverserelationshipwiththeconcentration ofserum cholesterol(Table3).Asimilartrendwasfoundbetween thisprobabilityandthemorphometric indexofbody condition. No apparent clinicalsigns were observed in anyofthestudiedwildcatsatthetimeofhandling.
Poissonregression indicatedthatsex,season,andbody conditiondidnotexplainasignificantfractionofdeviance inthenumberoftypesofantibodies foundinserum samples.
Similarityinthecomposition ofantibodiesagainst diseaseagents(number andidentity ofdetectedand undetectedantibodies)wassignificantly higherinpairsof femalewildcatsthaninpairsofmalesorheterosexualpairs (one-wayanalysisofvariance(ANOVA),F2,250=4.27,p=
0.015; Fig.1).Themean(±SE)simplematchingcoefficient
in pairs of the same sex (0.67±0.02) was significantly higherthaninwildcatpairsofdifferentsex(0.60±0.02;t=
2.48,df=251,p=0.014).Therewasaseasonalpatternof
Table1 Infectious agentsforwhichevidencesofcontactwereexaminedinasampleofEuropeanwildcat(F.silvestris)serafromsouthcentral
Spain,orderedbydecreasingprevalence
Agent / Positive/tested / Prevalence / 95%CIFelineleukemiavirus
Antibodies / 17/22 / 0.77 / 0.59–0.88
Antigenp27 / 3/20 / 0.15 / 0.04–0.37
Total / 18/22 / 0.81 / 0.61–0.93
Felinecalicivirus / 16/20 / 0.80 / 0.57–0.93
Chlamydophilasp. / 6/22 / 0.27 / 0.12–0.50
Felineherpesvirus-1 / 4/20 / 0.20 / 0.07–0.42
Felineparvovirus / 4/22 / 0.18 / 0.06–0.39
Felineimmunodeficiencyvirus / 0/22 / 0.00 / 0.00–0.15
Felinecoronavirus / 0/22 / 0.00 / 0.00–0.15
Table2 Serological resultsforwildcatssampledtwice
GM32(♀,Ad) / GM47(♀,Ad) / GM78(♂,Ad)1stsampling / 2ndsampling / 1stsampling / 2ndsampling / 1stsampling / 2ndsampling
May3rd, / May18th, / June14th, / August30th, / January12th, / March1st,
1991 / 1991 / 1992 / 1993 / 1994 / 1994
FeLVAb / Negative / → / Negative / Uncertain / → / Positive / Positive / → / Positive
FeLVp27 / Negative / → / Positive / Negative / → / Positive / Negative / → / Negative
FCV / Positive / → / Positive / Negative / → / Positive / Positive / → / Negative
FHV-1 / Negative / → / Negative / Negative / → / Negative / Positive / → / Positive
Chlamydophila
sp. / Negative / → / Negative / Positive / → / Negative / Positive / → / Positive
Code,sex,andagearegiven.AllanalyseswerenegativeinthesewildcatsforFIV,FPVandFCoV
similarity too.Thecompositionofantibodiesinwinter samplesdifferedfromthecompositioninsamplescollected inotherseasonsmorethanthecomposition ofspring– summerpairsofserumsamples andmorethanthe composition ofpairsofsamplescollected duringthesame season(one-wayANOVA,F5,247=6.71,p0.001;Fig.1). Themean(±SE)simplematchingcoefficientforpairsof wildcatscaughtduringthesameseason(0.68±0.02)was significantly higherthanforpairsofwildcatscaughtin differentseasons(0.61±0.02;t=2.55,df=251,p=0.011).
Discussion
Observedseroprevalences
Seroprevalencetothe differentstudieddiseaseagentsinthe wildcatpopulationofToledoMountainswaseitherinthe rangeof,orhigherthan,valuespreviously reportedin Europe(Artois andRemond1994;McOristetal.1991; Danielsetal.1999;Leuteneggeretal.1999;Fromontetal.
2000). ActiveinfectionsbyFeLV weredemonstratedinthe studiesquotedabove.TheFeLVantigenemiadetectedinthe presentstudywasintherangeofthedata byFromontetal.
(2000),McOristetal.(1991),andDanielsetal.(1999)but waslowerthanthatobserved byLeuteneggeretal.(1999). Ourresultsmay indicatethat, asobservedbyMcOristetal. (1991)andincontrastwiththeobservations inotherwild felids(e.g.Citino1986), FeLV maybeself-sustainedinthe studiedwildcatpopulation.However,transmission from domesticcatsmayalsooccur.Felineleukemiavirus isfatal indomesticcats,andmortalityrelatedtoFeLVinfection havebeenreportedinafree-livingcougar(Pumaconcolor; Jessupetal.1993)andinacaptivebobcat(Lynx rufus; Sleemanetal.2001).Similarcaseshavenotbeenrecorded intheEuropeanwildcat.Forexample,Leuteneggeretal. (1999)didnot find clinicalsignsinFeLV-infectedwildcats. Asarule,noevidentsignswereobserved inthewildcats duringthissurvey,eitherseropositiveorinfected.This may indicatethatmostofthewildcatsmaysurvivetheinfection. Nevertheless, someundetectedmortalityduetofeline leukemiamayoccur.
Eighteenpercentofthewildcatspresented antibodies againstFPV.Thisvaluewashigherthanthosereported by Leuteneggeretal.(1999)andOstrowskietal.(2003)by meansoftheimmunofluorescenceassay.Felineparvovirus causesfelinepanleukopenia(FPL),andmortalitydueto thissyndromehasbeenobservedinotherwildfelids(e.g.
Table3 Effectsofsexandseasonontheprevalenceofantibodies inwildcatssera
SexGpSeasonGpCondition
Females / Males / Winter / Spring / Summer / W/L / p / Chol / pFeLV / 0.72 / 0.86 / 0.542 / 0.462 / 0.86 / 0.69 / 0.75 / 0.703 / 0.703 / + / 0.556 / + / 0.601
FPV / 0.17 / 0.14 / 0.022 / 0.883 / 0.29 / 0.08 / 0.25 / 1.702 / 0.427 / − / 0.135 / − / 0.032
FCV / 0.76 / 0.67 / 0.214 / 0.644 / 0.80 / 0.69 / 0.75 / 0.231 / 0.891 / − / 0.731 / − / 0.797
FHV-1 / 0.12 / 0.50 / 3.452 / 0.063 / 0.80 / 0.08 / 0.00 / 11.527 / 0.003 / − / 0.957 / − / 0.139
Chamydophilasp. / 0.17 / 0.43 / 1.773 / 0.183 / 0.43 / 0.23 / 0.00 / 3.386 / 0.184 / − / 0.589 / − / 0.318
TheGstatisticindicatestheresultsoflogisticregressions(sex,df=1;season,df=2).Thesignofthelogitcoefficient anditssignificanceare shownfortheeffects oftwoindicesofbodycondition(df=1)
W/Lresidualsoftheregression log-weight vs.log-length,Cholserumcholesterolconcentration
Fig.1Mean(+SE)similarity inthecompositionofthepathogen communityinpairsofwildcatserumsamplesaccordingtosex(upper panel)andseason(lowerpanel)
inbobcat,Wassmeretal.1988;orcougar,Roelkeetal.
1993).Thisdiseaseismarkedly harmfulinkittens(Barker and Parrish2001).Thus,ifwildcatkittenssuccumboftento FPLasobserved inferalcats(vanRensburgetal.1987), thisdiseasemayhaveasubstantial impactonproductivity andthusonpopulation dynamics.Wefoundanegative relationship betweenindicesofwildcatconditionand exposuretoFPV.TheanalysisperformeddetectsIgG,thus indicatingpastinfection.Then,itisdifficult toexplainan effectofapastinfectionwithwildcatcondition atthetime ofsampling. Since,assaidabove,FPLisespecially harmfulinyoungcats,someincorrectdevelopmentdueto the diseasecouldpromoteasubsequentimpairedcondition. Anyway,relatively poorconditioninFPV-seropositive wildcats,togetherwiththeobserved lowseroprevalence, couldbeduetoFPV-associated morbidityormortality, althoughour resultsareinconclusiveinthis regard.Itisnot knownwhetherFPVisself-sustained inthewildcat populationordomesticcatsactasareservoir.Thisvirus
isextremely resistant andcansurviveformonthsinthe environment(Greene1998).Therefore,inadditiontodirect contactwithavirus-sheddingcarnivore,habitatcontamina- tionbydomestic catfecesmaybeasourceofinfection (Roelkeetal.1993).Additionaldifficultiestointerpretthe moderate FPVprevalencewefoundarisefrompotential specificity problems(Ostrowskietal.2003),sincethe ELISAtestutilizedheredoesnotallowdetermining if wildcatswereinfected bythefelineorthecanine parvovirus strain.Thus,notonlydomesticcatsbutalso dogsandredfoxes(Vulpesvulpes)couldserveassources ofinfection forwildcats,assuggestedfortheIberianlynx (Roelkeetal.2008).
Wedetectedevidence ofexposure tothethreemain agentsinvolvedinthefelinerespiratorysyndrome(FRS), i.e.FCV,FHV,andChlamydophila sp.ContactwithFCV andFHVwasalsodetected byArtoisandRemond(1994), Danielsetal.(1999),Leutenegger etal.(1999),and Ostrowski etal.(2003),buttheprevalences wefoundare markedlyhigherthan thosereportedinthesestudies.Asfar asweknow,wereporthereforthefirsttimeantibodies to Chlamydophilasp.(orantigenically relatedagents)ina wildfelinespecies.OnlyPaul-Murphy etal.(1994)tested butfailedtodetectantibodiestochlamydial agentsin cougars. Again,itisunknownwhethertheseagentsare endemicinthestudiedwildcatpopulation orwildcats becameinfectedaftercontactwithdomestic cats.Rileyet al.(2004)foundhighertiterstoFCVinbobcatsfromareas wheredomestic catsoccurred,suggestingthatFCVmight havebeenendemic indomestic catsbutnotinbobcats.In contrast,Roelkeet al. (1993)believedthatthevirusmaybe presentinFloridapanthers,withtransmissionsimilartothat in the domestic cat (a persistently infected mother- to-offspringpathway).We believe that the moderateto highprevalenceofdiseaseagentsrelatedwithFRSsuggests that(1)contactsbetweendomestic andwildcats,themost feasiblewayoftransmissionofFHVandChlamydophila, arefrequent, and(2)FCVmaybeendemicinthiswildcat population,themostlikelywayoftransmissionbeingfrom motherstokittens.Thepathologicalsignificanceofthese agentstowildcatsisunknown.Indomesticcats,morbidity ofFRSisconsiderable,althoughmortalityislow(Greene
1998).Moderateorhighseroprevalences suggestthatthis maybethecaseforwildcats, althoughsomeneonatal mortalitymayoccurtoo.
Thepresentstudyfailedtodemonstratewildcatcontact
withFIVandFCoV.Bothagentsseemtobeinfrequentin otherEuropeanwildcatpopulations(McOristetal.1991; Danielsetal.1999;Leuteneggeretal.1999;Fromontetal.
2000).FIVmightnotbepresent inthestudiedpopulation, althoughFIVantibodiesaremorelikelytobefoundin adult,malecats(Sellon 1998),andonlyfiveindividuals belonging to this cohort were included in the present
survey.TheprevalenceofFIV indomesticcatpopulations inSpainisaround8%(Solano-Gallegoetal.2006;Arjona etal.2000).Thus,theoccurrenceofunrecordedfatal cases offelineimmunodeficiencyinEuropeanwildcatsfromSpain cannotbediscarded.Theobservedlowseroprevalenceto FCoVmaybeduetoaprobableabsence oftheagentinthe domestic catsofthestudyarea.AccordingtoAddieand Jarret(1998),FCoVismorecommonindomesticcatsin catteriesthaninpetcatskeptsinglyorinferalorstraycats. Thedomesticcatsinhabitingourstudyareacanbebetter describedasferalcatsthanhouseholdcats.
Seasonandsex-relateddifferences
WefoundarelativelyhighseroprevalencetoFHVinwinter. Assumingthatinterbreeding maynotbeuncommon,we believethattransmission ofFHV(andperhapsofother agents)maybeenhancedduring theannual populationpeak ofwildcatsanddomesticcatsinsummer-autumn,whichmay explainahigherseroprevalenceinthefollowingwinter.No season-relateddifferencesinprevalenceofanyofthestudied agentswasrecordedinpreviousstudiesregardingwildcat (McOristetal.1991;Danielsetal.1999;Leuteneggeretal.
1999;Fromontetal.2000;Ostrowski etal.2003)orother wildfelines(e.g.,Paul-Murphyetal.1994).Inredfoxes fromAustralia,Robinsonetal.(2005)observed thatthe seroprevalence toCanineadenoviruspresentedthehighest seroprevalenceinwinter–spring, suggestingthatfoxesbecame infected inthematingseasonmoreprobablythanpriorto dispersal,whichisinagreementwithourhypothesisfor FHV. Relatedwiththis,theobservedhigherseroprevalenceof FHV andChlamydophila sp.inwildcatmalesmaybealsorelated withhigherratesofintra-andinter-species encounters because malestendtomovemoreduringthematingseason (Liberg,1988).
Wealsoobservedasimilarpatternofexposuretoagents in wildcatssampledin springand summer,whereasanimals sampledinwinterpresentedadifferent pattern.These resultsindicatethatthereareclearseasonal-relatedvaria- tionsintheexposuretotheagents.Wehypothesizethat, duringwinter,somemortalityofwildcatsmayberelated withtheexposuretooneormoreofthestudiedagents.This factcouldbelinkedwith thehigherseroprevalencetoFHV andChlamydophila sp.inwintersamples:selective mortality associated to these pathogens would explain whytheseagentsshowedlowerseroprevalence inthe followingspring–summer.Althoughbothpathogenscourse withlowmortality inadultdomesticcats(Gaskelland Dawson1998),nothingisknownabouttheclinical course oftheseinfectionsinfree-livingwildcats.Insomecases,a chronificationofthediseaseincatsoccurs,withclinical signsrangingfromsneezingandnasaldischargetocorneal edema and blindness(Gaskell and Dawson 1998). The
lattermaybecritical forsurvivalinthewild,andsome mortalityrelatedtotheseagentscannotbediscarded.
Interestingly, theidentityofagentstowhichfemales presentedantibodies,aswellastheidentityofagentsto whichfemaleswerenotexposed,weremoreconsistentthan thecompositionofinfectious agentsinmales.Inaddition, thesimilarityintheresultsoftestsformultipleagentsin pairsofthesamesexwassignificantly higherthanin wildcatpairsofdifferentsex.Asfarasweknow,nosimilar findings havebeenpreviously reportedforanywild species.Thisresult,farfromgivingsimpledifferencesin seroprevalence amongsexes,presentsevidenceofa different patternofexposureamongfemalesandmalesto thestudiedagents.Wesuggestthat,whereasthebehaviorof thefemales exposesthemtoaparticularsetofagentsand preventsexposuretoothers,thebehaviorofmales(witha highermovementpattern,especially amongjuvenilesduring dispersalandadultsduringmating)makesmoreunpredictable thetypeof diseaseagentstheyface.
Conclusions
Ourstudyconfirmedthatwildcatsincentral Spainwerein contact withfiveofthestudied commonfelinepathogens, someofwhichexhibitedhighseroprevalences.Ithasnot beendeterminedtowhatextenttheseagentsposeariskfor wildcat populations, althoughanegativerelationship betweenseroprevalenceandconditionwasfoundforFPV. Therefore, ourresultsindirectlysupportthehypothesisthat diseasesmaycontribute toreduceindividualfitness.In addition,duringtheperiodwhenwildcatswerecaptured,the extremelyendangeredIberianlynxwasstillpresentinToledo Mountains(Rodríguez etal.1996)andthuspotentially exposedtothestudiedpathogens.Sincethecausesofthe markedregressionoflynxpopulationsinthelast30years aremultipleandhavenotbeencompletely elucidated (Rodríguezand Delibes2002)andverylittleisknown about naturalcausesofmortality(Ferreras etal.1992;Rodríguez andDelibes2004),webelievethatdiseases mightbe consideredasapotentialcontributingfactorofdeclinein theToledoMountains lynxpopulationduringthe1980s.
Acknowledgments J.NicolásGuzmánandGiuliaCremaassisted withwildcattrappingandsamplecollection.WethankAmparoGomis at INAC(CiudadReal,Spain)forperformingbiochemicalanalyses,J. ManuelPérezdelaLastraatIREC(CiudadReal,Spain)forlaboratory assistanceduringseraanalyses,andJacquesDelbecque(Ingenasa)and SilviaSimón(EurovetVeterinariaS.L.)forinformationandadvice aboutimmunoassayperformance.Fieldworkwassupportedbythe SpanishMinistryofTransportation, andlabworkaswellas manuscriptpreparationwas fundedbygrantsfromJuntadeAndalucía toA.Rodríguez.Thetrappingandsamplingprotocolfollowedthe Spanishlawsofanimalwelfareinscientificresearch(RealDecreto
1201/2005).
References
AddieDD,JarretO(1998)Felinecoronavirus infections.In:Greene CE(ed)Infectious diseasesofdogandcat.W.B.Saunders, Pennsylvania,pp63–75
ArjonaA,EscolarE,SotoI,BarqueroN,MartínD,Gómez-LucíaE (2000)Seroepidemiologicalsurveyofinfectionbyfelineleukemia virusandimmunodeficiencyvirusinMadridandcorrelationwith someclinicalaspects.JClinMicrobiol38:3448–3449
ArtoisM,RemondM(1994)Viraldiseasesasathreattofree-livingwild cats(Felissilvestris)incontinentalEurope.VetRec134:651–652
BarkerIK,ParrishCR(2001)Parvovirusinfections.In:WilliamsES, BarkerIA(eds)Infectious diseasesofwildanimals.3rdedn. IowaStateUniversity Press,Iowa,pp131–146
CitinoSB(1986)TransientFeLVviraemiainacloudedleopard.J Zoo
WildlMed17:5–7
DanielsMJ,GolderMC,JarrettO,Macdonald DW(1999)Feline virusesinwildcatsfromScotland.JWildlDis35:121–124
DelibesM,RodríguezA,Ferreras P(2000)Actionplanforthe conservationoftheIberianlynxinEurope(Lynxpardinus).Nature andEnvironmentSeries 111.CouncilofEurope,Strasbourg
FerrerasP,AldamaJJ,BeltránJF,DelibesM(1992) Rates andcauses
ofmortalityinafragmentedpopulationofIberianlynxFelis pardina Temminck, 1824. Biol Conserv 61:197–202.doi:10.1016/0006-3207(92)91116-A
Forsythe WA,MillerER,HillGM,RomsosDR,Simpson RC(1980)
Effectsofdietaryproteinandfatsourceonplasmacholesterol parameters,LCATactivityandaminoacidlevelsandontissue lipidcontentofgrowingpigs.JNutr110:2467–2479
FromontE,SagerA,LegerF,BourguemestreF,JouqueletE,StahlP, PontierD,ArtoisM(2000)Prevalenceandpathogenicity of retrovirusesinwildcatsinFrance.VetRec146:317–319
García-PereaR(2002)FelissilvestrisSchreber,1775.In:PalomoLJ,
GisbertJ(eds)Atlasdelosmamíferosterrestres deEspaña. Madrid,DirecciónGeneraldeConservacióndelaNaturaleza- SECEM-SECEMU:pp.294–297
GaskellRM,DawsonS(1998)Felinerespiratory disease.In:Greene
CE(ed)Infectious diseasesofdogandcat.W.B.Saunders, Pennsylvania,pp106–115
GreeneCE(1998)FelinePanleukopenia.In:GreeneCE(ed)Infectious diseases ofdogandcat.W.B.Saunders, Pennsylvania,pp52–57
IUCN(2007)Felissilvestris. In:EuropeanMammalAssessment. environment/nature/conservation/species/ema/ species/felis_silvestris.htm.DownloadedonMarch31st,2008
JessupDA,PettanKC,LowenstineLJ,PedersenNC(1993)Feline leukemiavirusinfectionandrenalspirochetosisinfree-ranging cougar(Felisconcolor).JZooWildlMed24:73–79
KrebsCJ(1998)Ecologicalmethodology,2ndedn.AddisonWesley
Longman,MenloPark,CA
LeuteneggerCM,Hofmann-LehmannR,RiolsC,LiberekM,WorelG, LupsP,FehrD,HartmannM,WeilenmannP,LutzH(1999)Viral infectionsinfree-livingpopulationsoftheEuropeanwildcat.J Wildl Dis35:678–686
LibergO(1988)Spatialorganisationandreproductivetacticsinthe domesticcatandotherfelids.In:TurnerDC,BatesonP(eds)The domesticcat:thebiologyofitsbehaviour.CambridgeUniversity Press,Cambridge,pp83–98
LozanoJ,VirgósE,MaloAF,HuertasDL,Casanovas JG(2003) Importanceofscrub-pastureland mosaicsforwild-livingcats occurrenceinaMediterranean area:implicationsforthe conservationofthewildcat(Felissilvestris). BiodiversConserv
12:921–935.doi:10.1023/A:1022821708594
McOristS,BoidR,JonesTW,EasterbeeN,HubbardAL,JarrettO (1991) Some viral and protozool diseases in the European wildcat(Felissilvestris).JWildlDis27:693–696
Ostrowski S,VanVuurenM,LenainDM,DurandA(2003)A serologicsurveyofwildfelidsfromCentralWestSaudiArabia.J WildlDis39:696–701
Paul-MurphyJ,WorkT,Hunter D,McfieE,FjellineD(1994)Serologic surveyandserumbiochemicalreferencerangesofthefree-ranging mountainlion(Felisconcolor) inCalifornia.JWildlDis30:205–215
RacnikJ,Skrbinsek T,PotocnikH,KosI,TozonN(2008)Viral infectionsinwild-living EuropeanwildcatsinSlovenia.EurJ WildlRes54:767–770.doi:10.1007/s10344-008-0202-y
RileySP,FoleyJ,ChomelB(2004)Exposuretofelineandcanine
pathogensinbobcatsandgrayfoxesinurbanand ruralzonesofa nationalparkinCalifornia.JWildlDis40:11–22
RobinsonAJ,CrerarSK,Waight-SharmaN,MüllerWJ,BradleyMP
(2005)Prevalence ofserumantibodiestocanineadenovirusand canineherpesvirusintheEuropeanredfox(Vulpesvulpes)in Australia.AustVetJ83:356–361.doi:10.1111/j.1751-0813.2005. tb15634.x
RodríguezA,CarbonellE(1998)Gastrointestinal parasitesofthe Iberianlynxandotherwildcarnivores fromcentral Spain.Acta Parasitol43:128–136
RodríguezA,DelibesM(2002)Internalstructureandpatternsof
contraction in the geographic range of the Iberian lynx. Ecography25:314–328.doi:10.1034/j.1600-0587.2002.250308.x RodríguezA,DelibesM(2004)Patternsandcausesofnon-natural mortalityintheIberianlynxduringa40yearperiodofrange
contraction.BiolConserv118:151–161. doi:10.1016/j. biocon.2003.07.018
Rodríguez A, Crema G, Delibes M (1996) Use of non-wildlife
passagesacrossahighspeedrailwaybyterrestrialvertebrates.J ApplEcol33:1527–1540.doi:10.2307/2404791
RoelkeME,Forrester DJ,JacobsonER,KolliasGV,BarrMC, EvermannJF,PirtleEG(1993)Seroprevalence ofinfectious diseaseagentsinfree-rangingFloridapanthers(Felisconcolor coryi).JWildlDis29:36–49
RoelkeM,JohnsonWE,MillánJ,PalomaresF,RevillaE,Rodríguez A,CalzadaJ,FerrerasP,León-Vizcaíno L,DelibesM,O’Brien SJ(2008)Exposuretodiseaseagentsintheendangered Iberian lynx(Lynxpardinus).EurJWildlRes54:171–178.doi:10.1007/ s10344-007-0122-2
ScottME(1988)Theimpactofinfection anddiseaseonanimal populations:implicationsforconservationbiology.ConservBiol
2:40–65.doi:10.1111/j.1523-1739.1988.tb00334.x
SellonRH(1998)Felineimmunodeficiency virusinfection.In: GreeneCE(ed)Infectious diseasesofdogandcat.W.B. Saunders,Pennsylvania,pp84–96
SleemanJM,KeaneJM,JohnsonJS,BrownRJ,WoudeSV(2001) Felineleukemiavirusinacaptivebobcat.JWildlDis 37:194–200
SmithKF,SaxDF,Lafferty KD(2006)Evidencefortheroleof infectiousdiseaseinspeciesextinctionandendangerment.Conserv Biol20:1349–1357.doi:10.1111/j.1523-1739.2006.00524.x
Solano-Gallego L,HegartyB,EspadaY,LlullJ,BreitschwerdtE (2006)Serological andmolecularevidenceofexposureto arthropod-borne organismsincatsfromnortheasternSpain.Vet Microbiol 118:274–277.doi:10.1016/j.vetmic.2006.07.010
SuganoM,RobaK(1993)Dietaryproteinandlipidmetabolism:a multifunctionaleffect.AnnNY AcadSci676:215–222. doi:10.1111/j.1749-6632.1993.tb38736.x
TorresJ, CasanovaJC, Feliú C, GisbertJ, ManfrediMT (1989)
Contribuciónalconocimiento delacestodofauna deFelis silvestris Schreber,1776(Carnivora:Felidae)enlaPenínsula Ibérica.RevIberParasitol49:307–312
VanRensburg PJJ,SkinnerJD,vanAardeHJ(1987)Effectsoffeline
panleucopaeniaonthepopulationcharacteristicsofferalcatson
MarionIsland.JApplEcol24:63–73.doi:10.2307/2403787
Wassmer DA,Guenther DD,LayneJN(1988)Ecology ofthebobcat inFlorida.BullFlaStateMusBiolSci3:159–228