Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white-nose syndrome

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Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white-nose syndrome

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Title:
Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white-nose syndrome
Series Title:
Ecology and evolution
Creator:
Johnson, Joseph S.
Reeder, DeeAnn M.
Lilley, Thomas M.
Czirják, Gábor Á.
Voigt, Christian C.
McMichael, James W. III
Meierhofer, Melissa B.
Seery, Christopher W.
Lumadue, Shayne S.
Altmann, Alexander J.
Toro, Michael O.
Field, Kenneth A.
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Wiley
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English
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Bats -- Mortality ( lcsh )
White-nose syndrome ( lcsh )
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serial ( sobekcm )

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Abstract:
White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans (Pd) that affects bats during hibernation. Although millions of bats have died from WNS in North America, mass mortality has not been observed among European bats infected by the fungus, leading to the suggestion that bats in Europe are immune. We tested the hypothesis that an antibody-mediated immune response can provide protection against WNS by quantifying antibodies reactive to Pd in blood samples from seven species of free-ranging bats in North America and two free-ranging species in Europe. We also quantified antibodies in blood samples from little brown myotis (Myotis lucifugus) that were part of a captive colony that we injected with live Pd spores mixed with adjuvant, as well as individuals surviving a captive Pd infection trial. Seroprevalence of antibodies against Pd, as well as antibody titers, was greater among little brown myotis than among four other species of cave-hibernating bats in North America, including species with markedly lower WNS mortality rates. Among little brown myotis, the greatest titers occurred in populations occupying regions with longer histories of WNS, where bats lacked secondary symptoms of WNS. We detected antibodies cross-reactive with Pd among little brown myotis naïve to the fungus. We observed high titers among captive little brown myotis injected with Pd. We did not detect antibodies against Pd in Pd-infected European bats during winter, and titers during the active season were lower than among little brown myotis. These results show that antibody-mediated immunity cannot explain survival of European bats infected with Pd and that little brown myotis respond differently to Pd than species with higher WNS survival rates. Although it appears that some species of bats in North America may be developing resistance to WNS, an antibody-mediated immune response does not provide an explanation for these remnant populations.

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K26-05073 ( USFLDC DOI )
K26.5073 ( USFLDC handle )

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Antibodiesto Pseudogymnoascusdestructans arenot sufcientforprotectionagainstwhite-nosesyndrome JosephS.Johnson 1 ,DeeAnnM.Reeder 1 ,ThomasM.Lilley 2 ,G abor A.Czirj ak 3 ,ChristianC.Voigt 3 JamesW.McMichaelIII 1 ,MelissaB.Meierhofer 1 ,ChristopherW.Seery 1 ,ShayneS.Lumadue 1 AlexanderJ.Altmann 1 ,MichaelO.Toro 1 &KennethA.Field 1 1 DepartmentofBiology,BucknellUniversity,Lewisburg,Pennsylvania17837 2 DepartmentofBiology,UniversityofTurku,Turku,Finland 3 LeibnizInstituteforZooandWildlifeResearch,Berlin,Germany Keywords Antibody-mediatedimmunity,antifungal immunity, Corynorhinus ,diseaseecology, Eptesicus ,hibernation, Myotis Perimyotis wildlifedisease,WNS. Correspondence KennethA.Field,DepartmentofBiology, BucknellUniversity,Lewisburg,PA17837. Tel:570.577.3814; Fax:570.577.3537; E-mail:keld@bucknell.edu FundingInformation Fundingforthisprojectwasprovidedbythe UnitedStatesFishandWildlifeServicegrant F12AP01210(DMRandKAF)andthe WoodtigerFoundation(DMR). Received:18February2015;Accepted:22 March2015 EcologyandEvolution 2015;5(11):2203 – 2214 doi:10.1002/ece3.1502 Abstract White-nosesyndrome(WNS)isafungaldiseasecausedby Pseudogymnoascus destructans ( Pd )thataffectsbatsduringhibernation.Althoughmillionsofbats havediedfromWNSinNorthAmerica,massmortalityhasnotbeenobserved amongEuropeanbatsinfectedbythefungus,leadingtothesuggestionthatbats inEuropeareimmune.Wetestedthehypothesisthatanantibody-mediated immuneresponsecanprovideprotectionagainstWNSbyquantifyingantibodiesreactiveto Pd inbloodsamplesfromsevenspeciesoffree-rangingbatsin NorthAmericaandtwofree-rangingspeciesinEurope.Wealsoquantied antibodiesinbloodsamplesfromlittlebrownmyotis( Myotislucifugus )that werepartofacaptivecolonythatweinjectedwithlive Pd sporesmixedwith adjuvant,aswellasindividualssurvivingacaptive Pd infectiontrial.Seroprevalenceofantibodiesagainst Pd ,aswellasantibodytiters,wasgreateramonglittlebrownmyotisthanamongfourotherspeciesofcave-hibernatingbatsin NorthAmerica,includingspecieswithmarkedlylowerWNSmortalityrates. Amonglittlebrownmyotis,thegreatesttitersoccurredinpopulationsoccupyingregionswithlongerhistoriesofWNS,wherebatslackedsecondarysymptomsofWNS.Wedetectedantibodiescross-reactivewith Pd amonglittle brownmyotisna   vetothefungus Weobservedhightitersamongcaptivelittle brownmyotisinjectedwith Pd .Wedidnotdetectantibodiesagainst Pd in Pd infectedEuropeanbatsduringwinter,andtitersduringtheactiveseasonwere lowerthanamonglittlebrownmyotis.Theseresultsshowthatantibody-mediatedimmunitycannotexplainsurvivalofEuropeanbatsinfectedwith Pd and thatlittlebrownmyotisresponddifferentlyto Pd thanspecieswithhigher WNSsurvivalrates.AlthoughitappearsthatsomespeciesofbatsinNorth AmericamaybedevelopingresistancetoWNS,anantibody-mediatedimmune responsedoesnotprovideanexplanationfortheseremnantpopulations. Introduction White-nosesyndrome(WNS)isafungaldiseaseresponsibleforprecipitousdeclinesinbatpopulationsinNorth America(Lorchetal.2011;Blehert2012;Reederand Moore2013;Fricketal.2015).SinceitsdiscoveryinNew Yorkin2006,millionsofbatshavediedfromWNS,with mortalitycontinuingatanalarmingrateasthedisease spreadsacrosstheUnitedStatesandCanada(Coleman andReichard2014;Fricketal.2015).Populationdeclines inexcessof90%havebeenestimatedforseveralspecies, leadingtopredictionsofregionalandspecies-levelextinctionsinthenearfuture(Fricketal.2010,2015;Turner etal.2011). WNSiscausedbythepsychrophilicfungus Pseudogymnoascusdestructans ( Pd ),originallyclassiedas Geomycesdestructans in2009(Gargasetal.2009;Minnis andLindner2013).Suitabletemperaturesfor Pd growth overlapwithtemperaturesinsidebathibernacula(Webb etal.1996;Humphriesetal.2002;Verantetal.2012), 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd. ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse, distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited. 2203

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allowingthefungustogrowinthewinterhabitatofmany batspecies. Pd invadesthedermisandepidermisofbats whiletheyhibernate(Blehertetal.2009),andinfectionis likelyfacilitatedbythereductioninimmunefunction typicalofhibernation(Boumaetal.2010a).Fungal colonizationcausesfataldisruptionsinbehavior(Brownlee-BouboulisandReeder2013;Wilcoxetal. 2014 )and physiology(Verantetal.2014),includingenergyand waterbalance(Cryanetal.2010,2013).Thecriticaldisruptioninenergybalanceisillustratedbythehibernation ecologyoflittlebrownmyotis( Myotislucifugus )that, whenaffectedbyWNS,exhibitanincreaseinfrequency ofarousalsfromhibernation,depletingfatreservesnecessarytosurvivewinter(Reederetal.2012;Warneckeetal. 2012).LittlebrownmyotissufferingfromWNSarealso moredehydratedthanhealthybats,suggestingevaporative waterlosscouldatleastpartiallyexplainthisincreasein arousals(Cryanetal.2013). Pd isnotnativetoNorthAmerica;itisbelievedto havebeenintroducedfromEurope,whereitiswidespread(Puechmailleetal.2011;Warneckeetal.2012; Wibbeltetal.2013).Europeanisolatesof Pd causemortalityinNorthAmericanbats(Warneckeetal.2012),but Europeanbatsinfectedwith Pd donotappeartohavethe samepathology(Wibbeltetal.2013).Thediscoveryof Pd growingonEuropeanbatsduringhibernationledto thesuggestionthatEuropeanbatsmaybeimmuneto WNS(Puechmailleetal.2010).However,somemammal immuneresponseshavebeenshowntobesuppressed duringhibernation(Boumaetal.2010a).Hibernating bats,therefore,likelyhavealimitedabilitytomountan immuneresponseto Pd duringtheperiodofactiveinfection.Thereductionofimmunefunctionduringhibernationshouldnotbeinterpretedtomeanthataprotective immuneresponseisnotpossible,however,asbothcellandantibody-mediated(humoral)immuneresponsescan occurduringhibernation(Maniero2002;Boumaetal. 2013).Whileantibody-mediatedimmuneresponsesto fungicanhelpclearinfections,theycanalsoleadtotoleranceofchronicinfection(CasadevallandPirofski2012a, b;W   uthrichetal.2012).Thus,thealternativehypothesis thatactivationofanimmuneresponseduringwintercontributestopathologyandmortalityamongNorthAmericanspeciesmustalsobeconsidered. Thecapacitytomountanimmuneresponseto Pd may differamongbatspecies.Forexample,thelittlebrown myotisisasmallbat(6 – 14g)thatexhibitsrelativelylong periodsoftorporbetweenperiodicarousalsfromhibernationcomparedtolargerspeciessuchasthebigbrownbat ( Eptesicusfuscus )(BrackandTwente1985;Twenteetal. 1985;Reederetal.2012).Thisgreaterfrequencyofarousalsinbigbrownbatsmayresultingreaterimmunecompetenceandabilitytorespondto Pd duringwinter, possiblyexplainingthelowerWNSmortalityrates reportedforbigbrownbats(Turneretal.2011;Frank etal.2014). Thepurposeofourstudywastoexaminetheroleof antibody-mediatedimmuneresponsesto Pd incaptive andfree-rangingbats.Wehypothesizedthatcaptivelittle brownmyotisexposedto Pd duringhibernationwould havegreaterantibodytitersinthespringthanbatsnot exposedtothepathogen.Asecondarygoalofourcaptive studywastodeterminewhetherseroprevalenceandtiters couldbeexperimentallyincreasedbyinjectingcaptivelittlebrownmyotiswithlive Pd .Amongfree-rangingbats, ourgoalsweretodeterminewhetherfree-rangingbatsin NorthAmericaandEuropeproduceantibodiesagainst Pd ,whenantibodylevelspeak,andwhetherantibodyproductionvariesamongspeciesandgeographicregions.We alsotestedwhetherantibodyseroprevalence,denedasa bloodsamplewithdetectableantibodiesreactiveto Pd andtiterarecorrelatesofWNSsurvival.Wehypothesized thatEuropeanspecieswouldexhibitgreaterseroprevalenceandtitersthananyNorthAmericanspecies.Within NorthAmerica,wehypothesizedthatpopulationsinhabitingregionswithlongerhistoriesofWNSwouldexhibit thegreatestseroprevalenceandtiters,andthattiters wouldpeakshortlyaftertheendofhibernation.Finally, wehypothesizedthatNorthAmericanspecieswithhigher WNSsurvivalratesandwinterecologiesfavoringmore frequentarousalsfromhibernationwouldexhibitthe greatestseroprevalenceandtiters.MaterialsandMethodsThisstudywascarriedoutonbatsfromnonendangered speciesinstrictaccordancewiththerecommendationsin theGuidefortheCareandUseofLaboratoryAnimalsof theNationalInstitutesofHealth.Allmethodswere approvedbytheInstitutionalAnimalCareandUseCommitteeatBucknellUniversity(protocolDMR-016),the AnimalEthicsCommitteeoftheUniversityofTurku (licensenumberESAVI/3221/04.10.07/2013),andthe LeibnizInstituteforZooandWildlifeResearchBerlin.CaptivestudiesWeestablishedacaptivepopulationoflittlebrownmyotisna   veto Pd in2013.Wecapturedbatsinmist-nets (Avinet,Inc.,Dryden,NY)placedoutsideknownWNSfreeroostsinMontana,USA,andtransportedtoourlaboratoryatBucknellUniversityinPennsylvania,USA.Followingarrival,26batswererandomlyselectedforan immunizationtrialtodeterminewhetherantiPd antibodytiterscouldbeboostedthroughinjectionof Pd preparations.Tenofthesebatsweregivenintraperitoneal2204 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.AntibodiesareInsufcientforWNSProtection J.S.Johnson etal .

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injectionsof6 9 106live Pd cellssuspendedin0.1mLof phosphatebufferedsaline(PBS)emulsiedin0.1mLof theNovartisadjuvantMF59,whichhasbeensuccessfully usedingeneratingprotectiveantifungalimmunityinmice (Torosantuccietal.2005).Asacontrol,16batsthatwere housedseparatelyweregivenintraperitonealinjections containingonly0.2mLPBS.Batsweregivenintraperitonealboosterinjectionsofeither0.2mLPBS(control bats)or6 9 106live Pd cellssuspendedin0.2mLof PBS(immunizedbats)at1and3weeksfollowinginitial injections.Wecollectedplasmasamplesfromall26bats 6weeksfollowinginitialinjections.Wecollectedblood intoheparinizedglassmicrohematocritcapillarytubes (KimballChaseLifeScience,Vineland,NJ)afterpuncturingaveinintheuropatagiumusinga27.5-gaugesterile needle(ReederandWidmaier2009).Capillarytubeswere immediatelycentrifugedtoseparateplasmafromblood cells,andplasmawasstoredat 80 C. Foraseparatecaptiveinfectionexperiment,wecollected147littlebrownmyotisna   veto Pd fromWNS-free hibernaculainMichiganandIllinoisinNovemberof 2012andbroughtthembacktoourlaboratory(Johnson etal.2014).Batswereeithercutaneouslyinoculatedwith Pd ( n = 118)orshaminoculated( n = 29)withPBS,and hibernatedfor5monthsincaptivity. Pd -inoculatedbats werehibernatedinaseparatechamberfromcontrolbats, andthetwogroupswerehousedseparatelyuponarousal fromhibernation. Pd usedforinoculationswasobtained fromanisolateharvestedfromalittlebrownmyotisin Pennsylvaniain2010.Inordertodeterminewhetheror notsurvivingWNSresultsinthegenerationofantibodies against Pd ,wecollectednonterminalbloodsamplesfrom 63survivingbats(control: n = 19; Pd inoculated: n = 44) at2,6,and10weeksfollowingtheendofhibernation. Bloodwascollectedandstoredasdescribedabove.FieldstudiesWecapturedbatsatnightusingmist-nets,orcaptured roostingbatsbyhandduringthedayinordertocollect nonterminalbloodsamplesforouranalysis.Wecollected bloodasdescribedabove,exceptthatplasmawasstored ondryiceuntiltransferredtoourlaboratory,whereit wasstoredat 80 C.Bloodwasonlycollectedfromadult bats.Inadditiontocollectingblood,wescoredthewings fordamageconsideredtobeasecondarysymptomof WNS(ReichardandKunz2009)andswabbedthewings andmuzzlestolaterdeterminethenumberof Pd cells present(Johnsonetal.2014). Wecollectedbloodfromfree-ranginglittlebrownmyotis,northernmyotis( M.septentrionalis ),bigbrownbats, tri-coloredbats( Perimyotissubavus ),Ranesque's big-earedbats( Corynorhinusranesquii ),eveningbats ( Nycticeiushumeralis ),andeasternredbats( Lasiurus borealis )capturedatMammothCaveNationalPark,Kentucky,USA(seeTableS1).Easternredbatsweretheonly speciesinourstudynotknowntohibernateincaves. SampleswerecollectedfromlateMaythroughearlyJune (spring)of2013and2014,andduringlateJuly(summer) of2013.Sampleswerealsocollectedfromlittlebrown myotisduringthespringinNewYork,Pennsylvania,and Montana(seeTableS1).Wealsocollectedsamplesfrom Daubenton'smyotis( M.daubentonii )insouthwestern Finlandduringthespringandsummerof2013.Batsin Finlanddidnotshowanysignofwingdamagetypicalof WNSsurvivorsinNorthAmerica(Meteyeretal.2009; ReichardandKunz2009). Wecollectedterminalbloodsamplesfromlittlebrown myotisandtri-coloredbatscollectedfromtwoWNSpositivecavesinKentucky,USA,inMarch(winter)2014. Thesebatswereselectedforsamplingbaseduponthe presenceofvisiblefungus.Weswabbedthemuzzlesand wingsofbatstoconrmthepresenceof Pd byqPCR (Johnsonetal.2014).Batswerearousedfor 1hbefore beingeuthanizedusingisouranefollowedbydecapitation.Wealsocollectedterminalwintersamplesfroma Europeanspecies,greatermouse-earedmyotis( M.myotis ),with( n = 7)andwithout( n = 7)visiblefungal growth,presumedtobe Pd ,fromthreehibernaculain NorthernBavaria,Germany,duringMarch2012.Bats wereimmediatelyeuthanizeduponcollectionandwere notarousedpriortocollectionofbloodsamples.Blood wasprocessedandstoredasdescribedabove.MeasuringantiPd antibodytitersPlasmaantibodytitersweremeasuredusinganenzymelinkedimmunosorbentassay(ELISA)inU-bottom 96-wellplateswith100,000formalin-xed Pd conidiasuspendedin200 l LPBScontaining0.05%Tween-20 (PBST)and1%bovineserumalbumin(PBST + BSA).The concentrationof Pd cellswasdeterminedusingahemocytometer.Plateswerespuninacentrifugeat600 g for 5min,andthesupernatantdiscarded.Wethenadded 50 l LofbatplasmadilutedinPBST + BSAatconcentrationsof1:100and1:1000towellsandincubatedona plateshakerfor1hat20 – 24 C.Cellswerethenwashed twicewithPBSbeforeadding50 l LofbiotinylatedproteinA/G(BioVision,Inc.,Milpitas,CA)dilutedin PBST + BSAataconcentrationof1:10,000andincubated onaplateshakerfor1hat20 – 24 C.Cellswerethen washedtwicewithPBSbeforeadding50 l Lstreptavidin peroxidasepolymer,ultrasensitive(Sigma-Aldrich,St. Louis,MO)dilutedinPBST + BSAataconcentrationof 1:2000andincubatedfor30minonaplateshakerat20 – 24 C.CellswerethenwashedthreetimeswithPBSbefore 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.2205J.S.Johnson etal AntibodiesareInsufcientforWNSProtection

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incubatingwith100 l Ltetramethylbenzidine(eBioscience,SanDeigo,CA).Thereactionwasterminated after15minusing10 l Lof10%trichloroaceticacid. Absorbancewasmeasuredat450nmonamicroplate spectrophotometer(BioTek l Quant,Winooski,VT). Backgroundabsorbancewassubtractedfromallsamples usingnegativecontrolwellsthatreceivedPBST + BSA insteadofplasma.Serumfromarabbitimmunizedand boostedwithlyophilized Pd (LAMPIREBiologicalLaboratories,Pipersville,PA)wasusedforapositivecontrol andtovalidatetheassay.Sampleswithabsorbance < 0.1 unitsabovebackground(3standarddeviations)were assignedatiterof10andconsiderednot-detectablefor antiPd antibodies.Fortheremainingsamples,thebat wasconsideredtobeseropositiveandtiterwascalculated bymultiplyingtheabsorbancebythedilution.Thetiter fromthesamplediluted1:100wasusedunlessboth1:100 and1:1000titerswereabove50,andthen,thelargesttiter ofthetwoconcentrationswasassigned.Becausesamples wereanalyzedondifferentdays,positivetiterswerestandardizedacrossassaysusingtheaverageabsorbancevalue oftriplicatesamplesoftherabbitantiPd positivecontrol atadilutionof1:1000.Allplasmasamplesweremeasured ontwoseparatedaysandproducedsimilarresultson eachdayafterstandardization.StatisticalanalysisWeusedchi-squareteststocompareseroprevalence(the proportionofsampleswithdetectableantibodiesagainst Pd )betweencaptivelittlebrownmyotiscutaneously inoculatedwith Pd andshaminoculatedwithPBS.Bats testingpositiveforantiPd antibodiesatanyofthethree timepointswereconsideredpositiveforantibodiesfor thiscomparison.Wetestedourhypothesisthattiters wouldpeakshortlyafterhibernationusingalongitudinal nonparametricalternativetoarepeatedmeasuresanalysis,Friedman'stest,becausetiterdatacouldnotbe transformedtomeettheassumptionsofparametrictests. WeusedWilcoxonrank-sumtestsasamethodofmeans comparisonsbetweentimepoints.WealsousedWilcoxonrank-sumteststocompareantibodytitersbetween thetwogroupsateachtimepointtotestthehypothesis thattiterswouldbegreatestamonglittlebrownmyotis inoculatedwith Pd .Similarly,wecomparedseroprevalencebetweenlittlebrownmyotisimmunizedwithlive Pd andbatssham-injectedusingchi-squaretestsand comparedtitersbetweenthetwogroupsusingaWilcoxonrank-sumtest. Weusedchi-squareteststocompareseroprevalence andKruskal – Wallisteststocompareantibodytitersto testeachofourhypothesespertainingtowildpopulations.Whenappropriate,meanscomparisonsweremade usingaWilcoxonrank-sumtestor2 9 2chi-squaretests withasequentialBonferroni – Holmcorrectionforeach pairoftreatments.Totestthehypothesisthatantibody seroprevalenceandtiterswouldbegreatestinpopulations withlongerhistoriesofWNS,wecomparedlittlebrown myotissampledduringspringinMontana(noknown WNShistory)topopulationsinKentucky(WNS-positive since2011),Pennsylvania(WNS-positivesince2008),and NewYork(WNS-positivesince2006).Datacollectedin Kentuckyduringthespringseasonsof2013and2014 werecombinedfollowingvericationthatseroprevalence andtiterdidnotvarybetweenyears(TableS2).Wealso comparedseasonaldifferencesintitersforspeciessampledatthesamelocationatdifferenttimesofyear.To testthehypothesisthatEuropeanspecieswouldexhibit greaterseroprevalenceandtiter,wecomparedgreater mouse-earedmyotissamplestosamplesfromNorth Americanspeciesalsocollectedduringhibernationand comparedDaubenton'smyotistolittlebrownmyotis populationsinNewYork,ourNorthAmericansampling locationwiththelongesthistoryofWNS,alsosampled duringspring. Changesin Pd loadsquantiedbyPCRonfree-rangingbatscapturedinKentuckybetween2013and2014 werecomparedforeachspeciesindividuallyusingaWilcoxonrank-sumtest.Kruskal – Wallistestswereusedto compare Pd loadsamongspeciescapturedinKentucky andtocompare Pd loadsonlittlebrownmyotiscapturedinKentucky,Pennsylvania,andNewYork.Because Pd loadsinseveralspeciesdifferedbetweenyears,comparisonamongspeciescapturedinKentuckywaslimited tospeciescapturedin2014,andonlylittlebrownmyotiscapturedin2014werecomparedtobatscapturedin NewYorkandPennsylvania.Forillustratingcomparisonsacrosstimeandbetweenspecies,dataforsomelittlebrownmyotisgroupsarefoundinmorethanone gure.ResultsTotestwhetherlittlebrownmyotismountanantibody responseto Pd wheninfectedwiththefungus,wecomparedantiPd antibodylevelsincaptivebatspreviously infectedwith Pd ( n = 44)touninfectedconspecics ( n = 44).Fifty-sevenof63(90%)samplesfromcaptive littlebrownmyotiscollectedfromWNS-negativecaves testedpositiveforantiPd antibodiesuponarousalfrom hibernation.Therewasnodifferenceinseroprevalence ( v2= 0.08,df = 1, P = 0.77)ortiter(week2: Z = 0.63, P = 0.53;week6: Z = 1.40, P = 0.16;week10: Z = 0.05, P = 0.96)betweengroupscutaneouslyinoculatedwith Pd orshaminoculatedwithPBS.AntiPd antibodytitersdifferedacrossweeksamonginoculatedbats2206 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.AntibodiesareInsufcientforWNSProtection J.S.Johnson etal .

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( v2= 15.6,df = 2, P < 0.001;Fig.1),withtitersatweek 2beingsignicantlygreaterthanatweek6( v2= 9.8, df = 2, P = 0.002)andweek10( v2= 10.5,df = 2, P = 0.001).Todeterminewhetherinjectionwith Pd could increaseantiPd antibodylevels,weinjectedlittlebrown myotiswithlive Pd mixedwithanadjuvant.Compared tobatsthatwereinjectedwithPBS,injectedbatsexhibitedsignicantlygreaterantiPd antibodytiters (Z = 2.3, P = 0.02),butnotseroprevalence( v2= 3.3, df = 1, P = 0.07)(Fig.2). Next,wedeterminedwhetherdifferentspecieshave variableantiPd antibodylevelsthroughouttheyear. Comparinglittlebrownmyotisandtri-coloredbatssampledduringthespring,summer,andwinterinKentucky (Fig.3A),wefoundlittlebrownmyotisexhibitedgreater seroprevalence( v2= 18.0,df = 2, P < 0.001,TableS3) andtiters( v2= 29.5,df = 2, P < 0.001,seeTableS3) duringspringthanduringwinterorsummer,butnosignicantseasonaldifferencesweredetectedamongtri-coloredbats(seroprevalence: v2= 1.3,df = 2, P = 0.52; titer: v2= 1.2,df = 2, P = 0.56).Amongthreespecies sampledduringthespringandsummer(Fig.3B),northernmyotisexhibitedgreaterseroprevalence( v2= 8.0, df = 1, P = 0.005)andtiter( Z = 2.5, P = 0.01)during springcomparedtosummer,asdidRanesque'sbigearedbats( v2= 4.2,df = 1, P = 0.04;titer: Z = 2.2, P = 0.03).Bigbrownbatsexhibitedgreatertiters ( Z = 2.4, P = 0.02),butnotseroprevalence( v2= 3.2, df = 1, P = 0.08)duringspring. Pd loadsvariedamong speciessampledinKentuckyin2014( v2= 30.0,df = 4, P < 0.001),withlittlebrownmyotishavingthegreatest abb 10 20 30 40 50 60 70 80 90 100 200 300 2610Weeks after emergenceAnti Pd titer Figure1. ComparisonofantiPd antibodytitersin44littlebrown myotis( Myotislucifugus )collectedfrom Pd -negativecaves cutaneouslyinoculatedwith Pd andhibernatedincaptivityfor 5months.AntiPd titerswerehighestshortlyaftertheendof hibernationandthendeclined.Dataarepresentedwithquartiles, median(blackline),andwhiskersthatrepresent1.5timesthe interquartilerange.Titers 10areconsiderednegativefor Pd reactive antibodies.Titersdifferedsignicantlybetweenweeksnotsharing commonletters( P < 0.05). ab 10 20 30 40 50 60 70 80 90 100 200 300 ShamImmunizedAnti Pd titer Figure2. ComparisonofantiPd antibodytitersin26littlebrown myotis( Myotislucifugus )collectedfromoutsidetheWNS-affected regionofNorthAmericaandimmunizedwith Pd emulsiedwith adjuvantorPBS(sham).AntiPd titersweregreaterinimmunized bats,asdenotedbydifferentletters( P < 0.05).Dataarepresentedas quartiles,median(blackline),andwhiskersthatrepresent1.5times theinterquartilerange.Titers 10areconsiderednegativefor Pd reactiveantibodies. 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.2207J.S.Johnson etal AntibodiesareInsufcientforWNSProtection

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Pd loads(TableS4).ComparingantiPd antibodiesin NorthAmericanspeciessampledinthesamelocation (Kentucky)atthesametime(spring),seroprevalence ( v2= 60.8,df = 4, P < 0.001)andtiter( v2= 74.03, df = 4, P < 0.001)signicantlyvaried,withlittlebrown myotisexhibitinghigherseroprevalenceandtitercomparedtoallotherspecies(TableS5).Easternredbatsand eveningbatswerenotincludedincomparisonsdueto samplesizebutarereportedinTableS1.Antibodies against Pd wereneverdetectedamongredbats. TodeterminewhetherantiPd titerscorrelatewith lengthofexposureofapopulationtoWNS,wemeasuredantiPd antibodylevelsinfourNorthAmerican populationsoflittlebrownmyotisduringthespring.Seroprevalence( v2= 40.0,df = 3, P < 0.001)andtiter ( v2= 17.8,df = 3, P < 0.001)signicantlyvaried,with littlebrownmyotisexhibitinghighertitersinNewYork thaninallstatesexceptPennsylvania(Fig.4,TableS6). AmonglittlebrownmyotissampledinNewYorkand Pennsylvania,onlyonebathadnoticeablewingdamage, receivingaReichardscoreof1(ReichardandKunz 2009).Theextentof Pd infectionappearedtoincrease amongbatsinKentuckybetween2013and2014.Seven of20(35%)oflittlebrownmyotiscapturedinKentuckyin2013hadawingdamagescoreof1,withthe remaining65%batsexhibitingnodamage,but61% ( n = 11)ofbatssampledatthesamelocationin2014 hadmoderate(damageindex = 2)orseveredamage (value = 3).Furthermore,themediannumberof Pd cells(genomicequivalents)detectedbyqPCRonlittle brownmyotis( Z = 4.3, P < 0.001),northernmyotis ( Z = 4.3, P < 0.001),bigbrownbats( Z = 4.9, P < 0.001),andtri-coloredbats( Z = 3.2, P = 0.02) increasedbetween2013and2014.Wealsodetected greater Pd loadsonlittlebrownmyotiscapturedin Kentuckyduring2014thaninNewYorkorPennsylvania(TableS7). TodeterminewhetherantiPd antibodylevelsdiffer betweenNorthAmericanandEuropeanspecies,wecomparedantibodylevelsintwospeciesofEuropeanbatsto M.lucifugus inKentuckyandNewYork.Winterseroprevalence( v2= 20.2,df = 2, P < 0.001,TableS8)andtiters ( v2= 18.8,df = 2, P < 0.001,TableS8)differedbetween NorthAmericanandEuropeanspecies,withnoantibodiesagainst Pd detectedamonggreatermouse-earedmyotis inGermany(Fig.5,TableS8).Wewereabletodetect antiPd antibodiesamongDaubenton'smyotissampled duringthespringinFinland,buttitersweresignicantly lower( Z = 2.4, P = 0.02)andwerelesslikelytobe seropositive( v2= 9.8,df = 1, P = 0.002)thanlittle brownmyotissampledinNewYorkduringthesame timeperiod(Fig.5).SummersamplesfromDaubenton's myotiswerenotincludedinanalysesduetolowsample size,butarereportedinTableS1. M. lucifugus P. subflavus 10 20 30 40 50 60 70 80 90 100 200 300 a bb SpringSummerSpringSummerSpringSummerSpringSummer WinterSpringSummerWinter Anti–Pd titer(A) E. fuscus M. septentrionalis C. rafinesquii 10 20 30 40 50 60 70 80 90 100 Anti–Pd titer(B)aaa ababab Figure3. ComparisonofantiPd antibodytitersinvefree-rangingbatspeciessampledinKentucky,USA.(A)AntiPd titersweregreaterin littlebrownmyotis( Myotislucifugus )thanintri-coloredbats( Perimyotissubavus )( P < 0.05)andweregreatestduringthespringinlittle brownmyotis.(B)Bigbrownbats( Eptesicusfuscus ),northernlong-earedmyotis( M.septentrionalis ),andRanesque'sbig-earedbats ( Corynorhinusranesquii )hadgreaterantiPd titersinspringthaninsummer,andallhadlowertitersthanlittlebrownbatsinthespring ( P < 0.05).Dataarepresentedasquartiles,median(blackline),andwhiskersthatrepresent1.5timestheinterquartilerange.Titers 10are considerednegativefor Pd reactiveantibodies.Withineachspecies,titersdifferedsignicantlybetweenseasonsnotsharingcommonletters ( P < 0.05).2208 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.AntibodiesareInsufcientforWNSProtection J.S.Johnson etal .

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DiscussionWepresenttherstevidenceofanantibody-mediated immuneresponseto Pd inbats.Contrarytoourprediction,wedidnotobservegreaterseroprevalenceortiters ofantiPd antibodiesinEuropeanthaninNorthAmericanspecies,andnotably,wedidnotdetectantibodies against Pd amonggreatermouse-earedmyotisboth infectedanduninfectedwith Pd duringhibernation.Similarly,antiPd antibodylevelsinasecondEuropeanspecies,Daubenton'smyotis,werealsofoundtobelower thaninlittlebrownmyotisofNorthAmerica,although their Pd exposurestatuswasunknown.Thus,batsinEurope,whichhavepresumablylivedwith Pd forthousands ofyears,havelowerantibodytitersthanspeciesinNorth Americathathaveonlyrecentlybecomeexposed.These dataprovidestrongevidencethatanantibody-mediated immuneresponseisnotthemechanismofsurvival amongEuropeanbatsinfectedby Pd .Thisconclusionis furthersupportedbyourndingthatlittlebrownmyotis, aspecieswithWNSmortalityrates > 90%(Turneretal. 2011),havehigherantiPd antibodytitersthananyNorth AmericanorEuropeanspecies,whilesomespecieswith lowermortalityrateshadlowertiters.Furthermore,antibodiesreactiveagainst Pd wereidentiedinlittlebrown myotisthathadneverbeenexposedtothefungus.Were antibodiesinvolvedindefenseagainst Pd ,onewould expectthatlittlebrownmyotiswouldexperiencehigher survivalratesgiventheprevalenceofantibodiesinna   ve populations. Consistentwithourpredictions,however,populations oflittlebrownmyotisinhabitingNewYorkandPennsylvania,whereWNShasbeenpresentsince2006and 2008,respectively,hadgreaterseroprevalenceandtiters thanna   veandmorerecentlyexposedpopulations.LittlebrownmyotisinNewYorkandPennsylvaniaalso exhibitedlessseverewingdamageandhadfewer Pd cellsontheirskincomparedlittlebrownmyotisfrom Kentucky,whereWNSarrivedmorerecently.Thepresenceof Pd ontheskinofbatsinPennsylvaniaand NewYork,alongwiththepresenceof Pd inhibernaculaacrossbothstates,stronglysuggeststhatthesebats hadsurvivedawinterexposedto Pd ,yetlackedthe secondarysymptomsseeninsurvivorselsewhere(ReichardandKunz2009;presentstudy).Thissuggeststhat thesebatsbelongtoremnantpopulationspossessing behavioraland/orphysiologicaltraitsthataidinsurvivingWNS.Ourresultsfromcaptivelittlebrownmyotis andfree-rangingEuropean Myotis speciesleadusto hypothesizethatantibodiesarenotthemechanism behindsuchsurvival,butinsteadthathighantiPd antibodytitersinthesebatsarecorrelatesofsurvival.Itis alsopossiblethattheincreasedtitersobservedinnortheasternpopulationsoflittlebrownmyotisreectgreater exposurebylivinginareasofendemic Pd exposure, butthisisnotconsistentwithwhatwasobservedfor otherspeciesinthesamehibernacula,orwithwhat wasseeninEuropean Myotis ThecorrelationbetweenantiPd titerandsurvivalmay besimilartothatfoundinmiceexposedtothefungal pathogen Candidaalbicans ,whereantibodieswerefound tobepredictorsoftheabilityofmicetosurviveinfection oncetitersexceedacertainthreshold,eventhoughcellmediatedimmuneresponseswerethemechanismofsuccessfulhostdefense(Spellbergetal.2008).Although monoclonalantibodieshavebeenshowntohaveprotectiverolesinimmune-defenseagainstfungalpathogens, evidencethatnaturallyproducedantibodieshasaprotectiveeffectislacking(CasadevallandPirofski2012a,b; W   uthrichetal.2012). InthecaseofWNS,theopportunityforantibodiesto playaroleinhostdefenseisfurtheredhamperedbythe downregulationinimmunefunctionubiquitously observedamongmammalianhibernators(Boumaetal. 2010a).Inrodents,forexample,hibernationisassociated withleukopenia(Boumaetal.2010a,b,2011),reductions incomplementactivity(Maniero2002),anddecreased antibodyproduction(BurtonandReichman1999;Bouma etal.2013).Despitethisreduction,however,hibernating woodchucks( Marmotamonax )produceantibodiesduring 10 20 30 40 50 60 70 80 90 100 200 300 Montana abb,cc KentuckyPennsylvaniaNew YorkAnti–Pd titer Figure4. ComparisonofantiPd antibodytitersdeterminedforlittle brownmyotis( Myotislucifugus )capturedinspringacrossNorth America.AntiPd titersarelowest,butstilldetectable,inMontana (WNS-negative),followedbyKentucky(WNS-positivesince2011), thenPennsylvania(WNS-positivesince2008),andthehighesttiters werefoundinNewYork(WNS-positivesince2006).Dataare presentedasquartiles,median(blackline),andwhiskersthat represent1.5timestheinterquartilerange.Titers 10areconsidered negativefor Pd reactiveantibodies.Titersdifferedsignicantly betweenlocationsnotsharingcommonletters( P < 0.05). 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.2209J.S.Johnson etal AntibodiesareInsufcientforWNSProtection

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hibernation,evenwhenprimaryexposureoccursduring hibernation(Beaudoinetal.1969),andnodeclinein antibodyresponsestoaT-cell-dependentantigenwas observedinastudyofhibernating13-linedgroundsquirrels( Ictidomystridecemlineatus )(Boumaetal.2013).Similarly,weobservedthatlittlebrownmyotishave detectablelevelsofantiPd antibodiesduringwinter,and insomecasesexhibitedrelativelyhightiters. Ourresultsfromfree-ranginglittlebrownmyotissampledinthewinterandspring,andbatsexposedto Pd in captivity,suggestthatantiPd antibodylevelspeakshortly afteremergencefromhibernation.Thus,circulatingantibodiesagainst Pd inlittlebrownmyotispeakafterbats havelefttheirhibernacula,outsideofwhichthefungusis unabletogrowduetotemperaturerestrictionson Pd growth(Verantetal.2012).OtherstudiesofWNSaffectedbatssuggestboththepresence(Meteyeretal. 2012;Mooreetal.2013)andabsence(Meteyeretal. 2009)ofaninammatoryimmuneresponseto Pd during winter(seealsothediscussioninBrookandDobson 2015).Althoughourresultssuggestthatmuchofthe immuneresponseto Pd occursinspring,consistentwith theworkofMeteyeretal.(2012),itiscriticaltonote thatwesampledvisiblysickbatsduringwinter,suggestingthesebatswereunlikelytosurvive.Thus,studiesof littlebrownmyotispopulationssurvivingwithWNSare neededtobetterassesscirculatingantibodyduringwinter. ItisnotablethatseverallittlebrownmyotispopulationsfaroutsidetheWNS-affectedzonetestedpositive forantiPd antibodies.Littlebrownmyotismust,therefore,produceantibodiesthatarecross-reactivewith Pd andotherpathogens.Cross-reactivityofantibodiesto variousfungalpathogensisnotuncommon,withantibodybindingoftenoccurringatcommonmolecularsurfacepatternssharedacrossmanyspecies(Rappleyeetal. 2007).Thus,antibodiesthatrecognizethesesurfacemolecules – acommonpatternrecognizedbyantifungalantibodies(W   uthrichetal.2012) – willrecognize Pd .Agreat numberoffungalspeciesarepresentinbathibernacula (Lorchetal.2013),servingaspossiblesourcesofprimary exposuretomolecularpatternstowhichantibodiescan beproduced. Notallspeciesproducedantibodiesagainst Pd ,however,andantiPd antibodytitersvariedsignicantly amongspecies.Thesedifferencesamongspeciesmaybe linkedtodifferencesinwinterecologyorphysiology.We predictedthatbigbrownbatsandRanesque'sbig-eared batswouldexhibithigherantibodytitersbecausethese specieshaverelativelylowWNSmortalityrates(Turner etal.2011),arousemorefrequentlyfromhibernation (BrackandTwente1985;Twenteetal.1985;Johnson etal.2012),andbecauseimmunefunction(atleastin rodents)isknowntoberapidlyrestoredduringperiodic arousals(Maniero2002;Boumaetal.2011).Contraryto thisprediction,however,thesespeciesexhibitedlower seroprevalenceandtitersthanthesmallerlittlebrown myotis.Anothersmallspecieswithinfrequentarousals andhighWNSmortality,tri-coloredbats(Brackand Twente1985;Twenteetal.1985;Turneretal.2011), hadthelowesttitersandseroprevalenceofanyNorth Americanspecies.Thus,noconsistentrelationshipwas observedbetweenantibodytiterandbodysizeorwinter arousalfrequency.DifferencesinWNSsusceptibility acrossNorthAmericanspeciescannotbeexplained bydifferencesinabilitytomountahumoralimmune response. M. lucifugus M. daubentonii 10 20 30 40 50 60 70 80 90 100 200 300 ab New York FinlandAnti–Pd titer Anti–Pd titer M. lucifugus M. myotis 10 20 30 40 50 60 70 80 90 100 Kentucky Germany(B) (A)ab Figure5. ComparisonofantiPd antibody titersinfree-rangingbatspeciesinNorth AmericaandEurope.(A)AntiPd titerswere greaterinlittlebrownmyotis( Myotislucifugus ) inNewYork,USA,thaninDaubenton'smyotis ( M.daubentonii )inFinlandduringspring. (B)AntiPd titersweregreaterinlittlebrown myotishibernatinginKentucky,USA,thanin Daubenton'smyotishibernatinginGermany. Dataarepresentedasquartiles,median(black line),andwhiskersthatrepresent1.5timesthe interquartilerange.Titers 10areconsidered negativefor Pd reactiveantibodies.Titers differedsignicantlybetweenspeciesnot sharingcommonletterswithineachpanel ( P < 0.05).2210 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.AntibodiesareInsufcientforWNSProtection J.S.Johnson etal .

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Antifungalimmuneresponsesarenotalwaysprotective andantibodies,inparticular,canbeassociatedwith immunepathologyinsteadofprotection.Protection dependsonthetypeofTcellsactivatedandcytokinesproduced.Forexample,immuneresponsesdominatedbythe Th2phenotypearefrequentlycharacterizedaspermissive orexacerbatingtheeffectsoffungaldisease(Cencietal. 1997;Jainetal.2009;Haraguchietal.2010)whileTh17 cellsgenerallypromoteprotectionfromfungalinfection (Contietal.2009;Hern andez-SantosandGaffen2012). Unliketheroleofantifungalantibodiesindefense,cellmediatedimmuneresponseshavewell-knownrolesin clearingfungalinfectionsinmammals(Spellbergetal. 2008;Hern andez-SantosandGaffen2012).Giventhatwe wereunabletodetectantibodiesagainst Pd ingreater mouse-earedmyotisinfectedwith Pd duringwinter,and Pd isassociatedwithpathologybutnotmortalityinthis species(Pikulaetal.2012),wehypothesizethatcell-mediatedimmuneresponsesaidinhostdefenseinthisspecies. Furthermore,wehypothesizethatcell-mediatedimmune responsesarealsoinvolvedinhostdefenseinNorthAmericanspecieswithrelativelylowWNSmortalityrates,such asbigbrownbatsandRanesque'sbig-earedbats(Frank etal.2014).Inourstudy,thesespeciesexhibitedsignicantlylowerseroprevalenceandtitersofantiPd compared tolittlebrownmyotisinKentucky,despitepresenceof Pd ontheskinofallspecies.Although Pd waspresentand antiPd antibodytiterswerelowinbigbrownbatsand Ranesque'sbig-earedbats,weobservednowingdamage amongindividualsofthesespeciesinKentucky. Thisstudyprovidesstrongevidencethatantibodymediatedimmunityisnotthemechanismbehindsurvival ofEuropeanorNorthAmericanbatsinfectedwith Pd Thus,ourndingthatantiPd antibodytiterscanbe increasedinlittlebrownmyotisthroughvaccinationmust beconsideredinthecontextofthisroleofantibody-mediatedimmunity.Inmice,immunizationagainstfungal pathogensshowsthatimmunizationcanconferprotection ifcell-mediatedimmuneresponsesaretheresultofvaccinationandthatprotectionwasalsoassociatedwith highantibodytiters(Spellbergetal.2008).Thus, althoughantibody-mediatedimmuneresponsesarenota mechanismforpromotingsurvivalofWNS,immunizing batsmaystillconferprotectionagainst Pd ifcell-mediated immuneresponsesalsoresult. Inconclusion,ourstudyoncirculatingantibodies againstthefungusthatcausesWNSprovidesevidence thatanantibody-mediatedimmuneresponseisinsufcienttoexplainsurvivalofNorthAmericanorEuropean batsinfectedwith Pd .Althoughantibodiesagainst Pd are correlatesofprotectionforlittlebrownmyotis theirpresencecannotexplainthesurvivalofremnantpopulations oflittlebrownmyotisinthenortheasternUnitedStates (asdocumentedbyDobonyetal.2011andReichard etal.2014).Thelow Pd loadsandabsenceofsecondary injuries(ReichardandKunz2009;Meteyeretal.2012) weobservedamonglittlebrownmyotisinremnantpopulationsofsurvivorsinNewYorkandPennsylvaniasuggestthatthesepopulationsarephysiologicallydifferent frompopulationsinKentucky,where Pd hasmore recentlybeenintroduced.Theseresultsprovidehopethat littlebrownmyotisareadaptingtolifewithWNS.AcknowledgmentsFundingforthisprojectwasprovidedbytheUnited StatesFishandWildlifeServicegrantF12AP01210(DMR andKAF)andtheWoodtigerFoundation(DMR).The fundershadnoroleinstudydesign,datacollectionand analysis,decisiontopublish,orpreparationofthemanuscript.WewouldliketothankKCDeRuff,MPucciarello, SMReeder,EJRogers,MHSchwartz,LESigler,MEVodzak,andHWWintersofBucknellUniversityfortheir invaluableassistancewiththisresearch.WearealsogratefultotheanimalcarestaffatBucknell,especiallyCJ Rhone,GCLong,andMGavitt,forhelpcaringfor captivebats.DataAccessibilityAlldataareincludedinthesupplementarymaterial.ConictofInterestNonedeclared. ReferencesBeaudoin,R.,D.Davis,andK.Murrell.1969.Antibodiesto larvalTaeniacrassicepsinhibernatingwoodchucks Marmota monax .Exp.Parasitol.24:42 – 46. Blehert,D.S.2012.Fungaldiseaseandthedevelopingstoryof batwhite-nosesyndrome.PLoSPathog.8:e1002779. Blehert,D.S.,A.C.Hicks,M.Behr,C.U.Meteyer,B.M. Berlowski-Zier,E.L.Buckles,etal.2009.Batwhite-nose syndrome:anemergingfungalpathogen?Science 323:227. Bouma,H.R.,H.V.Carey,andF.G.M.Kroese.2010a. Hibernation:theimmunesystematrest?J.Leukoc.Biol. 88:619 – 624. Bouma,H.R.,A.M.Strijkstra,A.S.Boerema,L.E.Deelman, A.H.Epema,R.A.Hut,etal.2010b.Bloodcelldynamics duringhibernationintheEuropeanGroundSquirrel.Vet. Immunol.Immunopathol.136:319 – 323. Bouma,H.R.,F.G.M.Kroese,J.W.Kok,F.Talaei,A.S. Boerema,A.Herwig,etal.2011.Lowbodytemperature 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.2211J.S.Johnson etal AntibodiesareInsufcientforWNSProtection

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TableS5. ComparisonofantiPd antibodyseroprevalence andtitersforvebatspeciessampledduringspring2013 and2014atMammothCaveNationalPark,Kentucky, USA. TableS6. ComparisonofantiPd antibodyseroprevalence andtitersforlittlebrownmyotissampledatvariouslocationsintheUnitedStatesduringspring2013-2014. TableS7. Comparisonof Pd loadsdetectedonlittle brownmyotiscapturedatdifferentregionsoftheUnited Statesduring2013-2014. TableS8. ComparisonofantiPd antibodyseroprevalence andtitersforthreebatspeciessampledduringhibernation(March2012and2014).2214 2015TheAuthors. EcologyandEvolution publishedbyJohnWiley&SonsLtd.AntibodiesareInsufcientforWNSProtection J.S.Johnson etal .


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