Experimental Infection of Tadarida brasiliensis with Pseudogymnoascus destructans, the Fungus That Causes White-Nose Syndrome


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Experimental Infection of Tadarida brasiliensis with Pseudogymnoascus destructans, the Fungus That Causes White-Nose Syndrome

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Experimental Infection of Tadarida brasiliensis with Pseudogymnoascus destructans, the Fungus That Causes White-Nose Syndrome
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mSphere
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Verant, Michelle L.
Meteyer, Carol U.
Stading, Benjamin
Blehert, David S.
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Pseudogymnoascus ( local )
Fungal Disease ( local )
Tadarida Brasiliensis ( local )
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White-nose syndrome (WNS) is causing significant declines in populations of North American hibernating bats, and recent western and southern expansions of the disease have placed additional species at risk. Understanding differences in species susceptibility and identifying management actions to reduce mortality of bats from WNS are top research priorities. However, the use of wild-caught susceptible bats, such as Myotis lucifugus, as model species for WNS research is problematic and places additional pressure on remnant populations. We investigated the feasibility of using Tadarida brasiliensis, a highly abundant species of bat that tolerates captivity, as the basis for an experimental animal model for WNS. Using methods previously established to confirm the etiology of WNS in M. lucifugus, we experimentally infected 11 T. brasiliensis bats with Pseudogymnoascus destructans in the laboratory under conditions that induced hibernation. We detected P. destructans on all 11 experimentally infected bats, 7 of which exhibited localized proliferation of hyphae within the epidermis, dermis, and subcutaneous tissue, similar to invasive cutaneous ascomycosis observed in M. lucifugus bats with WNS. However, the distribution of lesions across wing membranes of T. brasiliensis bats was limited, and one discrete “cupping erosion,” diagnostic for WNS, was identified. Thus, the rarity of lesions definitive for WNS suggests that T. brasiliensis does not likely represent an appropriate model for studying the pathophysiology of this disease. Nonetheless, the results of this study prompt questions concerning the potential for free-ranging, migratory T. brasiliensis bats to become infected with P. destructans and move the fungal pathogen between roost sites used by species susceptible to WNS.
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mSphere, Vol. 3, no. 4 (2018-01-01).

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ExperimentalInfectionof Tadaridabrasiliensis with Pseudogymnoascusdestructans ,theFungusThatCauses White-NoseSyndrome MichelleL.Verant,a,b* CarolU.Meteyer,cBenjaminStading,a,b* DavidS.BlehertbaDepartmentofPathobiologicalSciences,UniversityofWisconsin„Madison,SchoolofVeterinaryMedicine, Madison,Wisconsin,USAbU.S.GeologicalSurvey,NationalWildlifeHealthCenter,Madison,Wisconsin,USAcU.S.GeologicalSurvey,NationalCenter,Reston,Virginia,USA ABSTRACT White-nosesyndrome(WNS)iscausingsigni“cantdeclinesinpopulationsofNorthAmericanhibernatingbats,andrecentwesternandsouthernexpansionsofthediseasehaveplacedadditionalspeciesatrisk.Understandingdifferences inspeciessusceptibilityandidentifyingmanagementactionstoreducemortalityof batsfromWNSaretopresearchpriorities.However,theuseofwild-caughtsusceptiblebats,suchas Myotislucifugus ,asmodelspeciesforWNSresearchisproblematic andplacesadditionalpressureonremnantpopulations.Weinvestigatedthefeasibilityofusing Tadaridabrasiliensis ,ahighlyabundantspeciesofbatthattoleratescaptivity,asthebasisforanexperimentalanimalmodelforWNS.Usingmethodspreviouslyestablishedtocon“rmtheetiologyofWNSin M.lucifugus ,weexperimentally infected11 T.brasiliensis batswith Pseudogymnoascusdestructans inthelaboratory underconditionsthatinducedhibernation.Wedetected P.destructans onall11experimentallyinfectedbats,7ofwhichexhibitedlocalizedproliferationofhyphae withintheepidermis,dermis,andsubcutaneoustissue,similartoinvasivecutaneous ascomycosisobservedin M.lucifugus batswithWNS.However,thedistributionoflesionsacrosswingmembranesof T.brasiliensis batswaslimited,andonlyonediscretecuppingerosion,ŽdiagnosticforWNS,wasidenti“ed.Thus,therarityoflesionsde“nitiveforWNSsuggeststhat T.brasiliensis doesnotlikelyrepresentan appropriatemodelforstudyingthepathophysiologyofthisdisease.Nonetheless, theresultsofthisstudypromptquestionsconcerningthepotentialforfree-ranging, migratory T.brasiliensis batstobecomeinfectedwith P.destructans andmovethe fungalpathogenbetweenroostsitesusedbyspeciessusceptibletoWNS. IMPORTANCE White-nosesyndrome(WNS)isafungaldiseasethatiscausingsevere declinesofbatpopulationsinNorthAmerica.Identifyingwaystoreducetheimpactsofthisdiseaseisaprioritybutisinhibitedbythelackofanexperimentalanimalmodelthatdoesnotrequiretheuseofwild-caughtbatspeciesalreadyimpactedbyWNS.Wetestedwhether Tadaridabrasiliensis ,oneofthemostabundant speciesofbatsintheAmericas,couldserveasasuitableanimalmodelforWNSresearch.While T.brasiliensis batsweresusceptibletoexperimentalinfectionwiththe fungusunderconditionsthatinducedhibernation,thespeciesexhibitedlimitedpathologydiagnosticforWNS.Theseresultsindicatethat T.brasiliensis isnotlikelya suitableexperimentalmodelforWNSresearch.However,therecoveryofviable WNS-causingfungusfromexperimentallyinfectedbatsindicatesapotentialforthis speciestocontributetothespreadofthepathogenwhereitcoexistswithother speciesofbatsaffectedbyWNS. KEYWORDS Pseudogymnoascusdestructans , Tadaridabrasiliensis ,WNS,bat, cutaneousinvasiveascomycosis,experimentaldiseasemodel,white-nosesyndromeReceived 3May2018 Accepted 30July 2018 Published 29August2018 Citation VerantML,MeteyerCU,StadingB, BlehertDS.2018.Experimentalinfectionof Tadaridabrasiliensis with Pseudogymnoascus destructans ,thefungusthatcauseswhite-nose syndrome.mSphere3:e00250-18. https://doi .org/10.1128/mSphere.00250-18 . Editor J.AndrewAlspaugh,DukeUniversity MedicalCenter ThisisaworkoftheU.S.Governmentandis notsubjecttocopyrightprotectioninthe UnitedStates.Foreigncopyrightsmayapply. AddresscorrespondencetoMichelleL.Verant, michelle_verant@nps.gov,orDavidS.Blehert, dblehert@usgs.gov. * Presentaddress:MichelleL.Verant,National ParkService,BiologicalResourcesDivision, WildlifeHealthBranch,FortCollins,Colorado, USA;BenjaminStading,VeterinaryCenterat Fishhawk,Lithia,Florida,USA. Mexicanfree-tailedbatsarenotasuitable experimentalmodelforwhite-nosesyndrome researchbutmaycontributetospreadofthe fungalpathogeninareaswheretheyco-exist withWNS-susceptiblebatspecies.@mverant RESEARCHARTICLE ClinicalScienceandEpidemiology crossm July/August2018Volume3Issue4e00250-18 msphere.asm.org 1

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S incetheywere“rstobservedin2006( 1 ),white-nosesyndrome(WNS)andthe causativefungalpathogen, Pseudogymnoascusdestructans ,continuetospreadand threatentheconservationstatusofbatsacrossNorthAmerica( 2 ).Thisemergent diseasehascausedpopulationsofseverelyimpactedspeciestodeclinesigni“cantly, andsomeareconsequentlyatriskforextinction( 3 ).Batspeciesthathaveexhibitedthe highestratesofmortality,suchasthelittlebrownbat( Myotislucifugus ),thetri-colored bat( Perimyotissub”avus ),andthenorthernlong-earedbat( Myotisseptentrionalis ),are thosewhichobligatelyhibernateduringwinter. Wingskinistheprimarytissuecolonizedby P.destructans ,andthecold,torpidstate ofhibernatingbatsprovidesidealconditionsforapsychrophilicfungalpathogen,such as P.destructans ,tocolonizeandinvadetheepidermisofthewingmembrane( 4…6 ). Destructionoftheepidermalbarrierofthewingby P.destructans inducesphysiologic disruptions( 7…9 )andalteredhibernationbehaviors( 10…12 )thatcollectivelycontribute tothehighratesofmortalitycharacteristicofWNS. Characterizingdiseaseprocessesandidentifyingpotentialmanagementstrategies aretopresearchprioritiesformitigatingimpactsofWNSonbats.However,remnant populationsofsusceptiblespeciesofbats,suchas M.lucifugus ,likelycannotsustain continuedremovalortakeŽofindividualanimalsforresearchpurposes.Thesecolonies ofbatssurvivingwithWNSadditionallyincluderesistantindividuals( 13 ),andfurther pressuresontheseremnantpopulationscouldimpactthepotentialforlong-term recovery( 14 ).Somespeciesofhibernatingbats,suchas M.lucifugus ,arealsodif“cult tomaintainincaptivity.Thus,anexperimentalanimalmodelforWNSisneeded. Experimentalmodelsarewidelyusedinhumanandveterinarymedicinetoinvestigatediseaseprocessesandtoassessthesafetyandef“cacyofpotentialtreatmentsor vaccines.Forhumandiseases,thesemodelsoftenuseanimalsthatnaturallyorexperimentallyexperiencesimilardiseaseprocesses.Additionally,modelanimalspeciesare ideallythosewhichcanbeeasilymaintainedandpropagatedincaptivityorare abundantinthewild.Forexample,themouseisawidelyusedanimalmodelfor in”ammatorydisordersinhumansduetostrikingsimilaritiesingeneexpression( 15 ), andinvertebrateshavedemonstratedutilityforstudyingfungalvirulencefactorsand forevaluatingcompoundsforantifungalproperties( 16 ). Inthisstudy,weinvestigatedthefeasibilityofusing Tadaridabrasiliensis (Mexicanor Brazilianfree-tailedbat)asanexperimentalanimalmodelforWNS. Tadaridabrasiliensis isoneofthemostabundantspeciesofinsectivorousbatsintheAmericasandranges throughoutthesouthernUnitedStates,Mexico,CentralAmerica,andsouthwestern SouthAmerica( 17 ).Additionally,smallcoloniesof T.brasiliensis batshavebeen successfullymaintainedincaptivity( 18 ).Thespeciesisprimarilyconsideredtobe migratory( 19 ),butsomepopulationsandindividualsforgoseasonalmigrationsand overwinter,potentiallyusingshort-termtorpororhibernationduringadverseenvironmentalconditionsasanalternatesurvivalstrategy( 20…22 ).IntheUnitedStates,the largestpopulationsof T.brasiliensis bats,upto15millionanimals,congregateincaves andunderbridges( 23 ).However, T.brasiliensis batsmayalsoroostinsmalleraggregationswithincaves,trees,andattics( 24 ),andthespecieshasbeenfoundtoshare roostsiteswithhibernatingspeciesofbats( 25 ).Giventheseattributes,wild T.brasiliensis batscouldbeexposedto P.destructans eitherthroughdirectcontactwith infectedbatsorbycontactwithenvironmentalreservoirsof P.destructans inroostsites. If T.brasiliensis batsexposedto P.destructans weretosubsequentlyenterprolonged torporduringanadverseweatherevent,theymightbefurthersusceptibletocolonizationandepidermalinvasionby P.destructans .Together,theseattributessuggestthat T.brasiliensis couldbeasuitableexperimentalmodelforWNS. Wetestedwhether T.brasiliensis batscouldbecomeinfectedwith P.destructans and developcutaneousinvasiveascomycosis,pathologyoftheskincharacteristicofWNS, followingexperimentalinfectionandinductionofhibernationinthelaboratory.Althoughprolongedperiodsoftorpormaynotre”ecttypicalbehaviorofthisspeciesin thewild,theseconditionswerechosentoreplicatepreviouslaboratoryexperiments thatdemonstratedthedevelopmentofWNSfollowingexperimentalinfectionofVerantetal. July/August2018Volume3Issue4e00250-18 msphere.asm.org 2

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M.lucifugus batswith P.destructans ( 7 , 26 ).Altogether,theexperimentdescribedherein assessedthesusceptibilityof T.brasiliensis batstoinfectionby P.destructans ,the suitabilityofthisspeciesasamodelforWNSresearch,andthepotentialforthis migratoryspeciesofbattocontributetothespreadof P.destructans . RESULTS Weidenti“edthepresenceofDNAfrom P.destructans byreal-timePCRoftissue specimensfromallexperimentallyinfectedbatsatthetimeofdeath(35to84days followingtheinitiationoftheexperiment)( Table1 ).While P.destructans wasisolated inpureculturefromoneexperimentallyinfectedbat,cultureattemptsfromwingskin samplesofotherinfectedbatswereovergrownbyyeastidenti“edas Debaryomyces sp. priortoobservingfungalcolonieswithmorphologysuggestiveof P.destructans .Ofthe experimentallyinfectedbats,fourhadlocalizedareaswithvisiblewhitehyphaeonthe wingsurface.Thepresenceofvisiblefungalhyphaewasassociatedwithlocalizedareas oftissuecontractionandbrightgreen-blue”uorescenceintwobats( Fig.1 shows representativeimagesfromonebat).Thesesectionswerespeci“callytrimmedfor histopathology( Table1 ,I-3andI-9).Onlyoneoftheexperimentallyinfectedbats(I-11) hadUV”uorescencecharacteristicofWNS,althoughthis”uorescencewasrestrictedto asmallareaintheplagiopatagiumandtherewerenovisiblewhitehyphaeonthewing surface( Fig.2 ).Allnoninfectedcontrolbatswerenegativefor P.destructans byTABLE1 Summaryofresultsfor Tadaridabrasiliensis bats,includingexperimentallyinfectedbatsandsham-treatednegativecontrols GroupBat Survival postinfection (days) Fluorescence observedunder ultravioletlight P.destructans detectedbyPCR No.ofwingsections: Examined(no.of UV-targetedsections) Withlocalizeddermal invasion(severity) Negative control C-149NoNo80 C-282NoNo60 C-361NoNo80 C-484NoNo8(2)0 InfectedI-151NoYes60 I-235NoYes80 I-375YesYes8(2)1(severe)bI-461YesYes81(severe) I-551NoYes90 I-684YesYes94(moderate) I-751YesYes92(moderate) I-835NoYes80 I-984(E)aYesYes9(2)1(severe),1(moderate)b , cI-1035(E)NoYes71(severe) I-1144YesYes91(moderate) aE,batwaseuthanized.bAsectionwastargetedforsamplingbasedon”uorescenceobservedunderUVlight.cAsingleareaofseverefungalinvasionofskinfrommuzzlewasalsoobserved. FIG1 Imagesofonewingmembranefroma Tadaridabrasiliensis batexperimentallyinfectedwith Pseudogymnoascusdestructans .ThewingshownisfrombatI-9( Table1 ).Areasofcontractedtissuewithwhitehyphae(black arrows)areseenunderilluminationbyvisiblelight(A)andareassociatedwithareasof”uorescence(whitearrows) apparentunderilluminationbyUVlightat365nm(B).ExperimentalWhite-NoseSyndromein Tadaridabrasiliensis July/August2018Volume3Issue4e00250-18 msphere.asm.org 3

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real-timePCRatthetimeofdeath(49to84daysfollowinginitiationoftheexperiment), andtheydidnotexhibit”uorescenceofwingmembranescharacteristicofWNS ( Table1 ). Onhistologicexamination,epidermalsectionsfromnoninfected T.brasiliensis bats wereunremarkable,andnoconidiawithmorphologytypicalof P.destructans were present( Fig.3A ,inset).Weobservedevidenceofcutaneousinvasiveascomycosisas describedforWNS( 27 )in7ofthe11 T.brasiliensis batsthatwereexperimentally infectedwith P.destructans ,includingwithinwingsectionsthatweretargetedfor histopathologybyscreeningunderUVlight( Table1 ).Additionally,threeexperimentallyinfectedbatshadmultiplecurvedconidiacharacteristicof P.destructans onornear theepidermalsurface,oneofwhichhadclustersofgerminatingconidiawithemerging hyphaelocatedsuper“cialtotheepidermis( Fig.3A ). The“rstcaseofcutaneousinvasiveascomycosisdetectedinvolvedlimitedinfection (e.g.,oneaffectedsectionofwingtissuewithoneregionofinfection)ona T.brasiliensis batthatwasmoribundandeuthanizedonday35followinginitiationoftheexperiment.Despitethepresenceofcutaneousinvasiveascomycosisin7oftheexperimentallyinfected T.brasiliensis bats,thedistributionoftheselesionsinhistologicsections ofwingmembranewassimilarlylimited( Table1 ).Oftheaffectedsections,generally onlyoneortworegionsinthesectioncontainedinvadinghyphae.Asinglesection fromoneofthe7batsexaminedhadadiscretecuppingerosionŽoftheepidermis ( Fig.3B ),astypicalforWNSinobligatelyhibernatingspecieslike M.lucifugus ( 4 ).Most otherlesionshadfocallyextensiveinvasionoftheepidermis,dermis,andsubcutaneous tissuewithdenseproliferationsofirregularfungalhyphae,consistentwithmorphology describedfor P.destructans ( Fig.3C ).Nearlycompleteobliterationoftheepidermaland dermalboundariesbydenseaggregatesofhyphaewasalsocommonintheselocalized areasofinfection( Fig.3D ).Onlyoneexperimentallyinfectedbathadhyphalinvasion ofthemuzzle,andnohistologicchangeswereobservedinearsections. Whenheldunderconditionsintendedtoinducehibernation,7.7°C(standarddeviation[SD] 0.9°C)and91.8%(SD 0.8%)relativehumidity, T.brasiliensis bats FIG2 Imagesofwingmembranesfrom Tadaridabrasiliensis and Myotislucifugus batsthatwereexperimentally infectedwith Pseudogymnoascusdestructans .(A,B)Thewingshownisfrom T.brasiliensis batI-11fromthisstudy ( Table1 ).Thewingappearsnormalundervisiblelight(A),butareasof”uorescencecharacteristicofwhite-nose syndrome(white-boxedarea)areapparentunderilluminationbyUVlightat365nm(B).(C,D)Thewingshown isfroman M.lucifugus batfromapreviousexperiment( 7 ).Whitehyphalgrowth(black-boxedarea)canbeseen onthesurfaceofthewingundervisiblelight(C),andareasof”uorescencecharacteristicofwhite-nosesyndrome arevisibleacrossthemajorityofthewingsurfaceunderilluminationbyUVlightat365nm(D).Verantetal. July/August2018Volume3Issue4e00250-18 msphere.asm.org 4

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demonstratedtheuseoftorpor-arousalcycles( Table2 ).Allbatsforwhichtemperature datawereavailable( n 11)spentamedianof6.8days(range,5.2to10.7days)per torporboutan d6h(range,3.6to7.6h)perarousal.Experimentallyinfectedbatshad shortertorporboutsandlongerarousalsthannoninfectedcontrolanimals,butthese differenceswerenotstatisticallysigni“cant(torpor, U 19, P 0.19,and r 5.7; arousal, U 3, P 0.08,and r 0.9[Mann-Whitney U test]).Theratesofmortalitywere similarbetweengroupsandwerepositivelyassociatedwiththebodymassindexatthe startoftheexperiment[ r(13) 0.89, P 0.001]. DISCUSSION Inthiscontrolledlaboratoryexperiment,wedemonstratedthatexperimentalinfectionof T.brasiliensis batswith P.destructans underconditionsofinducedhibernation causedcutaneousinvasiveascomycosiswithepidermallesionscharacteristicofWNS ( 4 ).Thelesionswecharacterizedinexperimentallyinfected T.brasiliensis batswere similartothoseobservedinbatsoftheobligatehibernatorspecies M.lucifugus infected with P.destructans inthewildorunderequivalentexperimentalconditions( 10 , 26 ). FIG3 Cutaneousinvasiveascomycosisinrepresentativehistologicsectionsofwingmembranesfrom Tadarida brasiliensis batsexperimentallyinfectedwith Pseudogymnoascusdestructans .(A)Characteristiccurvedconidiaof P. destructans areevidentsuper“cialtotheepidermis.Acrosssectionofnormalwingtissuefromanoninfected controlbatisprovidedforcomparison(inset).(B)Onesectionofwingmembranecontainedadiscretecupping erosionoftheepidermis“lledwithhyphae,adiagnosticcharacteristicofwhite-nosesyndrome.(C)Focal proliferationofirregularhyphaeontheskinsurfacewithinvasionoftheepidermal,dermal,andsubcutaneous tissue.(D)Denseaggregatesofhyphaeobscuringtheepidermalanddermalboundariesoffocallesions.TABLE2 Durationoftorporandarousalperiodsfor Tadaridabrasiliensis bats experimentallyinfectedwith Pseudogymnoascusdestructans orshamtreated GroupNo.ofbats Torporduration(days) [median(range)] Arousalduration(h) [median(range)] Negativecontrol38.1(6.8…9.9)4.5(3.6…5.7) Infected86.1(5.3…10.7)6.4(4.3…7.6) ExperimentalWhite-NoseSyndromein Tadaridabrasiliensis July/August2018Volume3Issue4e00250-18 msphere.asm.org 5

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However,thedistributionofpathologyoverwingmembranesof T.brasiliensis batswas muchmorelimitedthanin M.lucifugus bats.WeobservedminimalUV”uorescenceof wingmembranesin T.brasiliensis batsthatcouldbeconsideredcharacteristicofWNS, andrareepidermallesionswereidenti“edthroughextensivehistopathologicexaminationofmultiplesectionsofwingfromeachbat. In M.lucifugus andotherspeciesofhibernatingbats,WNSiscon“rmedbythe presenceoflesionsdescribedascuppingerosions,Žwhicharecharacterizedbybundlesofirregularfungalhyphaeformingadiscreteinterfacewithhosttissue( Fig.4A )( 4 ). Theselesionsaregenerallyreadilyobservedacrossthewingmembrane.Incontrast, amongexperimentallyinfected T.brasiliensis bats,cuppingerosionswereidenti“edin onlyonewingsectionfromasinglebat.Whenpresent,lesionsin T.brasiliensis bats primarilyconsistedofextensivehyphalinvasionandproliferationinthefocalareaof affectedwingskin.ThisdeepdermalinvasionistypicallyassociatedwithsevereWNS in M.lucifugus bats( Fig.4B )( 4 ).Therareoccurrenceofhistologiclesionsin T.brasiliensis batsinfectedwith P.destructans andtherelativelackofcuppingerosionsmayreduce thesensitivityofhistologicassessmentforWNSinthisspecies. Thecasede“nitionforWNSstatesthatbothhistologiclesions(i.e.,epidermal cuppingerosions)and P.destructans mustbepresenttocon“rmadiagnosisforWNS ( 28 ).Basedonthisde“nition,1of11 T.brasiliensis batsexperimentallyinfectedwith P. destructans developedWNS.However,WNSinobligatelyhibernatingbatsinNorth Americaisamultistagediseaseprocessthatisassociatedwithaberrantbehaviors, systemicphysiologiceffects,andmortality( 7…9 , 11 , 12 , 29 ).Inthisstudy,thelimited distributionofpathologyandlackofdifferencesintorpor-arousalpro“lesorsurvivalin experimentallyinfected T.brasiliensis batscomparedtocontrolssuggestthatthe pathogenesisofWNSinthisspecieslikelydiffersfromwhathasbeendescribedin obligatelyhibernatingspeciesofbats. Wedetectedthe“rstevidenceofcutaneousinvasiveascomycosisina T.brasiliensis batthatdied35dayspostinfection,ashortertimeframethandescribedforthe developmentofpathologyin M.lucifugus bats( 26 ).Furthermore,epidermallesionsin T.brasiliensis batsweregenerallyobservedinonlyoneofseveralsectionsofwing membraneexaminedfromeachbatandincludedonlyoneortwofocalareasof invadinghyphaeperaffectedsection.Thisisdistinctfromtheoftendiffusedistribution ofepidermallesionsandhyphalproliferationobservedinwingmembranesofobligatelyhibernatingspeciesofbatswithWNS.Additionally,themediantorporbout durationsfor T.brasiliensis batsinthisstudy(6.1to8.1days)( Table2 )wereshorterthan whathasbeendescribedfor M.lucifugus bats(approximately9to16days)insimilar publishedexperiments( 7 , 29 ).Whileweobservedmortalityofbatsduringthisstudy, theratesweresimilarintheinfectedandcontrolgroupsandstarted35daysfollowing initiationoftheexperiment.Incontrast,previousreportsindicatedthatmortalityfrom WNSin M.lucifugus batsunderexperimentalandnaturalconditionsdoesnotoccur untilapproximately120daysfollowinginfection( 26 , 29 ).Thus,themortalityobserved FIG4 Histologicsectionsofwingmembranesfrom Myotislucifugus batsexperimentallyinfectedwith Pseudogymnoascusdestructans inapreviousexperiment( 7 ).(A)Epidermalcuppingerosionsdiagnostic forwhite-nosesyndrome(WNS).(B)Denseproliferationofhyphaeandinvasionofepidermal,dermal, andsubcutaneoustissuesofwingmembranefromabatwithsevereWNS.Verantetal. July/August2018Volume3Issue4e00250-18 msphere.asm.org 6

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in T.brasiliensis batsexperimentallyinfectedwith P.destructans likelyresultedfrom causesotherthanWNS,suchasinef“cientuseofenergyduringinducedhibernation comparedtotheenergyuseofanobligatelyhibernatingspecies.Thebodymassindex ofabatatthestartofthisexperimentwaspositivelycorrelatedwiththenumberof daysabatremainedaliveposttreatment,suggestingthatsurvivaltimewasrelated,at leastinpart,toenergyreserves. Experimentalanimalmodelsareavaluabletoolforinvestigatingdiseaseprocesses andpotentialtreatments.Todate,researchonWNShasprimarilyreliedupontheuse ofwild-caughtmembersofbatspeciesthatarenaturallysusceptibletothediseasebut arenotreadilyamenabletolong-termmaintenanceincaptivity.Alternatively,tissue explantsfrombatwingshaveshownsomeutilityfortestingpotentialinhibitorsof P. destructans ( 30 ),butitremainsnecessarytocollectexplantsfromwild-caughtbats. Basedontheresultsofthisstudy, T.brasiliensis mayhavepotentialasanexperimental modelforinvestigatingaspectsof P.destructans persistenceandproliferationonbats orfortestinghypothesesonvariationsinspeciessusceptibilityto P.destructans and WNS.However,giventheobservedatypicalmanifestationof P.destructans infectionin T.brasiliensis andshorterdurationofsurvivalunderconditionsofinducedhibernation, theuseofthisspeciesisnotlikelyappropriatetostudythephysiologiceffectsand pathogenesisofWNSortotestpotentialtreatmentsdesignedtoreducemortality. Thisexperimentleavesunansweredquestionsregardingthesusceptibilityofwild T.brasiliensis batstoinfectionwith P.destructans undernaturalconditions.Ourresults re”ectexperimentallyinducedconditionsthatwereintendedtoprovidethehighest likelihoodofinfectionbyreplicatingpriorexperimentswith M.lucifugus bats(e.g.,see references 26 and 29 ).Incontrast, T.brasiliensis batsarenotobligatehibernators,but individualsofthisspecieshavebeenshowntousetorportoconserveenergyovershort periodsandmayhibernateinsomelocations( 22 , 31 ).Differentenvironmentalconditions(e.g.,temperatureorhumidity)withinroostsmayin”uencesusceptibilitytoWNS, ashasbeendescribedforotherspecies( 32 ).Ifwild T.brasiliensis batsaresusceptible toinfectionwith P.destructans ,itmaybechallengingtode“nitivelydiagnoseWNSin thisspeciesinaccordancewiththecurrentcasede“nition,ifepidermalpathologyin naturallyinfectedbatsislimitedsimilarlytothatdescribedforthebatsinthisstudy. Therecoveryofviable P.destructans fromanexperimentallyinfectedbatsuggests that T.brasiliensis batshavethepotentialtoharborthefungusundercertainconditions.Thelowrecoveryrateofviable P.destructans frominfectedbatsinthisstudymay haveresultedfromthelimitedextentofepidermalfungalinfections;recoverywasalso hamperedbyovergrowthofcultureplatesbymorerapidlygrowingyeast(e.g., Debaromyces sp.)presentonthewingmembranes.Regardless,ourresultsindicatea potentialfor T.brasiliensis batstocontributetomovementof P.destructans between roostsitesthatmayalsoharborotherbatspeciessusceptibletoWNS.Thisriskis highlightedbyarecentreportofPCR-baseddetectionofthepresenceof P.destructans on T.brasiliensis batsataroostincentralTexasthatisoccupiedbyapproximately3 million T.brasiliensis batsfromMaythroughOctoberofeachyear( 33 ).Suchdense aggregationsofbats,typicalforcoloniesofthisspecies,havethepotentialtofacilitate highpathogentransmissionrates( 34 )undersuitableenvironmentalconditions.Additionally, T.brasiliensis batshavebeenshowntomigrateupto1,500kmseasonally( 35 ) andtravelupto50kmduringdailymovements( 36 ),whichcouldfacilitatepathogen dispersal.However,”uctuatingenvironmentaltemperaturesandactivitypatternsare likelytoin”uencethepersistenceandviabilityof P.destructans on T.brasiliensis batsin nature. Understandingthepotentialfor T.brasiliensis batstobecomeinfectedwith P. destructans orcontributetothespreadofWNSistimely,giventhecontinuedspreadof thisdiseaseandthepathogenwithintherangeofthisbatspeciesinthesouthern UnitedStates( 37 ).Additionalsurveillancefor P.destructans andWNSinwild T.brasiliensis batsalongtheleadingedgeofWNSwillhelptoelucidatetherolethisabundant andfar-rangingspeciesmayhaveinthemovementofthisfungalpathogenacross westernandsouthernNorthAmerica.ExperimentalWhite-NoseSyndromein Tadaridabrasiliensis July/August2018Volume3Issue4e00250-18 msphere.asm.org 7

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MATERIALSANDMETHODS Bats,husbandry,andpermissions. ThisexperimentwasconductedinaccordancewithU.S. GeologicalSurveyNationalWildlifeHealthCenter(NWHC,Madison,WI)InstitutionalAnimalCareandUse Committee(IACUC)experimentalprotocol121025.Apparentlyhealthy,adultmale T.brasiliensismexicana bats( n 15)werecaughtinBrazosCounty,Texas,in2013underTexasParksandWildlife DepartmentpermitnumberSPR-1104-610andTexasA&MUniversityIACUCapprovalnumber2012-130. AfteracclimatingtocaptivityinTexas,thebatsweretransferredtoandheldattheU.S.GeologicalSurvey NationalWildlifeHealthCenter(NWHC,Madison,WI).AtNWHC,thebatsweremaintainedunderanimal biosafetylevel3conditionsin”ightcagesfora30-dayquarantineperiod,duringwhichbloodsamples werecollectedandthebatsweretopicallytreatedforparasiteswithselamectin(Revolution;Zoetis,Inc., Parsippany-TroyHills,NJ).Auniqueelectronicmicrochipidenti“cationunit(AvidIdenti“cationSystems, Inc.,Folsom,LA)wassubcutaneouslyinjectedbetweenthescapulaeofeachanimal.Batsweremaintainedonmealworms( Tenebriomolitor )supplementedwithvitaminsandanomegafattyacidmixture, andwaterwasprovided adlibitum .Thelightcyclewassetto12hoflightperday,invertedfromthe naturalcycletoallowmonitoringofbatactivitiesduringdaytimehours.Onemonthpriortoinitiationof the P.destructans infectiontrialdescribedherein,apoxvirusinfectionstudywascompletedatNWHC ( 38 ),involvingallbatssubsequentlyusedinthisstudy.Atthestartofthisinfectiontrial,nopoxviral activitywasevidentinanybat,basedonlackofactivityfromtheluciferasemarkergeneinsertedinto thepoxvirus,andnobatsshowedsignsofclinicalillness.Additionally,batswereassumedtobenegative for P.destructans priortothestartofthisinfectiontrialduetotheiroriginandtimespentactivein captivity. Infectiontrial. AniBBattemperaturelogger(AlphaMach,Sainte-Julie,Quebec,Canada)was attachedtothedorsalsurfaceofeachbattoassesstorpor-arousalpatternsaspreviouslydescribed( 7 ). Arousalthresholdsforeachindividualwerede“nedas10%ofmaximumskintemperature( 10 ).The lengthoftherightforearmandmassofeachbatwasmeasuredandusedtocalculatethebodymass index(forearmlength/mass)atthestartofthestudy.Batswererandomlyassignedtoinfected( n 11) andcontrol( n 4)groups.Conidiaof P.destructans (5 105,suspendedin20lphosphate-buffered salinesolutioncontaining0.5%Tween20[PBST])wereappliedtothemuzzleandthedorsalsurfaceof thewingsofeachbatintheinfectedgroupaspreviouslydescribed( 26 ).Batsinthecontrolgroupwere treatedsimilarlybutwithvehiclesolution(PBST)lackingconidia.Infectedandcontrolgroupswerethen placedinseparatemeshenclosures(25incheshighby14.5incheswideby14.5inchesdeep;Apogee Reptaria,Dallas,TX)separatedbyaplasticdividerwithinanenvironmentalchamber(modelnumber I-36NL;PercivalScienti“c,Perry,IA)maintainedat7.7°C(SD 0.9°C)and91.8%(SD 0.8%)relative humiditytoinduceandsupportextendedhibernation.Waterwasprovided adlibitum usingagravity-fed waterbowlonthe”oorofeachenclosure.Batsweremonitoredtwicedailythroughawindowinthe doorofthechamber,withtheinteriorilluminatedbyredlight.Anybatobservedonthe”oorofan enclosurefortwoconsecutivemonitoringcheckswasremovedfromthechamberforassessment. Moribundbatswereeuthanized,anddiagnosticsampleswerepromptlycollectedfromcarcassesas describedbelow. Evaluationfor P.destructans andWNS. Thepresenceof P.destructans oneachbatatthetimeof deathwasdeterminedbyreal-timePCRanalysisofwingskin( 39 )andculture-basedassessmentofa sectionofskin(approximately3cmby3cm)fromtheleftwingplacedontoSabourauddextroseagar containingchloramphenicolandgentamicinandincubatedat10°Cforupto1month.Theentire membraneoftherightwingwasexaminedusingahandheldUVlamp(modelnumberUVL-56,365nm; UVP,Inc.,Upland,CA)toidentifyareasoforange-yellow”uorescenceunderUVilluminationaspreviously describedforWNS( 40 ). FollowingexaminationunderUVlight,theentiremembranefromtherightwingofeachbatwas removedandprocessedforhistopathologyanalysisaspreviouslydescribed( 4 ).Brie”y,thewing membranewascutinto1-cmstripsandrolledinoverlappingspiralsarounddowels(approximately2cm by0.25cm)ofcolorlessdentalorthodonticparaf“n.Theparaf“ndowelswithwingtissuewerethen placedinnumericallycodedcassetteswithoutnotationoftreatmentgroupandsubmergedin10% neutralbufferedformalinforatleast24h.Eachparaf“ndowelwithtissuewasthentrimmedtoproduce 0.5-cmcrosssections,yieldingapproximatelyeightwhorlsofwingtissuefromeachbat.Thesecross sectionswerethenplacedcut-sidedowninthecodedcassetteforprocessingandembeddinginparaf“n; 4-msectionswerethenplacedonsimilarlycodedglassslidesandstainedusingtheperiodicacid-Schiff (PAS)method.Sectionsofmuzzleandearwerealsosampledforhistopathologyandprocessedtogether withthewingmembrane. Histologicsectionswereexaminedforepidermallesions(cutaneousinvasiveascomycosis[ 27 ]) consideredcharacteristicofWNS,whichincludecuppingerosionoftheepidermisbydenseirregular fungalhyphaethatformadiscreteinterfacewiththehosttissue( 4 ).Thedegreeoferosionandulceration andtheextentofdistributionoflesionsoverthesurfaceareaofthewingsectionsexaminedwereused todeterminetheseverityofinfection( 10 ). Dataavailability. DatasupportingtheresultsofthisstudyanddatafromiBBattemperatureloggers areavailablefromtheU.S.GeologicalSurveyScienceBaseCatalogat https://doi.org/10.5066/P93WAKH3 ( 41 ). ACKNOWLEDGMENTS ThisresearchwasfundedbytheU.S.GeologicalSurveythroughacooperative agreementwiththeUniversityofWisconsin„Madison.FundswereusedfordirectVerantetal. July/August2018Volume3Issue4e00250-18 msphere.asm.org 8

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projectcostsonly.PartialsupportforM.L.V.andB.S.wasprovidedbytheNational InstitutesofHealththroughRuthL.KirschsteinNationalResearchServiceAward institutionaltraininggrantnumberT32RR023916fromtheNationalCenterforResearchResourcesandbytheU.S.GeologicalSurvey. WethankMikeSmotherman,TexasA&MUniversity,forprovidingthebatsusedfor thisproject.WeadditionallythanktheNWHCanimalcarestaffforexpertiseanddaily assistancewithanimalhusbandry. Useoftrade,product,or“rmnamesisfordescriptivepurposesonlyanddoesnot implyendorsementbytheU.S.government. M.L.V.andB.S.werevolunteersattheU.S.GeologicalSurvey,NationalWildlife HealthCenter,Madison,Wisconsin,USA. B.S.,D.S.B.,andM.L.V.conceivedtheideasanddesignedthestudy;B.S.andM.L.V. collectedthedata;C.U.M.andM.L.V.analyzedthedata;andC.U.M.,M.L.V.,andD.S.B. wrotethemanuscript.Allauthorscontributedcriticallytothedraftsandgave“nal approvalforpublication. 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28.USGSNationalWildlifeHealthCenter.2014.Diagnosticcategoriesfor reportingcasesofbatwhite-nosesyndrome(WNS).USGeological SurveyNationalWildlifeHealthCenter,Madison,WI. https://www .nwhc.usgs.gov/disease_information/white-nose_syndrome/Case %20De“ntions%20for%20WNS.pdf . 29.WarneckeL,TurnerJM,BollingerTK,LorchJM,MisraV,CryanPM, WibbeltG,BlehertDS,WillisCKR.2012.InoculationofbatswithEuropean Geomycesdestructans supportsthenovelpathogenhypothesisfor theoriginofwhite-nosesyndrome.ProcNatlAcadSc iUSA 109: 6999…7003. https://doi.org/10.1073/pnas.1200374109 . 30.CornelisonCT,KeelMK,GabrielKT,BarlamentCK,TuckerTA,PierceGE, CrowSA,Jr.2014.Apreliminaryreportonthecontact-independent antagonismof Pseudogymnoascusdestructans by Rhodococcusrhodochrous strainDAP96253.BMCMicrobiol14:246. https://doi.org/10.1186/ s12866-014-0246-y . 31.HerreidCF.1963.Survivalofamigratorybatatdifferenttemperatures. JMammal44:431…433. https://doi.org/10.2307/1377227 . 32.LangwigKE,FrickWF,HoytJR,PariseKL,DreesKP,KunzTH,FosterJT, KilpatrickAM.2016.Driversofvariationinspeciesimpactsforamultihostfungaldiseaseofbats.PhilosTransRSocLondBBiolSci371: 20150456. https://doi.org/10.1098/rstb.2015.0456 . 33.TexasParksandWildlifeDepartment.2018.Funguscausingwhite-nose syndromespreadsintocentralTexas.TexasParksandWildlifeDepartment,Austin,TX. http://tpwd.texas.gov/newsmedia/releases/print.phtml ?req 20180404a .Accessed20April2018. 34.AndersonRM,MayRM.1979.Populationbiologyofinfectiousdiseases: partI.Nature280:361…367. https://doi.org/10.1038/280361a0 . 35.BernardoVR,CockrumEL.1962.Migrationintheguanobat Tadarida brasiliensismexicana (Saussure).JMammal43:43…64. https://doi.org/10 .2307/1376879 . 36.DavisRB,HerreidCF,ShortHL.1962.Mexicanfree-tailedbatsinTexas. EcolMonogr32:311…346. https://doi.org/10.2307/1942378 . 37.USFishandWildlifeService.2018.Whereisitnow?USFishandWildlife Service,Washington,DC. https://www.whitenosesyndrome.org/about/ where-is-it-now Accessed:20March,2018. 38.StadingBR,OsorioJE,Velasco-VillaA,SmothermanM,Kingstad-BakkeB, RockeTE.2016.Infectivityofattenuatedpoxvirusvaccinevectorsand immunogenicityofaraccoonpoxvectoredrabiesvaccineintheBrazilian free-tailedbat( Tadaridabrasiliensis ).Vaccine34:5352…5358. https://doi .org/10.1016/j.vaccine.2016.08.088 . 39.MullerLK,LorchJM,LindnerDL,OConnorM,GargasA,BlehertDS.2013. Batwhite-nosesyndrome:areal-timeTaqManpolymerasechainreactiontesttargetingtheintergenicspacerregionof Geomycesdestructans . Mycologia105:253…259. https://doi.org/10.3852/12-242 . 40.TurnerGG,MeteyerCU,BartonH,GumbsJF,ReederDM,OvertonB, BandouchovaH,Bartonic kaT,MartínkováN,PikulaJ,ZukalJ,BlehertDS. 2014.Nonlethalscreeningofbat-wingskinwiththeuseofultraviolet ”uorescencetodetectlesionsindicativeofwhite-nosesyndrome.JWildl Dis50:566…573. https://doi.org/10.7589/2014-03-058 . 41.VerantML,MeteyerCU,StadingB,BlehertDS.2018.Experimental infectionofTadaridabrasiliensiswiththefungusthatcauseswhite-nose syndrome:hibernationdata.USGeologicalSurvey,Reston,VA. https:// doi.org/10.5066/P93WAKH3 .Verantetal. July/August2018Volume3Issue4e00250-18 msphere.asm.org 10


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