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Literature Cited

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Abstract DiscussionMethods

Introduction

SeroprevalenceofLymeDisease(Borreliaburgdorferi) inBirdsinCentralIllinoisLoganJ.Bader1,Dr.TravisE.Wilcoxen1,2,JaneSeitz2,&JacquesNuzzo2

1MillikinUniversityBiologyDepartment2IllinoisRaptorCenterDecatur,IL

Manyzoonoticdiseases,suchasLymedisease,canbetransmittedfromwildlifehostsandvectorstoavianpopulations.LymediseaseisacquiredthroughexposuretothebacteriumBorreliaburgdorferi. WhilepaststudiesofLymediseaseinaviancommunitiesremainlargelydescriptive,thisstudyaimstogainanunderstandingofthedevelopmentofantibodiesagainstB.burgdorferi inraptors,songbirds,wadingbirds,andwaterfowl.WehypothesizedthatbirdsthatforageingrassyhabitatswouldpossessthemostelevatedlevelsofIgYantibodiesagainstB.burgdorferiduetomoreexposuretoticks,theprimaryvectorofLyme.Wecollectedbloodsamplesfrom363birdsof17variousfamiliesandperformedanenzyme-linkedimmunosorbentassay(ELISA)todetectthepresenceofIgYantibodiesspecifictoB.burgdorferiineachsample.Ofthesample,11ofthe17familiespossessedIgYtoLyme.FamilieswiththehighestseroprevalenceofIgYspecifictoB.burgdorferi includedCorvidae (Crows,Jays;42.8%); Turdidae (Robins,Bluebirds;28.5%);and Columbidae (Doves,Pigeons;25%).Overall,wefoundthatvariationintheseroprevalenceofLymewassignificantamongtaxonomicgroups.Whileitisclearthatsongbirdspossessthelargestnumberofseropositiveindividuals,therewasnosignificantassociationofhabitatorseasononseroprevalenceofB.burgdorferiinourpopulation.

• Therewasasignificanteffectoftaxon(Family)onseroprevalenceofIgYspecificforB.burgdorferi (X2 =33.757,df =16,p=0.006;Fig.5).

• SeroprevalenceofIgYwasnotpredictedbyhabitat(X2 =0.117,df =6,p=0.679;Fig.6)orseasoncaptured(X2 =0.064,df =3,p=0.698;Fig.7).

IthankDr.Wilcoxenforhisassistanceandinputthroughoutthisproject.IwouldalsolikethankJaneSeitzandJacquesNuzzo fromtheIllinoisRaptorCenterforallowingmetogathersamplesonsite,aswellastheMillikinUniversityBiologyDepartmentfortheopportunitytoparticipateinundergraduateresearch.

RaptorAdmissionsandBloodSampling• Wecollectedbloodsamplesfromeachraptoradmittedfromtheyear

2015throughSummer2018attheIllinoisRaptorCenter(Decatur,IL),aswellasfromsongbirdscapturedinmistnetsintheDecaturarea.

• Allindividualsunderwentphysicalexamination,includingthesearchforanyparasites,specificallyticksastheyserveasvectorsofB.burgdorferi.

• Westudiedatotalof363birdsfrom17families.

Enzyme-LinkedImmunosorbentAssay(ELISA)• Plasmasampleswereisolatedfrombloodusingcentrifugation.• EachsamplewasplatedontoawellcoatedwithB.burgdorferi antigenas

thepositiveantigen(OSP-Aprotein)andkidneybeanlectinprotein(phytohaemagglutinin,PHA)asthenegativeantigen.

• Allbirdswithapositivetonegative(P/N)antigenratioof1.987orgreaterwereconsidered“seropositive.”

Statistics• WeassesseddifferencesinquantitiesofIgYbetweentaxon,habitattype,

andseasonusingageneralizedlinearmodel.

• Thestrengthoftheseroprevalence(SP)ofIgYantibodywasindicatedbasedonthepercentageofindividualswithapositiveELISAresultfromeachfamily(Table1):

• HighSP:SignificantIgYlevelsin25%ormoreofindividuals• IntermediateSP:SignificantIgYlevelsin10to25%ofindividuals• LowSP:SignificantIgYlevelsin10%orlessofindividuals• NodetectionofIgYspecificforB.Burgdorferi.

• Weusedap-valueof0.05orlesstodeterminestatisticalsignificance.

Figure4.Anexampleofa96-wellELISAplate.Thisimagedoesnotdisplayanyresultfromthestudyandisintendedforvisualpurposesonly.

• ThemajorityofresearchinvolvingtheacquisitionofLymediseasebybirdsremainslargelydescriptive(Lossetal.2016).Additionally,veryfewstudieshaveinvolvedraptors(Scottetal.2014).

• ThecausativeagentofLymeisBorreliaburgdorferi,whichisidentifiedbyitshelicalshapeandexpressionofavarietyofsurfaceproteins(Andone etal.2013).Specifically,OSP-Aisastrongantigenthatelicitstheimmuneresponseinthehost(Caimano etal.2016).

• TheprimaryvectorofLymeisthetickspeciesIxodesaffinis(Hameretal.2011).OnsetofinfectionoccurswhenB.burgdorferiisingestedbythetick(Magnarelli2011,Lossetal.2016,Tillyetal.2008).

• I.affinisisgenerallyfoundindisturbedforesthabitat(Nadolny andGaff2018).

• ReservoirsofB.burgdorferitransmittheagentforlongperiodswithoutshowingobvioussymptomsofthedisease(Isogai etal.1994).

• MigratorybirdsareaknownvectorofLymedisease(Andone etal.2013).

• Thepurposeofthisstudywastoanalyzewhetherdifferencesintaxonomy(family),habitat,andseasonalteredtheseroprevalenceofimmunoglobulinY(IgY)toB.burgdorferi inbirdsofCentralIllinois.

Figure1.TheenzooticcycleofB.burgdorferi.LarvalticksmustacquireB.burgdorferibyfeedingonaninfectedvertebrate(Caimano etal.2016).

Figure5.TheinteractionoffamilygroupandtheseroprevalenceofIgYspecifictoB.burgdorferiwithinapopulationofraptorsandsongbirdsinCentralIllinois.

Figure6.AcomparisonoftheseroprevalenceofIgYspecificforB.burgdorferiamongthefollowinghabitats:bottomland(BL),tallgrass/lowwoodymix(DG),edge(E),forest(F),opengrassland(OG),urban(U),wetland(W).

Figure7.AcomparisonoftheseroprevalenceofIgYspecificforB.burgdorferiamongseasons.

• WedeterminedthatourELISAwasabletodetectthepresenceofIgYantibodiesagainstB.burgdorferi inbirds.

• Positivetonegative(P/N)antigenratioof1.987orgreater.• SamplesthatwerenotseropositiveoftendisplayedP/Nratios

greaterthanzero.Thisisreflectedinthebindingofnaturalantibodiestothenegativeantigen.

• OurhypothesisthatseroprevalenceofIgYtoB.burgdorferi wouldbehighestamonggrasslandandedgehabitattypeswasnotsupported.

• Generalisttickspeciesarestronglydependentonhostavailabilityanddiversityforsurvival(Legeretal.2012).

• Tickdensitypositivelyimpactsspreadofvector-borneillnessesacrossaregion.Habitatconnectivitymaybeastrongerpredictoroftickdensitythanhabitattype(Nadolney andGaff2018).

• OurhypothesisthatseroprevalenceofIgYantibodyvaryamongseasonswasnotsupported.

• Variabletoconsiderinthefuture,asclimatechangehasbeenforecastedtoleadtoanoverallincreaseintickhabitat(NadolneyandGaff2018).

1. Loss,S.,B.Noden,G.Hamer,andS.Hamer.2016.Aquantitativesynthesisoftheroleofbirdsincarryingticksandtick-bornepathogensinNorthAmerica. Oecologia 182(4):947-959.

2. Scott,J.,J.Anderson,andL.Durden. 2014.FirstdetectionofLymediseasespirocheteBorreliaburgdorferiintickscollectedfromaraptorinCanada. JournalofWildlifeRehabilitation 34(2):11-16.

3. Andone,I.,C.Daia,O.Berdilă,V.Popa,V.,M.Popa,G.Onose,andD.Blendea.2013.ActualsyntheticoverviewonBoreliosis (Lymedisease). Infectio.Ro 34(2):18-24.

4. Caimano,M.J.,D.Drecktrah,F.Kung,andD.S.Samuels.2016.InteractionoftheLymediseasespirochetewithitstickvector. CellularMicrobiology 18(7):919–927.

5. Hamer,S.A.,G.J.Hickling,J.L.Sidge,M.E.Rosen,E.D.Walker,andJ.I.Tsao.2011.Diverseborreliaburgdorferistrainsinabird-tickcrypticcycle. Applied&EnvironmentalMicrobiology 77(6):1999-2007.

6. Magnarelli,L.A.2011.Theroleofvertebratehostsintick-borneinfections. ClinicalMicrobiologyNewsletter 33(3):17-20.

7. Tilly,K.,P.A.Rosa,andP.E.Stewart.2008.BiologyofinfectionwithBorreliaburgdorferi.InfectiousDiseaseClinicsofNorthAmerica 22:217-234.

8. Nadolny,R.M.andH.D.Gaff.2018.Modellingtheeffectsofhabitatandhostsontickinvasions.LettersinBiomathematics5(1):2-29.

9. Isogai,E.,S.Tanaka,I.S.Braga,C.Itakura,H.Isogai,K.Kimura,andN.Fujii.1994.ExperimentalBorreliagarinii infectionofJapanesequail. InfectionandImmunity 62(8):3580–3582.

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Hypotheses• WehypothesizedthatIgYseroprevalencespecifictoB.burgdorferi wouldvary

amongtaxon(Families),duetovariationinbehaviorandhabitatusethatmightleadtovariationinexposuretovectors.

• WehypothesizedthatseroprevalenceofIgYantibodywouldvaryamonghabitat,withhighestvaluesingrasslandandedgehabitatswherevectorsmostcommonlyreside.

• WehypothesizedthatseroprevalenceofIgYtoB.burgdorferi wouldvaryamongtheseasoninwhichthebirdwascaptured,reflectingvariationinvectoractivity.

DependentVariable:• IgYresult(Y/N)

IndependentVariables:• Taxon(family)• Habitat• Season

Figure2.Fourspecies(familynamesinparentheses)analyzedforIgYantibodies;(lefttoright)GreatHornedOwl(Strigidae),BarredOwl(Strigidae),Red-tailedHawk(Accipitridae),andNorthernCardinal(Cardinalidae).

Figure3.AdiagramoftheELISAtechniqueusedinthisstudy.ThetargetproteininourstudywasOSP-A,theantigenelicitedbyB.burgdorferi.AdditionofTMBsubstrateproducescolorationafterreactionwiththeenzymeattachedtothesecondaryantibody.Retrievedfromhttps://ruo.mbl.co.jp/bio/e/support/method/elisa.html.

NY

NY

NY

HighSeroprevalence(25%orhigher)

IntermediateSeroprevalence(Between10and25%)

LowSeroprevalence(10%orless)

NotDetected

Corvidae (Crows,Jays;42.8%) Fringillidae(Finches;18.8%) Strigidae(Owls;8.2%) Caprimulgidae (Nighthawks,Whip-poor-whils)

Turdidae (Robins,Bluebirds;28.5%)

Picidae(Woodpeckers;17.6%) Accipitridae(Hawks,Eagles;8.0%)

Cardinalidae (Cardinals,Buntings)

Columbidae (Doves,Pigeons;25.0%)

Passeridae(HouseSparrows;16.7%) Emberizidae (Sparrows,Juncos)

Carthartidae(Vultures;14.2%) Falconidae (Falcons,Kestrels)

Anatidae(Ducks,Geese;12.5%) Rallidae (Coots,Rails)

Ardeidae(Herons,Egrets;11.1%) Sittidae (Nuthatches)

Table1.TheseroprevalenceofIgYantibodiesspecifictoB.burgdorferi in17familiesofbirdswithinCentralIllinois.

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• OurhypothesisthatIgYseroprevalencetoB.burgdorferi wouldvaryamongtaxon(families)wassupported.

• Highestseroprevalencewereamongsongbirdfamilies,includingCorvidae,Turdidae,andColumbidae (Fig.5,Table1).

• RaptorspeciesweregenerallylowinIgYseroprevalencewiththeexceptionofCarthartidae (vultures;Table1).

• Lymeexposuremayoccurmoreinbirdsthatfrequentlyforageontheground;B.burgdorferiisamicroaerophilicbacterium(Hameretal.2011,Caimano etal.2016).

• Itisuncommonforraptorstoharborticksviadirectattachment(Lossetal.2016).

• Futurestudyconsiderations• I.affinisparasitizesbirdsasimmatures(Nadolney andGaff2018).• PathwaysofB.Burgdorferi transmissionotherthandirecttick

attachment:• RoleofpredationonB.burgdorferiinfectioninbirds.• Birdtobirdinteractions,suchasfeeding,territory

defense,etc.• Stresshormonequantificationinseropositiveversus

seronegativebirds.

• Althoughphysicalsymptomswerenoteasilydetectableamongseropositivebirds,frequentantibodypresencesuggeststhatavianfamiliesarecapableofformingadaptiveimmuneresponsestoLyme.