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Animals2022,12,2829.https://doi.org/10.3390/ani12202829www.mdpi.com/journal/animals
Article
OccurrenceofBorreliasp.amongWildLivingInvasiveand
NativeMesocarnivoresinPoland
JoannaHildebrand
1
,KacperJurczyk
2
,MarcinPopiołek
1
,KatarzynaBuńkowska‐Gawlik
1
andAgnieszkaPerec‐Matysiak
1,
*
1
DepartmentofParasitology,FacultyofBiologicalSciences,UniversityofWrocław,51‐148Wrocław,Poland
2
DepartmentofTropicalMedicineandParasitology,MedicalUniversityofGdańsk,
M.Skłodowskiej‐Curie3a,80‐210Gdańsk,Poland
*Correspondence:agnieszka.perec‐matysiak@uwr.edu.pl
SimpleSummary:Vertebratehosts,especiallywildlivinganimals,arepivotaltothecirculationand
maintenanceofBorreliaspp.MesocarnivoresareinvolvedinLymediseaseecologyinsylvaticand
suburbanecosystems.Inthisstudy,weaimedtoexaminetherelativeimportanceofsixmedium‐
sizedcarnivorespecies,raccoon,redfox,raccoondog,Europeanbadger,pinemartenandstone
marten,ashostsofBorreliaspp.andinvestigatedtheirroleinthisspirochaete’stransmissioncycle.
Wealsoaimedtotracethereservoircompetenceoftheseinvasiveandnativepredatorsandtheeco‐
epidemiologyofBorreliaspp.inthecontextofadilutioneffect.Inallexaminedcarnivorespecies,
theoccurrenceofBorreliawasrecorded,andtheresultssuggestthatraccoonsmayplayaroleas
reservoirhostsforthesespirochaetalbacteria.Theroleofinvasivespeciesseemstobeworthyof
furtheranalysiswithreferencetothecirculationofvector‐bornepathogensaswellasinthecontext
ofthe“dilutioneffect”hypothesis.
Abstract:Wildlivingmesocarnivores,bothintroducedandnativespecies,areabletoadaptwellto
peri‐urbanenvironments,facilitatingcross‐speciespathogentransmissionwithdomesticanimals,
andpotentiallyhumans.Individualtissuesamplesderivedfrom284specimensofsixcarnivore
species,i.e.,raccoon,raccoondog,redfox,Europeanbadger,pinemartenandstonemarten,were
usedformolecularinvestigationswiththenestedPCRmethod.Theanimalsweresampledinthe
RuszówForestDistrict(Poland).Weaimedtoexaminetherelativeimportanceofthestudied
mesocarnivoresashostsofBorreliaspp.andinvestigatedtheirroleinthisspirochaete’stransmission
cycle.Wealsoaimedtotracethereservoircompetenceoftheseinvasiveandnativepredatorsand
borreliosiseco‐epidemiologyinthecontextofadilutioneffect.TheoverallprevalenceofBorrelia
spp.inthetestedcarnivoreswas8.8%.AlmostalloftheconsensussequencesofthepartialflaBgene
sharedidentitywithasequenceofspecificBorreliaspecies,i.e.,B.afzelii,B.gariniiandB.burgdorferi.
Ourresultssuggestthatraccoonsmayplayaroleasreservoirhostsforthesespirochaetalbacteria.
Theroleofinvasivespeciesseemstobeworthyoffurtheranalysiswithreferencetothecirculation
ofvector‐bornepathogensaswellasinthecontextofthe“dilutioneffect”hypothesis.
Keywords:Borrelia;wildlivingcarnivores;invasivespecies;zoonosis
1.Introduction
ThegenusBorreliacomprisesarthropod‐bornespirocheteswhicharepathogensof
humanandanimals.Vertebratehosts,especiallywildlivinganimals[1],arepivotaltothe
circulationandmaintenanceofBorreliaspp.Thelevelofcompetencevariesbetweenhosts,
withrodentsappearingtobeimportanthosts[2,3],whereas,e.g.,roedeer(Capreolus
capreolus)seemtobezooprophylacticor“dilution”hosts[4].Accordingtothedilution
effecthypothesis,diversehostcommunitiesinhibittheparasiteabundancethrough
variousmechanisms,suchasregulatingpopulationsofsusceptiblehostsorinterfering
Citation:Hildebrand,J.;Jurczyk,K.;
Popiołek,M.;Buńkowska‐Gawlik,K;
Perec‐Matysiak,A.Occurrenceof
Borreliasp.amongWildLiving
InvasiveandNativeMesocarnivores
inPoland.Animals2022,12,2829.
https://doi.org/10.3390/ani12202829
AcademicEditor:MariaLuisa
Menandro
Received:19August2022
Accepted:17October2022
Published:18October2022
Publisher’sNote:MDPIstays
neutralwithregardtojurisdictional
claimsinpublishedmapsand
institutionalaffiliations.
Copyright:©2022bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(https://creativecommons.org/license
s/by/4.0/).
Animals2022,12,28292of7
withparasitetransmission[5–7].Nevertheless,theroleofcarnivoresas hosts inBorrelia
spp.ecologyremainsunclear.Thecurrentknowledgeofthecomplexinteractionbetween
thisdiversepathogenanditsvectorsaswellasdifferentreservoirandnon‐reservoirhosts
isstillincomplete.
Mesocarnivoressuchasraccoon(Procyonlotor),redfox(Vulpesvulpes),raccoondog
(Nyctereutesprocyonoides),Europeanbadger(Melesmeles),pinemarten(Martesmartes)and
stonemarten(Martesfoina)serveashostsforseveraltickspecies[8–11]andtheir
significantinvolvementinLymediseaseecologyinsylvaticandsuburbanecosystemsis
likely.Thecastorbeantick(Ixodesricinus)isknownasoneofthemainvectorsofBorrelia
spp.inCentralEuropeandhasquiteoftenbeenfoundontheseanimals[12–14].
InPoland,raccoonsandraccoondogsareconsideredinvasiveandalienspecies[15].
Theymayintroducenewpathogenstotheenvironmentandmayserveaspotentialhosts
formanyinfectiousagentsimportanttodomesticanimalsandhumans.Someofthe
predatorshaveadirectimpactonthehostcompetence,e.g.,redfoxesreduce,byhunting,
populationsofrodents,beingoneofthemostnumerousreservoirsofBorreliaburgdorferi
s.l.[16].
Here,weaimedtoexaminetheimportanceofsixmedium‐sizednativeandinvasive
speciesofcarnivorehostsofBorreliaspp.withoverlappingrangesandtoinvestigatetheir
roleinthespirochaetetransmissioncycle. Wealsoaimedtocheckthereservoir
competenceofthesepredatorsandtostudytheborreliosiseco‐epidemiology.
2.MaterialsandMethods
2.1.SampleCollection
Atotalof586tissuesamples(ear,spleen,liver)derivedfrom284specimensofsix
carnivorespecies,i.e.,raccoon(n=51),raccoondog(n=50),redfox(n=50),European
badger(n=53),pinemarten(n=27)andstonemarten(n=53),wereusedformolecular
study(Table1).TheanimalsweresampledintheRuszówForestDistrict(Poland),inthe
westpartoftheLowerSilesianWilderness,beingthelargestlowlandforestcomplexin
Europeandtheuniquelocationofco‐occurrenceofthenativeandinvasivecarnivore
speciesinEurope.Thetissuesamplesobtainedthroughcollaborationwithotherprojects
werecollectedmainlyduringapredatorcontroloperationcarriedoutasapartofthe
capercaillie(Tetraourogallus)reintroductionprogramintheLowerSilesianForest[17,18].
ThetissuesamplesweredeliveredtothelaboratoryoftheDepartmentofParasitology,
UniversityofWrocław,andstoredat−20°C.
Table1.Tissuesamplesofcarnivorehostsusedinthepresentstudy.
CarnivoreHost
TotalNo.of
TestedSamplesNo.ofSampleTypes
EarBiopsySpleen Liver
Procyonlotor153515151
Nyctereutesprocyonoides150505050
Vulpesvulpes150505050
Melesmeles5353‐‐
Martesmartes2727‐‐
Martesfoina5353‐‐
Total586284151151
Animals2022,12,28293of7
2.2.MolecularAnalyses
DNAwasextractedusingtheBio‐TraceDNAPurificationKit(EURx,Poland),
followingthemanufacturer’sinstructions.DNAconcentrationswereestimatedwitha
NanoDrop2000spectrophotometer(NanodropTechnologies,Wilmington,DE,USA).The
moleculardetectionofBorreliaspp.wasperformedinthreesteps,i.e.,(1)qPCRsonall
obtainedtissuesamplesofinvasive(raccoonandraccoondog)andnativespecies(red
fox);(2)nestedPCRstargetingtheflaBgeneonselectedsamplesofallcarnivorespecies
co‐occurringinthestudyarea;(3)conventionalPCR targetingthe5S‐23SrDNAintergenic
spacerregiononpositivesamplesfortheflaBgene.
Firstly,qPCRwiththeospAgeneasamarkerandprimersandprobessuch as B‐
OspA_modF,B‐OspA_borASandB‐OspAmodPattowasapplied[19,20]. The qPCR was
performed on all tissue samples(ear,liverandspleen)obtainedfromraccoons,raccoondogs
andredfoxes.ThepresenceofBorreliaspp.DNAwasdeterminedbytheamplificationof
a600bpfragmentoftheflaBgeneinnestedPCR,withtwoprimersets:132f,905rand
220f,824r[21].AstheqPCRresultswereconfirmedbynestedPCRs,weperformedonly
nestedPCRsforotherco‐occurringcarnivorespeciessuchasbadgersandpineandstone
martens(mustelids).Inthelaststep,theflaB‐positivesampleswerefurtherexamined
usingconventionalPCRassaysamplifyingpartsofthe5S‐23SrDNAintergenicspacer
region(IGS)withprimersIGS_AandIGS_B[22].Negativecontrolswithnuclease‐free
distilledwateraswellaspositivecontrols(B.afzeliifromIxodesricinus)wereincludedin
eachPCRreaction.InthePCRs,wefollowedtheprotocolsdescribedintheabove‐
mentionedliterature.
AllnestedPCR‐positiveampliconswerepurifiedusingExo‐BAP(EURx)and
sequencedinbothdirectionsbyMacrogen(Amsterdam,theNetherlands)withthe
primersusedforDNAamplification.ThenucleotidesequenceswereeditedusingDNA
BaserSequenceAssemblysoftware(HeracleBioSoft,Mioveni,Romania)andcompared
toeachotherandwithcorrespondingsequencesdepositedinGenBankusingtheBasic
LocalAlignmentSearchTool(BLAST)program(http://blast.ncbi.nlm.nih.gov/Blast.cgi,
accessedon30September2022).TherepresentativesequencesweredepositedinGenBank
underaccessionnumbersOP559180–OP559187.
3.Results
Thesamplesobtainedfromskin(ear),spleenandliverofraccoons,raccoondogsand
foxeswerecheckedforthepresenceofBorreliaDNAusingqPCRandnestedPCR(14vs.
16).Apartfromonecase,allpositivesampleswereobtainedforearisolates(16/151;
10.6%).Theonlyothersample,whichprovedtobepositiveintheqPCR,originatedfrom
raccoonliver.Followingtheresultsobtainedforraccoons,raccoondogsandfoxes,the
nestedPCRswasperformedforco‐occurringmustelidsonly.Theoverallprevalenceof
Borreliaspp.Inalltestedcarnivoreswas8.8%,consideringskinsamples(25/284)(Table2),
andtheinfectionratesvariedsignificantlybetweenthesehostspecies.Almostall
consensussequencesofthepartialflaBgenewhichwereanalyzedwiththeBLASTmethod
revealedsharedidentitywithasequenceofspecificBorreliaspecies,i.e.,B.afzelii(23),B.
garinii(1)andB.burgdorferi(1)(Figure1).
Thehighestprevalence(23.5%)wasobservedforraccoons(12/51)ascomparedto
raccoondogsandredfoxeswithasinglepositivesample(prevalenceof2%each)(Table
2).ThemajorityofpositivesampleswereBorreliaafzelii(100%identitywith,e.g.,
CP018262,DQ016619,KF894068,KX646195),onesamplefromfoxwasidentifiedasB.
garinii(100%homologywithafewsequences,i.e.,KF894068,MF150061).
ThebadgersprovedtobemoresusceptibletoBorreliainfection.Of53testedskin
samples,eightwerepositive(15.1%),whileDNAofBorreliasp.wasfoundintwosamples
obtainedfrompinemarten(27tested,i.e.,7.4%)andonefromstonemarten(53tested,i.e.,
1.9%).TheBLASTanalysisofsevensequencesshowed100%similaritytoseveralBorrelia
afzeliisequences;onewas100%identicaltoaBorreliaburgdorferisequence(acc.no.
Animals2022,12,28294of7
KF836508)fromIxodesricinusfromPoland(LowerSilesia)and99.8%identicaltoother
sequences(KX646200,KR782218)fromI.ricinusfromPolandaswellasonesequence(acc.
no.CP077727)fromBelarus.
TheinterspecificdivergenceofIGSsequencesofBorreliaafzeliiwas0.0–2.3%(0–6bp
outof264bp),andthreegroupscouldbedistinguishedwhenanalyzingthenucleotide
similarity.Thecomparisonbetweentheselinesandthehomologicalsequencesavailable
inGenBankshowed100%to99.2%similaritytosamplesderivedfromhumans(CP018262,
CP002933,JX888444),IxodesticksfromRussia,Estonia,Germany(CP009212,KX418638,
AY772053,MW489224,OL848283)aswellastosequencesobtainedfromrodenttissues
fromFranceandRomania(KY273112,KY123663).
Table2.Borreliaspeciesidentifiedinsamplesobtainedfromwildlivingmesocarnivoresbasedon
flaBgene.
CarnivoreHostNo.ofTested
Specimens
No.ofPositive
EarBiopsy
Samples
Prevalence(%)
(95%ConfidenceInterval)
DetectedBorreliaSpecies
(No.ofSequencedSamples)
Procyonlotor511223.5(15.9–33.1)Ba(12)
Nyctereutesprocyonoides5012.0(0.1–10.7)Ba(1)
Vulpesvulpes5012.0(0.1–10.7)Bg(1)
Melesmeles53815.1(8.8–23.8)Ba(7),Bb(1)
Martesmartes2727.4(1.3–23.7)Ba(2)
Martesfoina5311.9(0.2–7.4)Ba(1)
Total284258.8(6.3–12.2)Ba(23),Bb(1),Bg(1)
Ba—B.afzelii;Bb—B.burgdorferi;Bg—B.garinii.
Figure1.Distribution of Borreliaspecies identifiedinexaminedcarnivores.
4.Discussion
ThemajorityofdataonBorreliaspp.inraccoonsoriginatefromtheUSAandare
basedontheresultsofserologicaltesting[23–26].Additionally,theresultsofstudies
carriedoutbyTuftsetal.[27]showedthepresenceofB.burgdorferis.l.in2.6%ofexamined
samples.Serologicalstudiesconcerningthisspirochaetalinfectioninraccoonsfrom
introducedareas,carriedoutinJapan,revealedthepresenceofB.afzelii(0.1%)andB.
garinii(0.1%)[27,28].Ourresults,withthefirstmolecularevidenceofB.afzeliiinfection,
attaining24.0%intheEuropeanpopulationofintroducedraccoons,indicatethis
carnivoreasapotentialreservoirhostfortheaforementionedspirochaetalspecies.
Animals2022,12,28295of7
MolecularstudiescarriedoutbyWodeckaetal.[10]onEuropeanraccoondogsin
Polandrevealedthat11.9%oftestedanimalswerepositiveforB.garinii(dominant
species),followedbyB.afzeliiandB.valaisiana.Oursurveyindicatedamuchlowerlevel
ofBorreliainfection—2.0%inraccoondogs,beingdetectedasB.afzeliionly.Ontheother
hand,astudycarriedoutinSouthKorea,onnativecarnivores,resultedinthefirstreport
ofBorreliatheileri(0.7%)inraccoondogs[29].
Inthepresentstudy,badgersyieldedahigherprevalenceofBorreliaspp.(15.1%)
comparedtoothertestedspeciesofnativecarnivores,althoughBorreliaafzeliiandB.
burgdorferiweredetectedaswell.ResearchbyWodeckaetal.[10]providedevidencefor
Borreliainfectioninbadgerswithasimilarlevelofprevalence.AnalysesofallPCR‐
positiveblood,earbiopsyandliversamplesrevealedthat12%ofbadgerswereinfected
withborreliae.GernandSell[30]detectedthepresenceofbothB.afzeliiandB.valaisiana
in19.4%oftissuesamplesfromtheearsofbadgersinSwitzerland.ThePCR‐positive
tissuesamplesidentifiedinbadgersrevealedthattheywereinfectedexclusivelywithB.
afzelii.Incontrast,duringresearchconductedinBelgiumandtheNetherlands,Borrelia
burgdorferi(s.l.)wasdetectedin0.9%ofliversamplesofEuropeanbadgers[11].
DNAofB.burgdorferi(s.l.)wasfoundintissuesofothermusteloidspecies,i.e.,stone
andpinemartens.Borreliaburgdorferi(s.l.)wasdetectedin3.9%ofspleensamplesofpine
martensand2.9%ofearbiopsiesofstonemartens[11].Tothebestofourknowledge,this
studyisthefirstmolecularidentificationofB.afzeliiinstonemarten(7.4%)andinpine
marten(1.9%).
Todate,therehavebeenonlyafewreportsfromEuropeontheoccurrenceofB.
burgdorferis.l.intissuesofredfoxesinGermany[31,32],Romania[33],Norway[34]and
Poland[35].ResultsofourstudyshowedthattheprevalenceofBorreliaspp.was2.0%and
B.gariniiwastheonlyspeciesdetectedinredfoxesinthissurvey.Borreliagariniiwasalso
solelynoticedinfoxesinGermany,buttherecordedprevalencewasmuchhigher—24%
[31].InPoland,theanalysisoftissuesof243animalsshowedthat23.5%ofthemcontained
DNAofBorreliaspp.,whereasB.gariniiwasidentifiedin91%oftheinfectedfoxes[35].
Ontheotherhand,onlyB.afzeliiandB.burgdorferiweredetectedintissuesofredfoxesin
Romania[33].
5.Conclusions
Tothebestofourknowledge,thisisoneof thefewandthemostcomprehensive
studiesundertakentoassesstheimportanceofintroducedandnativecarnivoresinthe
ecologyofBorreliaspp.worldwide.Inallexaminedcarnivorespecies,theoccurrenceof
Borreliawasrecorded.WeidentifiedB.afzelii(themostabundantgenospecies),B.garinii
andB.burgdorferiinstudiedanimals.ThehighestlevelofBorreliaprevalenceestimatedin
raccoonssuggeststheycanplayaroleasreservoirhostsforthesespirochaetalbacteria.
Thesignificanceofinvasivespeciesisworthfurtheranalysis,withspecialreferencetothe
circulationofvector‐bornepathogensaswellasinthecontextofthe“dilutioneffect”
hypothesis.
AuthorContributions:Conceptualization,J.H.,A.P‐M.;methodology,J.H.,K.B.‐G.;material
collection,M.P.;molecularstudy,K.J.,K.B‐G.;resultsanalysis;J.H.,K.B.‐G.,A.P‐M.;writing—
originaldraftpreparation,J.H.,K.J.,K.B.‐G.,A.P.‐M.,writing—reviewandediting,J.H.,A.P.‐M.All
authorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:TheresearchwaspartiallyfinancedbytheNationalScienceCentre,Poland,underthe
MINIATURA‐2project(grantno.2018/02/X/NZ6/01983).
InstitutionalReviewBoardStatement:TheapprovaloftheEthicsCommitteewasnotrequired
becausethematerialfortheresearchwasobtainedfromthepredatorcontroloperation.
InformedConsentStatement:Not applicable
DataAvailabilityStatement:Thedatapresentedinthisstudyareavailableonrequestfromthe
correspondingauthor.
Animals2022,12,28296of7
Acknowledgments:Thecarnivores’carcasseswerecollectedduringthepredatorcontroloperation
conductedasapartoftheprogramtoreintroducethecapercaillie(Tetraourogallus)intheLower
SilesianForest,financedbytheEuropeanCommission,theNationalFundforEnvironmental
ProtectionandWaterManagementandthePolishStateForests(grantLIFE11NAT/PL/428).Weare
gratefultoJanuszKobielski,HeadoftheRuszówForestDistrict,andDorotaMertafortheirhelpin
collectingthematerial.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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