Borrelia burgdorferi sensu lato-infected Ixodes ricinus collected from vegetation near the Arctic Circle

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ContentslistsavailableatScienceDirect

Ticks and Tick-borne Diseases

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / t t b d i s

Original article

Borrelia burgdorferi sensu lato-infected Ixodes ricinus collected from vegetation near the Arctic Circle

Dag Hvidsten

^a,∗

, Frode Stordal

^b

, Malin Lager

^c

, Bjørg Rognerud

^b

, Bjørn-Erik Kristiansen

^d

, Andreas Matussek

^c

, Jeremy Gray

^e

, Snorre Stuen

^f

aDepartmentofMicrobiologyandInfectionControl,UniversityHospitalofNorthNorway,Sykehusveien1,9038Tromsø,Norway

bDepartmentofGeosciences,UniversityofOslo,P.O.Box1047Blindern,0316Oslo,Norway

cDepartmentofLaboratoryServices,DivisionofMedicalServices,CountyHospitalRyhov,Sjukhusvägen1,55185Jönköping,Sweden

dResearchGroupofHost–MicrobeInteractions,DepartmentofMedicalBiology,FacultyofHealthSciences,UiT–TheArcticUniversityofNorway,P.O.Box 6050Langnes,9037Tromsø,Norway

eUCDSchoolofBiologyandEnvironmentalScience,UniversityCollegeDublin,Dublin4,Ireland

fNorwegianUniversityofLifeSciences,DepartmentofProductionAnimalClinicalSciences,Kyrkjevegen332/334,4325Sandnes,Norway

a r t i c l e i n f o

Articlehistory:

Received18December2014 Receivedinrevisedform14June2015 Accepted2July2015

Availableonline6July2015

Keywords:

Ixodesricinus

Borreliaburgdorferisensulato Borreliaspp.

Prevalence Tickdensity ArcticCircle

a b s t r a c t

ThisistheﬁrststudytodeterminethedensityofquestingIxodesricinusinnorthernNorway.Itwasper- formedattwositesinBrønnøy,whichhasbeenknownforitstickpermissivehabitatsfordecadesand isoneofthenorthernmosthabitatswithanabundantI.ricinuspopulationintheworld.FromAprilto November2011,allstagesofhost-seekingI.ricinuswerecollectedfromthetwosites.Theoverallpreva- lenceofnymphsinfectedwithBorreliaburgdorferisensulatowas21%andthatofadultticks46%.Therates ofthegenospeciesBorreliaafzelii,Borreliagarinii,andBorreliavalaisianaweresimilartoﬁndingsinmost otherstudiesinScandinavia,withB.afzeliibyfarthemostprevalentat76%.ThehighBorrelia-infection prevalenceinticksfromBrønnøymayexplainthehighincidencerateofreportedLymeborreliosisinthe municipality.

1. Introduction

The common tick Ixodes ricinus, one of the most impor- tantvectorsinEuropetransmittinginfectiousagents tohumans (Estrada-Pe ˜na,2008),harboursBorreliaburgdorferisensulato,the causativeagentofLymeborreliosis.I.ricinusticksareexpanding theirgeographicdistribution(Grayet al.,2009; Hvidstenet al., 2014;JaensonandLindgren,2011;Jenkinsetal.,2012;Joreetal., 2011;Medlocketal.,2013),andtick-borneinfectionsarecurrently agrowingconcerninScandinavia.In thelasttenyears,approx- imately250–350cases(5–7per100,000population)ofsystemic infectionofLymeborreliosishavebeenreportedannuallytothe Norwegiansurveillancereportingsystemforcommunicabledis- eases(MSIS,http://www.msis.no).InnorthernNorway,with10%of theinhabitantsinthecountry,onlyanaverageofﬁvecaseshasbeen reportedannually.InthemunicipalityofBrønnøy(Fig.1),10cases ofLymeborreliosiswerereportedtoMSISfrom2007through2013.

∗Correspondingauthor.

E-mailaddress:[email protected](D.Hvidsten).

TheseBrønnøycasesaccountfor29%ofallreportedcasesinregion ofnorthernNorwayoverthisperiod,althoughthemunicipalityhas only1.6%oftheinhabitantsintheregion.Thesediseasedatasug- gestthatBrønnøyhastick-permissivehabitatsthatdistinguishit fromotherpartsofnorthernNorway.

In the1930s,a studyof ticksfromfarm animalsin Norway consisted of the collection of nymphs and adults of I. ricinus northwardstoBrønnøy(Tambs-Lyche,1943).Mehl(1983)found nymphalandadultI.ricinusticksbyﬂaggingasfarasBrønnøy;

furthernorth,inadditiontoﬂagging,heexaminedhostanimals, burrowsandbirdnests,butwasnotabletodetectticksotherthan IxodestriangulicepsandIxodesuriae.Usingmultipledatasources, Joreetal.(2011)concludedthatI.ricinushasreachedcoastalareas asfarnorthasHarstadatapproximately69^◦N(Fig.1).Inthelast decades,I.ricinushasbeenfoundsporadicallyinallthreecounties ofnorthernNorway(Nilssen,2010).Whethertheseticksrepresent residentpopulationsortransientpopulationsintroducedbymigra- torybirdsorlargemammals,suchasdeer,remainsunknown.Two recentstudiescollectedticksfrompetsandfoundthattheden- sityofticksandtheBorreliainfectionrateintheticksweremuch higherinBrønnøythanfurthernorth(Hvidstenetal.,2014;Jenkins http://dx.doi.org/10.1016/j.ttbdis.2015.07.002

4.0/).

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etal.,2012).However,tickscollectedfromthevegetationaremore relevanttotheriskoftickbiteanddiseaseinhumans.Further- more,analysisofunfedticksforB.burgdorferis.l.mightresultina highersensitivityofdetectioncomparedwithanalysisofengorged ticksbecausethebloodofcatsanddogsmayhavea borreliaci- daleffectwhichwouldhaveanegativeeffectondetection(Hovius etal.,1998;Hvidstenetal.,2014).

ThepresentstudyaimedtodetermineI.ricinusactivityinthe municipalitywiththenorthernmostknownestablishedtickpopu- lationinNorway.Wealsoaimedtodeterminethetickdensityand theprevalenceofB.burgdorferis.l.toestimatetheriskofBorrelia infection.Toourknowledge,repeatedsamplingsofI.ricinusticks withtheﬂaggingmethodhavenotbeenperformedpreviouslyso closetotheArcticCircle.

2. Materialsandmethods 2.1. Studyareas

Brønnøyis a municipality(population 7900 in 2015) in the southernpartofthenorthernNorwayregion(population480,700 in2015)(Fig.1).Itislocatedonthewesterncoastlineandconsists ofbothmainlandandnumerousislands.Host-seekingtickswere collectedattwosites,located4kmnorthwardsand9kmsouth- wards,respectively,ofthecitycentreofBrønnøysund.Thesites werechosenamongseveralotherswheretickattachmentondogs andhumanshadbeenreported.Site1(Mosheim,65^◦30N,12^◦14E, 10mabovesealevel)isaﬂatrecreationalareaonthemainland oftenusedbythelocalpopulation.Thegroundconsistsofgrassand

Fig.1. NorthwesternEurope.Norway:ThemunicipalityofBrønnøyincludesthecityofBrønnøysund(65^◦28N,12^◦12E),whichislocatedapproximately120kmsouthof theArcticCircle(66^◦33N)inthecountyofNordland.TheregionofnorthernNorwayconsistsof(towardsnortheast)thecountiesofNordland(red),Troms(orange)and Finnmark(yellow).Coordinates:Farsund(58^◦05N,06^◦48E);Harstad(68^◦48N,16^◦32E).

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heather;partlyopenbutwithscatteredmixeddeciduoustreessuch asbirch(Betulapubescens),juniper(Juniperuscommunis)andwil- low(Salixspp.).Therearescatteredblueberry(Vacciniummyrtillus) andbogbilberry(Vacciniumuliginosum)bushes;thesoildrainageis goodandthevegetationlitterismoderate.Site2(Torget,65^◦23N, 12^◦05E,25mabovesealevel)issituatedclosetoawell-known touristpathonanislandof16.4km².Thesamplingareaavailable isaforestlocatedonagentleslopeofafewhundredsquaremetres.

Theareaconsistsofheterogeneousdeciduoustreesincludinggrey alder(Alnusincana)androwan(Sorbusaucuparia)inadditionto thespeciesmentionedatsite1.Thegroundiscoveredbyfallen branches,denseblueberryandbilberrybushesandisdampened byaverysmallstream.

Vertebratehostsatthetwositesthatfeedadultticksinclude farmanimals,Eurasianelk(Alcesalces), androedeer(Capreolus capreolus).Inadditiontolargeanimals,vertebratehoststhatfeed immatureticksinclude thecommonshrew(Sorexaraneus), the bankvole(Myodesglareolus)andtheﬁeldvole(Microtusagrestis).

Watervoles(Arvicolaamphibius)arefoundontheislands.

2.2. Climateofthestudyarea

Tocharacterisetheclimatic conditionsin Brønnøy, we used adatasetdevelopedbytheNorwegianMeteorologicalInstitute, basedona time seriesfrom1957.A geostatisticalmethodwas appliedtoobservations froma largenumber of meteorological stationsto create airtemperature and precipitation mapson a 1km×1kmregulargrid(Orskaugetal.,2011;Tveitoetal.,2005);

seefurtherdetailsin Lindholmetal.(2012). AccordingtoKöp- pen’sclimatezonestheclimateinBrønnøyisclassiﬁedassubarctic maritime(designatedasDfb)(Peeletal.,2007).

We have analysed the period 1961–2012. During this period, 2011 was the warmest year, and the spring months (March–April–May)of2011werethesecondwarmest(Fig.2).The yearlyaveragenormaltemperatureofthe30-yearnormalperiod 1961–1990was4.7^◦Catthetwosites,increasingby0.9^◦Cinthefol- lowing20-yearperiod(1991–2010).Themeanannualprecipitation inBrønnøywas1849mm(1981–2010),andthedurationofsnow cover(>5cm)atbothsitesinthedecade2003–2012wasapproxi- matelyindicatedbytherangeof50–100daysyearly(http://www.

senorge.no).Thenumberofdayswithaveragetemperatureabove 5^◦C(averageoftheyears2008–2012)intheareasnorthwardsand southwardsofBrønnøyisdepictedinFig.3,showingthatBrønnøy andnearbyislandshavearelativelywarmclimatecomparedto districtsinlandandfurthernorth.Overtheperiod1981–2010,the lengthofthegrowingseason(definedasstartingwhenthetem- peratureonfiveconsecutivedaysexceeds5^◦C,andendsafterfive consecutivedaysoftemperaturebelow5^◦C)inBrønnøywas179 days,butincreasedto189daysinthedecade2001–2010.Asshown above,2011wasanexceptionallywarmyear,resultingalsoinavery longgrowingseason(241days).

2.3. Tickcollectionandtickdensity

Samplingswereperformedusingawhiteflannelcloth(hereafter called‘flag’)of1.40m×0.70m(approximately1m²).Theleading edgeoftheflagwasnailedbydrawing-pinstoawoodenplank, whichwasattachedtoamopbase.Aleadchainof65gwassewn intothetrailingedgeoftheflag;thechainfunctionedtokeepthe flag’ssurface closetothegroundwhen movingtheflagonthe ground.Thejointofthetelescopichandlemakesiteasytomove itineverydirectioninthevegetation,alsosideways.Thecollec- torsmovedwiththeflagstretchedbeforethemata velocityof 0.5–1m/s,andtheflagwasinspectedonbothsidesevery10m.At site1,theflagwasmovedinatransectlineof140m,whichcovered asurfaceof100m²;foreachsampling,anewdirectionwastaken

2 3 4 5 6 7 8

1960 1970 1980 1990 2000 2010

Temperature (oC)

Years

Yearly average Running 5 year average Normal 1961-1990 Normal 1971-2000 Normal 1981-2010

1 2 3 4 5 6 7

1960 1970 1980 1990 2000 2010

Temperature (oC)

Years

Average March-April-May Running 5 year average Normal 1961-1990 Normal 1971-2000 Normal 1981-2010

Fig.2. Averagetemperatureatsite2(Torget)haschangedfrom1961to2010.The toppanelshowstheannualaveragetemperatureandthebottompanelshowsthe averagespringtemperature(March,AprilandMay).Theredlinerepresentsthe 5-yearrunningaverageandthebluedotsrepresenteachyear.Averageannualand averagespringtemperatureareshownforthree30-yearperiods:1961–1990(green line),1971–2000(purpleline),and1981–2010(orangeline).

fromthesamestartingpoint.Althoughitispreferabletosample ticksperunitarea,thisissometimesimpossible,asatsite2,which wascharacterisedbyfallenbranchesanddensebushes.Therefore, atsite2ticksweresampledfor30-minperiods,inwhichsamp- lingtimeandinspectionswereincluded,bothvaryingaccording tothenumberoftickscollected.Iftheweatherconditionswere suitable(i.e.,airtemperature≥7^◦Cand≤20^◦C;norainorstrong wind),weaimedtosampletickseverysecondweek.Airtempera- tureandrelativehumidityweremeasuredwithTesto610(Testo, Egg,Switzerland)at1mabovegroundlevel.

InJuneandAugust2012,wecollectedticksonsubsequentdays atbothsites.Toobtainsomemeasureofcomparisonforthesites, ticksweresampledfromsquaresof10m×10matbothsites,and onthesamedayswerealsosampledfor30-minperiodsatsite2.

Thecollectedticksweretransferredto2.5mlplastictubeswith 1mlethanol70%,andthereafterstoredat4–8^◦CuntilDNAextrac- tionandBorreliaPCR(1–10larvae,onenymph,oroneadulttickper tube)wereperformed.Theorderinwhichthetubeswerechosen foranalysiswasrandom.Tickspeciesandstagesweredetermined usingastereomicroscope(20–80×)(Arthur,1963;Filippova,1977;

Hillyard,1996).

2.4. DetectionandcharacterisationofBorreliaspp.inI.ricinus Reversetranscribedtotalnucleicacidwaspreparedfromticks asdescribedpreviously(Jenkinsetal.,2012).Reversetranscrip- tionincreasesthesensitivityoftheLUX16SrRNAreal-timePCR test usedfor genuslevel detectionof Borrelia spp. 10–100-fold (Wilhelmsson etal.,2010)resultingin asensitivityof lessthan onecellperPCRreaction(Wilhelmssonetal.,2013).Tickscontain- ingBorreliaspp.werefurthercharacterisedtodetermineBorrelia genospeciesbynested PCRampliﬁcationand sequencingofthe

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Fig.3. ColoursketchmapofthesurroundingregionofBrønnøy.Thecolourcolumn(150–200days)displaysaveragenumberofdaysabove5^◦C(averageoftheyears 2008–2012).

5S–23Sas wellas16S–23Sintergenic spacerregions, following Posticetal.(1994)andBunikisetal.(2004).Sequencingwasper- formedbyGATCBiotech(GATCBiotechAG,Konstanz,Germany).

ChromatogramswereanalysedusingtheRipSeqwebapplication (iSentio,Bergen,Norway),whichallowsspeciesdeterminationin samplescontaininguptothreespeciessimultaneously(Kommedal etal.,2009).IntickswherecharacterisationtoBorreliagenospecies levelfailed,asecondreal-timePCRtesttargetingﬂaBwasdoneto conﬁrmBorreliaDNApresence(Jenkinsetal.,2012).

2.5. Statisticalanalysis

Thedensityoftickswasdeterminedasthenumberoftickscol- lectedbyonepersonper100m²orper30min.Peaktickdensity inthe2011seasonwasdeterminedasthesite’shighestnumberof ticksbyonepersonper100m²orper30min.ThedifferenceinB.

burgdorferis.l.prevalencefornymphalandadultticks,separately atsite1andsite2,werecalculatedwithFisher’sexacttest(http://

graphpad.com).Wealsodidthesamecalculationswithnymphs atsite1vs.site2,andwithadultsatsite1vs.site2.Statistical signiﬁcancewassetatP<0.05.

3. Results

3.1. Numbersofticks

In2011,582I.ricinusticks(116larvae,319nymphsand147 adults)werecollectedfrom19Aprilto12November(207days).

Noothertickspecieswasfound.Atsite1,atotalof139tickswere collectedonsevenvisits(from27Juneto14October)outof10.

Atsite 2,443tickswerecollectedonall10samplingoccasions.

Themediannumberofdaysbetweensamplingsatsite1and2was 23and21,respectively(atbothsites,range13–35).Peaknymphal densityatsite1was10nymphs/100m²on20June,andatsite2,

itwas53nymphs/30minon6July.Smallnumbersoflarvaewere foundatbothsites,andalthoughtherewereinsufﬁcientdatato reliablyassessseasonality,itwasevidentthatthepeaks,suchas theywere,occurredinJuneandAugust/September.

In June 2012, tick numbers (nymphs/adults) collected from 10m×10msquaresatsite1and2were3/3and25/26,respectively, and in August 2012, 8/0 and 30/15, respectively. The time-basedmethod(30-minperiods)wasusedatsite2onthesame days,andticknumbers(nymphs/adults)were41/17inJuneand 17/33inAugust.

3.2. TheprevalenceanddiversityofBorreliaburgdorferisensu lato

Since,asexpected,Borreliawasnotdetectedinanyofthelarvae;

thefollowingcalculationsapplytonymphsandadultticks(Table1).

Intotal,135(29%)ofthetickscontainedBorreliaspp.asdetected byreal-timePCR;site1,40%(32/81) andsite 2,27%(103/385).

Genospeciesweredeterminedin97%(131/135)oftheticks.Bor- reliaafzeliiwasdetectedin76%(102/135),Borreliagariniiin18%

(24/135)andBorreliavalaisianain4%(5/135)(Table1).Therewasa significantdifferenceinBorreliaprevalencebetweenallticksatsite 1(40%,32/81)andallticksatsite2(27%,103/385)(P=0.03).Thedif- ferenceintheBorreliaprevalencebetweennymphs(21%,67/319) andadults(46%,68/147)atbothsitesandatsite2(20%,55/278, and45%,48/107, respectively),wassignificant(P<0.001).There werenoothersignificantdifferencesinBorrelia-infectionpreva- lence(nymphsoradultsatsite1vs.site2;nymphsvs.adultsatsite 1).

4. Discussion

Thisstudycomplementsanearlier,moreextensiveoneinnorth- ernNorwayinwhichticks(almostalladults)werecollectedfrom

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Table1

BorreliagenospeciesinnymphalandadultIxodesricinus;sampledfromtwositesinBrønnøy,northernNorway.

Site^a Stage^b No.ticks Borrelia positive,total

B.afzelii B.garinii B.valaisiana Borreliasp.,ND^c

n n % n %ofBorrelia

positive

n %ofBorrelia positive

1 N 41 12 29 12 100 0 0 0 0 0 0

2 N 278 55 20 49 89 3 5 2 4 1 2

1+2 N 319 67 21 61 91 3 4 2 3 1 1

1 A 40 20 50 14 70 5 25 0 0 1 5

2 A 107 48 45 27 56 16 33 3 6 2 4

1+2 A 147 68 46 41 60 21 31 3 4 3 4

1+2 A+N 466 135 29% 102 76% 24 18% 5 4% 4 3%

aTheBorreliaprevalenceinallticksatsite1(40%)wassigniﬁcantlydifferentfromallticksatsite2(27%)(P=0.03).

b N,nymphs;A,adultticks;Borreliaspecieswasnotfoundinlarvae(n=116).

c ND;Borreliagenospeciesnotidentiﬁed.

pets,mainlydogs.In thatstudy,I.ricinusappeared tobeespe- cially prevalentin Brønnøyand its adjacentmunicipalities and wereinfectedwithatleastthreeB.burgdorferis.l.genospecies.The presentstudyfocusedentirelyonBrønnøyand wasdesignedto providedataontickactivityattwodifferentsitesinthemunicipal- ity,andalsoontherisktopublichealthexpressedastherelative abundanceofunfedtickscollectedfromthevegetationandthe frequencyoftheirinfectionwithB.burgdorferis.l.Collectingticks directlyfromthevegetationbyﬂagging,ratherthanfromhosts, permitsa more direct measure locally of humantick bite risk, especiallysince,incontrasttocollectionsofadultticksfromdogs andcats,nymphsarethemostcommonstageobtainedbyﬂag- gingandareresponsibleforthevastmajorityofhumantickbite (Maiwald et al.,1998; Robertson et al., 2000). Asin theprevi- ousstudy(Hvidstenetal.,2014),B.afzeliiwasthemostcommon genospecies;theinfectionrateswithB.burgdorferis.l.werehigher thanreported inother studiesin Norway (Jenkins etal., 2001;

Paulauskasetal.,2008;SolengandKjelland,2013)exceptforFar- sund(58^◦05N,06^◦48E)inthesouthernmostpartofNorwaywhere theprevalencewas31.3%in2007(Kjellandetal.,2010).

Inthepresentstudy,itwasnecessarytocollectticksusingtwo differentmethods,area-basedandtime-based.Whileitispossible, ingeneral,toestablisha relationshipbetweenthetwomethods (Grayetal.,1998),butthisisinﬂuencedbytickdensityandvegeta- tionstructure,anditshouldbenotedthatthetime-basedapproach isusuallymorevariablethanthearea-basedmethod.Thesimilar area-basedsampling(i.e.,10m×10m)atthetwositesresultedin 5–6timesasmanyticksbeingcapturedatsite2thanatsite1.The reasonsforthehigherticknumbersatsite2arenotclear,because theclimateissimilarandlargehostssuchasroedeerandEurasian elkarepresentatbothsites.However,moretreesandthedenser undergrowthatsite2provideamorefavourablemicro-climatefor theticks.Thedatafrombothsitesprovidesampleevidenceofa signiﬁcantriskoftickbite,withnumberssampledreachinglev- elsmatchingthoserecordedfurthersouth(Jenkinsetal.,2001;

Kjelland,2011;LindströmandJaenson,2003).

Wefoundthattickquestingcontinuedforlongeratsite2(207 days)thanat site 1.In general,thequesting activitymayhave beenunusuallylonginthe2011season,correlatingwiththelong growingseasonthatyear.Tickactivityisknowntobepositivelycor- relatedwithhightemperaturesinthespringmonths(Perretetal., 2000),andin2011theaveragetemperatureinMarch–May(5.5^◦C) wasthesecondhighestintheperiod1961–2012,whichmayhave beeninﬂuencedthetickquestingbehaviour(Fig.2).Temperatureis oneofthemostimportantcuesfortickactivation(Belozerov,1982;

Gray,1991),buttheexacttemperaturethresholdforquestingtick activityisunknowninmanyplaces.Arecentstudycomparingthe questingactivityofI.ricinuspopulationsfromdifferentlatitudesin

UnitedKingdomandFrance,foundthatatlowertemperatures,ticks ofnorthernpopulationsshowed20%–30%morequestingactivity thanthoseofsouthernpopulations(Gilbertetal.,2014).InBrønnøy, themeanannualtemperatureislow(4.7^◦C)andthereforethelocal tickpopulationmaybeadaptedtoquestatlowtemperatures.

Although sampling was not frequent enough to accurately assess seasonalactivity, thepresence and peaks of thevarious tickstages(datanotshown)seemedtoaccordwellwiththefind- ingsofmostotherstudiesonI.ricinusinthatnymphsandadults appearinspringandareactiveuntillateautumn,withadecline inactivityinlateJulyandAugustbeforeaslightriseinSeptember, whilethelarvaetendtopeakinearlysummerandearlyautumn (DobsonandRandolph,2011;Gray,1991).However,moredetailed studieson seasonalactivity arerequired, particularlysince the influence of photoperiod which is known to regulate seasonal activity(Belozerov,1982),maybeparticularlyimportantatthis northernlatitude.IntheArcticCircle,thedaylengthof24hdaylight atsummersolsticechangestopolarnightatthewintersolstice.In thesunlitpartoftheyear,thenearlyconstantchangeindaylength inBrønnøyisapproximately7minperday(increasingbeforeand decreasingafterthesummersolstice),whichisconsiderablylonger thanthepeakvalueofdaylengthchangeseenat56^◦N(e.g.,Edin- burgh;Moscow)and49^◦N(e.g.,Paris;Volgograd),whichis4.8and 3.7min,respectively.Itisinterestingtonotethatprobablynoother tickpopulationisinfluencedbyastrongerphotoperiodsignalthan theticksclosetotheArcticCircle,butasyetnostudiesondiapause inthisparticulartickpopulationhavebeenundertaken.

Borreliaspp.weredetectedin40%and27%oftheticks(nymphs andadults)fromsite1and2,respectively.Theoverallproportionof nymphscontainingBorreliaspp.was21%andthatofadultticks46%.

Inapreviousstudyfrom2010to2011,whentickswerecollected frompets,asimilarBorrelia-infectionprevalenceof42%wasfound intheunfedadultticksfromBrønnøyandadjacentmunicipalities (Hvidstenetal.,2014).

InapreviousstudyfromBrønnøy(SolengandKjelland,2013), theBorrelia-infectionprevalencewas11%and33%innymphsand adults,respectively.TheproportionofBorrelia-infectedtickscar- ryingB.afzeliiwas95%,whichishigherthaninthepresentstudy (76%).InthepreviousstudyfromnorthernNorway(Hvidstenetal., 2014),mostofthetickswereadultscollectedfrompets,andthe prevalenceofB.afzeliiinnymphsandadultswas65%.Theeven higherprevalenceofB.afzeliiinnymphsaloneinthepresentstudy (91%)indicatesthattheyweremainlyinfectedbyrodentsbecause B.afzeliiisknowntohaveapredilectionforthesehosts(Hanincova etal.,2003).

In conclusion, this studyrecorded thepresence of allactive stagesofI.ricinusattwodifferentsitesinBrønnøy.Thequesting activityperiodlastedsevenmonths(19April–12November),and

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thepeakdensityof53nymphsper30-minsamplingperiodwas highconsideringthatthestudysiteisonly130km southofthe ArcticCircle.ThehighBorrelia-infectionprevalenceinticksfrom recreationalareasmayexplainthehighincidenceofreportedLyme borreliosisinthemunicipalitycomparedwithsurroundingareas, andthediscernibleriskofdiseasethatthisstudyreports,should alertthelocalhealthauthorities. Furtherstudiesare neededon therisk ofdiseasetransmissionand alsoonseasonaltick activity,includingthe roleof photoperiod,in thenorthernmosttick populations.

Conﬂictofinterest

Allauthorsstatenoconﬂictofinterest.

Acknowledgements

AspecialthankyoutoHenrietteHanssenandHalvorAsperwho introducedustotick-infestedareasinBrønnøyandthencollected theticksduringtheseason2011.WealsoacknowledgeReidarMehl forprovidingvaluableinformationfromhisearliersamplingsat Brønnøy,andEivindGrøttingWærstedforassistancewiththetem- peraturedata.WethankOlafDienusfortheﬁnallabwork.Wealso thankSteinarPedersenforalwaysbeingourvaluableliaisontothe municipalityofBrønnøy.

ThisworkwassupportedbygrantSFP912-10fromtheNorth- ern Norway Regional Health Authority (Helse Nord RHF) and grantSLS-100221fromtheScandinavianSocietyofAntimicrobial Chemotherapy.(ThenewdesignationforthisorganizationisNordic SocietyofClinicalMicrobiologyandInfectiousDiseases[NSCMID].)

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