Serological evidence of tick-borne encephalitus virus infection in moose and deer in Finland: sentinels for virus circulation

RESEARCH Open Access Se ological e idence o ick-bo ne encephali is i us in ec ion in moose and dee in Finland: sen inels o i us ci cula ion Elina Ton e i 1* , Pikka Jokelainen 2,3,4 , Juho Ma ala 5 , Jy ki Pusenius 5 and Olli Vapalah i 1,2,6 Abs ac Backg ound: The incidence o ick-bo ne encephali is (TBE) in humans has inc eased in Finland, and he disease has eme ged in new oci. These oci ha e been in es iga ed o de e mine he ci cula ing i us sub ype, he ick hos species and he ecological pa ame e s, bu coun ywide epidemiological in o ma ion on he dis ibu ion o TBEV has been limi ed. Me hods: In his s udy, we sc eened se a om hun e -ha es ed wild ce ids o he p esence o an ibodies agains ick-bo ne encephali is i us (TBEV) wi h a hemagglu ina ion inhibi ion es . The posi i e esul s we e con i med by a neu alisa ion assay. Resul s: Nine (0.74 %) o 1213 moose, one (0.74 %) o 135 whi e- ailed dee , and none o he 17 oe dee we e ound se oposi i e o TBEV. A close geog aphical cong uence be ween se oposi i e ce ids and ecen ly epo ed human TBE cases was obse ed: nine o he en se oposi i e animals we e om known endemic a eas. Conclusions: Ou esul s con i m he local ci cula ion o TBEV in se e al known endemic a eas. One se oposi i e moose had been sho in an a ea whe e human TBE cases ha e no been epo ed, sugges ing a possible new ocus. Moose appea o be a use ul sen inel animal o he p esence o TBEV in he aiga egion. Keywo ds: Moose, Roe dee , Se ology, Tick-bo ne encephali is, Whi e- ailed dee , Zoonosis Backg ound Tick-bo ne encephali is i us (TBEV) is a zoono ic a bo- i us. In 1991–2010, i caused up o 12,733 annual e- po ed human cases o ick-bo ne encephali is (TBE) in Eu ope [1]. The e a e h ee known gene ic sub ypes o he i us: Eu opean, Sibe ian and Fa -Eas e n [2]. The sub ypes di e in hei endemic egions and ecological p e e ences, and possibly in hei pa hogenici y [3]. The i us is main ained in an enzoo ic cycle o icks, which a e ec o s and hos s o he i us, and hei e - eb a e hos s. Non- i emic ansmission (NVT) o TBEV be ween icks co- eeding on small e eb a es is consid- e ed impo an o he main enance o he i us [4]. Due o he complex ecology o TBEV, i s geog aphical dis i- bu ion is mul i ocal [5]. La ge mammals a e seconda y hos s o TBEV as hey suppo he ick popula ions by p o iding blood meals. The NVT compe ence o la ge mammals is con o e sial and la gely unknown: goa s a e no NVT-compe en , whe eas he NVT po en ial o oe dee (Cap eolus cap eolus), which can be hea ily in es ed by co- eeding la ae and nymphs, has no been s udied [6–10]. Ne e - heless, oe dee and ed dee (Ce us elaphus) appea o be key hos s o icks and he eby impo an o TBEV main enance [8–10]. In Sweden, he inc ease in TBE incidence has been associa ed wi h changes in ce - id popula ions, especially oe dee popula ions [11]. TBE is usually subclinical in middle-sized and la ge mammals [12]. Howe e , in Sweden, a moose (Alces alces) cal wi h se e e beha io al abno mali ies and * Co espondence: [email p o ec ed] 1 Depa men o Vi ology, Uni e si y o Helsinki, Facul y o Medicine, Helsinki, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2016 Ton e i e al. Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Ton e i e al. Pa asi es & Vec o s (2016) 9:54 DOI 10.1186/s13071-016-1335-6 subsequen ly diagnosed encephali is was ound o be TBEV-posi i e [13]. Finland is a he no he n bounda y o he cu en dis- ibu ion a ea o TBEV [14]. Two sub ypes o he i us, Eu opean and Sibe ian, as well as he wo main hos ick species, Ixodes icinus and I.pe sulca us, ha e been de- sc ibed [15].Be ween 2007 and 2014, a o al o 273 hu- man TBE cases we e epo ed in Finland [16, 17]. The cases occu ed mainly in known endemic a eas, whe e human TBE cases ha e been diagnosed o decades, and whe e TBEV an ibodies we e de ec ed in ca le al eady in he 1960s [16, 18]. The incidence o human TBE has inc eased, and he disease has eme ged in new oci [16]. Bank ole (Myodes gla eolus) appea s o be he domin- an small mammal in he s udied TBEV oci in Finland in he bo eal aiga egion [14, 19]. La ge and middle- sized mammalian species suppo ing he local main en- ance o TBEV ha e no been in es iga ed. The e a e h ee wild ce id species wi h subs an ial pop- ula ions in Finland. Since he1970's, he moose popula ion has comp ised 60,000 o 200,000 indi iduals ha a e e enly dis ibu ed ac oss he coun y [20, 21]. The whi e- ailed dee (Odocoileus i ginianus) popula ion has 40,000 o 50,000 indi iduals, ha inhabi mainly sou h-wes e n Finland [22]. The oe dee popula ion o 10,000 o 20,000 indi iduals is dis ibu ed spa sely ac oss he coun y, wi h he highes numbe s in he sou h-wes [23]. In his s udy, we sc eened se a o hese h ee wild ce - id species o TBEV-speci ic an ibodies o es ima e he se op e alence and o de e mine he geog aphical dis i- bu ion o TBEV in Finland. We discuss he sui abili y o moose as a sen inel animal o he p esence o TBEV and as an indica o o local isk o human TBE in ec ions. Me hods E hics s a emen No animals we e killed o he pu pose o his s udy. The animals we e sampled pos mo em, a e hey had been legally killed by hun e s. The samples we e s o ed, coded, and all da a we e ea ed con iden ially. S udy design and sampling The se um bank was collec ed du ing he hun ing sea- son o 2008–2009, p ima ily o a na ionwide c oss- sec ional se ological s udy on Toxoplasma gondii [24]. The sample is a con enience sample, and he sampling was no a ge ed o TBEV oci. Al oge he 2917 sam- pling packages we e dis ibu ed o he game manage- men dis ic s, which subsequen ly dis ibu ed hem o he hun e s. Each package included wo plas ic VACU- ETTE® Se um Clo Ac i a o blood sample ubes (G eine Bio-One GmbH, K emsmüns e , Aus ia) and a ques ionnai e co e ing he game managemen dis ic , and species, sex and age g oup o he animal sampled. All he animals included in he s udy we e legally hun ed o human consump ion. The olun a ily pa ici- pa ing hun e s collec ed he samples om he animals and illed ou he ques ionnai e o each animal. The pa icipa ion a e was 47 %. Samples we e sen by mail and a i ed a he labo a o y wi hin 4 days o sampling, be ween Sep embe 15 h 2008 and Feb ua y 2nd 2009. Upon a i al, he samples we e coded, and se a we e sepa a ed by cen i uga ion, di ided in o aliquo s and s o ed a - 20 °C un il analyzed. Only he code numbe o each sample was known by he pe sons pe o ming he es s. A o al o 1371 ce id samples we e included in his s udy. The samples om 1213 moose o igina ed om all 15 game managemen dis ic s. The dee samples, om 135 whi e- ailed dee and 17 oe dee , had been col- lec ed only om he sou h-wes e n dis ic s whe e hese ce ids a e he mos nume ous. The species o six sam- ples was unspeci ied. Se ological me hods Fo he se ological analysis o he p esence o an i- TBEV an ibodies, we used an in-house hemagglu ina ion inhibi ion (HI) es [25] using he ollowing wo- old di- lu ions: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640. Ti e s we e de e mined acco ding o hese dilu ions, howe e , in cases whe e he las dilu ion showed pa ial inhibi ion o hemagglu ina ion, he i e was de e mined as he alue be ween he las clea dilu ion and he bo de line dilu ion and is p esen ed in Table 1 by showing bo h he lowe and highe dilu ions. Posi i e esul s we e u he con i med by a apid ocus- o ming inhibi ion es o neu alizing an ibodies using Swedish Eu opean sub ype s ain 93–783 [25]. The es was pe o med in dilu ions 1:5 and 1:20. The samples ha es ed posi i e in he HI es and posi i e o bo de line in he neu alisa ion es we e de ined as se oposi i e. In addi ion, he samples we e sc eened wi h in-house HI es s o he p esence o an ibodies agains wo o he la i i uses ha could cause c oss- eac i e an ibody esponses: Wes Nile i us (WNV), which has no been ound in Finland, and Lam- mi i us (LAMV) which has been ound in mosqui oes in Finland [26]. S a is ical analyses Two-by- wo ables and es s a is ics we e used o p e- limina y compa isons [27]. Logis ical eg ession analyses wi h S a a 11.0 (S a aCo p, College S a ion, Texas, US) we e used o e alua e e ec s o a ailable explana o y a iables (game managemen dis ic , sex, species, age g oup) on he ou come a iable, TBEV-se oposi i i y. Ton e i e al. Pa asi es & Vec o s (2016) 9:54 Page 2 o 8 Resul s Al oge he 28 samples es ed posi i e o an i-TBEV an ibodies wi h he HI es (Table 1), and i e s anged om 15 o 640. These se a we e u he subjec o con- i ma o y analysis by he TBEV neu alisa ion es . Ten samples showed posi i e (<10/20) o bo de line esul wi h dilu ions 1:5 (NT 5) o 1:20 (NT 20) and we e hus de ined as se oposi i e (Table 1). The samples showing TBEV-speci ic an ibodies by he neu alisa ion es had highe HI- i e s han hose samples ha es ed posi i e only on he HI es . The se oposi i e animals consis ed o i e adul male moose, wo adul emale moose, wo young male moose, and one adul male whi e- ailed dee . The en se oposi i e animals we e om six di e en game managemen dis ic s, and hei geog aphical dis- ibu ion appea ed o co ela e closely wi h he known human in ec ion si es: nine se oposi i e animals o igi- na ed om a eas whe e human cases ha e been de- sc ibed (Fig. 1). The se op e alences in moose and Table 1 Basic da a and se ological esul s o he wild ce ids hun ed in 2008–2009 in Finland ha es ed se oposi i e in he sc eening o an ibodies agains ick-bo ne encephali is i us by hemagglu ina ion inhibi ion es Indi idual Species Age Sex Dis ic a HI Neu alisa ion HI HI TBEV TBEV WNV LAMV NT 5 NT 20 NT esul 1. moose adul M 8 640 20/20 20/20 neg neg 20–40 2. moose adul M 1 160–320 0/20 6/20 pos <10 neg 3. moose cal M 8 160–320 9/20 17/20 pos neg neg 4. moose adul M 5 80–160 0/20 20/20 pos neg neg 5. moose nd. F 8 80 4/20 15/20 pos neg neg 6. moose adul M 1 80 0/20 10/20 pos neg neg 7. moose adul M 13 80 20/20 20/20 neg neg neg 8. moose adul M 3 80 2/20 17/20 pos neg neg 9. moose cal M 14 40–80 10/20 20/20 pos/neg neg neg 10. moose adul M 7 40 0/20 13/20 pos neg 20–40 11. moose cal F 13 40 20/20 20/20 neg neg neg 12. moose adul M 14 <40 20/20 20/20 neg neg neg 13. moose adul F 10 20–40 20/20 20/20 neg neg neg 14. moose cal M 7 20 20/20 20/20 neg neg neg 15. dee b adul M 1 20 12/20 20/20 pos/neg neg neg 16. moose cal M 4 20 20/20 20/20 neg neg neg 17. moose cal F 3 20 16/20 20/20 neg neg 10 18. moose adul F 12 20 18/20 20/20 neg neg neg 19. moose adul M 5 <20 20/20 20/20 neg neg neg 20. moose adul F 1 10–20 20/20 20/20 neg neg neg 21. moose adul M 8 10–20 14/20 20/20 pos/neg 10 10–20 22. moose adul M 1 10–20 20/20 20/20 neg neg neg 23. moose adul M 7 10–20 17/20 20/20 neg neg neg 24. moose cal M 3 10–20 20/20 20/20 neg neg neg 25. moose adul F 3 10–20 20/20 20/20 neg neg neg 26. moose adul M 1 10–20 20/20 20/20 neg neg neg 27. moose adul F 8 10–20 16/20 20/20 neg neg neg a Dis ic s p esen ed in Fig. 1. The game adminis a i e dis ic s a e desc ibed acco ding o The Finnish Wildli e Agency b Whi e- ailed dee NT 5 neu alisa ion es , dilu ion 1:5 NT 20, neu alisa ion es , dilu ion 1:20 HI hemagglu ina ion inhibi ion es TBEV ick-bo ne encephali is i us WNV Wes -Nile i us LAMV Lammi i us Ton e i e al. Pa asi es & Vec o s (2016) 9:54 Page 3 o 8 whi e- ailed dee we e 0.74 %, and he se op e alences did no di e signi ican ly be ween he species, sexes and age g oups (Table 2). Logis ic eg ession analyses e- ealed no mul i a iable associa ion. None o he samples es ed posi i e o WNV. Th ee o ou samples ha we e weakly posi i e o LAMV, showed lowe i e s o his i us han o TBEV in he hemagglu ina ion es (Table 1). One o he LAMV- posi i e samples showed a a he high hemagglu ina ion i e , bu his sample was s ill also posi i e o TBEV ac- co ding o he TBEV neu alisa ion es (Table 1). Discussion P esence o an i-TBEV-an ibodies in moose has been p e iously in es iga ed appa en ly only in Sweden in 1962 and mo e ecen ly in No way [13, 28]. In he 15 13 7 9 14 5 3 12 11 6 10 2 4 1 8 30°E21°E 68°N 65°N 62°N Ce id samples: Pos_Hl + neu Neg HI high, neu neg. HI low, neu neg. Human pos 2007-08 Human pos 2009-13 62 neg. A c ic Ci cle Fig. 1 Geog aphical dis ibu ion o he hun ing loca ions o he es ed wild ce ids, he animals de ined as se oposi i e o an ibodies agains ick-bo ne encephali is i us, and he cases o human ick-bo ne encephali is in Finland. Da a on hun ing loca ion we e a ailable o 79.2 % o he animals. The numbe s 1–15 e e o he game managemen dis ic s Ton e i e al. Pa asi es & Vec o s (2016) 9:54 Page 4 o 8 Swedish s udy, he se op e alence among 75 indi iduals was 44 %, which is almos six y imes highe han he Finnish es ima e ob ained in his s udy. The neu alisa- ion es was used in bo h s udies, bu he esul s a e no compa able because mos o he animals included in he Swedish s udy had been sho in a known endemic a ea, whe eas his s udy co e ed he whole o Finland excep o he highly endemic Åland islands. In he No wegian s udy, which used a comme cial ELISA me hod, he p e alence was 52 %, bu he sample size (N= 27) was small and he geog aphical ange limi ed. In his s udy, we used an in-house HI es as he p i- ma y sc eening es . Because la i i uses cause c oss- eac ions in se ological analyses, he posi i e esul s we e con i med by a labou -in ensi e, bu mo e speci ic neu- alisa ion es . To exclude some known c oss- eac i e la i i uses, we es ed he TBEV-posi i e samples (in he HI es ) also o Wes Nile i us (WNV) and Lammi- i us (LAMV). Low i e s o LAMV-speci ic an ibodies we e de ec ed in ou animals in Sou he n and Cen al Finland, while he e was no indica ion o WNV ha ing been in oduced in he ce id popula ions. Finding se ological e idence o TBEV in ec ion in ce ids indica es p e ious TBEV in ec ion. A sys emic in ec ion is c i ical o he de elopmen o he speci ic an ibody esponse. Non- i emic ansmission (NVT) in oden s be ween co- eeding icks is conside ed impo an o he main enance o he i us and i does no e- qui e sys emic in ec ion [29, 30]. Acco ding o ou unpublished obse a ions and he ea lie esul s by S edmy e al. [13], moose can be hea ily in es ed by Ixodes icks. The ole o moose as seconda y hos s o TBEV in he aiga egion and hei po en ial o NVT du ing ick co- eeding ha e no been in es iga ed, bu hey a e likely o ha e a ole in ampli ying he ick popula ions and po en ially in in oducing in ec ed icks o new a eas. In Cen al and Sou he n Eu ope as well as in la ge pa s o Sweden, he oe dee is a key hos o icks, and a good indica o o he occu ence o human TBEV in ec ions [8, 10, 11, 31–34]. The a ailable es i- ma es o TBEV se op e alence in oe dee and ed dee om o he Eu opean coun ies ange om 2.4 o 40 %, wi h ma ked local a ia ion [31, 32, 35–38]. Due o he low numbe o oe dee samples in ou s udy, he ole o oe dee in he sp ead o ci cula ion o TBEV in Finland could no be e alua ed. In Finland, moose is he mos abundan and mos widesp ead wild ce id, while he la ges popula ions and he e ec i e dispe sal o oe dee and whi e- ailed dee a e mainly limi ed o sou h-wes Finland due o ha sh win e condi ions and hick snow co e in o he pa s o he coun y. Howe e , small popula ions o oe dee exis in Lapland in sho e and i e bank a eas ha Table 2 P e alence o an ibodies agains ick-bo ne encephali is i us in wild ce ids hun ed in 2008–2009 in Finland n a n posi i e Se op e alence (%) 95 % con idence in e al (Mid-P Exac ) Moose Male 672 7 1.04 0.46–2.05 Female 512 2 0.39 0.07–1.29 Adul 729 7 0.96 0.42–1.89 Cal 453 2 0.44 0.07–1.45 All 1213 9 0.74 0.36–1.36 Whi e- ailed dee Male 77 1 1.30 0.07–6.24 Female 57 0 0.00 0.00–5.12 Adul 71 1 1.41 0.07–6.75 Cal 64 0 0.00 0.00–4.57 All 135 1 0.74 0.04–3.60 Roe dee Male 11 0 0.00 0.00–23.84 Female 6 0 0.00 0.00–39.30 Adul 14 0 0.00 0.00–19.26 Cal 3 0 0.00 0.00–63.16 All 17 0 0.00 0.00–16.16 All 1371 10 0.73 0.37–1.30 a Backg ound in o ma ion was una ailable o some o he animals in es iga ed Ton e i e al. Pa asi es & Vec o s (2016) 9:54 Page 5 o 8 ha e less snow [39, 40]. In Sweden, he geog aphic ex- pansion o I. icinus has ollowed he inc ease and dis- pe sal o oe dee [11]. In Finland, he dispe sal o whi e- ailed dee is no as e ec i e and widesp ead as ha o oe dee [41]. In e es ingly, acco ding o unpub- lished da a o Finnish game au ho i ies and he incidence and geog aphical dis ibu ion o human TBE [16], he eme gence o new human cases seemed o coincide wi h he sp ead and inc ease o oe dee and whi e- ailed dee popula ions a he u n o he millenium in Finland. On he o he hand, he same a eas ha e had la ge moose popula ions and he e a e se e al o he species ha may also suppo he ick popula ions [42–44]. Mos o he s udies a ailable ha e ocused on single seconda y hos species. Sampling o all ce id species in known endemic a eas migh elucida e hei oles in he sp ead and ci cu- la ion o TBEV. Collec ing samples om ce ids in co-ope a ion wi h hun e s p o ed success ul and use ul o e alua ing he epidemiology o zoono ic diseases ([24] and his s udy). To analyse he ole o moose o he local epidemiology and o he moni o ing o TBEV locally and coun y- wide, epea ed hun e -ha es ed sampling om ce ids could be used alongside he con inuous moni o ing o human TBE. Good sample sizes a e achie able, and sampling animals ha we e killed o ano he eason is e hically sound. The e we e mo e males han emales among he sampled animals, and male ce ids domina e also in he annual hun ing s a is ics in Finland [21, 45]. Eigh o he en se oposi i e animals we e male. Male moose and young indi iduals mig a e ac oss a wide ange and migh he e o e ha e mo e possibili ies o encoun e an in ec ed ick and also o ca y icks o new loca ions [46]. In oe dee , highe TBEV-an ibody p e alence has been seen in males e en a e he mo- bili y is excluded [32]. In his s udy, he posi i e e- sul s we e de ec ed mainly in a eas, which a e known o be endemic o TBEV. Among he animals included in his s udy, 37.9 % we e cal es (less han 1 yea old), which is a smalle p opo - ion han in he game s a is ics o Finland [21]. All ani- mals included in he s udy had expe ienced a leas one ick- eeding season, bu he exac ages o he adul ani- mals we e no de e mined. Assuming ha he an ibodies pe sis , i canno be es ima ed when he in ec ions we e acqui ed. Compa ing he an ibody p e alence in old in- di iduals wi h he p e alence in young indi iduals could be used o de e mine whe he he incidence o in ec ions is inc easing in he a ea. In his s udy, he p e alences we e simila , sugges ing ecen in oduc ion, o inc eas- ing in ec ion p essu e. The only p e ious coun ywide se ological su ey on he dis ibu ion and p e alence o TBEV in ec ion in Finland was done in 1960s by sc eening TBEV an i- bodies in ca le se um samples [18]. The se oposi i e ca le we e om a eas whe e human cases ha e been di- agnosed o decades. In his s udy, se e al se oposi i e wild ce ids we e shown o inhabi he same a eas. Du ing he wo p e ious ick- eeding seasons be o e he collec ing o he ce id samples, human cases had been epo ed in eigh a eas in Finland (Fig. 1). In his s udy, se oposi i e ce ids we e de ec ed in six o he eigh a eas. Finding no se oposi i e ce ids in wo o he eigh a eas may be explained by sampling bias: o ex- ample, he Helsinki ocus is an island wi h no hun ing ac i i y. Finding no se oposi i es a ound he oci sup- po s he p e ious obse a ions ha dis ibu ion o TBEV is highly ocal. One se oposi i e moose male cal (indi idual 9, Table 1) was om Kainuu, an a ea wi h no epo s o human TBE cases. As a cal unde 1 yea o age ollows a cow, and cows wi h cal es usually do no do dispe sal mig a ions, i is mos likely ha his indi idual had en- coun e ed he i us locally. This esul could hus sug- ges a possible new TBEV ocus, which calls o u he moni o ing o he a ea o TBEV ci cula ion and po en- ial human cases. In Finland, he human TBE cases ha e eme ged mainly nea by wa e –in he a chipelago, coas al egions, and nea big lakes [16]. This dis ibu ion o human TBE cases may be due o he high densi y o summe co ages by wa e as well as long exposu e imes du ing he sum- me holidays, which o e lap wi h he ick eeding season. O he explana ions o he p oximi y o human TBE cases o wa e include he ecological ac o s ha cause he highly ocal dis ibu ion o TBEV in a ou able mic oclima ic condi ions. Summe habi a s o ce ids a e o en also nea by wa e [47, 48] and moose a e known o use wa e o cool o [49]. Fu he mo e, dis- pe sal o oe dee has ollowed he seasho es, i e s, and lakes [40]. These habi a p e e ences migh enhance he po en ial ole o ce ids in main aining ick popula ions ha enable TBEV ci cula ion. Du ing ecen yea s, human TBE cases ha e been e- po ed in new a eas in Finland, and he s ic ly ocal pa - e n may be changing owa ds a coalescing dis ibu ion [14–16, 50]. The esul s o his s udy sugges ha moose could se e as sen inels and indica o s o isk o human TBEV in ec ions in he aiga egion whe e dee , commonly used as sen inels o TBEV isk o humans, a e no wide- sp ead. Su eying an i-TBEV an ibodies in wild ce ids could be a use ul ool o moni o ing his zoonosis. Conclusions An i TBEV-an ibodies we e de ec ed in en ee- anging ce ids in Finland. The in ec ions we e p esumably au och honous, na u ally acqui ed by he ce ids om Ton e i e al. Pa asi es & Vec o s (2016) 9:54 Page 6 o 8 hei local na u al en i onmen . Nine ou o en se o- posi i e animals had been hun ed in a eas whe e human TBE cases ha e occu ed, con i ming he local ci cula- ion o he i us in he oci. Ou s udy also ound one possible new ocus, as one se oposi i e moose male cal was om an a ea wi h no epo s o human TBE cases. Ou obse a ion o he geog aphical cong uence be ween he human cases and se oposi i e moose sugges ha moose could se e as sen inels and indica o s o TBEV isk o humans. Abb e ia ions HI: hemagglu ina ion inhibi ion es ; LAMV: Lammi i us; NVT: non- i emic ansmission; TBE: ick-bo ne encephali is; TBEV: ick-bo ne encephali is i us; WNV: Wes Nile i us. Compe ing in e es s E. Ton e i has ecei ed a lec u ing ee om P ize L d o gi ing a lec u e in he In e na ional Scien i ic Wo king G oup on Tick-Bo ne Encephali is (ISW-TBE) mee ing, Janua y 2015. The o he au ho s decla e ha hey ha e no compe ing in e es s. Au ho s’con ibu ions ET, PJ and OV designed he s udy. PJ was esponsible o planning he o iginal sampling. ET did he labo a o y wo k. All au ho s con ibu ed o he w i ing p ocess o he manusc ip : ET and OV con ibu ed especially o he TBE aspec s, ET, PJ and OV o zoono ic and epidemiological aspec s, and JM and JP o ecological and game managemen aspec s. All au ho s accep ed he inal e sion o his manusc ip o be submi ed o publica ion. Acknowledgemen s The au ho s would like o exp ess hei g a i ude o I ina Suomalainen, Uni e si y o Helsinki and Si kka Vene, The Public Heal h Agency o Sweden, o hei help wi h he se ological assays, and o he hun e s and he s a o Ve e ina y Pa hology and Pa asi ology, Depa men o Ve e ina y Biosciences, Facul y o Ve e ina y Medicine, Uni e si y o Helsinki o he con ibu ions o he o iginal sampling. We hank Vesa Ni ala (Na u al Resou ces Ins i u e Finland) o d awing hemap. The wo k was inancially suppo ed by he Finnish Founda ion o Ve e ina y Resea ch, S a egic De elopmen Fund o he Es onian Uni e si y o Li e Sciences, Academy o Finland, and he Helsinki Uni e si y Hospi al unds. 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Ticks Tick Bo ne Dis. 2016;7(1):216–23. • We accep p e-submission inqui ies • Ou selec o ool helps you o ind he mos ele an jou nal • We p o ide ound he clock cus ome suppo • Con enien online submission • Tho ough pee e iew • Inclusion in PubMed and all majo indexing se ices • Maximum isibili y o you esea ch Submi you manusc ip a www.biomedcen al.com/submi Submi you nex manusc ip o BioMed Cen al and we will help you a e e y s ep: Ton e i e al. Pa asi es & Vec o s (2016) 9:54 Page 8 o 8