Birds as potential reservoirs of tick-borne pathogens: first evidence of bacteraemia with Rickettsia helvetica

RESEARCH Open Access Bi ds as po en ial ese oi s o ick-bo ne pa hogens: i s e idence o bac e aemia wi h Ricke sia hel e ica Sándo Ho nok 1* , Dá id Ko á s 2,3 , Tibo Csö gő 3,4 , Ma ina L Meli 5 , EnikőGönczi 5 , Zsó ia Hadnagy 1 , Nó a Takács 1 , Róbe Fa kas 1 and Regina Ho mann-Lehmann 5 Abs ac Backg ound: Bi ds ha e long been known as ca ie s o icks, bu da a om he li e a u e a e lacking on hei ole as a ese oi in he epidemiology o ce ain ick-bo ne disease-causing agen s. The e o e, he aim o his s udy was o e alua e he p esence o h ee eme ging, zoono ic ick-bo ne pa hogens in blood samples and icks o bi ds and o assess he impac o eeding loca ion p e e ence and mig a ion dis ance o bi d species on hei ick in es a ion. Me hods: Blood samples and icks o bi ds we e analysed wi h TaqMan eal- ime PCRs and con en ional PCR ollowed by sequencing. Resul s: Du ing he sp ing and au umn bi d mig a ions, 128 blood samples and 140 icks (Ixodes icinus, Haemaphysalis concinna and a Hyalomma specimen) we e collec ed om bi ds belonging o 16 species. The p e alence o ick in es a ion and he p esence o ick species we e ela ed o he eeding and mig a ion habi s o a ian hos s. Bi ds we e shown o be bac e aemic wi h Ricke sia hel e ica and Anaplasma phagocy ophilum, bu no wi h Candida us Neoeh lichia miku ensis. The p e alence o icke siae was high (51.4%) in icks, sugges ing ha some o hem may ha e acqui ed hei in ec ion om hei a ian hos . Conclusion: Based on he p esen esul s bi ds a e po en ial ese oi s o bo h I. icinus ansmi ed zoono ic pa hogens, R. hel e ica and A. phagocy ophilum, bu hei epidemiological ole appea s o be less impo an conce ning he la e , a leas in Cen al Eu ope. Keywo ds: G ound eeding bi ds, Mig a o y bi ds, Ticks, Ricke sia hel e ica,Anaplasma phagocy ophilum, Candida us Neoeh lichia miku ensis Backg ound Bi ds can ly o e la ge dis ances in he cou se o a ew days, pa icula ly du ing hei seasonal mig a ion. Addi ion- ally, hey ha e long been known o hei epidemiological ole as ca ie s o ha d icks (Aca i: Ixodidae), implying ha ick-bo ne pa hogens in icks a ached o a ian hos s can be anspo ed o geog aphically dis an places [1]. The majo i y o ick-bo ne pa hogens a e biologically ansmi ed, which means ha hese mic oo ganisms in- ec hei ha d ick ec o , whe e hey mul iply and/o de- elop p io o ansmission o ano he e eb a e hos [2]. On he o he hand, ixodid la ae, nymphs and adul emales suck blood on hei hos only once. Co espond- ingly, i la ae o nymphs acqui e ick-bo ne pa hogens wi h hei blood meal, hey will be able o in ec ano he hos only du ing a subsequen de elopmen al s age, which is e e ed o as anss adial ansmission [2]. Tick-bo ne pa hogens aken up by adul emale icks will only be able o gain access o ano he hos , i hey pass h ough he o a ies/o a o he emale icks o he nex gene a ion (i.e. wi h anso a ial ansmission) [2]. Each species o ick-bo ne pa hogen is able o in ec a ce ain ange o e eb a e hos o ese oi species. The epidemiological signi icance o hese hos s/ ese oi s is ha hey can p o ide he sou ce o in ec ion o u he ick ec o s, he eby ensu ing he main enance o ick- * Co espondence: [email p o ec ed] 1 Depa men o Pa asi ology and Zoology, Facul y o Ve e ina y Science, Szen Is án Uni e si y, Budapes , Hunga y Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2014 Ho nok e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly c edi ed. 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. Ho nok e al. Pa asi es & Vec o s 2014, 7:128 h p://www.pa asi esand ec o s.com/con en /7/1/128 bo ne pa hogens in na u e. Con e sely, he ick- o- ick ansmission o pa hogens may no always necessi a e he in ec ion o he hos / ese oi because ick-bo ne agen s may sp ead be ween icks eeding close o each o he ia he so-called co- eeding mechanism [3]. The mos common ick species associa ed wi h a ian hos s in a ious Eu opean coun ies is Ixodes icinus [4-6]. Acco ding o he phenomenon o hos -size es ic- ion, usually he la ae and nymphs a e he only s ages o I. icinus o suck blood on bi ds, whe eas all h ee s ages, i.e. la ae, nymphs and adul s, eed on small, medium and la ge-sized mammals, espec i ely [7]. Compa ed o o he ick species o he genus, I. icinus is known as a compe en ec o o he highes numbe o impo an zoono ic bac e ia [8]. Among hese Ricke sia hel e ica is conside ed an eme ging ick-bo ne pa hogen and is suspec ed o be in ol ed in cases o human disease [9]. On he con a y, he majo i y o I. icinus-bo ne pa h- ogens may elici clinical signs in bo h humans and ani- mals, as exempli ied by Anaplasma phagocy ophilum, he causa i e agen o human, equine and canine g anulocy ic anaplasmosis, and o ick-bo ne e e in uminan s [10]. Recen ly, I. icinus and i s hos s we e demons a ed o play an epidemiological ole in he main enance and ans- mission o Candida us Neoeh lichia miku ensis, a newly desc ibed zoono ic pa hogen [11]. In icks, Ricke sia spe- cies ha e bo h anss adial and anso a ial ansmission [12], whe eas membe s o he Anaplasma aceae amily (including A. phagocy ophilum and Ca. Neoeh lichia miku ensis) lack he la e [2]. In he abo e con ex , he aim o he p esen s udy was h ee old: (1) o in es iga e whe he he eeding p e e ence and/o mig a ion dis ance o di e en bi d species in lu- ences hei ick bu den and hus hei chances o becoming in ec ed wi h ce ain ick-bo ne pa hogens; (2) o e alua e whe he bi ds can ac ually become in ec ed/bac e aemic wi h h ee eme ging, zoono ic ick-bo ne pa hogens (R. hel e ica,A. phagocy ophilum,Ca. Neoeh lichia miku ensis); and (3) o compa e he a e o in ec ion wi h ick-bo ne pa hogens among bi ds and hei icks, wi h ele ance o ansmission e icacy o hese agen s. Me hods Sample collec ion Bi ds we e mis -ne ed du ing he sp ing and au umn mig a ion pe iods o 2013 a heÓcsaBi dRingingS a ion, Hunga y ( o six days in Ap il, i e days in May and wo days in June, as well as o ou days a he end o Augus and he beginning o Sep embe ). The loca ion and ime pe iods we e chosen by conside ing he mig a- ion s opo e o bi ds in Hunga y [13]. On he sampling days, 50 s anda d Eco one mis -ne s (Gdynia, Poland), 12 m in leng h, 2.5 m in heigh and wi h 16 × 16 mm holes, we e used om 7:00 o 12:00 a.m. The whole body o each cap u ed bi d was sc u inized o he p esence o icks. All icks we e emo ed wi h ine o ceps, and pu in o 70% e hanol in sepa a e ials acco ding o hei hos s. Tick species we e de e mined acco ding o s anda d keys [7], and he eeding p e e ence (g ound le el o abo e) and mig a ion habi we e assigned o bi d species based on o ni hological clues [13]. In addi ion, all ick ca ie bi ds, as well as a simila numbe o ick- ee indi iduals o he same bi d species we e blood sampled om he b achial ein using a ine (28G) needle and 0.5 ml sy inge (Kendall Monojec : Tyco Heal hca e G oup Lp., Mans ield, MA, USA). Ticks we e s o ed a oom empe a u e, whe eas blood samples we e kep a -20°C. E hical app o al The s udy was ca ied ou acco ding o he na ional ani- mal wel a e egula ions (28/1998), and was app o ed by he E hics Commi ee o he Facul y o Ve e ina y Science (SZIU). Sample p epa a ion Ticks we e d ied, washed h ee imes (in de e gen con- aining wa e , in ap wa e and in dis illed wa e ) and minced a he bo om o 1.5 ml Eppendo ubes in 100 μl PBS wi h poin ed scisso s. Be ween each sample, he scisso s we e washed and lame s e ilized o decon am- ina ion. Samples we e hen incuba ed o e nigh a 56°C in issue lysis bu e con aining p o einase-K. DNA was ex ac ed om his lysa e and om 20-100 μlo he blood samples using he QIAamp DNA Mini Ki (QIAGEN, Hilden, Ge many) ollowing he manu ac- u e ’s ins uc ion and using ex ac ion con ol. The quan i y and quali y o bi d blood DNA was assessed by PCR, which ampli ied pa o he 18S RNA gene [14]. These samples we e consequen ly used a a dilu ion o 1:10. PCR me hods All PCR assays we e pe o med using he app op ia e posi i e and nega i e con ols. The ex ac ion con ols we e nega i e in all es s. Candida us Neoeh lichia miku ensis A mul iplex eal- ime TaqMan PCR was applied o de ec pa o he 16S RNA gene [15]. The assay is based on p obes ha indica e posi i i y on he amily (Anaplasma aceae), genus (Neoeh lichia)andspecies (Ca. Neoeh lichia miku ensis) le el. Anaplasma phagocy ophilum A TaqMan PCR was employed ha ampli ies pa o he gene encoding a majo su ace p o ein (msp2)o A. phagocy ophilum [16]. The p obe was modi ied as 6-FAM-TGGTGCCAGGGTTGAGCTTGAGAT Ho nok e al. Pa asi es & Vec o s 2014, 7:128 Page 2 o 7 h p://www.pa asi esand ec o s.com/con en /7/1/128 TG-TAMRA (5′-3′). The assay consis ed o 40 cycles, and he esul s we e conside ed posi i e i he h esh- old cycle (C ) alue was below 39. Ricke siae The p esence o icke siae was e alua ed by wo TaqMan PCRs, de ec ing ei he pa o he 23S RNA gene o R. hel e ica o pa o he gl Agene o o he icke siae [14]. The i s es was speci ic o R. hel e ica,and heC alue was conside ed an in e se measu e o he bac e ial load [14]. In addi ion, o demons a e he p esence o R. monacensis in icks, a con en ional PCR ampli ying a 381 bp long po ion o he gl Agene [17] was pe o med, ollowed by gel elec opho esis (1.5% aga ose) and sequen- cing o he posi i e samples (Mac ogen Inc., Ko ea). S a is ical analysis Exac con idence in e als (CIs) o he p e alence a es a he 95% le el we e calcula ed acco ding o S e ne’s me hod [18]. Sample p e alence da a we e analyzed using Fishe ’s exac es . Di e ences we e conside ed sig- ni ican when P < 0.05. Resul s Tick in es a ion o bi ds Du ing he s udy, 1219 bi ds we e cap u ed. Tick in es a- ions we e ound in 68 o hese bi ds, belonging o 16 species (Table 1). Al oge he 140 icks we e emo ed om he 68 bi ds. Ixodes icinus was ound on he majo i y o ick-in es ed bi ds (63 o he 68 indi iduals: 92.6%, CI: 83.7- 97.6%). This was also he mos abundan ick species (121 o he 140 icks: 86.4%, CI: 79.6-91.6%) and was ep esen ed by 42 la ae, 78 nymphs and one adul emale. Eigh bi ds we e in es ed wi h Haemaphysalis concinna,amoun ing o a p e alence o 11.8% (CI: 5.2-21.9%). The abundance o his species (wi h 13 la ae and i e nymphs) was 12.6% (CI: 7.8-19.6%). Fou bi ds we e co-in es ed wi h I. ici- nus and Ha. concinna. In addi ion, one Wood Wa ble (Phylloscopus sibila ix) ca ied a Hyalomma la a, which is no indigenous o Hunga y. The p e alence o ick in es a ion was compa ed be- ween wo ca ego ies o bi d species: hose ha p e e en- ially eed om he g ound and hose ha eed abo e g ound le el (Table 1). The e we e signi ican ly (P < 0.0001) mo e ick-in es ed bi ds among hose p e e en- ially eeding om he g ound, han among hose eeding abo e he g ound le el, wi h a 23.5% (27 o 115, CI: 16.1- 32.3%) and 5.3% (41 o 771, CI: 3.8-7.1%) p e alence o ick in es a ion, espec i ely. Nei he Ha. concinna la ae no nymphs we e ound on bi ds ha eed on he g ound (Table 1), indica ing a signi ican di e ence in compa ison wi h I. icinus when conside ing ei he he p e alence (P = 0.018) o in ensi y o ick in es a ion (i.e. he o al numbe o icks pe bi d; P < 0.0001) (Table 1). Conside ing bi d species acco ding o hei mig a ion cha ac e is ics (Table 1), he p e alence o ick in es a ion Table 1 P e alence and in ensi y o ick in es a ion among bi ds acco ding o hei eeding p e e ence and mig a ion habi s Bi d species ( ick in es ed/all) Feeding p e e ence ( ick in es ed/all) Dis ance o mig a ion ( ick in es ed/all) Numbe o bi ds in es ed wi h …(numbe o icks) Ixodes icinus Haemaphysalis concinna Hyalomma sp. La a Nymph La a Nymph La a Luscinia mega hynchos (4/36) G ound le el (27/115) Long (6/44) 2 (5) 4 (5) Luscinia luscinia (1/7) 1 (5) 1 (2) An hus i ialis (1/1) 1 (1) 1 (4) E i hacus ubecula (13/52) Sho (16/59) 7 (9) 8 (14) Tu dus philomelos (2/6) 1 (3) 2 (5) Tu dus iliacus (1/1) 1 (1) Tu dus me ula (5/12) Local (5/12) 2 (2) 4 (11) Ac ocephalus sci paceus (15/129) Abo e g ound le el (41/771) Long (23/283) 2 (3) 12 (14) 2 (2) 3 (4) Ac ocephalus schoenobaenus (4/90) 2 (2) 2 (2) Locus ella luscinioides (1/26) 1 (5) Syl ia communis (2/19) 1 (1) 2 (2) Phylloscopus sibila ix (1/19) 1 (1) Syl ia a icapilla (12/372) Middle (13/464) 6 (10) 8 (8) 1 (1) 1 (1) Phylloscopus collybi a (1/92) 1 (1) Cocco h aus es cocco h aus es (2/18) Sho (5/24) 2 (2) P unella modula is (3/6) 1 (1) 3 (7) 1 (5) Ho nok e al. Pa asi es & Vec o s 2014, 7:128 Page 3 o 7 h p://www.pa asi esand ec o s.com/con en /7/1/128 was 41.7% in he case o local bi d species (5 o 12, CI: 15.2-72.3%) and 25.3% in he case o sho dis ance mi- g an s (21 o 83, CI: 16.4-36%). Bo h o hese a es a e sig- ni ican ly (P < 0.003) highe han hose obse ed among middle and long dis ance mig an s, wi h 2.8% (13 o 464, CI: 1.5-4.7%) and 8.9% (29 o 327, CI: 6-12.5%) p e alence o ick in es a ion, espec i ely. A Hyalomma la a was e- mo ed om a bi d species wi h long dis ance mig a ion (Table 1). Tick-bo ne pa hogens in blood samples o bi ds Blood samples we e collec ed om all 68 ick-in es ed and 60 ick- ee bi ds o he same species. Among he 128 blood-sampled bi ds six we e ound o be PCR posi- i e wi h R. hel e ica (4.7% p e alence, CI: 1.7-9.9%): hese consis ed o i e Robins (E i hacus ubecula) and one Dunnock (P unella modula is). The ange o C alues (33-40) e lec ed low o medium le els o bac e - ial loads. Ano he Dunnock ha bo ed icke siae o he han R. hel e ica ( he species could no be de e mined due o a high C alue o 41). All icke sia-posi i e bi ds we e cap u ed in Ap il. The blood sample o ano he bi d (Redwing: Tu dus iliacus), also caugh in Ap il, was PCR posi i e o A. phagocy ophilum (C alue 32.2). A u he 25 bi ds ca - ied uniden i ied membe (s) o he Anaplasma aceae amily (C alues abo e 31.4), bu none we e posi i e o Candida us Neoeh lichia miku ensis. Tick-bo ne pa hogens in icks collec ed om bi ds Al oge he , 72 o he 140 bi d icks (51.4%, CI: 42.8-60%) we e PCR posi i e o icke siae: 61 ha bou ed R. hel e ica (43.6%, CI: 35.2-52.2%), and 11 con ained R. monacensis (7.9%, CI: 4-13.6%). R. hel e ica was ound in bo h I. icinus and Ha. concinna la ae and nymphs, bu R. monacensis was only de ec ed in I. icinus. The p opo ions o icke sia-posi i e I. icinus la ae (22 o 42: 52.4%) and nymphs (42 o 78: 53.8%) we e simila . Se en o he 18 Ha. concinna icks ( h ee la ae, ou nymphs) ca ied R. hel e ica (38.9%, CI: 17.3-64.3%). Conce ning he six R. hel e ica PCR-posi i e bi ds, no icks we e ound on ou o hem, and a PCR-nega i e ick was ound on ano he . An addi ional R. hel e ica bac e - aemic bi d ca ied 11 icks, o which wo (an I. icinus nymph and a Ha. concinna la a) u ned ou o be PCR posi i e. A he same ime, he e we e 38 R. hel e ica PCR-nega i e bi ds, om which 59 R. hel e ica PCR- posi i e icks (including 17 I. icinus and 2 Ha. concinna la ae) we e collec ed. In he A. phagocy ophilum-speci ic PCR, only one I. icinus nymph ( emo ed om a PCR-nega i e bi d) was posi i e. Con e sely, he bi d ha es ed posi i e o A. phagocy ophilum ca ied a PCR-nega i e ick. Discussion The p e alence o ick in es a ion among bi ds, as well as he species o icks a aching o hem, may depend on se e al ac o s ha , in u n, in luence he epidemio- logical ole o a ian ese oi s and hos s om he poin o iew o ick-bo ne diseases. One o hese ac o s is he eeding loca ion p e e ence o bi ds. In he p esen s udy, he p e alence o ick in es - a ion was signi ican ly associa ed wi h he habi o g ound eeding in bi ds, simila ly o p e ious obse a ions [19]. Howe e , he esul s shown he e also e ealed ha his co ela ion may signi ican ly depend on he ick species, as imma u e s ages o Ha. concinna occu ed exclusi ely on bi ds ha p e e en ially eed abo e he g ound. The di - e ence no ed he ein be ween Ha. concinna and I. icinus may be ela ed o he hos seeking beha iou o ele an imma u e s ages. I has been epo ed ha Haemaphysalis nymphs ques a a ious heigh s on ege a ion acco ding o he body size o he local dee popula ion [20]. Simi- la ly, in he p esen s udy, Ha. concinna la ae and nymphs in es ed bi ds ha eed abo e g ound le el mos likely because he p e e ed hos s o imma u e s ages o his ick species in Hunga y a e oe dee [21]. Con e sely, I. icinus subadul s appa en ly had lowe ques ing heigh s (being highly p e alen on g ound eeding bi ds) in associ- a ion wi h oden s as p ima y hos s [22]. In he p esen s udy, i was also demons a ed ha he dis ance o mig a ion signi ican ly in luences he ick bu dens o bi ds, conce ning bo h he p e alence and he species composi ion. Fi s , he p e alence o ick in- es a ion was signi ican ly highe among local bi ds and sho dis ance mig an s, mos likely due o hei p es- ence/a i al du ing he main ick season, as sugges ed in o he s udies [4]. Bi d species wi h middle o long dis- ance mig a ion habi s a e known o a i e in Hunga y in May and June [13] (i.e. a he end o a e he main ick season). On he o he hand, a Hyalomma la a (which is no indigenous o Hunga y) was emo ed om a long dis ance mig an cap u ed in his s udy. In Eu ope, bi ds depa ing om sou he n loca ions o no he n coun ies may ca y Medi e anean ha d ick species (e.g. wo-hos Hyalomma spp. ha s ay on he hos as la ae and nymphs o 12-26 days [23]). Consequen ly, he epidemiological signi icance o bi ds wi h middle o longe dis ances o mig a ion is he po en ial impo o exo ic ick species, as p e iously epo ed [6] and ob- se ed he e. The mos signi ican esul o he p esen s udy is he molecula e idence o bac e aemia in bi ds caused by R. hel e ica. Acco ding o a p e ious epo , when 130 wild bi ds we e e alua ed o he p esence o icke siae, none we e ound o be icke saemic [24]. In ano he s udy 3% o 131 pooled bi d blood samples we e PCR posi i e o unknown icke siae [25]. Da a a e also a ailable on he Ho nok e al. Pa asi es & Vec o s 2014, 7:128 Page 4 o 7 h p://www.pa asi esand ec o s.com/con en /7/1/128 p olonged, expe imen ally elici ed icke saemia o R. icke sii and R. canadensis in bi ds [1,26]. The e o e, o he bes o ou knowledge, his is he i s epo o R. hel e ica in he blood o any a ian species. Bo h bi d species wi h PCR-posi i e blood samples ( he Robin and he Dunnock) a e known o hei synan h opic li e (i.e. ha ing u ban and subu ban habi a s) [13]. Because he e we e no icks on he majo i y o R. hel e ica bac e aemic bi ds in he p esen s udy, and om one PCR-posi i e bi d only a PCR-nega i e ick was collec ed, hese da a sugges icke saemia pe sis s a e he de ach- men o he ec o ick in ele an a ian hos s. A he same ime, only wo o he 11 icks emo ed om a R. hel e ica bac e aemic bi d we e PCR posi i e. Consequen ly, he ansmission o icke siae om bi ds o ick ec o s may occu wi h low e icacy. I is known ha he bac e ial load o R. icke sii in he ci cula ion o ce ain e eb a e ese oi hos s is enough o in ec icks, whe eas icke - saemia in o he (also suscep ible) hos s may be oo low o his [27]. Al e na i ely, in e mi en icke saemia was also epo ed in case o some Ricke sia spp. [28]. Howe e , mo e han hal (51.4%) o he icks collec ed om bi ds in his s udy we e ound o be PCR posi i e o icke siae, mos ly o R. hel e ica. This p e alence is signi ican ly highe han he in ec ion a es epo ed in ques ing icks collec ed om he ege a ion (usually 4-16%, based on da a om many coun ies: [9]). The p e a- lence o PCR posi i i y among Ha. concinna icks om bi ds in his s udy (se en o 18 indi iduals) was also signi ican ly (P < 0.001) highe , han he minimum and pool p e alence obse ed in he case o ques ing Ha. concinna icks collec ed om he ege a ion in Hunga y (one o 53 pools we e posi i e: [29]). The high p e alence o R. hel e ica among imma u e icks o bi ds in he p esen s udy may be explained by se e al po en- ial causes. Fi s , PCR posi i i y in la ae (especially on PCR-nega i e bi ds) can be pa ly a ibu ed o he anso a ial ansmission o R. hel e ica, as p e iously epo ed [30]. Second, he signi ican ly highe p e alence among he imma u e s ages epo ed he e, when com- pa ed o adul s in o he s udies, may indica e he low anss adial main enance o R. hel e ica (al hough he simila in ec ion p e alence among la ae and nymphs as shown he e, o bo h I. icinus and Ha. concinna,a gues agains his). Thi d, some o he icks may ha e acqui ed icke siae h ough co- eeding [1,3]. Las , bu no leas , some o he PCR-posi i e icks collec ed om PCR- nega i e bi ds may e en ha e acqui ed hei R. hel e ica in ec ion om hei a ian hos (PCR nega i i y o a bi d a he ime o blood sampling does no exclude icke - saemia on o he days o i s ick’s blood sucking). In addi ion o he bi ds posi i e o icke siae, he e was one Redwing (Tu dus iliacus) wi h de ec able A. phagocy ophilum bac e aemia. Acco ding o da a in he li e a u e, his may be he second piece o di ec e i- dence o a na u al a ian in ec ion o bac e aemia wi h his zoono ic pa hogen, indica ing ha bi ds may play a ole in he epidemiology o g anulocy ic anaplasmosis. In he ini ial s udy aising his possibili y [10], he p e a- lence o A. phagocy ophilum in ec ion among bi ds was signi ican ly highe (22%) han shown he e, and, in e es - ingly, i was he highes in Blackbi ds (Tu dus me ula), which a e axonomically closely ela ed o he species ound o be PCR posi i e in he p esen s udy. On he o he hand, da a om he li e a u e a e con adic o y when assessing he ole o bi ds as ese oi s o A. phagocy ophilum in compa ison wi h small mammals. In one epo , icks emo ed om bi ds con ained his pa hogen wi h a signi ican ly highe p e alence com- pa ed o hose om he long known ese oi s (i.e. small mammals) [5]. Howe e , ano he s udy (assessing he ese oi compe ence based on he molecula analysis o A. phagocy ophilum in ick la ae om di e en mam- malian and a ian hos s) sugges ed a mo e impo an es- e oi ole o small mammals compa ed o bi ds [31]. The e was only one A. phagocy ophilum-posi i e ick on a bi d ha es ed nega i e in he ele an PCR. The ick collec ed om he PCR-posi i e bi d was also ound o be nega i e o A. phagocy ophilum. This is no su - p ising in ligh o he ac ha he p e alence o A. phagocy ophilum may equen ly be e y low in ques ing icks (0.8%: [32]). This zoono ic pa hogen was no e en ound in any o a la ge numbe o icks collec ed om small mammals (known ese oi s) in Hunga y [22]. Though Candida us Neoeh lichia miku ensis was e- cen ly demons a ed in icks o he same egion whe e bi ds o his s udy we e sampled [33], no PCR posi i i y was ound o his zoono ic pa hogen in bi ds. Ne e he- less, his may be he i s sc eening o Ca. Neoeh lichia miku ensis among bi ds (hea ily in ec ed wi h i s ec o I. icinus). In he same mul iplex PCR high numbe s o bi d blood samples we e posi i e o membe (s) o he Anaplasma aceae amily. The species could no be u - he iden i ied due o he low le el o in ec ion (high C alues). This obse a ion is in line wi h he p esence o Anaplasma spp. in bi d blood samples as epo ed p e i- ously [25]. Conclusions This is he i s molecula e idence o R. hel e ica in ec- ion in a ian hos s. Based on he p esen esul s, bi ds a e po en ial ese oi s o bo h I. icinus ansmi ed zoono ic pa hogens, R. hel e ica and A. phagocy ophilum, bu hei epidemiological ole appea s o be lowe con- ce ning he la e , a leas in Cen al Eu ope. Ou da a also sugges ha icke saemic bi ds may p o ide a sou ce o in ec ion o I. icinus and Ha. concinna,bu wi h low e icacy. Ho nok e al. Pa asi es & Vec o s 2014, 7:128 Page 5 o 7 h p://www.pa asi esand ec o s.com/con en /7/1/128 Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Au ho s’con ibu ions SH iden i ied he icks, ex ac ed he DNA and w o e he manusc ip . DK collec ed he blood samples and p o ided o ni hological in o ma ion. CST made possible bi d cap u e and sample collec ion and supe ised o ni hological wo k. MM designed and, in pa , pe o med he molecula analyses. ZSH collec ed he icks. EG and NT pe o med mos o he molecula analyses. RF supe ised pa asi ological wo k. RH-L ini ia ed and supe ised he s udy. All au ho s ead and app o ed he inal e sion o he manusc ip . Acknowledgemen s This s udy was pa ially unded by EU g an FP7-261504 EDENex and is ca alogued by he EDENex S ee ing Commi ee as EDENex 213. The con en s o his a icle a e he sole esponsibili y o he au ho s and do no e lec he iews o he Eu opean Commission. Molecula biology wo k was pa ially pe o med using he logis ics o he Cen e o Clinical S udies a he Ve suisse Facul y o he Uni e si y o Zu ich. The su ey was also o ganized unde he amewo k o he Eu NegVec COST Ac ion TD1303. Au ho de ails 1 Depa men o Pa asi ology and Zoology, Facul y o Ve e ina y Science, Szen Is án Uni e si y, Budapes , Hunga y. 2 Depa men o E olu iona y Zoology and Human Biology, Deb ecen, Hunga y. 3 Bi d Ringing S a ion, Ócsa, Hunga y. 4 Depa men o Ana omy, Cell and De elopmen al Biology, Eö ös Lo án Uni e si y, Budapes , Hunga y. 5 Clinical Labo a o y and Cen e o Clinical S udies, Ve suisse Facul y, Uni e si y o Zu ich, Zu ich, Swi ze land. Recei ed: 5 Decembe 2013 Accep ed: 4 Ma ch 2014 Published: 28 Ma ch 2014 Re e ences 1. Hasle G: T anspo o ixodid icks and ick-bo ne pa hogens by mig a o y bi ds. F on Cell In ec Mic obiol 2013, 3:48. 2. 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Coipan EC, Jah a i S, Fon ille M, Maassen CB, an de Giessen J, Takken W, Takumi K, Sp ong H: Spa io empo al dynamics o eme ging pa hogens in ques ing Ixodes icinus.F on Cell In ec Mic obiol 2013, 3:36. 33. Ho nok S, Meli ML, Gönczi E, Ho mann-Lehmann R: Fi s e idence o Candida us Neoeh lichia miku ensis in Hunga y. Pa asi Vec o s 2013, 6:267. doi:10.1186/1756-3305-7-128 Ci e his a icle as: Ho nok e al.:Bi ds as po en ial ese oi s o ick-bo ne pa hogens: i s e idence o bac e aemia wi h Ricke sia hel e ica.Pa asi es & Vec o s 2014 7:128. Submi you nex manusc ip o BioMed Cen al and ake ull ad an age o : • Con enien online submission • Tho ough pee e iew • No space cons ain s o colo figu e cha ges • Immedia e publica ion on accep ance • Inclusion in PubMed, CAS, Scopus and Google Schola • Resea ch which is eely a ailable o edis ibu ion Submi you manusc ip a www.biomedcen al.com/submi Ho nok e al. Pa asi es & Vec o s 2014, 7:128 Page 7 o 7 h p://www.pa asi esand ec o s.com/con en /7/1/128