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
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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
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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)
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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
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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. S ich RW, Schae e JJ, B eme WG, Needham GR, Ji apalapong S: Hos
su eys, ixodid ick biology and ansmission scena ios as ela ed o he
ick-bo ne pa hogen, Eh lichia canis.Ve Pa asi ol 2008, 158:256–273.
3. Randolph SE, Ge n L, Nu all PA: Co- eeding icks: Epidemiological
signi icance o ick-bo ne pa hogen ansmission. Pa asi ol Today 1996,
12:472–479.
4. Olsén B, Jaenson TG, Be gs öm S: P e alence o Bo elia bu gdo e i sensu
la o- in ec ed icks on mig a ing bi ds. Appl En i on Mic obiol 1995,
61:3082–3087.
5. F anke J, F i zsch J, Tomaso H, S aube E, Do n W, Hildeb and A:
Coexis ence o pa hogens in hos -seeking and eeding icks wi hin a
single na u al habi a in Cen al Ge many. Appl En i on Mic obiol 2010,
76:6829–6836.
6. Ho nok S, Csö gőT, de la Fuen e J, Gyu anecz M, P i igyei C, Meli ML,
K eizinge Z, Gönczi E, de Me a IG F, Ho mann-Lehmann R: Synan h opic
bi ds associa ed wi h high p e alence o ick-bo ne icke siae and wi h
he i s de ec ion o Ricke sia aeschlimannii in Hunga y. Vec o Bo ne
Zoono ic Dis 2013, 13:77–83.
7. Hillya d PD: Ticks o No h-Wes Eu ope.InSynopses o he B i ish Fauna
(New Se ies), Volume Volume 52. Edi ed by Ba nes RSK, C o he s JH.
Sh ewsbu y: Field S udies Council; 1996.
8. Es ada-Peña A, Jongejan F: Ticks eeding on humans: a e iew o eco ds
on humanbi ing Ixodoidea wi h special e e ence o pa hogen
ansmission. Exp Appl Aca ol 1999, 23:685–715.
9. Sp ong H, Wielinga PR, Fon ille M, Reusken C, B andenbu g AH, Bo gs eede
F, Gaasenbeek C, an de Giessen JW: Ixodes icinus icks a e ese oi
hos s o Ricke sia hel e ica and po en ially ca y lea-bo ne Ricke sia
species. Pa asi Vec o s 2009, 2:41.
10. de La Fuen e J, Na anjo V, Ruiz-Fons F, Hö le U, Fe nández De Me a IG,
Villanúa D, Almazán C, To ina A, Ca acappa S, Kocan KM, Go áza C:
Po en ial e eb a e ese oi hos s and in e eb a e ec o s o
Anaplasma ma ginale and A. phagocy ophilum in cen al Spain.
Vec o Bo ne Zoono ic Dis 2005, 5:390–401.
11. Jah a i S, Fon ille M, Henge eld P, Reusken C, Schol e EJ, Takken W,
Heyman P, Medlock J, Heylen D, Kle e J, Sp ong H: P e alence o
Neoeh lichia miku ensis in icks and oden s om No h-wes Eu ope.
Pa asi Vec o s 2012, 5:74.
12. Pe lman SJ, Hun e MS, Zcho i-Fein E: The eme ging di e si y o Ricke sia.
P oc Biol Sci 2006, 273:2097–2106.
13. Csö gőT, Ka cza Z, Halmos G, Gyu ácz J, Magya G, Szép T, Schmid A,
Banko ics A, Schmid E: Hunga ian Bi d Mig a ion A las. Budapes : Kossu h
Publishing Z ; 2009.
14. Bo e i FS, Pe e en A, Meli ML, Ca o i V, Willi B, Wengi N, Ho nok S,
Honegge H, Hegglin D, Woel el R, Reusch CE, Lu z H, Ho mann-Lehmann
R: Molecula In es iga ions o Ricke sia hel e ica in ec ion in dogs,
oxes, humans, and Ixodes icks. Appl En i on Mic obiol 2009,
75:3230–3237.
15. Mau e FP, Kelle PM, Beu e C, Joha C, Ache mann Y, Guble J, Bi che D,
Ka e U, Feh J, Zimme li L, Bloembe g GV: Close geog aphic associa ion
o human neoeh lichiosis and ick popula ions ca ying “Candida us
Neoeh lichia miku ensis”in eas e n Swi ze land. J Clin Mic obiol 2013,
51:169–176.
16. Cou ney JW, Kos elnik LM, Zeidne NS, Massung RF: Mul iplex eal- ime
PCR o de ec ion o Anaplasma phagocy ophilum and Bo elia
bu gdo e i.J Clin Mic obiol 2004, 42:3164–3168.
17. S enos J, G a es SR, Unswo h NB: A highly sensi i e and speci ic eal- ime
PCR assay o he de ec ion o spo ed e e and yphus g oup
Ricke siae. Am J Med Hyg 2005, 73:1083–1085.
18. Reiczigel J: Con idence in e als o he binomial pa ame e : some new
conside a ions. S a Med 2003, 22:611–621.
19. Ishigu o F, Takada N, Masuzawa T, Fukui T: P e alence o Lyme disease
Bo elia spp. in icks om mig a o y bi ds on he Japanese mainland.
Appl En i on Mic obiol 2000, 66:982–986.
20. Tsunoda T, Ta suzawa S: Ques ing heigh o nymphs o he bush ick,
Haemaphysalis longico nis, and i s closely ela ed species, H.
mageshimaensis: co ela ion wi h body size o he hos . Pa asi ology 2004,
128(P 5):503–509.
21. Ho nok S, Ho á h G, Jongejan F, Fa kas R: Ixodid icks on uminan s, wi h
on- hos ini ia ed moul ing (apolysis) o Ixodes,Haemaphysalis and
De macen o la ae. Ve Pa asi ol 2012, 187:350–353.
22. Rigó K, Gyu anecz M, Tó h AG, Föld á i G: De ec ion o Bo elia
bu gdo e i SensuLa oandAnaplasma phagocy ophilum in small
mammals and ec opa asi es in Hunga y. Vec o Bo ne Zoono ic Dis 2011,
11:1499–1501.
23. Fa kas R, Es ada-Pena A, Jaenson TGT, Pascucci I, Madde M: Basic biology
and geog aphical dis ibu ion o ick species in ol ed in he ansmission
o animal pa hogens, including zoonoses.InTicks and Tick-Bo ne Diseases:
Geog aphical dis ibu ion and con ol s a egies in he Eu o-Asia egion.
Edi ed by Salman M, Ta es-Call J. Walling o d, UK: Published by CABI (CAB
in e na ional); 2003:6–26.
24. S ańczak J, Racewicz M, Michalik J, Cieniuch S, Siko a B, Sko acki M:
P e alence o in ec ion wi h Ricke sia hel e ica in eeding icks and hei
hos s in wes e n Poland. Clin Mic obiol In ec 2009, 15(Suppl 2):328–329.
25. Ioannou I, Chochlakis D, Kasinis N, Anayio os P, Lyssand ou A,
Papadopoulos B, Tselen is Y, Psa oulaki A: Ca iage o Ricke sia spp.,
Coxiella bu ne ii and Anaplasma spp. by endemic and mig a o y wild
bi ds and hei ec opa asi es in Cyp us. Clin Mic obiol In ec 2009,
15(Suppl 2):158–160.
26. Bu gdo e W: Obse a ions on Ricke sia canada a ecen ly desc ibed
membe o he yphus g oup icke siae. J Hyg Epidemiol Mic obiol
Immunol 1968, 12:26–31.
27. Bozeman FM, Shi al A, Humph ies JW, Fulle HS: Ecology o Rocky
Moun ain spo ed e e . II. Na u al in ec ion o wild mammals and bi ds
in Vi ginia and Ma yland. Am J T op Med Hyg 1967, 16:48–59.
28. Kelly PJ, Ma hewman LA, Mason PR, Cou ney S, Ka sande C, Rukwa a J:
Expe imen al in ec ion o dogs wi h a Zimbabwean s ain o Ricke sia
cono ii.J T op Med Hyg 1992, 95:322–326.
29. Ho nok S, Meli ML, Pe e en A, Fa kas R, Willi B, Beugne F, Lu z H,
Ho mann- Lehmann R: Molecula in es iga ion o ha d icks (Aca i:
Ixodidae) and leas (Siphonap e a: Pulicidae) as po en ial ec o s o
icke sial and mycoplasmal agen s. Ve Mic obiol 2010, 140:98–104.
30. Bu gdo e W, Aeschlimann A, Pe e O, Hayes SF, Philip RN: Ixodes icinus:
ec o o a hi he o undesc ibed spo ed e e g oup agen in
Swi ze land. Ac a T op 1979, 36:357–367.
31. Keesing F, He sh MH, Tibbe s M, McHen y DJ, Due S, B unne J, Killilea M,
LoGiudice K, Schmid KA, Os eld RS: Rese oi compe ence o e eb a e
Ho nok e al. Pa asi es & Vec o s 2014, 7:128 Page 6 o 7
h p://www.pa asi esand ec o s.com/con en /7/1/128
hos s o Anaplasma phagocy ophilum.Eme g In ec Dis 2012,
18:2013–2016.
32. 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.
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