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ick-borne pathogens in ticks collected from breeding and migratoryirds in Switzerland
lena Lommanoa, Charles Dvorákb, Laurent Vallottonc,d, Lukas Jennie, Lise Gerna,∗
Institute of Biology, Laboratory of Eco-Epidemiology of Parasites, Emile-Argand 11, 2000 Neuchâtel, SwitzerlandRue Louis Ruchonnet 14, 1337 Vallorbe, SwitzerlandDepartment of Mammalogy and Ornithology, Natural History Museum of Geneva, Geneva, SwitzerlandJaman’s Group of Faunal Studies, Lausanne, SwitzerlandSwiss Ornithological Institute, Seerose 1, 6204 Sempach, Switzerland
r t i c l e i n f o
rticle history:eceived 22 May 2012eceived in revised form 25 May 2014ccepted 4 July 2014vailable online xxx
From 2007 to 2010, 4558 migrating and breeding birds of 71 species were caught and examined forticks in Switzerland. A total of 1205 specimens were collected; all were Ixodes ricinus ticks except oneIxodes frontalis female, which was found on a common chaffinch (Fringilla coelebs) for the first timein Switzerland. Each tick was analysed individually for the presence of Borrelia spp., Rickettsia spp.,Anaplasma phagocytophilum and tick-borne encephalitis virus (TBEV). Altogether, 11.4% of birds (22species) were infested by ticks and 39.8% of them (15 species) were carrying infected ticks. Bird speciesbelonging to the genus Turdus were the most frequently infested with ticks and they were also carryingthe most frequently infected ticks. Each tick-borne pathogen for which we tested was identified withinthe sample of bird-feeding ticks: Borrelia spp. (19.5%) and Rickettsia helvetica (10.5%) were predomi-nantly detected whereas A. phagocytophilum (2%), Rickettsia monacensis (0.4%) and TBEV (0.2%) were onlysporadically detected. Among Borrelia infections, B. garinii and B. valaisiana were largely predominant fol-lowed by B. afzelii, B. bavariensis, B. miyamotoi and B. burgdorferi ss. Interestingly, Candidatus Neoehrlichia
mikurensis was identified in a few ticks (3.3%), mainly from chaffinches.
Our study emphasizes the role of birds in the natural cycle of tick-borne pathogens that are of humanmedical and veterinary relevance in Europe. According to infection detected in larvae feeding on birdswe implicate the common blackbird (Turdus merula) and the tree pipit (Anthus trivialis) as reservoir hostsfor Borrelia spp., Rickettsia spp. and A. phagocytophilum.
Wild birds play a significant role in the ecology and circulation ofick-borne pathogens. They can disperse infected tick vectors overong distances and across geographical barriers (Olsen et al., 1995;oupon et al., 2006) but they can also act as reservoir hosts in nat-ral foci of disease (Humair et al., 1998). In Europe, passerine birdsre hosts of immature Ixodes ricinus ticks, which are vectors of aide range of zoonotic pathogens. Therefore they can transport andisseminate infected ticks along their migration routes. Migratory
Please cite this article in press as: Lommano, E., et al., Tick-borne paSwitzerland. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.
irds have been found to harbour I. ricinus ticks infected by tick-orne encephalitis virus (TBEV) (Ernek et al., 1968; Waldenströmt al., 2007), A. phagocytophilum (Alekseev et al., 2001b; Paulauskas
et al., 2009; Hildebrandt et al., 2010), Borrelia spp. (Olsen et al.,1995; Gylfe et al., 1999; Poupon et al., 2006; Dubska et al., 2009),Rickettsia spp. (Elfving et al., 2010; Movila et al., 2011) and Can-didatus Neoehrlichia mikurensis (Spitalská et al., 2006). All thesepathogens have been described in questing ticks in Switzerlandwhere they represent a threat for human health (Lommano et al.,2012a).
The introduction of infected ticks by birds into new areas canallow emergence of endemic foci of disease if biotic and abiotic con-ditions are favourable for the maintenance of the pathogen in ticksand vertebrate hosts. In Switzerland, birds have been suspectedto play a role in the emergence of new TBE foci (Lommano et al.,2012b). In fact, at the beginning of 2000, new TBE endemic areas
thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
have been identified in the Western part of the country outside aperimeter where all clinical cases had been reported during the 30previous years (Lommano et al., 2012b). Genotyping of TBEV strainsdetected in questing I. ricinus ticks revealed that they were closely
elated to Swiss strains previously identified in the North and theast of the country, two regions distant from the new TBE endemicrea (Lommano et al., 2012b; Burri et al., 2011a).
Implication of birds in the maintenance of tick-borne pathogennzootic cycles has been widely demonstrated for Borrelia spp. inurope (Humair et al., 1998; Hanincová et al., 2003; Poupon et al.,006; Michalik et al., 2008), in North America (Levine et al., 1991;amer et al., 2011) as well as in Asia (Nakao et al., 1994). In Europe,
he reservoir competence of pheasants (Phasianus colchicus), com-on blackbirds (Turdus merula), song thrushes (Turdus philomelos)
nd puffins (Fratercula arctica) for B. garinii and B. valaisiana waslearly demonstrated (Humair et al., 1998; Kurtenbach et al., 1998;ylfe et al., 1999; Taragel’ova et al., 2008). However, the role ofild birds in the circulation and transmission of Rickettsia spp., A.
hagocytophilum and TBEV is still discussed. They are suspected toe reservoir hosts for A. phagocytophilum (Daniels et al., 2002) andickettsia spp. (Spitalská et al., 2011; Elfving et al., 2010) but theirxact role is still unclear. Concerning TBEV, the avian contributionn the maintenance cycle of TBEV was early suspected (Ernek et al.,968; Hoogstraal, 1972) but could never be clearly demonstrated.
A reservoir host is defined by its capacity to infect ticks feedingn it (Kahl et al., 2002). One method that helps to identify reser-oirs in nature is the comparison of infection rate of questing larvaend nymphs with larvae fed on the suspected host in the sameabitat. A higher infection rate in larvae feeding on tested host is
strong indication that the host is reservoir, particularly in casehe pathogen is not transmitted transovarially from the female tohe eggs. The goal of the present study was to evaluate the rolef avian hosts in the circulation and dissemination of tick-borneathogens like A. phagocytophilum, TBEV, Candidatus N. mikurensiss well as pathogens belonging to the genus Borrelia (B. burgdorferiensu lato complex, B. miyamotoi) and the genus Rickettsia. All theseathogens, except Rickettsia spp., are rarely transmitted transovar-
ally meaning that high infection rate in larvae from birds woulduggest that birds act as reservoirs.
aterials and methods
ird trapping and tick sampling
Birds were captured using Japanese mist nets during the breed-ng period and fall migration. Breeding birds were captured in 2008,009 and 2010 at two sylvatic locations situated between 600nd 650 m above sea level: Bois de l’Hôpital (Canton of Neuchâ-el, 47◦01.00′N, 6◦56.00′E) and Agiez (Canton of Vaud, 46◦43.23′N,◦29.90′E). Both sites are mixed and deciduous forests and wereubject to previous studies concerning prevalence of pathogens inuesting ticks (Jouda et al., 2004; Lommano et al., 2012a,b; Moránadenas et al., 2007). At one site (Agiez), TBEV is known to occur in
ree-living ticks (Lommano et al., 2012b). All birds were carefullyxamined for ticks, especially around the eyes and the beak. Permitsnly allowed banding of some Turdus spp. and Erithacus rubecula.n injured hawfinch (Coccothraustes coccothraustes) found 200 m
rom Bois de l’Hôpital and carrying ticks, was included in the study.From August to October 2007, 2008 and 2009, birds migrat-
ng southwestward (fall migration) were caught and banded byrnithologists during regular ringing work at the Col de Jaman (VD),
Pre-Alpine pass situated at 1512 m above sea level (46◦26.95′N,◦58.41′E), an altitude in Switzerland that is too high for estab-
ishment of I. ricinus population (unpublished data). All attachedicks were removed with forceps, identified to stage and species
Please cite this article in press as: Lommano, E., et al., Tick-borne paSwitzerland. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.
Cotty, 1985) and stored at −20 ◦C until further analysis, using aeparate vial for each bird. Engorgement status was recorded forost ticks. Ticks collected in 2007 were stored in RNAlater® solu-
ion (Qiagen, Hombrechtikon, Switzerland) at 4 ◦C for one week and
PRESSne Diseases xxx (2014) xxx–xxx
then at −20 ◦C until nucleic acid extraction. All birds were releasedimmediately after tick removal.
Nucleic acid extraction
All feeding ticks were individually disrupted and homogenizedwith a mixer mill MM 300 (Retsch, Arlesheim, Switzerland) during5 min in tubes containing 50 �l Tris–EDTA buffer (pH = 8) and a 3-mm ball. Ticks collected in 2007 and stored in RNAlater® solution(Qiagen) were first dried on a paper towel. Lysis was performedby adding 1.5 ml of lysis buffer (bioMérieux, France) in each tubeand incubated during 30 min. Total nucleic acid extraction was per-formed using the NucliSENS® easyMAGTM (bioMérieux, France) andthe protocol provided by the manufacturer. Negative controls, con-sisting of reagents without tick, were included in each extractionsession. A total of 120 �l of eluted nucleic acid was directly dividedinto 5 �l and 10 �l aliquots ready for further amplifications andstored at −20 ◦C. The remaining eluted nucleic acid was dividedinto two aliquots and stored at −20 ◦C (DNA) and −80 ◦C (RNA).
Borrelia spp.
A real-time PCR was used to amplify and detect a 132 bp frag-ment of the flagellin gene of Borrelia spp. (Schwaiger et al., 2001), asdescribed in Herrmann and Gern (2010, 2012). The real-time PCRmixture (25 �l) consisted of 0.4 �M of each primer (FlaF1A andFlaR1), 0.2 �M of TaqMan probe, 0.025 U of KAPATaqTM Hotstart(Kapabiosystems by Labgene Scientific, Switzerland), 10× buffer(provided by the manufacturer), 200 �M dNTPs, 500 �M MgCl2 and5 �l of template DNA. Isolate of B. burgdorferi ss (B31), B. garinii(NE11), B. afzelii (NE632) and B. valaisiana (VS116) were used as pos-itive controls. Each positive sample in real-time PCR was analysedby PCR and RLB in order to identify B. burgdorferi sl genospecies aspreviously described (Herrmann and Gern, 2010, 2012). Amplifica-tion of the intergenic spacer region between 5S and 23S rRNA geneswas performed (Alekseev et al., 2001a). The PCR reaction mixture(25 �l) consisted of each primer (23S-Bor and B5S-Bor), 0.75 U ofTaq polymerase (Qiagen, Hombrechtikon, Switzerland), dNTPs, 10×buffer (provided by the manufacturer) and 5 �l of template DNA.Isolate of B. burgdorferi ss (B31), B. garinii (NE11), B. afzelii (NE632),B. lusitaniae (PotiB1, PotiB2) and B. valaisiana (VS116) were usedas positive controls. A touchdown program (Burri et al., 2007)was used. For Borrelia identification by RLB, PCR products werehybridized to 15 genospecies-specific probes (Gern et al., 2010).
Rickettsia spp.
For the detection and identification of Rickettsia spp., a PCRtargeting a 345 bp fragment of the 23S–5S rRNA internal spacer(Jado et al., 2006) coupled with RLB hybridization was used, asdescribed in Lommano et al. (2012a). Each PCR mixture (25 �l)consisted of 0.5 �M of each primer (RCK/23–5F and RCK/23–5R),200 �M dNTPs, 1.5 U of Taq polymerase (Qiagen), 10× buffer (pro-vided by the manufacturer) and 5 �l of template DNA. DNA of R.conorii, kindly provided by Simona Casati (Instituto di Microbi-ologia, Ticino, Switzerland) and Olivier Péter (Institut Central desHôpitaux du Valais, Sion, Switzerland) was used as positive con-trol. RLB was performed with modifications in the temperatures ofhybridization (48 ◦C) and washing steps (52 ◦C) (Burri et al., 2011b,
thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
modified from Jado et al., 2006). Probes for RLB are described inLommano et al. (2012a). To identify the Rickettsia species of somesamples, citrate synthase gene (gltA) was amplified (Bernasconiet al., 2002) and sequenced.
For the screening of A. phagocytophilum, a 77 bp fragment of thesp2 gene was amplified and detected using a real-time PCR mod-
fied after Courtney et al. (2004) as described in Lommano et al.2012a). The real-time PCR mixture (25 �l) consisted of 0.72 �M ofach primer (ApMSP2f and ApMSP2r), 0.12 �M of TaqMan probepMSP2p-FAM, 0.75 U of KAPATaq Hotstart (Kapabiosystems byabgene Scientific, Switzerland), 200 �M dNTPs, 6 mM MgCl2, 10×uffer (provided by the manufacturer) and 5 �l of template DNA.ositive control consisted of 3 �l of A. phagocytophilum (Webstertrain, kindly provided by Ana Sofia Santos, CEVDI, Portugal). Forurther sequencing and characterization, each positive sample wasubmitted to another amplification reaction targeting the partialroESL heat shock operon (Katargina et al., 2012). Primers HS1 andS6 were used for the first PCR amplification (Sumner et al., 1997).roducts of amplification (1 �l) of the primary reaction were useds template for the nested PCR using primers HS43 and HSVR (Lizt al., 2000; Lotric-Furlan et al., 1998). The results of PCR amplifi-ations (∼1297 bp) were assessed using a 1.5% agarose gel stainedith red gel (Brunschwig, Basel, Switzerland) and visualized underV light.
BEV
TBEV RNA reverse transcription and amplification of the non-oding region localized in 3′ (NCR3′) were performed in an iCyclerBiorad, Reinach, Switzerland) (Lommano et al., 2012b) and modi-ed from Schwaiger and Cassinotti (2003). Reaction volume (25 �l)onsisted of 12.5 �l of reaction mix (containing dNTP’s, 0.04 mMach), primers and probes: 3 �M of F-TBE1, 0.6 �M of R-TBE1nd 0.8 �M of probe TBE-WT, 0.5 �l Superscript III Platinum TaqInvitrogen, Basel, Switzerland, Superscript III Platinum One-stepuantitative system) and 10 �l of template RNA. The TBEV RNAxtract was first reverse transcribed into complementary DNAcDNA) at 42 ◦C for 30 min and then incubated for 10 min at 95 ◦C.irectly after reverse transcription, NCR3′ was amplified at 95 ◦C
or 15 s, 60 ◦C for 1 min during 45 cycles (Schwaiger and Cassinotti,003). A human TBEV isolate was used as positive control (providedy P. de Mendonc a, Ludwig-Maximillians-Universität, München,ermany). Negative control, consisting of 10 �l RNase-free water
Qiagen, Hombrechtikon, Switzerland) was included in each run.
andidatus N. mikurensis
A selected batch of ticks (n = 215, 124 larvae, 90 nymphs and 1emale I. frontalis) from 8 different bird species was screened for theresence of Candidatus N. mikurensis. Analysed ticks included allicks from common chaffinches (n = 69) and breeding birds (excepthe ticks from the hawfinch) (n = 72), and some randomly chosenicks (n = 75) (Tables 3 and 4). For the detection of Candidatus N.
ikurensis, a ∼500 bp fragment of the 16S rRNA gene spanninghe V1 region of Anaplasma spp. and Ehrlichia spp. was amplifiedSchouls et al., 1999, modified by Bekker et al., 2002). Each PCR mix-ure consisted of 0.2 �M of each primer (16S8FE and BGA1B-new),00 �M dNTPs, 0.63 U of Taq polymerase (Qiagen, Hombrechtikon,witzerland), 1 mM MgCl2, 10× buffer (provided by the manufac-urer) and 5 �l of template DNA. Positive control consisted of 3 �l ofNA (Webster strain, kindly provided by Ana Sofia Santos, CEVDI,
Please cite this article in press as: Lommano, E., et al., Tick-borne paSwitzerland. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.
ortugal). A touchdown PCR program described in Tonetti et al.2009) was performed. RLB technique with specific probes was usedo identify Candidatus N. mikurensis (Schouls et al., 1999; Bekkert al., 2002).
PRESSne Diseases xxx (2014) xxx–xxx 3
DNA sequencing
DNA of pathogens that could not be identified at the specieslevel with RLB and other samples that we wanted to characterizewere purified with a purification kit (Promega, Madison, USA) andsequencing of both strands was performed by Microsynth AG (Bal-gach, Switzerland). Sequences were assembled and corrected withthe SeqMan program of the DNASTAR package (Lasergene, Madison,USA). Each corrected sequence was then compared with availablesequences from the international databank (NCBI BLAST) with theuse of ClustalW 2.0.12 (Thompson et al., 1994).
Phylogenetic analyses
In order to compare and relate A. phagocytophilum sequencesobtained here with others, a phylogenetic tree based on the groESLoperon was constructed using Bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and PHYLIP 3.69 (http://evolution.gs.washington.edu/phylip/getme.html) (Felsenstein, 1993). First 1000bootstrap replicates of the sequence data (SEQBOOT) wereexecuted. Then, distance matrices were calculated by usingKimura’s two-parameter model (DNADIST) and analysed bythe neighbour-joining algorithm (NEIGHBOR). Alternatively, theDNAPARS program was used to find the trees with maxi-mum parsimony. The bootstrap support percentages of particularbranching points were calculated from these trees (CONSENSE). Theresulting phylogenetic tree was presented using the program Tree-View 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html)(Page, 1996).
For tree reconstruction, partial DNA sequences (776 bp) of thegroESL operon of different A. phagocytophilum strains, an Ehrlichiaplatys strain (outgroup) and four sequences obtained from host-seeking I. ricinus ticks from Western Switzerland were chosen fromthe NCBI GenBank database.
Statistical analysis
The �2 test was used to analyse the differences in the prevalenceof tick-borne pathogens in ticks, without correction.
Results
Tick infestation of birds
Breeding birds (n = 33), belonging to 11 species, were caughtat two sylvatic sites, Agiez and Bois de l’Hôpital, from 2008to 2010 (17 capture events), and examined for ticks (Table 1).Eighty-one ticks (28 larvae and 53 nymphs) were removed from16 infested birds, giving a mean infestation prevalence of 48.5%(16/33) (no. infested/examined birds). Mean density (no. collectedticks/examined birds (Kahl et al., 2002)) was 2.45 ticks per bird(81/33) and infestation intensity (no. collected ticks/infested birds(Kahl et al., 2002)) reached 5.06 ticks (81/16) (Table 1). Maximumnumber of ticks collected on a single individual was 17. All tickswere determined as I. ricinus, except two damaged nymphs belong-ing to Ixodes spp.
Migratory birds (n = 4525), belonging to 71 species, were caughtat Col de Jaman (VD) from 2007 to 2009 and examined for ticks(Table 2). Only one bird (not infested by ticks) was recaptured. Atotal of 1124 attached ticks (722 larvae, 401 nymphs and 1 female)was collected from 504 infested birds, giving a mean prevalence of11.1% (504/4525). Mean density was 0.25 tick per bird (1124/4525)
thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
and infestation intensity reached 2.23 ticks (1124/504) (Table 2).The maximum number of ticks collected from a single individualwas 15. The majority of ticks were determined as I. ricinus (n = 1091)except one female I. frontalis attached on a common chaffinch
Fringilla coelebs) and 32 specimens, partly damaged, belonging toxodes spp.
Altogether, 520 breeding and migratory birds (22 species)ere carrying 1205 ticks (750 larvae, 454 nymphs and 1 female)
Tables 1 and 2). The redwing (Turdus iliacus) (64.3%), the commonlackbird (T. merula) (52.4%) and the song thrush (T. philome-
os) (27.6%) were the most frequently infested species, and theost heavily infested birds were the common blackbird (3.45
icks/infested bird), the redwing (2.78) and the tree pipit (Anthusrivialis) (2.73). If we consider birds infested by immature I. rici-us stages only (n = 519), 226 (43.5%) were infested by larvae, 16231.2%) by nymphs and 131 (25.2%) were co-infested by both stages.he song thrush (42.9%, 18/42) and the common blackbird (34.1%,5/44) were the most frequently co-infested species and they werelso carrying more nymphs than larvae, followed by tree pipits30%, 36/120) that, in contrast, were infested by more larvae thanymphs (Tables 1 and 2).
ick-borne infections
A total of 1205 ticks (750 larvae, 454 nymphs and 1 female) col-ected from 16 breeding and 504 migratory birds were individuallycreened for the presence of Borrelia spp., Rickettsia spp., A. phago-ytophilum, TBEV and Candidatus N. mikurensis. Altogether, 39.8%207/520) of infested birds were carrying infected ticks. Globally,0.8% (371/1205) of ticks collected from birds were infected with at
east one pathogen species (Table 3) and 5.8% (70/1205) were har-ouring more than one pathogen. Nymphs (175/454, 38.5%) wereignificantly more infected than larvae (196/750, 26.1%) (�2 test,
< 0.0001) though relative infection rate of larvae versus nymphsiffered by pathogen (Table 3). The single female I. frontalis wasot infected and therefore we considered only immature ticks inhe infection rates mentioned below. Ticks from breeding birdsere more infected (44.4%, 36/81) than those from migrating birds
29.8%, 335/1124) but this was not significant (Table 3).Ticks collected from birds belonging to Turdus species were
ighly infected by pathogens (136/228, 59.6%) and ticks collectedrom tree pipits (95/328, 29%) were significantly more infectedhan ticks collected on European robins (81/484, 16.7%) (�2 test,
< 0.0001) (Table 3).The most prevalent pathogens in ticks collected from breed-
ng and migratory birds were Borrelia spp. (19.5%), followed byickettsia spp. (12.3%), Candidatus N. mikurensis (3.3%), A. phago-ytophilum (2%) and TBEV (0.2%) (Table 3).
ickettsia spp.
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Rickettsial infections were detected in 12.3% of ticks (148/1204)Table 3). Larvae (13.6%, 102/750) were more infected than nymphs10.1%, 46/454), but this was not significant. Prevalence of Rickettsiapp. in ticks feeding on migrants (11.9%, 134/1123) was lower than
32 (0;32) 166 (3;3) 6
81 (28;53) 5.06
in ticks feeding on breeding birds (17.3%, 14/81) without statisti-cal significance (Table 3). The common blackbird (11/44, 25%) andthe tree pipit (28/120, 23.3%) were the species the most frequentlyinfested with Rickettsia-infected ticks. However, in ticks from treepipits (15.9%, 52/328) the prevalence was higher than the one inticks from common blackbirds (11.8%, 18/152). Interestingly, theprevalence reached 75% (9/12) in ticks from one tree pipit and 40%(6/15) in ticks from one common blackbird. Larvae from tree pip-its (17.3%, 42/243) were more infected than nymphs (11.8%, 10/85)and in addition larvae feeding on individuals infested by larvae onlyshowed the same infection prevalence (17.9%) that larvae feedingwith nymphs (16.3%).
Among Rickettsia spp. infections identified with RLB (n = 148),R. helvetica was predominant with 126 occurrences (85.1%, 85 lar-vae and 41 nymphs) (Table 3). R. monacensis (3.4%) was identifiedin ticks from five migratory birds: two robins (two larvae), oneEuropean serin (Serinus serinus) (one larva), one common blackbird(one nymph) and one song thrush (one nymph). GenBank acces-sion numbers for the 23S–5S partial sequences of R. monacensis are:JQ838250, JQ838251, JQ838252 and JQ838253. Seventeen Rickettsiaspp. infections (11.5%, 14 larvae and three nymphs) were identi-fied at the genus level only. Sequencing part of the gltA gene of2/17 Rickettsia sp. obtained from larvae feeding on two Europeanrobins (accession numbers JQ922509 and JQ922510) revealed 100%homology with another unidentified Rickettsia sp. sequence fromone tick feeding on one European robin (JN849396). European robinwas the main host for ticks infected with Rickettsia sp. that couldnot be identified at the species level (64.7%, 10/14 larvae and 1/3nymphs).
Candidatus N. mikurensis
Seven of the 215 ticks (3.3%) screened for the presence of Can-didatus N. mikurensis were infected. The pathogen was detectedexclusively in nymphs and from migrants (Table 3). Interestingly,85.7% (6/7) of infected nymphs were from four common chaffinchesand 24% (6/25) of nymphs feeding on that species were infected,with a prevalence reaching 21.4% (3/14) in ticks from one individ-ual.
A. phagocytophilum
A. phagocytophilum DNA was detected in 24 ticks feeding oneleven birds: seven common blackbirds, two European robins,one song thrush and one chaffinch (Table 3). All infected larvae(8/8) and 75% (12/16) of infected nymphs were feeding on com-
thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
mon blackbirds, resulting in a global prevalence of 13.2% (20/152)(Table 3). The highest prevalence (54.5%, 6/11) was observed inticks collected from one common blackbird. Seven of the eightA. phagocytophilum-infected larvae were carrying simultaneously
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Table 2Prevalence and intensity of tick infestation of migratory birds captured at the Col de Jaman in summer–autumn 2007, 2008 and 2009.
Table 3Prevalence of Borrelia spp., Rickettsia spp., A. phagocytophilum (A. phago), TBEV and Candidatus N. mikurensis (C. N. mikurensis) in immature I. ricinus ticks (L, larva; N, nymph) from breeding and migratory birds.
Bird species No. birds with infectedticks/no. infested birds (%)
No. tested ticks No. infected ticks No. ticks infected with atleast 1 pathogen species (%)
L N Borrelia spp. Rickettsia spp. A. phago TBEV C. N. mikurensis L N
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F gocytf
oa
i(nlwa(bbnl
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ig. 1. Phylogenetic tree based on partial groESL operon sequences (776 bp) of A. pharom GenBank database. Only bootstrap values of >60% are shown.
ther pathogen species, namely B. garinii (n = 2), B. valaisiana (n = 5)nd R. helvetica (n = 1).
From the three groESL operon sequences obtained by sequenc-ng (∼1200 bp), two (from nymphs collected from one chaffinchaccession number JX082324) and one European robin (accessionumber JX082325)) were 100% identical to each other. On the phy-
ogenetic tree based on the groESL operon (776 bp), they clusteredith sequences belonging to lineage 2, obtained from I. ricinus
nd roe deer (Capreolus capreolus) and described as nonpathogenicKatargina et al., 2012) (Fig. 1). The third sequence (accession num-er JX082323), obtained from one larva feeding on one commonlackbird, showed only 97% homology with the two others (22/777ucleotides difference) and formed a separate branch on the phy-
ogenetic tree (Fig. 1).
BEV
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Two larvae feeding on two European robins and one nymphrom one common blackbird (all captured at Col de Jaman) werenfected by TBEV (Table 3). Sequencing of TBEV NS5 gene failed forll three samples. None of the ticks feeding on birds captured at
ophilum found in ticks feeding on birds (bold), in comparison to sequences obtained
Agiez (known as TBE endemic area) (n = 33) and at Bois de l’Hôpital(n = 48) was infected by TBEV (Table 3).
Borrelia spp.
Borrelia spp. was detected in 30.9% (25/81) and 18.7% (210/1123)of ticks collected from breeding and migratory birds, respectively(Table 4), giving a global prevalence of 19.5% (235/1204). Nymphs(26.9%, 122/454) were significantly more infected than larvae(15.1%, 113/750) (�2 test, p < 0.0001) and that was observed forboth breeding (37.7% vs 17.9%), although not significant, and migra-tory birds (25.4% vs 15%, p < 0.0001). Conversely, larvae feeding oncommon blackbirds (65.9%, 27/41) and tree pipits (19.3%, 47/243)were more infected than nymphs (50.4%, 56/111 and 16.5%, 14/85,respectively) but this was not significant. The common blackbird(75%, 33/44) was the species carrying the most frequently Borre-lia spp. infected ticks and 54.6% (83/152) of ticks feeding on that
thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
species were infected (Table 4).Six different Borrelia genospecies were identified in ticks from
migratory birds (Table 4). B. garinii (n = 91) and B. valaisiana (n = 75)were the most frequent followed by B. afzelii (n = 18), B. bavariensis
Table 4Prevalence of Borrelia genospecies in immature I. ricinus ticks (L, larva; N, nymph) collected from breeding and migratory birds. B.b. ss: B. burgdorferi sensu stricto; Borrelia sl: Borrelia spp. matching only with the SL1 probe (RLB);Borrelia sp.: Borrelia sp. that could not be identified with RLB but was positive with real-time PCR.
Bird species No. of tested ticks No. of infected ticks (%) No. ticks with mixedinfections (L + N)
L N Borrelia spp. B. afzelii B. garinii B. valaisiana B. b. ss B. bavariensis B. miyamotoi Borrelia sl. Borrelia sp.
n = 8), B. miyamotoi (n = 6) and B. burgdorferi ss (n = 3) (Table 4).n addition, 34 Borrelia infections could be identified at the genusevel only, among which 33 were positive with real-time PCR only24 larvae and 9 nymphs) and one was real-time PCR positivend hybridized only on the genus probe (SL) with RLB (Table 4).equencing of the latter was not successful. In ticks collected fromreeding birds (n = 81), five genospecies were identified with B.arinii and B. valaisiana being predominant (Table 4). In contrast toicks collected from migrants, B. afzelii was not detected in theseicks.
Considering Borrelia species in ticks from migrant and breed-ng birds, B. garinii was predominantly identified in infected ticksrom tree pipits (74.5%, 35/47 identified Borrelia species) whereas B.alaisiana was predominant in infected ticks from common black-irds (75%, 60/80 identified Borrelia species) (Table 4). Among B.fzelii infections, 94.4% (17/18) were detected in nymphs and inter-stingly most of them (66.7%, 12/18) in only slightly engorged ticks.. bavariensis was more frequently found in larvae (88.9%, 8/9) than
n nymphs (11.1%, 1/9) and most B. bavariensis infected larvae (5/8)ere collected from tree pipits.
Mixed Borrelia infections involving more than one Borreliaenospecies were detected in 30/235 (12.8%) ticks (collectedrom breeding and migrating birds): 2 different genospecies werebserved in 29 ticks (10 larvae and 19 nymphs) (Table 4) andhree genospecies (B. garinii, B. valaisiana and B. miyamotoi) werebserved in one larva from one tree pipit. Most mixed infec-ions (with two different genospecies) associated B. garinii and. valaisiana (21/30). The other associations were B. afzelii/B.alaisiana (n = 3), B. valaisiana/B. bavariensis (n = 2), B. afzelii/B.arinii (n = 1), B. garinii/B. miyamotoi (n = 2) and B. valaisiana/B.iyamotoi (n = 1). Mixed infections were predominantly observed
n ticks from common blackbirds (53.3%, 16/30). Interestingly, inddition to the fact that ticks from tree pipits were mainly infectedy B. garinii, no mixed infection, except the triple infection (seebove), was found in these ticks.
o-infections
Among all infected ticks (n = 371), we observed more co-nfections involving different pathogen species (n = 43) than mixednfections (with more than one Borrelia species) (n = 30) (seebove). Most co-infections were B. garinii/R. helvetica (n = 19)nd B. valaisiana/A. phagocytophilum (n = 10). Interestingly, most. garinii/R. helvetica associations were found in ticks from treeipits (13/19) whereas most B. valaisiana/A. phagocytophilum asso-iations were detected in ticks from common blackbirds (9/10).o-infections involving B. valaisiana, A. phagocytophilum and R. hel-etica were found in two ticks from common blackbirds: one larvand one nymph (which was harbouring B. garinii as well).
nfection prevalence of birds
Considering birds as infected when at least two infected larvaeere feeding on them, T. merula was the bird species that showed
he highest prevalence of infection (20.5%), followed by A. trivialis15.8%) (Table 5). Both bird species were mainly infected by Borreliapp. (15.9% and 10%, respectively), although 5.8% of tree pipits werenfected by Rickettsia spp. and 4.6% of common blackbirds by A.hagocytophilum.
iscussion
Please cite this article in press as: Lommano, E., et al., Tick-borne paSwitzerland. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.
In Europe, birds are hosts of various tick species, among which I.icinus is the most common (Papadopoulos et al., 2002). I. ricinus ishe vector of many tick-borne pathogens of medical and veterinaryelevance. Here, I. ricinus was clearly the dominant tick species on
PRESSne Diseases xxx (2014) xxx–xxx 9
birds. One I. frontalis female was found on one chaffinch for the firsttime in Switzerland. In addition to be hosts for ticks, birds play animportant role in the dispersal of ticks and tick-borne pathogenicagents (Olsen et al., 1995; Elfving et al., 2010). Here we report theoccurrence of eleven tick-borne pathogens in bird-feeding ticks: B.garinii, B. valaisiana, B. afzelii, B. bavariensis, B. miyamotoi, B. burgdor-feri ss, R. helvetica, R. monacensis, A. phagocytophilum, TBEV andCandidatus N. mikurensis.
Birds as sources/reservoirs of tick-borne pathogens
Birds are known reservoir hosts for B. garinii and B. valaisiana(Humair et al., 1998; Kurtenbach et al., 1998; Hanincová et al., 2003;Taragel’ova et al., 2008). Therefore the predominance of B. gariniiand B. valaisiana among the six Borrelia genospecies identified inticks attached to birds in this study is not surprising (Humair et al.,1998; Kurtenbach et al., 1998). Unlike all other bird species, larvaefrom common blackbirds and tree pipits were more infected thannymphs. Since questing larvae usually show a very low prevalenceof infection (Richter et al., 2012) this may indicate these bird speciesas sources of infection and accordingly as reservoir hosts. Thus, treepipits seemed to play a role in the transmission of Borrelia spp., andmore precisely of B. garinii since most larvae feeding on them wereinfected with this genospecies.
In addition, to these two bird-associated Borrelia genospecies,two rodent-associated genospecies, B. bavariensis (Huegli et al.,2002; Margos et al., 2009) and B. afzelii (Humair et al., 1999) wereidentified in bird-feeding ticks. The detection of B. bavariensis inmost feeding larvae from tree pipits suggests that tree pipits maybe competent reservoirs for this genospecies. Concerning B. afzelii,it was identified in 18 ticks. Interestingly, B. afzelii is currently moreand more frequently reported in bird-feeding ticks, and even inlarvae (Humair et al., 1998; Poupon et al., 2006; Taragel’ova et al.,2008; Dubska et al., 2009; Franke et al., 2010) suggesting that birdscan act as reservoir hosts for this genospecies (Franke et al., 2010).We suggest that some B. afzelii variants, showing signs of adap-tation to birds, can survive in the gut of ticks during feeding onbirds and that birds could be reservoirs for these B. afzelii vari-ants. This suggestion could be paralleled with the observation inrodents (Huegli et al., 2002) of OspA serotype 4 spirochetes of B.garinii, a bird associated species (Humair et al., 1998), which arenow recognized as belonging to B. bavariensis (Margos et al., 2009).Interestingly, specific lineages of B. burgdorferi sensu stricto in bird-feeding ticks were recently reported in North America (Brinkerhoffet al., 2010; Mathers et al., 2011) suggesting host specialization inNorth American system as well.
B. miyamotoi, a relapsing-fever like spirochete, was detected inthree larvae and four nymphs feeding on birds. This microorganism,that is pathogenic for humans, has been previously described in dif-ferent tick species collected from various hosts, including birds, aswell as in host tissues in Eurasia and North America (see Burri et al.,2014). In Switzerland, Apodemus and Myodes rodents (captured atthe same site as the studied breeding birds) have been identified inthe laboratory as reservoirs transmitting B. miyamotoi to 23.8% ofxenodiagnostic ticks (Burri et al., 2014). Due to low infection preva-lence of B. miyamotoi in bird-derived ticks and because B. miyamotoiis known to be transmitted transovarially (Scoles et al., 2001), ourobservations require additional studies to evaluate the reservoirstatus of birds in Europe.
The contribution of birds in the natural cycle of Rickettsia spp.remains unclear although birds are known to transport Rickettsia-infected ticks (Spitalská et al., 2011; Elfving et al., 2010; Movila
thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
et al., 2011). In this study, we report the presence of Rickettsia spp.in bird-feeding ticks for the first time in Switzerland. The preva-lence observed (12.3%) is in line with what was reported frommigratory birds in Sweden (11.3%) (Elfving et al., 2010) and with
prevalence observed in free-living ticks in Switzerland (10.2%)Lommano et al., 2012a). Besides I. ricinus, which is vector andeservoir of R. helvetica (Parola et al., 2005), it is not clear whichertebrate can act as reservoir host for the bacteria. A recent studyesigned small mammals as potential efficient source of infectionSchex et al., 2011) but the reservoir competence of some birdselonging to the genus Parus (family Paridae) for Rickettsia spp.as also suggested by others (Spitalská et al., 2011; Elfving et al.,
010). Here, the reservoir competence of tree pipits for R. helveticas suggested since 5.8% of tree pipits were infested by at least twonfected larvae and larvae (17.3%) were more infected than nymphs11.8%). Furthermore, larvae feeding with nymphs (16.3%) showedhe same infection prevalence as larvae feeding alone on host (with-ut nymphs) (17.9%), suggesting that co-feeding transmission is nothe main mean of transmission and that birds may be the source ofnfection. Common blackbirds seem also important in the life cyclef Rickettsia, since they are the species carrying the most frequentlynfected ticks (25% of birds) and because prevalence of ticks col-ected from one bird reached 40%. Rickettsiaemic birds, rather thano-feeding transmission, seemed to be the most probable source ofnfection for ticks. Thus, common blackbirds and tree pipits mightepresent efficient sources for infecting ticks with Rickettsia spp. inature.
In addition to R. helvetica, sequencing of gltA gene and 23S–5Snternal spacer of Rickettsia spp. infections in two larvae feeding onuropean robins showed gltA nucleotide sequences 100% similaro another sequence obtained from a tick feeding on one Europeanobin as well (L. Dubska, unpublished). Moreover, the Europeanobin was, in the present study, the main host (64.7%) for ticksnfected with unidentified Rickettsia sp., among them 10 larvae andne nymph. We could therefore wonder whether European robinsransmit a particular Rickettsia species to ticks attached on them.
A. phagocytophilum has been identified in ticks feeding on birds,nd even in larvae suggesting a potential role of birds as reser-oir hosts (Daniels et al., 2002; Paulauskas et al., 2009). In thistudy, only 2% of bird-feeding ticks were infected with A. phagocy-ophilum, which is in line with previous reports (Hildebrandt et al.,010; Franke et al., 2010). But surprisingly, 4.6% of common black-irds were infested by at least two infected larvae, 13.2% of ticksrom common blackbirds were carrying the bacteria and all infectedarvae were feeding on that bird species. Since transovarial trans-
ission of A. phagocytophilum (Ogden et al., 1998) and transmissiony co-feeding (Rar and Golovljova, 2011) seem to be inefficientr absent, infection of larvae should result from infectious bloodeals. At Bois de l’Hôpital, among breeding birds, we observed that
ird-feeding ticks (6.3%) and especially the ones feeding on com-on blackbirds (9.4%) (data not shown) were more infected by
. phagocytophilum than free-living ticks (2.8%) (Lommano et al.,012a). An infection prevalence of 54.5% was even observed inicks from one common blackbird, supporting the fact that com-
on blackbirds are a source of infection for ticks. This statement
Please cite this article in press as: Lommano, E., et al., Tick-borne paSwitzerland. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.
s strengthened by the finding of an unusual nucleotide sequencef A. phagocytophilum in one larva feeding on a common blackbird,hich may belong to a new genetic variant adapted to its avian host.
nterestingly, most A. phagocytophilum-infected larvae (7/8) were
0/42 0/42
simultaneously harbouring other pathogen species like B. garinii,B. valaisiana, for which birds are known reservoirs (Humair et al.,1998; Kurtenbach et al., 1998) and R. helvetica (see this study). Thismay suggest an immunosuppressive effect of A. phagocytophilumin birds, particularly in common blackbirds since an immunosup-pressive role of this bacteria, which allows secondary infectionsin mammals, has been previously described (Woldehiwet, 2010).However, this needs further investigation.
Wild rodents appear to be reservoir hosts for Candidatus N.mikurensis (Andersson and Raberg, 2011). Whether other verte-brates are of importance in the natural cycle of the parasite isstill unknown. To our knowledge this pathogen has been describedonly once in ticks feeding on birds (Spitalská et al., 2006). In thisstudy, the prevalence of Candidatus N. mikurensis in ticks from birdswas lower (3.3%) than the prevalence observed in questing ticksin Switzerland (6.4%) (Lommano et al., 2012a). Moreover, no larvawas infected by this pathogen suggesting that birds are less likelyto play a role as reservoir hosts. Nevertheless, the findings that 24%(6/25) of nymphs from chaffinches were infected with CandidatusN. mikurensis, that 6/7 infected ticks were obtained from this birdspecies and that 21.4% (0/8 larvae and 3/6 nymphs) of ticks fromone individual were infected, question about the exact role of thisbird species in the natural cycle of Candidatus N. mikurensis.
Birds as transporters of infected ticks
Due to low infection prevalence in bird-derived ticks, we werenot able to assess the reservoir potential of birds for a number ofpathogens including R. monacensis that was rarely identified (3.4%)and only in ticks from migrants. R. monacensis was identified in free-living ticks at Agiez (Lommano et al., 2012a), one of the site wherebreeding birds were caught, but its low prevalence at this site (<1%, Lommano et al., 2012a) may explain its absence in ticks (0/33)from birds at this site. The reservoirs for R. monacensis remain to beidentified although infected ticks were removed from one rodentin Switzerland (Burri et al., 2011b) and from birds in Germany,Sweden and Russia (Hildebrandt et al., 2010; Elfving et al., 2010;Movila et al., 2011). Our observation and these reports suggest thatbirds play a role in the transport of R. monacensis-infected ticks.
Similarly, in the present study, TBEV was detected in ticks, allcollected from migratory birds. None of the birds caught at the TBEVendemic site (Agiez) was carrying infected ticks. The low num-ber of bird-feeding ticks collected at this site (n = 33) in relationwith the low prevalence in questing ticks (0.34%, Lommano et al.,2012b) may explain the absence of TBEV in our samples. The factthat we detected TBEV in ticks from migratory birds can explainthe emergence of new TBE endemic area in Western Switzerland(Lommano et al., 2012b). However, the role of birds in the main-tenance of TBEV in endemic foci cannot be excluded. In fact, twolarvae from European robins were infected supporting the hypoth-esis of Waldenström et al. (2007) that this bird species may be
thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
reservoir of the virus. A co-feeding transmission of TBEV betweeninfected nymphs and uninfected larvae, a mechanism necessaryfor the maintenance of the virus in nature (Labuda et al., 1993),could not be excluded in this study. Although both infected larvae
ere not feeding with nymphs, it is possible that infected nymphsetached just before capture of the birds.
onclusion
Here, we showed that birds act as transporters of R. monacensis-nd TBEV-infected ticks and as reservoir hosts for Borrelia spp., Rick-ttsia spp. and A. phagocytophilum. Globally, 11.1% of birds werenfested by ticks with an intensity of 2.23 ticks per infested host.hese observations are similar to those from another previoustudy undertaken in Switzerland (Poupon et al., 2006) reporting arevalence of 18.2% and 2.32 ticks per infested migratory bird. Mostirds captured during the fall migration southward over the Alpsravelled through areas where I. ricinus ticks may have a secondeak of questing activity during this period of the year explain-
ng these infestations. According to our results, common blackbirdsere the most frequently and heavily infested birds and 83.3% of
nfested individuals were carrying infected ticks. They representlso an efficient source of infection since 70.7% of larvae feedingn this species were infected with at least one pathogen and 29.3%ere harbouring more than one pathogen. Moreover, they were
requently infested with both larvae and nymphs allowing for aotentially significant role in co-feeding transmission of tick-borneathogens.
Rodents are commonly designated as important hosts and reser-oirs of numerous tick-borne pathogens. However, our findingshat more than 30% of bird-feeding ticks were infected with at leastne of the eleven pathogens investigated and that almost 40% ofnfested birds were carrying infected ticks emphasize the impor-ant role of birds in the natural cycle of tick-borne pathogens thatre of human medical and veterinary relevance.
cknowledgments
Results are parts of the Ph.D. thesis of Lommano E. This work wasnancially supported by the Swiss National Foundation (grants no.20000-113936/1 and no. 310030-127064/1). Breeding birds wereaptured with permission of the Federal Office for the Environment.
We would like to thank ADMED (Laboratory of Medical Micro-iology, La Chaux-de-Fonds, Switzerland) for lending extractionachine and Reto Lienhard for his valuable advices. We particularly
hank Mégane Pluess for her precious technical assistance.We thank all the ornithologists working at Col de Jaman (Groupe
’études faunistiques de Jaman) for the precious help in collectingicks on birds, especially L. Maumary, R. Fürst, A. Gerber and S.rogin. We are thankful to R. Béguelin, C. Burri and E. Patalas (Insti-ute of Biology, University of Neuchâtel, Switzerland) and all othereople for their help in birds trapping; M. Dvorak, M. Quartier foraluable help and advice.
We are also grateful to A. S. Santos (CEVDI, Portugal), S.asati (Instituto di Microbiologia, Ticino, Switzerland), O. PéterInstitut central des Hôpitaux du Valais, Sion, Switzerland)nd P. de Mendonc a (Ludwig-Maximillians-Universität, München,ermany) for providing positive controls and I. Golovljova for therotocol for sequencing of A. phagocytophilum. Thank you to thewo reviewers for their useful comments.
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thogens in ticks collected from breeding and migratory birds inttbdis.2014.07.001
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