The Importance of Ixodes arboricola in Transmission of Rickettsia spp.,Anaplasma phagocytophilum,and Borrelia burgdorferi Sensu Lato in the Czech Republic,Central Europe

Introduction

F ree-living birds have the potential to disperse pathogenic microorganisms by three mechanisms, as biological carriers, mechanical carriers, and hosts or carriers of infected ectoparasites (Hubálek 2004). Birds play a role as reservoir of some species of the Borrelia burgdorferi sensu lato species complex and they can also carry ticks infected with Rickettsia spp. and Anaplasma phagocytophilum (Poupon et al. 2006, Špitalská et al. 2006, Ogden et al. 2008, Taragel'ová et al. 2008, Dubská et al. 2009, Ionnou et al. 2009, Elfving et al. 2010, Hildebrandt et al. 2010).

In total, 15 tick species have been reported in the Czech Republic (Dusbábek 1995). Wild birds were found parasitized by Ixodes (Pholeoixodes) arboricola (Schulze et Schlottke, 1929), Ixodes ricinus (Linnaeus, 1758), and Haemaphysalis concinna (Koch, 1844) (Hubálek et al. 1996, Literák et al. 2007).

Ticks of the family Ixodidae, obligate hematophagous ectoparasites, are vectors for medically important zoonoses worldwide. I. ricinus is a known vector for tick-borne encephalitis virus, RNA flaviviruses (causing louping ill), genospecies of the B. burgdorferi s.l. species complex (causing Lyme borreliosis), A. phagocytophilum, Anaplasma marginale (anaplasmosis), Ehrlichia equi (ehrlichiosis), R. helvetica, Rickettsia conorii (rickettsiosis, Boutonneuse fever), Coxiella burnetii (Q fever), Babesia divergens, Babesia bovis, and Babesia ovis (babesiosis). This tick species is endemic in most of Europe and North Africa.

I. ricinus is a three-host tick and immature stages parasitize mainly rodents, birds, and lizards (Gern and Humair 2002). Adult ticks have a host preference for larger vertebrates and occasionally bite humans (Tälleklint and Jaenson 1994).

I. arboricola is an ornithophilous tick with very narrow host specificity; it infests birds breeding or roosting in tree holes and also bats (Rosický 1953; Černý 1972; Filippova, 1977; Krištofík et al. 1993, 2003; Krumpál et al. 1995; Siuda et al. 2006). The life cycle of I. arboricola is not completely known (Siuda 1993). To the best of our knowledge, any association of this tick species with viruses, bacteria, or protozoa has not been described to date.

The third tick species included into our analyses is H. concinna, which infests a wide range of mammals but is also partly ornithophilous. For adults, very important hosts are Artiodactyla (Nosek et al. 1971). This tick species serves as a vector for tick-borne encephalitis virus and can also be infected with C. burnetii, Francisella tularensis, Rickettsia sibirica, Rickettsia hulinii, Ehrlichia spp., and Anaplasma spp. (Labuda and Randolph 1999, Khazova and Iastrebov 2001, Shpynov et al. 2004, Sréter-Lancz et al. 2006). The role of H. concinna as B. burgdorferi s.l. vector has been discussed (Sun and Xu 2003, Chu et al. 2008). It is widely distributed in the forests of Eurasia.

In the present study, we collected immature stages of three tick species, I. arboricola, I. ricinus, and H. concinna, from different species of birds in the Czech Republic. Infection of these ticks with bacteria such as Rickettsia, Anaplasma, and Borrelia was analyzed using molecular techniques. Our data show, for the first time, a high infection rate of I. arboricola with Rickettsia spp.

Materials and Methods

Results

A total of 549 subadult ticks, 412 I. arboricola (75.0%), 127 I. ricinus (23.1%), and 10 H. concinna (1.8%) were collected from 120 birds of 20 species (Table 1). A total of 96 I. arboricola nymphs were collected from five bird species: S. europaea (1.0%), E. rubecula (1.0%), Periparus ater (2.1%), C. caeruleus (2.1%), and P. major (93.8%). P. major was also the dominant host for I. arboricola larvae, as 91.8% of larvae were collected from this bird species. Twenty-two larvae (7.0%) were collected from P. palustris and 4 (1.3%) from C. caeruleus. I. ricinus ticks were found on all species of birds. However, not all bird species carried both stages (Table 1). Nymphs were not found on S. europaea, Anthus pratensis, and P. ater, and larvae were collected only from S. europaea, E. rubecula, Turdus merula, Acrocephalus scirpaceus, A. palustris, Carpodacus erythrinus, A. pratensis, P. modularis, C. caeruleus, and P. major. The majority of I. ricinus ticks were found on P. major, 45.1% of larvae and 22.4% of nymphs. H. concinna ticks (nine larvae, one nymph) were collected only from Acrocephalus schoenobaenus.

In I. arboricola ticks, 44.0% larvae and 24.5% nymphs harbored rickettsiae infections. Rickettsia DNA was detected only in I. arboricola ticks collected from P. major (43.4% of larvae and 25.6% of nymphs) and P. palustris (59.1% of larvae) and in nymph feeding on S. europaea. Rickettsiae-positive I. ricinus larvae (13.7%) were collected from P. major (8.7%), C. caeruleus (40.0%), and S. europaea (42.9%), and 2.6% of nymphs from E. rubecula (25.0%) and P. modularis (12.5%) (Table 3).

Sequence comparison of the amplified gltA gene fragment identified R. helvetica and other Rickettsia spp. (Fig. 1). R. helvetica was found in I. ricinus nymph from P. modularis (128 IRN; GenBank accession number: JF303666). Rickettsial species identification was based on sequence similarities between amplicons from the Rickettsia in I. ricinus and R. helvetica sequences available in GenBank. The PCR amplicon obtained in the present study was similar to those of R. helvetica, including a 97.3% similarity of a 265 bp stretch of the gltA gene to isolates of R. helvetica (GenBank accession numbers: EU779822, EU596563, DQ910785, and AM418450). Undescribed Rickettsia spp. were detected in I. arboricola larvae (37 IAL, 195 IAL; GenBank accession numbers: JF303665 and JF303663) and nymph (100 IAN; GenBank accession number: JF303664) collected from P. major. From these samples, the gltA, ompA, and sca4 genes were amplified and sequenced. PCR amplicons of the gltA gene were 98.6%–100% similar to that of Rickettsia heilongjiangii, Rickettsia aeschlimannii, Rickettsia raoultii, R. sibirica, and Candidatus Rickettsia antechini (GenBank accession numbers: EU365689, DQ235776, DQ365804, DQ124930, and DQ372954). Fragments of 632 and 650 bp of the ompA and sca4 genes, respectively, were successfully amplified, but these sequences did not show any similarity with available sequences in GenBank.

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FIG. 1.

Evolutionary relationship of Rickettsia spp. inferred from the comparison of gltA sequences by the neighbor-joining method. Sequences obtained in the present study (37 IAL, 100 IAN, 128 IRN, and 195 IAL) were compared with sequences downloaded from GenBank. The numbers at nodes are the bootstrap values obtained from 100 resamplings. Scale bar indicates a 1% difference in nucleotide sequences.

A. phagocytophilum was found in two I. ricinus nymphs collected from E. rubecula and P. major only.

Infections with B. burgdorferi s.l. were recorded in 1.3% larvae of I. arboricola acquired from P. palustris and P. major and in 11.8% larvae and 25.0% nymphs of I. ricinus collected from P. major, P. palustris, C. caeruleus, A. schoenobaenus, T. merula, C. erythrinus, S. atricapilla, P. modularis, and P. collybita. RLB hybridization based on 5S–23S intergenic spacer identified B. garinii, B. valaisiana, and mixed infection with these two genospecies (Table 4).

Coinfection of Rickettsia with A. phagocytophilum was established in one I. ricinus nymph and Rickettsia with B. garinii in two I. arboricola and one I. ricinus larvae.

Discussion

The role of birds in transmission of rickettsiae and A. phagocytophilum is still discussed and only limited data are available. In this study, we have investigated three tick species, I. arboricola, I. ricinus, and H. concinna, collected from birds in the Czech Republic for the presence of tick-borne pathogens. I. arboricola was the most abundant tick species recovered from birds, with highest infestations found on P. major. Both larvae and nymphs of I. arboricola and I. ricinus were found positive for Rickettsia and Borrelia. However, larvae of both I. arboricola and I. ricinus ticks showed higher infection prevalences with rickettsiae than nymphs. P. major and P. palustris were more likely to carry rickettsiae-infected larvae than other tested bird species. C. caeruleus, S. europaea, E. rubecula, and P. modularis were involved in the maintenance of rickettsiae in nature in this study. More precise identification of rickettsiae will be the aim of the next study.

Detection of rickettsiae in ticks collected from wild birds dates back to 1964, when Somov and Soldaton (1964) isolated R. sibirica from H. concinna in the Far East. In recent years, the European robin was found as a carrier of Rickettsia-positive I. ricinus nymph in Slovakia (Špitalská et al. 2006). In previous studies, R. helvetica- and Rickettsia monacensis-infected I. ricinus larvae and nymphs were found to be carried by E. rubecula, Turdus iliacus (Redwing [Linnaeus, 1766]), T. merula, Turdus philomelos (Song Trush [Brehm, 1831]), and P. major captured in Germany (Hildebrandt et al. 2010). Moreover, Elfving et al. (2010) identified Rickettsia infections in I. ricinus and Ixodes lividus larvae and nymphs from C. caeruleus and T. iliacus captured in Sweden. Partial sequences of the 17 kDa ompB, gltA, and ompA genes reported by Elfving et al. showed the greatest similarity to Rickettsia spp. strain Davousti, Rickettsia japonica, and Rickettsia heilongjiangensis. In Portugal, R. helvetica was identified in male Ixodes ventalloi (Gil Collado, 1936) collected from Asio flammeus (the Short-Eared Owl [Pontoppidan, 1763]); R. aeschlimannii was found in three nymphs of Hyalomma marginatum (Koch, 1844) collected from Alcedo athis (the Common Kingfisher [Linnaeus, 1758]), Athene noctua (the Little Owl [Scopoli, 1769]), and Bubo bubo (the Eurasian Eagle Owl [Linnaeus, 1758]); and Rickettsia massiliae was found in female Rhipicephalus turanicus (Pomerantsev, 1936) from Buteo buteo (the Common Buzzard [Linnaeus, 1758]) (Santos-Silva et al. 2006). Rickettsia spp. were identified in 1.5% of pools of blood from Fulica atra (the Eurasian coot [Linnaeus, 1758]), Phoenicopterus ruber (the American flamingo [Linnaeus, 1758]), and I. ventalloi collected on Alectoris chukar (the Chukar [Gray, 1830]) in Cyprus (Ionnou et al. 2009). All these studies suggest that ticks from wild birds could transmit rickettsiae. In addition, our results suggest a putative role of further species of ticks—I. arboricola feeding on P. major and P. palustris—in rickettsial transmission cycles.

There are several mechanisms by which tick species feeding on vertebrates can acquire pathogens. First, ticks could acquire the infection through a transovarial route. Although this is rare for Borrelia species, it has been documented for Rickettsia (Parola et al. 2005). Other potential routes are via cofeeding with infected I. arboricola and/or I. ricinus or ticks could acquire the bacteria from the blood of infected birds. Birds can be infested with more than one infected tick (Elfving et al. 2010). Pathogen spillover between tick species might occur when the life cycle of these two congeneric tick species, with contrasting habitat requirements, temporarily overlap. Heylen and Matthysen (2010) found that all I. ricinus immatures left the songbird hosts within 5.5 days, whereas in I. arboricola the time between attachment and detachment was long (up to 20 days) and highly variable. I. arboricola detaches during the night, the period when P. major rests in tree holes. In contrast, I. ricinus detaches during the day, the time when birds are most active.

In our study, A. phagocytophilum was identified only in two I. ricinus nymphs found on E. rubecula and P. major. All I. arboricola were negative. Alekseev et al. (2001) and Bjöersdorff et al. (2001) suggested that birds are important in the dispersal of Ehrlichia. They found 14.0% and 8.0%, respectively, Ehrlichia-positive I. ricinus nymphs collected from passerine birds (S. atricapilla, E. rubecula, T. merula, Phoenicurus phoenicurus [Redstart (Linnaeus, 1758)], Luscinia luscinia [Thrush Nightingale (Linnaeus, 1758)], and T. philomelos). The presence of A. phagocytophilum was also confirmed in I. ricinus nymphs from T. philomelos in Slovakia and in I. ricinus larvae (infections ranged from 1.3% to 6.3%) and nymphs (infection ranged 2.1%–10.5%) from E. rubecula, T. iliacus, and T. merula captured in Germany (Špitalská et al. 2006, Hildebrandt et al. 2010). Daniels et al. (2002) proposed that Turdus migratorius (the American robin [Linnaeus, 1766]) and Catharus fuscescens (the Veery [Stephens, 1817]) may be reservoirs for A. phagocytophilum by infecting larval Ixodes scapularis (Say, 1821) during feeding. Demonstration of the presence of Anaplasma spp. in I. ventalloi from A. chukar (the Chukar [Gray, 1830]) failed, but 37.7% of blood pools of 27 bird species in Cyprus were positive (Ionnou et al. 2009). All these reports support the importance of birds in spreading Anaplasma and Ehrlichia spp.

We studied the role of the bird-feeding tick I. arboricola in maintaining B. burgdorferi s.l. in natural transmission cycles. It has been demonstrated convincingly that some bird species play an important role as reservoirs of species belonging to the Lyme borreliosis (LB) group of spirochetes (Poupon et al. 2006, Ogden et al. 2008, Taragel'ová et al. 2008, Dubská et al. 2009). In these cycles, I. ricinus ticks constitute the main vector species. The most important host for I. ricinus larvae and nymphs are ground foraging birds, mainly thrushes (Hubálek et al. 1995, Taragel'ová et al. 2005, Dubská et al. 2009). Many passerine bird species can feed Borrelia-infected I. ricinus ticks, but only some species are competent reservoirs of the spirochete. Several bird species, for example, pheasants, blackbirds, and song thrushes, are competent reservoirs for B. garinii and B. valaisiana in Europe (Humair 2002, Kurtenbach et al. 2002, Michalik et al. 2008, Taragel'ová et al. 2008, Dubská et al. 2009). Several studies in Slovakia, Czech Republic, and Poland have shown that thrushes produce high numbers of Borrelia-infected I. ricinus larvae and nymphs and constitute the major competent reservoir bird species (Taragel'ová et al. 2008, Michalik et al. 2008, Dubská et al. 2009). I. arboricola ticks often coparasitize birds together with I. ricinus, but their vector competence has not yet been demonstrated. Our study provides the first data that suggest that this tick species is probably not competent in maintaining LB group spirochetes in nature. B. garinii and B. valaisiana DNA was detected in four larvae (1.3%) feeding on P. palustris and P. major. The low transmission efficiency suggests that these ticks may have acquired the infection via cofeeding with infected I. ricinus.

In our study, infections with tick-borne pathogens were determined for the first time for the tick species I. arboricola. A putative role of wild birds and their ectoparasites in the transmission of rickettsiae and borreliae was recorded, supporting their role in the distribution of zoonotic disease agents. However, the declining infection prevalence of Rickettsia spp. from larvae to nymphs requires further investigations of the mechanisms of transmission and maintenance of this tick-borne pathogen.