Detection of Babesia Sp. EU1 and Members of Spotted Fever Group Rickettsiae in Ticks Collected from Migratory Birds at Curonian Spit,North-Western Russia

Introduction

I xodes ricinus (L.) tick is a common ectoparasite in Europe. It is a well-known vector for many pathogenic human viruses, bacteria, and protozoa, causing zoonoses and circulating in the natural foci. Birds, mainly passerines (order Passeriformes), often host subadult ticks and represent a reservoir for human tick-borne pathogens (Hubalek 2004).

Migratory birds can act as long-distance vectors for several microbial agents of human disease. Borrelia garinii was detected in ground dwelling and sea birds in Eurasia, whereas Borrelia valaisiana and Borrelia burgdorferi sensu stricto were identified in different passerine birds in Europe (Dubska et al. 2009). The human pathogenic members of the family of Anaplasmataceae, spotted fever group (SFG) rickettsia, Coxiella burnetii, and Tick-borne encephalitis (TBE) virus have been detected in ticks from different species of migratory birds collected in Europe (Alekseev et al. 2001, Santos-Silva et al. 2006, Spitalska et al. 2006, Waldenström et al. 2007, Ioannou et al. 2009, Elfving et al. 2010). However, the involvement of birds in the ecology and epidemiology of babesiosis has so far been little studied. Skotarczak et al. (2006) investigated by PCR the prevalence of Babesia in ticks' and birds' blood from West-Central Poland.

In this study, the prevalence of SFG rickettsiae and Babesia sp. in I. ricinus ticks collected from the migratory birds at Curonian Spit, North-Western Russia, was investigated.

Materials and Methods

Birds (total 236) were caught in ornithological nets (permit no. DLOPiKog.4201/154/00) during spring 2008 at Rybachy Biological Station of Zoological Institute Russian Academy of Sciences, Curonian Spit, Kaliningrad enclave, Russian Federation (55^o09′N 20^o51′E). Tick nymphs were detached from passerine migratory birds at the end of the ringing procedure. All collected ticks were identified morphologically and stored individually in 70% ethanol. Total tick DNA was isolated by AxyPrep Blood Genomic DNA kit (AxygenBio). The success of DNA extraction was confirmed by PCR using primers T1B (5′-AAACTAGGATTAGATACCCT-3′) and T2A (5′-AATGAGAGCGACGGGCGATGT-3′) described by Beati and Keirans (2001), which amplifies a generic PCR product of 360 bp in 12S rDNA gene of all known tick species.

Babesia sp. was detected in ticks by PCR using primers BJ1 (5′-GTCTTG TAATTGGAATGATGG-3′) and BN2 (5′-TAGTTTATGGTTAGGACTACG-3′) (Casati et al. 2006). Amplified fragments corresponded to the 560 bp region of Babesia 18S rDNA. For detection of Rickettsia species, the following primers were used: Rp CS.877p (5′-GGGGACCTGCTCACGGCGG-3′) and Rp CS.1258n (5′-ATTGCAAAAAGTACAGTGAACA-3′), amplifying a 380 bp fragment of the gltA gene in all known SFG rickettsiae (Regnery et al. 1991).

All positive PCR products were purified using the QIAquick PCR purification kit (Qiagen, GmbH) and then directly sequenced to identify the species and strains.

Results

Altogether, 236 birds were captured, representing 8 species of Passeriformes: Turdus philomelos (59), Fringilla coelebs (68), Troglodytes troglodytes (28), Parus major (17), Sturnus vulgaris (26), Fringilla montifringilla (18), Sylvia borin (6), and Phylloscopus trochilus (14). Eighty-six of the captured birds (36.4%) hosted 126 nymphs (Table 1). All ticks were identified as I. ricinus. The DNA was successfully isolated from all of the tested ticks.

Babesia spp. were detected in two cases of 126 (1.6%) analyzed ticks collected from two specimens of T. philomelos (Table 1). The partial sequence of 18S rDNA had 100% similarity to human pathogenic Babesia sp. EU1. The SFG rickettsiae were detected in 19 of 126 (15.1%) ticks (Table 1). BLAST analysis of SFG rickettsiae gltA assigned sequences to human pathogenic Rickettsia helvetica (10.3%), Rickettsia monacensis (3.9%), and Rickettsia japonica (0.8%) with 98%–100% sequence similarity. The R. helvetica was detected in ticks detached from the three species of birds, T. philomelos, P. major, and F. coelebs, whereas R. monacensis and R. japonica were revealed in F. coelebs and S. vulgaris, respectively (Table 1).

Discussion

To the best of our knowledge, this is the first report describing SFG rickettsiae and Babesia sp. EU1 in ticks collected from the passerines in the North-Western part of Russia.

It was previously shown that the tick I. ricinus represents a potential vector and natural reservoir of R. helvetica and R. monacensis in Russia (Rudakov et al. 2003); however, the SFG rickettsiae-infected ticks have never been found in birds captured in Russia. The R. helvetica had the highest prevalence among the above-listed pathogens and was found in 10.4% of infected ticks. The R. helvetica-infected ticks were found only in three of eight captured passerine species (T. philomelos, F. coelebs, and P. major). The R. monacensis was detected only in ticks collected from F. coelebs. Only one tick detached from starling was infected by R. japonica. The identification of R. japonica in bird-feeding I. ricinus ticks is perhaps the most significant finding. This member of SFG rickettsiae is commonly associated with the tick species Dermacentor taiwanensis Sugimoto, Haemaphysalis flava Neumann, and, perhaps, Haemaphysalis longicornis Neumann from parts of Asia and Japan (Fournier et al. 2002). In North Europe, SFG rickettsiae were detected only in ticks collected from migratory birds in Sweden (Elfving et al. 2010).

Babesia sp. EU1 was found only in two ticks collected from the two exemplars of song thrush birds. Babesia spp. are piroplasmid protozoan parasites of human and animal red blood cells (Casati et al. 2006). In Europe, human cases of babesiosis have been reported over the past years and have been traditionally attributed to infections with the bovine parasite Babesia divergens transmitted by I. ricinus (Herwaldt et al. 2003, Casati et al. 2006). However, Herwaldt et al. (2003) reported the first molecular characterization of the new Babesia sp. EU1, isolated from patients in Southern Europe. Until now, tick-transmitted Babesia sp. EU1 has only been detected in roe deer, sheep, goats, and humans (Herwaldt et al. 2003, Casati et al. 2006). Skotarczak et al. (2006) were the first who tried to reveal Babesia sp. in I. ricinus ticks collected from nine passerine bird species as well as from questing ticks. They additionally tested blood samples of 84 bird specimens, from which ticks were detached. Specific DNA was not detected in any samples of either ticks or birds' blood.

The detection of Babesia sp. EU1 in tick species that is frequently found on humans and that have only fed on passerines suggests that some bird species may represent another reservoir with a potential risk for humans.

Interestingly, only nymphs were detached from birds during tick collection. This unusual situation was detected for the first time. Our previous studies indicated that larvae and nymphs are parasitizing together on birds in this region (Alekseev et al. 2001). From the literature published to date, it seems that some birds such as Parus caeruleus and Sitta europea (Skotarczak et al. 2006) or F. coelebs (Hanincová et al. 2003) can be the hosts only for I. ricinus nymphs, for at least within some periods of their migration. Dubska et al. (2009) reported about lower prevalence of larvae in song thrushes and dunnocks.

Our survey indicates that wild birds may play a significant role as a reservoir of babesiae and SFG rickettsiae and that ticks being infected by these pathogens may transmit them to humans. Future investigations are necessary to further characterize the role of birds in the epidemiology of these human pathogens.