Role of Birds in Dispersal of Etiologic Agents of Tick-borne Zoonoses, Spain, 2009

We amplified gene sequences from Anaplasma phagocytophilum, Borrelia garinii, B. valaisiana, B. turdi, Rickettsia monacensis, R. helvetica, R. sibirica sibirica, and Rickettsia spp. (including Candidatus Rickettsia vini) in ticks removed from birds in Spain. The findings support the role of passerine birds as possible dispersers of these tick-borne pathogens.

H ard ticks are a major vector of infectious diseases in industrialized countries. Several tick-borne bacterial diseases, such as Lyme disease, Mediterranean spotted fever, and tick-borne lymphadenopathy (also called Dermacentor-borne necrosis erythema and lymphadenopathy), are endemic to Spain. Furthermore, a few cases of human anaplasmosis and Rickettsia monacensis infection in humans have been diagnosed in Spain (1)(2)(3).
Birds are the preferred host for some tick species. As carriers of infected ticks, birds could be responsible for the spread of tick-borne bacteria that cause human anaplasmosis, Lyme disease, rickettsioses, and other diseases (4). Multiple studies support the conclusion or propose the hypothesis that birds play a role as reservoirs of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia spp. (4)(5)(6). Because the Iberian Peninsula plays a major role in the migratory routes of birds, we aimed to determine the presence and prevalence of A. phagocytophilum, B. burgdorferi sensu lato, and Rickettsia spp. in ticks removed from birds captured in northern Spain.

The Study
During April-October 2009, bird bandings were conducted in the protected area of Finca Ribavellosa in La Rioja, Spain (42°14′N, 2°54′W). Ticks were collected from birds and classifi ed through taxonomic keys (7) and molecular methods (8). DNA was individually extracted by using 2 incubations of 20 minutes each with ammonium hydroxide (1 mL of 25% ammonia and 19 mL of Milli-Q water that had been autoclaved) at 100°C and 90°C.
DNA extracts were used as templates for PCRs targeting fragment genes for tick classifi cation and for bacteria detection (Table 1). Two negative controls, 1 containing water instead of template DNA and the other with template DNA but without primers, and a positive control (a tick extract, A. phagocytophilum, B. burgdorferi sensu stricto, or R. slovaca) were included in all PCRs. Amplifi cation products were sequenced, and nucleotide sequences were compared with those available in GenBank by using a BLAST search (www.ncbi.nlm.nih.gov/blast/ Blast.cgi). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA4 (16 in online Technical Appendix, wwwnc.cdc.gov/EID/pdfs/11-1777-Techapp1.pdf).

Conclusions
The presence of Anaplasma, Borrelia, and Rickettsia species in ticks removed from passerine birds corroborates the role of these vertebrates in the epidemiology and dispersion of tick-borne pathogens in Spain and in other zones of the planet. Some of the parasitized birds in our study, such as the European robin (Erithacus rubecula) or Eurasian blackcap (Sylvia atricapilla), are considered migratory or partial migratory birds. In addition, these species share an ecologic niche and ectoparasites (horizontal transmission) with other migratory birds that cover long distances from Africa to the Eurasian region.
Except for I. arboricola, the tick species captured in this study previously had been found on birds in Spain (18 in online Technical Appendix). Nevertheless, I. arboricola ticks are commonly hosted by birds. The high prevalence of I. ricinus ticks was expected because it is the most frequent tick in this area, and the immature stages of this tick frequently parasitize birds.
I. ricinus ticks are the main vectors of A. phagocytophilum in Europe, and this microorganism has been detected on vegetation in the studied area (1). However, the low prevalence (0.5%) of A. phagocytophilum in the ticks in our study corroborates data from other studies (19,20 in online Technical Appendix). The presence of A. phagocytophilum in a larva in our study supports the role of birds as reservoirs of A. phagocytophilum.
The prevalence (13.1%) of B. burgdorferi in our samples is similar to prevalences reported in other studies in Europe in which I. ricinus is the main species of tick captured from birds (19 in online Technical Appendix). In Spain, B. garinii, B. valaisiana, and B. afzelii have been detected in ticks from birds (18 in online Technical Appendix). According to our data, the human pathogen B. garinii was the most prevalent species, as reported in birds from Europe (21 in online Technical Appendix). B. turdi was discovered in Asia. Although it has been recently detected in ticks from birds in Norway (22 in online Technical Appendix), its fi nding in Spain was unexpected.
Regarding Rickettsia species, R. monacensis and R. helvetica are among the human pathogens detected in our study. Both species have been identifi ed in ticks from birds in Europe (19,20,23 in online Technical Appendix). On the contrary, Candidatus Rickettsia vini, a potential new Rickettsia species, also detected in our study, has not been related to human disease (17 in online Technical Appendix). Several genospecies closely related to R. heilongjiangensis and R. japonica have been identifi ed in Ixodes spp. ticks removed from birds (23 in online Technical Appendix). R. sibirica sibirica, responsible for Siberian tick typhus in western People's Republic of China and in Siberia, was also amplifi ed in an I. ricinus larva in this study.
Our data confi rm the involvement of birds in the cycle of human tick-borne diseases. The fi ndings confi rm that birds can disperse vectors and microorganisms. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012