Summary: Birds are important in the ecology of Borrelia burgdorferi sensu lato (s.l.) because they are important hosts for vector tick immature stages and are known reservoirs for some Borrelia genospecies. The aim of our study was to assess the role of common passerine bird species as reservoirs for B. burgdorferi s.l. in Western Europe. We surveyed birds in enzootic areas in Portugal, where no information is available for birds as reservoirs for this aetiologic agent and where B. lusitaniae , for which few reservoirs have been identified, is the dominant genospecies. Twenty‐three birds (2.9%), including Turdus merula , T. philomelos , Parus major and Fringilla coelebs harboured infected ticks, but only Turdus sp. harboured infected tick larvae. In one study area, although B. lusitaniae was dominant in questing Ixodes ricinus , no ticks feeding on birds were infected with this genospecies, and B. valaisiana was the dominant genospecies in I. ricinus larvae feeding on birds. In the other area ticks collected from birds were mainly I. frontalis which were infected with B. turdi . Two skin biopsies (4.2%) from two T. merula were positive, one for B. valaisiana and the other for B. turdi . This is the first report for B. turdi in Western Europe.

Borrelia and Rickettsia bacteria are the most important tick-borne agents causing disease in Portugal. Identification and characterization of these circulating agents, mainly in recreational areas, is crucial for the development of preventive measures in response to the gradually increasing exposure of humans to tick vectors. A total of 677 questing ticks including Dermacentor marginatus, Rhipicephalus sanguineus, Ixodes ricinus, Hyalomma lusitanicum, H. marginatum, and Haemaphysalis punctata were collected in a Safari Park in Alentejo, Portugal, to investigate the prevalences of infection and characterize Borrelia and Rickettsia species. From a total of 371 ticks tested by PCR for Borrelia burgdorferi sensu lato (s.l.), of which 247 were tested for Rickettsia, an infection prevalence of 18.3% was found for B. lusitaniae and 55.1% for Rickettsia spp. Sequence analysis of positive amplicons identified the presence of B. lusitaniae (18.3%), R. monacensis strain IRS3 (51.7%), and R. helvetica (48.3%) in I. ricinus. R. slovaca (41.5%), R. raoultii (58.5%), and also B. lusitaniae (21%) were identified in D. marginatus ticks. One (5.9%) H. lusitanicum was infected with B. lusitaniae, and R. massiliae was found in one Rhipicephalus sanguineus. Coinfection was found in 7 (20%) I. ricinus and 34 (23.3%) D. marginatus ticks. We report, for the first time, simultaneous infection with R. helvetica and B. lusitaniae and also R. slovaca, the agent of TIBOLA/DEBONEL, with B. lusitaniae. Additionally, 6 isolates of B. lusitaniae were established, and isolates of Rickettsia were also obtained for the detected species using tick macerates cultured in mammalian and mosquito cell lines. This report describes the detection and isolation of tick-borne agents from a Portuguese Safari Park, highlighting the increased likelihood of infection with multiple agents to potential visitors or staff.