Genetic diversity of Borrelia burgdorferi sensu lato in ticks from mainland Portugal

To date Borrelia lusitaniae is the only genospecies of Borrelia burgdorferi sensu late isolated from Ixodes ricinus ticks collected in Portugal and Tu nisia. This suggests that the genospecies diversity of B. burgdorferi sensu late decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of B. burgdorferi sensu late from this region, 55 I. ricinus and 27 Hyalomma marginatum ques ting adults, collected during the spring of 1998 from a sylvatic habitat so uth of Lisbon, Portugal, were analyzed. Infection prevalences of 75% in I. ricinus ticks and 7% in H. marginatum ticks were detected by a nested PCR t hat targets the rrf (5S)-rrl (23S) spacer of B, burgdorferi sensu late, Res triction fragment length polymorphism (RFLP) analysis of the I. ricinus-der ived amplicons showed that the sequences in the majority of samples were si milar to those of B. lusitaniae type strains (76% for strain PotiB1, 5% fur strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samp les, The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic ana lysis of rrf-rrl spacer sequences revealed a diverse population of B. lusit aniae in questing adult I. ricinus ticks (the sequences did not cluster wit h those of any other genospecies), This population consisted of 10 distinct sequence types, suggesting that multiple strains of B. lusitaniae were pre sent in the local I. ricinus population. We hypothesize that B. lusitaniae has a narrow ecological niche that involves host species restricted to the Mediterranean Basin.