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

Abstract

To date Borrelia lusitaniae is the only genospecies of Borrelia burgdorferi sensu lato isolated from Ixodes ricinus ticks collected in Portugal and Tunisia. This suggests that the genospecies diversity of B. burgdorferi sensu lato decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of B. burgdorferi sensu lato from this region, 55 I. ricinus and 27 Hyalomma marginatum questing adults, collected during the spring of 1998 from a sylvatic habitat south 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 that targets the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. Restriction fragment length polymorphism (RFLP) analysis of the I. ricinus-derived amplicons showed that the sequences in the majority of samples were similar to those of B. lusitaniae type strains (76% for strain PotiB1, 5% for strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samples. The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic analysis of rrf-rrl spacer sequences revealed a diverse population of B. lusitaniae in questing adult I. ricinus ticks (the sequences did not cluster with those of any other genospecies). This population consisted of 10 distinct sequence types, suggesting that multiple strains of B. lusitaniae were present 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.

FIG. 1

MseI restriction patterns of B. burgdorferi sensu lato rrf-rrl spacer nested PCR products amplified directly from ticks or cultured strains. Lanes 1 and 10, D-15 marker; lanes 2 and 3, cultured B. burgdorferi sensu stricto (ZS 7) and B. lusitaniae (PotiB1), patterns A and E, respectively; lanes 4 and 5, tick-derived samples 58 and 151 (pattern E), respectively; Lane 6, tick-derived sample 132 (pattern F); lane 7, tick-derived sample 163 (patterns E and F); lane 8, tick-derived sample 156 (pattern H); lane 9, tick-derived sample 167 (pattern G).

FIG. 2

Aligned rrf-rrl spacer DNA sequences of B. burgdorferi sensu lato amplified directly from ticks or cultured strains or downloaded from GenBank. Gaps were introduced to obtain maximum homology. Only the last three bases of the 3′ end of the rrf gene and the first three bases of the 5′ end of the rrl gene are shown (in boldface).

FIG. 3

Neighbor-joining tree based on the comparison of rrf-rrl spacer sequences of B. burgdorferi sensu lato. The tree is drawn rooted at the midpoint for clarity. Sequences that are identical and that share the same branch are underlined. The numbers in the grey boxes are the results of 1,000 bootstrap resamplings (values of less than 70 are not shown). B. burgdorferi s.s., B. burgdorferi sensu stricto.

FIG. 4

Maximum likelihood phylogenetic tree based on the comparison of rrf-rrl spacer sequences of B. burgdorferi sensu lato. The tree was constructed by using the general reversible model of DNA substitution and a gamma distribution of rate variation among sites (drawn rooted at the midpoint). Sequences that are identical and that share the same branch are underlined. B. burgdorferi s.s., B. burgdorferi sensu stricto.