Fine-Scale Phylogeographic Structure of Borrelia lusitaniae Revealed by Multilocus Sequence Typing

Abstract

Borrelia lusitaniae is an Old World species of the Lyme borreliosis (LB) group of tick-borne spirochetes and prevails mainly in countries around the Mediterranean Basin. Lizards of the family Lacertidae have been identified as reservoir hosts of B. lusitaniae. These reptiles are highly structured geographically, indicating limited migration. In order to examine whether host geographic structure shapes the evolution and epidemiology of B. lusitaniae, we analyzed the phylogeographic population structure of this tick-borne bacterium using a recently developed multilocus sequence typing (MLST) scheme based on chromosomal housekeeping genes. A total of 2,099 questing nymphal and adult Ixodes ricinus ticks were collected in two climatically different regions of Portugal, being ∼130 km apart. All ticks were screened for spirochetes by direct PCR. Attempts to isolate strains yielded 16 cultures of B. lusitaniae in total. Uncontaminated cultures as well as infected ticks were included in this study. The results using MLST show that the regional B. lusitaniae populations constitute genetically distinct populations. In contrast, no clear phylogeographic signals were detected in sequences of the commonly used molecular markers ospA and ospC. The pronounced population structure of B. lusitaniae over a short geographic distance as captured by MLST of the housekeeping genes suggests that the migration rates of B. lusitaniae are rather low, most likely because the distribution of mediterranean lizard populations is highly parapatric. The study underlines the importance of vertebrate hosts in the geographic spread of tick-borne microparasites.

Inadequate Binding of Immune Regulator Factor H Is Associated with Sensitivity ofBorrelia lusitaniaeto Human Complement

“…Due to the limited number of isolates that have been tested (4,33), the resistance/sensitivity pattern of B. lusitaniae to human complement remains largely unknown. The association of B. lusitaniae with lizards as the most important reservoir hosts (13,46,58) implicates an adaptation of this genospecies to the hosts' complement system. All B. lusitaniae isolates analyzed in the present study were readily killed by complement-active human serum, arguing for the inability of this genospecies to infect and survive in the human host.…”

A NewBorreliaSpecies Defined by Multilocus Sequence Analysis of Housekeeping Genes

“…LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22,24,46,48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12,15,16,23,26,45).…”

“…In this study, we analyzed the intra-and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30,48).…”

“…Borrelia strains used in this study are listed in Table 1. DNA extraction, primers, and PCR conditions. Genomic DNA was extracted and purified from cultured isolates as described earlier (10,30,48). The loci analyzed comprised the "housekeeping" genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA), the 5S-23S IGS, ospA, and ospC.…”

“…Only the phylogenetic trees generated using MLSA were rooted. B. lusitaniae sequences were omitted from ospA and ospC analyses because of insufficient sequence overlap for phylogenetic analyses (48). For detailed information, see supplemental material.…”