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

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.

卢氏疏螺旋体（Borrelia lusitaniae）是旧世界分布的莱姆疏螺旋体病（Lyme borreliosis, LB）类群蜱传疏螺旋体物种，主要流行于地中海盆地周边国家。蜥蜴科（Lacertidae）蜥蜴已被确定为卢氏疏螺旋体的储存宿主，这类爬行动物具有显著的地理种群结构，提示其迁移能力有限。为探究宿主地理结构是否塑造卢氏疏螺旋体的进化与流行病学特征，本研究基于新近开发的染色体持家基因多位点序列分型（multilocus sequence typing, MLST）方案，对该蜱传细菌的系统地理种群结构展开分析。研究共在葡萄牙两个气候迥异、间距约130公里的区域，采集了2099只寻血型蓖麻硬蜱（Ixodes ricinus）若蜱与成蜱。通过直接PCR对所有蜱样本进行疏螺旋体筛查，并尝试分离菌株，最终共获得16株卢氏疏螺旋体培养物。本研究纳入了未受污染的培养物与感染蜱样本。MLST分析结果显示，不同区域的卢氏疏螺旋体种群构成遗传分化显著的种群。与之相反，常用分子标记外膜蛋白A（ospA）与外膜蛋白C（ospC）的序列未检测到明确的系统地理信号。基于持家基因MLST所捕捉到的卢氏疏螺旋体在短地理距离下的显著种群结构，提示该螺旋体的迁移速率较低，这极有可能是因为地中海蜥蜴种群的分布呈现高度邻域分布特征。本研究证实了脊椎动物宿主在蜱传微寄生虫地理传播中的重要作用。