Table_1_Novel Genotypes of Nidicolous Argas Ticks and Their Associated Microorganisms From Spain.docx

The knowledge of the distribution, richness and epidemiological importance of soft ticks of the genus Argas is incomplete. In Spain, five Argas species have been recorded, including three ornitophilic nidicolous ticks, but their associated microorganisms remain unknown. This study aimed to investigate ticks from bird nests and their microorganisms. Ticks were collected extensively from natural cavities and nest-boxes used by European rollers (Coracias garrulus) and little owls (Athene noctua) in Southeastern and Central Spain. Ticks were morphologically and genetically identified and corresponding DNA/RNA tick extracts were analyzed [individually (n = 150) or pooled (n = 43)] using specific PCR assays for bacteria (Anaplasmataceae, Bartonella, Borrelia, Coxiella/Rickettsiella, and Rickettsia spp.), viruses (Flaviviruses, Orthonairoviruses, and Phenuiviruses), and protozoa (Babesia/Theileria spp.). Six Argas genotypes were identified, of which only those of Argas reflexus (n = 8) were identified to the species level. Two other genotypes were closely related to each other and to Argas vulgaris (n = 83) and Argas polonicus (n = 33), respectively. These two species have not been previously reported from Western Europe. Two additional genotypes (n = 4) clustered with Argas persicus, previously reported in Spain. The remaining genotype (n = 22) showed low sequence identity with any Argas species, being most similar to the African Argas africolumbae. The microbiological screening revealed infection with a rickettsial strain belonging to Rickettsia fournieri and Candidatus Rickettsia vini group in 74.7% of ticks, mainly comprising ticks genetically related to A. vulgaris and A. polonicus. Other tick endosymbionts belonging to Coxiella, Francisella and Rickettsiella species were detected in ten, one and one tick pools, respectively. In addition, one Babesia genotype, closely related to avian Babesia species, was found in one tick pool. Lastly, Anaplasmataceae, Bartonella, Borrelia, and viruses were not detected. In conclusion, five novel Argas genotypes and their associated microorganisms with unproven pathogenicity are reported for Spain. The re-use of nests between and within years by different bird species appears to be ideal for the transmission of tick-borne microorganisms in cavity-nesting birds of semiarid areas. Further work should be performed to clarify the taxonomy and the potential role of soft Argas ticks and their microorganisms in the epidemiology of zoonoses.

关于软蜱（soft ticks）中锐缘蜱属（Argas）的分布、物种丰富度及流行病学重要性的认知仍不全面。在西班牙，目前已记录有5种锐缘蜱属物种，其中包括3种嗜鸟性巢栖蜱（ornithophilic nidicolous ticks），但与之相关的微生物仍未被探明。本研究旨在探究鸟类巢穴中的蜱虫及其携带的微生物。研究人员在西班牙东南部与中部地区，针对蓝胸佛法僧（European rollers, Coracias garrulus）与小鸮（little owls, Athene noctua）所使用的天然洞穴及巢箱开展了广泛的蜱虫采集工作。对采集到的蜱虫进行了形态学与分子遗传学鉴定，并将提取的蜱虫DNA/RNA分别以单份样本（n=150）与混合样本（n=43）为单位，采用特异性聚合酶链反应（PCR）检测技术，对以下病原体类群进行筛查：细菌类（无形体科（Anaplasmataceae）、巴尔通体属（Bartonella）、疏螺旋体属（Borrelia）、柯克斯体属/立克次体样菌属（Coxiella/Rickettsiella）以及立克次体属（Rickettsia）物种）、病毒类（黄病毒属（Flaviviruses）、正内罗病毒属（Orthonairoviruses）以及白蛉病毒属（Phenuiviruses））以及原虫类（巴贝斯虫属/泰勒虫属（Babesia/Theileria）物种）。本次研究共鉴定出6种锐缘蜱基因型，其中仅反射锐缘蜱（Argas reflexus，n=8）可确定至物种水平。另外两种基因型彼此亲缘关系较近，且分别与普通锐缘蜱（Argas vulgaris，n=83）、波兰锐缘蜱（Argas polonicus，n=33）高度同源；这两个物种此前从未在西欧地区有过记录。另有2种基因型（n=4）与此前在西班牙有记录的波斯锐缘蜱（Argas persicus）聚类。剩余1种基因型（n=22）与所有已报道的锐缘蜱属物种序列同源性均较低，其最接近的亲缘类群为非洲鸽锐缘蜱（Argas africolumbae）。微生物筛查结果显示，74.7%的蜱虫样本感染了隶属于弗氏立克次体（Rickettsia fournieri）与候选文氏立克次体（Candidatus Rickettsia vini）类群的立克次体菌株，此类感染主要出现在与普通锐缘蜱、波兰锐缘蜱亲缘关系较近的蜱虫个体中。此外，研究人员分别在10份、1份、1份混合蜱虫样本中检测到了隶属于柯克斯体属（Coxiella）、弗朗西斯菌属（Francisella）以及立克次体样菌属（Rickettsiella）的蜱虫内共生菌。同时，在1份混合蜱虫样本中发现了1种与鸟类巴贝斯虫属物种亲缘关系较近的巴贝斯虫基因型。最终，未在所有样本中检测到无形体科、巴尔通体属、疏螺旋体属病原体以及相关病毒类群。综上，本研究在西班牙境内报道了5种全新的锐缘蜱基因型，以及与之相关、致病性尚未明确的微生物。不同鸟类在年度间或同一年度内重复利用巢穴的行为，似乎为半干旱地区洞穴筑巢鸟类的蜱传微生物传播提供了理想的环境条件。未来仍需开展进一步研究，以明确锐缘蜱属软蜱的分类学地位，以及此类蜱虫及其携带微生物在人畜共患病流行病学中潜在的作用。