目的 了解内蒙古中西部草原蜱类的群落结构、携带病原体多样性及基因型。 方法 于2016—2019年春夏季,在内蒙古中西部草原,采用动物体表搜集法采集蜱标本,进行蜱种鉴定。解剖摘取蜱的唾液腺并提取基因组DNA,以斑点热立克次体柠檬酸合成酶A（gltA）、疏螺旋体以鞭毛蛋白B（flaB）、埃立克体属以外膜蛋白质1（omp1）、无形体属主要表面蛋白2（msp2）为靶基因进行PCR扩增初筛。立克次体gltA初筛阳性样品经限制性片段长度多态性（RFLP）分类,再根据蜱种和地区每类选20~30个代表性样品进行gltA、立克次体外膜蛋白A（rOmpA）基因测序。扩增序列测序后用BLAST、Clustal W和MEGA 7.0软件进行同源性分析,以邻接法构建系统进化树。 结果 共采集成蜱3 822只,经形态学特征和特异性18S rRNA基因分型法鉴定,隶属于2属3种,分别为草原革蜱、亚东璃眼蜱和边缘璃眼蜱,其中草原革蜱占55.7%（2 129/3 822）、亚东璃眼蜱占30.0%（1 147/3 822）,为该地区的优势蜱种。PCR检测结果显示,立克次体gltA基因阳性蜱1 899只,阳性率为49.7%（1 899/3 822）,gltA基因阳性样品根据RFLP结果分为两类,两类样品的gltA基因序列均为581 bp,与R. raoultii（DQ365804）或R. aeschlimanni（KT873466）的同源性为100%;两类样品的rOmpA基因均长367 bp,与R. raoultii（AH015610）或R. aeschlimanni（U83466）的同源性为100%,与gltA基因的结果相符。3 822只蜱中,R. raoultii和R. aeschlimanni的阳性率分别为37.2%（1 422/3 822）和12.5%（477/3 822）,其中草原革蜱中分别为58.5%（1 245/2 129）和11.1%（477/2 129）;亚东璃眼蜱中分别为15.4%（177/1 147）和0;边缘璃眼蜱中分别为0和44.0%（240/546）。疏螺旋体flaB基因阳性蜱28只,阳性率为0.7%（28/3 822）,其中草原革蜱中为0.8%（16/2 129）,亚东璃眼蜱为1.0%（12/1 147）。共获得疏螺旋体flaB基因序列10条,与莱姆病主要病原体B. garinii（AB035602）和B. afzelii PKo（NC008277）的同源性分别为90.6%~100%和95.6%~100%。亚东璃眼蜱中B. garinii和B. afzelii的阳性率分别为0.9%（10/1 147）和0.2%（2/1 147）,草原革蜱中均为0.4%（8/2 129）。3 822只蜱中omp1基因阳性1只,TA克隆后获得8个氨基酸序列相同的克隆和3个氨基酸序列存在差异的克隆,11个克隆的氨基酸序列与E. muris的同源性最高,但仅为65%~69%。3 822只蜱中均未检出无形体属菌群。系统进化树分析结果显示,3种蜱感染的立克次体均与R. raoultii和R. aeschlimanni聚在一簇。在获得的10条疏螺旋体菌群flaB基因序列中,源于草原革蜱和亚东璃眼蜱的1条序列与B. garinii聚在一簇,草原革蜱的另1条序列与B. afzelii聚在一簇,其余8条序列与B. garinii和B. afzelii的flaB基因序列处在不同的分支。草原革蜱感染的埃立克体属菌与目前已知的埃立克体属菌群关系较远,形成独自的聚类。 结论 内蒙古中西部草原存在草原革蜱、亚东璃眼蜱和边缘璃眼蜱,蜱类中广泛存在斑点热立克次体和莱姆病螺旋体的感染,是R. raoultii、R. aeschlimanni、B. garinii和B. afzelii潜在的自然疫源地。有必要加强该地区蜱媒传染病的预防控制工作。

Objective To investigate the structure of tick populations, the diversity of pathogens they carry, and the genotypes of tick-borne Rickettsia-related pathogens in central and western part of Inner Mongolia of China. Methods Tick samples were collected from body surface of animals in the grasslands in central and western Inner Mongolia in spring and summer between 2016 and 2019. The tick species was identified. The salivary glands of ticks were dissected and genomic DNA was extracted. PCR was performed to amplify citrate synthase A (gltA) in spotted fever Rickettsia, flagellin gene B (flaB) in Borrelia, outer membrane protein 1 (omp1) in Ehrlichia, and major surface protein 2 (msp2) in Anaplasma. The samples positive for Rickettsia gltA were classified by restriction fragment length polymorphism (RFLP), and then 20 to 30 representative samples were selected for each type for each species and region for gene sequencing of gltA and Rickettsia outer membrane protein A (rOmpA). Homology analysis was performed by BLAST, Clustal W and MEGA 7.0 softwares, and the phylogenetic tree was constructed using the adjoining method. Results A total of 3 822 ticks were collected, 3 genera of 2 species were identified by morphological observation and genotyping of the specific 18S rRNA, which were Dermacentor nuttalli, Hyalomma asiaticum and H. marginalis, of which D. nuttalli (55.7%, 2 129/3 822) and H. asiaticum (30.0%, 1 147/3 822) constituted the predominant species. PCR results showed that 1 899 ticks were positive for Rickettsia gltA, with positive rate of 49.7% (1 899/3 822). The gltA-positive samples were divided into two groups according to the RFLP results. The gltA sequence was 581 bp in both groups, and was 100% homologous to R. raoultii (DQ365804) or R. aeschlimanni (KT873466). The rOmpA length of the two groups was 367 bp, which was 100% homologous to R. Raoultii (AH015610) or R. aeschlimanni (U83466), consistent with the results of gltA. The positive rates of R. raoultii and R. aeschlimanni in the 3 822 ticks were 37.2% (1 422/3 822) and 12.5% (477/3 822), respectively; 58.5% (1 245/2 129) and 11.1% (477/2 129) in D. nuttalli, respectively; 15.4% (177/1 147) and 0 in H. asiaticum; and 0 and 44.0% (240/546) in H. marginalis, respectively. A total of 28 ticks (0.7%, 28/3 822) were positive for Borrelia flaB, with positive rate of 0.8% (16/2 129) in D. nuttalli and of 1.0% (12/1 147) in H. asiaticum. A total of 10 sequences of Borrelia flaB were obtained, with 90.6%-100% and 95.6%-100% homology to B. garinii (AB035602) and B. afzelii PKO (NC008277), the main pathogens of Lyme disease. The positive rates of B. garinii and B. afzelii were 0.9% (10/1 147) and 0.2% (2/1 147) in H. asiaticum, respectively, and were both 0.4% (8/2 129) in D. nuttalli. Of the 3 822 ticks examined, 1 tick was positive for Ehrlichia omp1. After TA cloning, 8 clones with the same amino acid sequence and 3 with different amino acid sequences were obtained. The amino acid sequences of the 11 clones had the highest homology with E. muris, although the homology was only 65%-69%. No Anaplasma microbiome was detected in the 3 822 ticks. The phylogenetic tree analysis showed that the Rickettsia that infected the three tick species clustered with R. raoultii and R. aeschlimanni. Among the 10 Borrelia flaB sequences obtained, one sequence from D. nuttalli and H. asiaticum clustered with B. garinii, another sequence from D. nuttalli also clustered with B. afzelii, and the remaining 8 sequences were in different branches from the flaB sequences of B. garinii and B. afzelii. The Ehrlichia infected by D. nuttalli was also far from the known Ehrlichia, forming a separate cluster. Conclusion D. nuttalli, H. asiaticum and H. marginalis are found in grasslands in the central and western part of Inner Mongolia. Infection of spotted fever Rickettsia and Lyme disease spirochete are widespread in the ticks, creating potential natural foci of R. raoultii, R. aeschlimanni, B. garinii, B. afzelii and Ehrlichia infection. It is necessary to strengthen the prevention and control of tick-borne infectious diseases in these areas.