新疆维吾尔自治区喀什地区亚洲璃眼蜱携带病原菌研究

Hyalomma asiaticum asiaticum tick-borne pathogenic bacteria in Kashgar Prefecture,Xinjiang Uygur Autonomous Region

目的采用16S rDNA V3-V4区高通量测序,并结合特异基因的PCR检测,研究新疆维吾尔自治区喀什地区亚洲璃眼蜱体内菌群特征及常见病原菌携带情况,为当地蜱传疾病防控提供理论依据。方法于2021年5月在喀什地区巴楚县采用布旗法采集游离蜱342只,经形态学和特异性16S rDNA检测鉴定蜱种;除228只用于分离培养外,其余114只提取全基因组DNA,采用Illumina Novaseq测序平台进行16S rDNA V3-V4区高通量测序,分析喀什地区亚洲璃眼蜱携带菌的种类及其相对丰度水平;根据新疆地区既往报道常见蜱媒病原菌分布情况及蜱种带菌特征,选取6种常见病原菌对其特异性基因进行PCR检测,分析各病原菌的携带情况,并与高通量测序结果进行比较分析。结果16S rDNA V3-V4区高通量测序分析显示1个样本携带贝纳柯克斯体;所有样本在属水平均检出立克次体属,16个样本检出无形体属,但缺少种水平注释;所有样本均未检出疏螺旋体。PCR法检测表明14只蜱携带伯氏疏螺旋体,7只蜱携带米氏疏螺旋体,3只蜱携带嗜吞噬细胞无形体,2只蜱携带贝纳柯克斯体,斑点热立克次体及查菲埃立克体未检出。结论与特异基因的PCR法相比,16S rDNA V3-V4区高通量测序能够同时检测多种病原体,但也存在其检测受限于数据库、无法检测到种水平等缺点。因此,在病原监测的实际应用中,两种方法应相互结合,才能更全面、准确的反应当地蜱媒病原菌的分布情况。

To analyze the microbial population characteristics and common pathogenic bacteria of Hyalomma asiaticum asiaticum in Kashgar Prefecture,Xinjiang Uygur Autonomous Region,high-throughput sequencing of the 16S rDNA V3-V4 region combined with PCR detection of specific genes was performed to provide a theoretical basis for the prevention and control of tick-borne diseases.In May 2021,342 free ticks were collected through flagging and trapping methods in Bachu County,Kashgar Prefecture.After morphological identification and specific 16S rDNA detection,228 ticks were used for isolation and culture;in the remaining 114 ticks,genomic DNA was extracted,and high-throughput sequencing of the 16S rDNA V3-V4 region was performed with the Illumina Novaseq sequencing platform to analyze the species and relative abundance of tick-borne pathogenic bacteria in Kashgar Prefecture.According to previous reports on the distribution of common tick-borne pathogenic bacteria and the characteristics of ticks in Xinjiang,we selected six common bacteria and detected their specific genes by PCR.The carriage status of each pathogen was analyzed and compared with the results of high-throughput sequencing.High-throughput sequencing analysis of 16S rDNA V3-V4 region indicated that one sample carried Coxiella burnetii;all samples carried Rickettsia;16 samples carried Anaplasma,but species-level annotation was lacking;and no Borrelia was detected in any samples.PCR detection indicated that 14 ticks carried Borrelia burgdorferi,seven ticks carried Borrelia miyamotoi,three ticks carried Anaplasma phagocytophilum,two ticks carried Coxiella burnetii spotted fever group rickettsia,and Ehrlichia chaffeensis was not detected.Compared with PCR tests of specific genes,high-throughput sequencing of the 16S rDNA V3-V4 region simultaneously detected multiple pathogens but had shortcomings such as limited database detection and the inability to perform species level detection.Therefore,the two methods should be combined in practical pathogen monitoring applications to more comprehensively and accurately reflect the distribution of local tick-borne pathogenic bacteria.