西藏羊蜱蝇中巴尔通体检测及gltA基因序列分析

Detection of Bartonella and Sequence Analysis of gltA Gene in Tibetan Melophagus ovinus

为了明确西藏部分地区绵羊体外寄生羊蜱蝇携带病原巴尔通体的感染情况,作者于2019年1—9月采集了西藏林芝、日喀则、那曲地区绵羊体外寄生羊蜱蝇298只,通过形态学鉴定、PCR扩增羊蜱蝇18S rRNA基因进行虫体鉴定,并对病原巴尔通体的gltA基因进行检测,将部分阳性PCR产物连接pMD^TM-18T后转入DH5α感受态细胞,将阳性结果进行测序并进行遗传进化分析。结果显示,雌性阳性率49.8%(113/227),雄性阳性率42.3%(30/71),雌雄之间差异不显著(χ^2=0.944,P=0.267);羊蜱蝇携带病原巴尔通体的总感染率为48.0%(143/298),林芝极显著高于日喀则、那曲地区(χ^2=13.801,P<0.01;χ^2=17.067,P<0.01),日喀则和那曲地区无显著差异(χ^2=0.084,P=0.771);散养模式羊蜱蝇携带阳性率44.3%(102/230),圈养阳性率85.0%(34/40),屠宰场阳性率23.3%(7/30),宿主散养和圈养、屠宰场存在极显著差异(χ^2=20.929,P<0.01;χ^2=24.38,P<0.01),宿主散养和屠宰场存在显著差异(χ^2=3.989,P=0.046);将测序结果上传GenBank数据库获得3个巴尔通体gltA基因登录号(MN623006、MN623007、MN623008);序列比对表明和云南、新疆巴尔通体相似性为99.6%~100%。本次研究首次检测出西藏羊蜱蝇携带病原巴尔通体,为了解西藏绵羊体外寄生虫携带病原巴尔通体和病原防控提供依据。

This study aimed to clarify the infection of the pathogenic Bartonella in the ectoparasite Melophagus ovinus in sheep in parts of Tibet.From January to September 2019,298 sheep parasites in Linzhi,Xigaze and Naqu were collected.The morphological identification and PCR amplification of the 18S rRNA gene of the Melophagus ovinus were used to identify the body and the pathogen gltA gane of Bartonella was detected,pMD^TM-18T was connected to some positive PCR products and transferred into DH5αcompetent cells.The positive results were sequenced and analyzed for genetic and evolutionary analysis.The results showed that the positive rate of female and male were 49.8%(113/227)and 42.3%(30/71),there was no significant difference between male and female(χ^2=0.944,P=0.267).The total infection rate of the pathogen Bartonella was 48.0%(143/298).Positive rate of Linzhi was significantly higher than that of Xigaze and Naqu area(χ^2=13.801,P<0.01;χ^2=17.067,P<0.01),there was no significant difference between Xigaze and Naqu areas(χ^2=0.084,P=0.771).The positive rate of Melophagus ovinus in free culture,captivity,and slaughterhouse were 44.3%(102/230),85.0%(34/40)and 23.3%(7/30),respectively.There were significant differences between captive culture and free host culture(χ^2=20.929,P<0.01)or slaughterhouse(χ^2=24.38,P<0.01).There was significant differences between host free culture and slaughterhouse(χ^2=3.989,P=0.046).The sequencing results were uploaded to GenBank database,and obtained three Bartonella gltA gene accession numbers,MN623006,MN623007 and MN623008.The sequence alignment showed that the homology of Bartonella in Yunnan and Xinjiang was 99.6%-100%.In this study,the pathogen Bartonella of Tibetan Melophagus ovinus was detected for the first time,which provided the basis for understanding the pathogen Bartonella carried by Tibetan sheep parasites in vitro and the prevention and control of the pathogen.