多方法组合调查新疆阿勒泰地区6种常见鼠传致病菌流行状况

Prevalence of 6 common rodent-borne pathogens identified using multiple methods in Xinjiang Altay prefecture,China

【背景】鼠传疾病是对人类危害较大的一种人兽共患病,全球化使得鼠传疾病流行区域不断扩大,出现了多种新发鼠传疾病的发生及旧传染病的复燃。【目的】调查新疆阿勒泰地区常见的鼠传致病菌在啮齿动物中的流行状况,为当地自然疫源性疾病防控提供科学依据。【方法】采用夹夜法捕获啮齿动物,无菌收集其脾脏和肾脏组织,提取基因组DNA。应用TaqMan探针法的荧光定量聚合酶链式反应(quantitative polymerase chain reaction,qPCR)检测巴尔通体(Bartonella spp.)、问号钩端螺旋体(Leptospira interrogans)、恙虫病东方体(Orientia tsutsugamushi)、莫氏立克次体(Rickettsia mooseri)、嗜吞噬细胞无形体(Anaplasma phagocytophilum)和土拉弗朗西斯菌(Francisella tularensis)6种常见的鼠传致病菌。采用16S rRNA基因的通用引物进行常规PCR扩增后,应用Illumina测序和Nanopore测序进一步检测致病菌,同时对脾脏组织进行巴尔通体体外分离培养。比较qPCR、16S rRNA基因测序和分离培养的结果。【结果】共捕获啮齿动物8种66只,其中,乌拉尔姬鼠(Apodemus uralensis)31只,占捕获总数的46.97%,其余为褐家鼠(Rattus norvegicus)、小家鼠(Mus musculus)等。qPCR检测常见的鼠传致病菌感染率为巴尔通体31.80%(21/66)、问号钩端螺旋体1.50%(1/66)、恙虫病东方体1.50%(2/66)、莫氏立克次体3.00%(1/66)和土拉弗朗西斯菌13.60%(9/66),未检出嗜吞噬细胞无形体。16S rRNA基因Illumina测序分析通过质控的23份样品检测出致病菌,以巴尔通体为主;16S rRNA基因Nanopore测序分析通过质控的11份样品检测出巴尔通体,其他5种常见的鼠传致病菌未检出。有11份脾脏组织分离培养出巴尔通体,感染率为16.67%(11/66)。【结论】新疆阿勒泰地区的啮齿动物可携带巴尔通体等多种鼠传致病菌,应关注并加强该地区相关传染病的防治工作。qPCR、细菌分离培养、16S rRNA基因测序3种检测方法可互相补充,更全面地了解当地啮齿动物携带致病菌的状况。

[Background]Rodent-borne diseases are a class of zoonoses harmful to humans.The epidemic areas of rodent-borne diseases keep expanding with the progress in globalization,and a variety of new rodent-borne diseases emerge while the old infectious diseases reoccur.[Objective]To investigate the prevalence of common rodent-borne pathogens in rodents in Altay prefecture of Xinjiang,and to provide a scientific basis for the prevention and control of local natural focus diseases.[Methods]The spleen and kidney tissue samples were collected aseptically from the rodents captured by night trapping method,and the genome DNA was extracted.Six common rodent-borne pathogens including Bartonella spp.,Leptospira interrogans,Orientia tsutsugamushi,Rickettsia mooseri,Anaplasma phagocytophilum,and Francisella tularensis were detected by fluorescence quantitative polymerase chain reaction(qPCR)with TaqMan probe.Illumina sequencing and Nanopore sequencing after routine PCR amplification with universal primers for the 16S rRNA gene were employed to further detect the pathogens.Meanwhile,the spleen tissue was used for the isolation and culture of Bartonella in vitro.The results of qPCR,16S rRNA gene sequencing,and bacterial isolation and culture were compared.[Results]A total of 66 rodents of 8 species were captured,of which 31(46.97%)rodents were Apodemus uralensis,and the rest were Rattus norvegicus,Mus musculus,etc.The infection rates of common rodent-borne pathogens detected by qPCR were as follows:Bartonella 31.80%(21/66),L.interrogans 1.50%(1/66),O.tsutsugamushi 1.50%(2/66),R.mooseri 3.00%(1/66),and F.tularensis 13.60%(9/66).A.phagocytophilum was not detected.The Illumina sequencing of 16S rRNA gene detected pathogens(mainly Bartonella)in the 23 samples passing the quality control.The Nanopore sequencing of 16S rRNA gene detected Bartonella in 11 samples passing the quality control,and did not detect the other 5 common rodent-borne pathogens.Bartonella was isolated from 11 spleen tissue samples,with the infection rate of 16.67%(11/66).[Conclusion]Rodents in the Altay prefecture of Xinjiang can carry a variety of rodent-borne pathogens such as Bartonella.We should pay attention to and strengthen the prevention and control of related infectious diseases in this region.Quantitative polymerase chain reaction,bacterial isolation and culture,and 16S rRNA gene sequencing can complement with each other to provide a comprehensive understanding of the local rodents carrying pathogens.