浙江省中部及南部地区巴贝虫自然疫源地调查

Investigation of the source regions of Babesia spp. infection in the central and south areas of Zhejiang Province

目的调查浙江省中部及南部地区巴贝虫动物宿主和传播媒介感染情况,对浙江省确诊的1例人感染巴贝虫的18S rRNA基因序列与Gen Bank中巴贝虫序列进行比对,了解巴贝虫亲缘关系和进化特征。方法采集浙江省确诊的病例居住地以及浙江省中部及南部地区的动物宿主抗凝血样和蜱虫,提取DNA,以巴贝虫18S rRNA基因序列引物进行PCR扩增、测序,并对测序序列进行分析比对,确认感染虫种;在GenBank数据库中查询巴贝虫属、种的18S rRNA基因序列信息,以及不同动物宿主、不同地理来源的田鼠巴贝虫种内18S rRNA序列信息,进行BLAST比对分析,并构建系统进化树。结果采集的261份生物样品中有37份扩增出阳性条带,其中动物宿主抗凝血阳性率为14.2%(32/225),蜱阳性率为13.9%(5/36)。对部分阳性样品进行测序,获得有效测序序列20份,经序列比对分析,共发现田鼠巴贝虫(Babesia microti)5份,均来自鼠类血样;此外,在鼠、牛等宿主/媒介体内还分别检测到肉孢子虫属(Sarcocystis sp.)4份、肝簇虫属(Hepatozoon sp.)2份,球虫(Coccidia sp.)1份、东方泰勒虫(Theileria orientalis)阳性5份、水牛泰勒虫(Theileria buffeli)2份和驽巴贝虫(Babesia caballi)1份。浙江省确诊的人巴贝虫序列与Gen Bank中不同宿主、地理来源田鼠巴贝虫18S rRNA序列的同源性为98.1%~99.8%,与日本、中国云南的人源以及福建、浙江杭州、天台、仙居的鼠源田鼠巴贝虫同属一个分支,而我国的CNMM-2株和新疆1647株与美国GI株和德国Jena株的遗传距离较近。结论在浙江省中部及南部地区的啮齿类小型哺乳动物中发现多份田鼠巴贝虫的自然感染。

Objective To investigate the infectious status of Babesia spp. in hosts and vectors in the central and south areas of Zhejiang Province, and analyze the 18S rRNA gene sequence from a patient confirmed to have human babesiosis in Zhejiang Province and align it with that in GenBank, in order to understand the genetic relationship and evolutionary characteristics of Babesia. Methods Anticogulated blood was collected from animal hosts and Babesia spp. vectors were collected. DNA was extracted from these samples and PCR was performed to amplify the 18S rRNA gene of Babesia. The PCR products were further sequenced and sequence alignment was performed to identify the species of Babesia. The family- and species-specific 18S rRNA sequences of Babesia spp., as well as those of B. microti from different animal hosts and from different geographical source regions were searched in the NCBI database and analyzed by BLAST. Phylogenetic trees were constructed by the MEGA5 software. Results Thirty-seven samples from a total of 261 produced positive bands. The positive rate of anticogulated blood from animal hosts was 14.2% （32/225）, and that of vectors was 13.9%（5/36）. The positive bands were sequenced, and 20 underwent successful sequencing. Results of sequence alignment revealed five samples with B. microti infection from rodents, four samples with Sarcocystis sp. infection, two with Hepatozoon sp. infection, one with Coccidia sp. infection, five with Theileria orientalis infection, two with T. buffeli infection and one B. caballi infection in rodent and cattle hosts/vetors. The 18S rRNA gene sequence of the confirmed human babesiosis in the Province had 98.1%-99.8% homology with the sequences of B. microti from different hosts and different geographical regions. And it was in the same branch as the human-source B. microti from Yunnan and Japan, and the rodent-source B. microti from Fujian, and Hangzhou, Tiantai and Xianju of Zhejiang. The Chinese CNMM-2 strain and Xinjiang 1647 strain of B. microti had a closer genetic distance with American GI strain and German Jena strain. Conclusion The B. microti was commonly found in rodents in the central and south areas of Zhejiang Province. Studies should be extended to other small mammals and domestic livestock, as well as to the biological vectors to see if they are carrying Babesia species pathogenic to humans.