青海地区多房棘球绦虫和石渠棘球绦虫遗传分化特征

Characteristics of genetic differentiation of Echinococcus multiloculariss and E.shiquicus in Qinghai region

目的 对青海地区多房棘球绦虫和石渠棘球绦虫遗传分化特征进行分析,为青海省棘球蚴病的防控提供理论依据。方法 在玉树、果洛、黄南藏族自治州等棘球绦虫主要自然疫源地捕捉小型哺乳动物,剖检采集包囊,提取包囊组织基因组DNA,PCR扩增细胞色素氧化酶1 (cox1)基因并测序,运用DnaSP v6、Iqtree、BEAST v2.7.4等软件进行单倍型分析、核苷酸多态性分析、系统进化树构建及棘球绦虫属分化时间估算。结果从2 864只小型哺乳动物中共获得55份棘球蚴包囊。55份包囊DNA样品均扩增出长度约800 bp的cox1条带,其中37份为多房棘球绦虫,18份为石渠棘球绦虫,青海田鼠多房棘球蚴患病率为1.96%(37/1 884),高原鼠兔石渠棘球蚴患病率为1.84%(18/980)。37条多房棘球绦虫cox1序列共有单倍型5个,以EmH3单倍型为主(占33/37),单倍体多样性指数为0.207,核苷酸多样性指数为0.033 55,共有156个变异位点。18条石渠棘球绦虫cox1序列共有单倍型8个,以EsH2单倍型为主(占8/18),单倍型多样性指数为0.778,核苷酸多样性指数为0.060 52;共有14个变异位点。多房棘球绦虫和石渠棘球绦虫的13个单倍型上传GenBank,单倍型EmH1~EmH5的登录号依次为OR821706、 OR821707、 OR830343、 OR830344、 OR826123, EsH1~EsH8登录号依次为OR835156、 OR835157、OR830376、OR830378、OR831110、OR875250、OR835161、OR841080。系统进化树分析显示,多房棘球绦虫的5个单倍型与多房棘球绦虫亚洲株聚为一支,石渠棘球绦虫的8个单倍型与GenBank中的石渠棘球绦虫聚为一支。基于cox1基因的分化时间结果显示,细粒棘球绦虫、多房棘球绦虫、石渠棘球绦虫、少节棘球绦虫和伏氏棘球绦虫的共同祖先存在于约5.67百万年前(Mya)(95%CI:4.72~6.66 Mya),细粒棘球绦虫、石渠棘球绦虫和多房棘球绦虫的平均分化时间约为2.02 Mya (95%CI:1.51~2.49 Mya)。结论 青海地区多房棘球绦虫和石渠棘球绦虫具有较高的遗传多样性,其中多房棘球绦虫以EmH3单倍型为主,石渠棘球绦虫以EsH2单倍型为主。

Objectivee To analyze the genetic differentiation characteristics of Echinococcus multilocularis and E.shiquicus in Qinghai region to provide theoretical support for the prevention and control of echinococcosis in Qinghai Province.Methodss Small mammals were captured in the main natural endemic areas of Echinococcus spp.in Yushu,Guoluo,and Huangnan Tibetan Autonomous Prefecture and were dissected to collect cysts.The genomic DNA from cysts tissue was extracted and the cytochrome oxidase 1(coxl)gene was amplified using PCR and sequenced.DnaSP v6,Iqtree,BEAST v2.7.4 and other software were used for haplotype analysis,nucleotide polymorphism analysis,construction of a phylogenetic tree,and estimation of the divergence time of the Echinococcus genus.Results A total of 55 hydatid cysts were obtained from 2864 small mammals.All 55 cyst samples were amplified for coxl bands with a length of approximately 800 bp,of whichh37 were E.multilocularis,and 18 were E.shiquicus,the prevalence of E.multilocularis in Neodon fuscus was 1.96%(37/1884).The prevalence of E.shiquicus in Ochotona curzoniae was 1.84%(18/980).In the 37 coxl sequences of E.multilocularis,there were 5 haplotypes in the 37 coxl sequences of E.multilocularis with EmH3 being the predominant one(33/37),the haplotype diversity index was 0.207,the nucleotide diversity index was 0.03355,and there were 156 variable sites.In the 18 coxl sequences of E.shiquicus,There were 8 haplotypes,with the EsH2 haplotype being the predominant one(8/18),the haplotype diversity index was 0.778,the nucleotide diversity index was 0.06052,and there were 14 variable sites.Thirteen haplotypes of E.multilocularis andE.shiquensiswereuploaded to GenBank.The accession numbers of haplotypes EmH1-EmH5 are OR821706,OR821707,OR830343,OR830344,OR826123,respectively.The accession numbers of haplotypes ESH1-ESH8are OR835156,OR835157,OR830376,OR830378,OR831110,OR875250,OR835161,OR841080.The phylogenetic tree shows that the 5 haplotypes of E.multilocularis were clustered together with the Asian strain of E.multilocularis,and the 8 haplotypes of E.shiquicus were clustered with E.shiquicus in the GenBank.The divergence time based on the coxl gene showed that the common ancestor of E.granulosus,E.multilocularis,E.shiquicus,E.oligarthrus and E.vogeli existed approximately 5.67 million years ago(Mya)(95%CI:4.72-6.66 Mya),and the average divergence time for E.granulosus,E.shiquicus and E.multilocularis was approximately 2.02 Mya(95%CI:1.51-2.49 Mya).Conclusion E.multilocularis and E.shiquicus in Qinghai region have high genetic diversity,with EmH3 haplotype dominating E.multilocularis and EsH2 haplotype dominating E.shiquicus.