青海人源两型棘球绦虫（棘球蚴）遗传多样性及分化时间研究

doi:10.12140/j.issn.1000-7423.2022.05.007

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (5): 610-615.doi: 10.12140/j.issn.1000-7423.2022.05.007

青海人源两型棘球绦虫（棘球蚴）遗传多样性及分化时间研究

毋德芳¹(), 付永², 任宾¹, 张耀刚¹, 许晓磊¹, 庞明泉¹, 樊海宁¹^,^*()

1.青海大学附属医院,青海省包虫病研究重点实验室,西宁 810001
2.青海省畜牧兽医科学院,西宁 810001

收稿日期:2021-12-30 修回日期:2022-02-26 出版日期:2022-10-30 发布日期:2022-10-24
通讯作者: 樊海宁
作者简介:毋德芳（1988-）,女,硕士研究生,从事棘球蚴病基础研究。E-mail： w627138884@163.com
基金资助:
国家自然科学基金(81960576);青海省应用基础研究项目(2021-ZJ-724)

Genetic diversity and differentiation time of human isolates of Echinococcus granulosus and E. multilocularis from Qinghai

WU De-fang¹(), FU Yong², REN Bin¹, ZHANG Yao-gang¹, XU Xiao-lei¹, PANG Ming-quan¹, FAN Hai-ning¹^,^*()

1. Affiliated Hospital of Qinghai University, Qinghai Research Key Laboratory for Echinococcosis, Xining 810001, China
2. Qinghai Academy of Animal Science and Veterinary Medicine, Xining 810001, China

Received:2021-12-30 Revised:2022-02-26 Online:2022-10-30 Published:2022-10-24
Contact: FAN Hai-ning
Supported by:
National Natural Science Foundation of China(81960576);Project of Science and Technology Department of Qinghai Province(2021-ZJ-724)

摘要/Abstract

摘要：

目的分析青海人源细粒棘球绦虫（细粒棘球蚴）和多房棘球绦虫（多房棘球蚴）遗传多样性、群体间遗传差异及分化时间,为青海省棘球绦虫溯源及防控提供科学依据。方法收集2016—2021年青海大学附属医院住院棘球蚴病患者肝病灶样品50份,提取基因组DNA,PCR扩增线粒体脱氢酶1（nad1）基因,采用Clustal X v2.0软件对序列进行多重比对,应用ArcGIS软件进行患者居住地的地理信息学构图,应用DnaSP v6软件对序列单倍型进行分析,应用Modeltest 3.7软件及PAUP*4.0B10软件运行计算最小最适核酸进化模型,用MrBayes-3.2.7软件构建贝叶斯系统进化树,通过BEAST v2.6.3软件用贝叶斯方法估算系统进化树各节点的分化时间。结果成功鉴定出48份棘球蚴病灶样品,获得长度为894 bp的nad1完整基因序列。其中13份样品被鉴定为细粒棘球绦虫G1基因型,35份样品被鉴定为多房棘球绦虫。所有序列均与GenBank中的序列具有大于99%的相似度。两种棘球绦虫分别鉴定出4个单倍型H1~H4：细粒棘球绦虫以H3为主导单倍型（占10/13）,分布在西宁、果洛、玉树、海东、海北和黄南等地;多房棘球绦虫以H2为优势单倍型（占51.4%,18/35）,分布在西宁、果洛、玉树、海东等地。系统发育进化树显示,细粒棘球绦虫与G1基因型聚为一支,多房棘球绦虫与亚洲株聚为一支。分化时间分析结果显示,棘球属中细粒棘球绦虫、多房棘球绦虫、福氏棘球绦虫、少节棘球绦虫最近共同祖先存在大约5.5百万年前（millions of years ago,Mya）（95%置信区间4.5~6.5 Mya）,细粒棘球绦虫和多房棘球绦虫分化的时间大约为2.5 Mya（95%置信区间2.3~4.1 Mya）。结论青海省人源细粒棘球绦虫和多房棘球绦虫均表现出较高的遗传多样性,细粒棘球绦虫为G1基因型,以H3为主导单倍型,多房棘球绦虫以H2为优势单倍型,二者均广泛分布于青海各地。棘球属中细粒棘球绦虫、多房棘球绦虫的亲缘关系较近且分化时间也较近。

关键词: 细粒棘球蚴, 多房棘球蚴, nad1基因, 单倍型, 分化时间

Abstract:

Objective To analyzed the genetic diversity, genetic differences between populations and differentiation time of Echinococcus granulosus and E. multilocularis of Qinghai isolates, in order to provide scientific basis for species tracing and prevention and control of Echinococcus in Qinghai Province, China. Methods For genetic analysis, 50 liver lesion samples were collected from hospitalized echinococcosis patients in the Affiliated Hospital of Qinghai University to extract genomic DNA and amplify mitochondrial dehydrogenase 1 gene (nad1). Sequence multiple alignment was performed using Clustal X v2.0 software. Geographic informatics mapping of patients’ residence was constructed using ArcGIS software. Sequence haplotype analysis was made with DnaSP v6 software. Modeltest 3.7 software and PAUP*4.0B10 software were used to calculate the minimum optimal nucleic acid evolution model. The Bayesian’s phylogenetic evolution tree was constructed with MrBayes-3.2.7 software. The differentiation time of each node in the phylogenetic tree was estimated with the Bayesian method using BEAST v2.6.3 software. Results We successfully identified 48 Echinococcus lesion samples specimen and obtained the full length of complete nad1 gene of 894 bp. Among them, 13 samples were identified as the G1 genotype of E. granulosus, and 35 samples as E. multilocularis. All the sequences showed > 99% similarity to those in GenBank. Four haplotypes were identified as H1-H4 in the two species respectively; H3 was the dominant haplotype in E. granulosus samples(10/13), which is present in Xining, Guoluo, Yushu, Haidong, Haibei and Huangnan. H2 haplotype was found dominant in E. multilocular samples (51.4%,18/35), which is present in Xining, Guoluo, Yushu, and Haidong. The phylogenetic tree showed that E. granulosus and G1 genotype clustered into one branch, and E. multilocularis and Asian strain clustered into one branch. The results of differentiation time showed that the nearest common ancestor of E. granulosus, E. multilocularis, E. vogeli and E. oligarthrus was about 5.5 Mya (95% confidence interval 4.5-6.5 Mya), and the differentiation time of E. granulosus and E. multilocularis was about 2.5 Mya (95% confidence interval 2.3-4.1 Mya). Conclusion Both human E. granulosus and E. multilocularis in Qinghai Province show high genetic diversity. E. granulosus was found of G1 genotype, with H3 as the dominant haplotype, while in E. multilocularis samles H2 is the dominant. The two speies are widely distributed throughout Qinghai Province. The two species of Echinococcus exhit closer genetic relationship and differentiation timing.

Key words: Echinococcus granulosus, Echinococcus multilocularis, nad1 gene, Haplotype, Differentiation time

中图分类号:

R383.33

毋德芳, 付永, 任宾, 张耀刚, 许晓磊, 庞明泉, 樊海宁. 青海人源两型棘球绦虫（棘球蚴）遗传多样性及分化时间研究[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 610-615.

WU De-fang, FU Yong, REN Bin, ZHANG Yao-gang, XU Xiao-lei, PANG Ming-quan, FAN Hai-ning. Genetic diversity and differentiation time of human isolates of Echinococcus granulosus and E. multilocularis from Qinghai[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(5): 610-615.

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分型 Species identification	序列数 No. sequence	单倍型数量 No. haplotype	单倍型多样性（HD）Haplotype diversity(HD)	核苷酸多样性（π） Nucleotide diversity(π)	单倍型（频率） Haplotype (Frequency)
细粒棘球绦虫 E. granulosus	13	4	0.423	0.000 69	EgH1（1）,EgH2（1）, EgH3（10）,EgH4（1）
多房棘球绦虫 E. multilocularis	35	4	0.588	0.000 76	EmH1（2）,EmH2（18）,EmH3（14）,EmH4（1）

青海人源两型棘球绦虫（棘球蚴）遗传多样性及分化时间研究

Genetic diversity and differentiation time of human isolates of Echinococcus granulosus and E. multilocularis from Qinghai

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