目的 了解新疆新源县和四川石渠县棘球绦虫线粒体细胞色素c氧化酶亚基1（cox1）基因的遗传多态性,探讨棘球绦虫在我国的传播动力和传播方向。 方法 2014-2018年,在新疆新源县、四川石渠县捕捉啮齿类动物并采集其肝组织样品,收集家畜病变脏器。提取肝组织和病变脏器组织的DNA,PCR扩增棘球绦虫cox1基因片段,经分子克隆、测序后,在NCBI数据库中比对以确定所感染的虫种。使用Clustal X2 和MEGA 7剪辑序列,使用DnaSP v5和Arlequin 3.5计算核苷酸多态性和中性检验,分析新源县和石渠县棘球绦虫的遗传多样性。以石渠棘球绦虫为外群,用MrBayes 3.2.4建立基于cox1基因的贝叶斯系统进化树,总结分析虫种的基因型。采用Network 5.0绘制棘球绦虫cox1基因的单倍型网络图,分析单倍型结构。 结果 在新疆新源县捕获普通田鼠122只,收集绵羊病变脏器5个;在四川石渠县捕获青海田鼠144只、经营田鼠44只和高原鼠兔135只,收集牦牛病变脏器6个。其中从5只绵羊、4头牦牛的病变脏器样品DNA扩增获得细粒棘球绦虫cox1基因片段40条（新源县26条,石渠县14条）,从8只普通田鼠、14只青海田鼠、5只经营田鼠和2只高原鼠兔获得多房棘球绦虫线粒体cox1基因59条（新源县20条,石渠县39条）,扩增产物长度均为875 bp。细粒棘球绦虫在两地的遗传分化程度（Fst = 0.088 63）高于多房棘球绦虫（Fst = 0.000 88）,新源县细粒棘球绦虫遗传多样性（0.002 58 ± 0.000 46）低于石渠县（0.005 88 ± 0.000 58）,而其多房棘球绦虫遗传多样性（0.002 28 ± 0.000 46）高于石渠县（0.001 37 ± 0.000 30）。贝叶斯系统进化树显示,两地的细粒棘球绦虫基因型为G1型,多房棘球绦虫为亚洲型。序列比对发现25个细粒棘球绦虫cox1基因单倍型（新源县15个,石渠县9个,共同单倍型1个）和29个多房棘球绦虫cox1基因单倍型（新源县13个,石渠县15个,共同单倍型1个）,两种棘球绦虫的单倍型网络图均为环绕主单倍型（共同单倍型）的辐射状结构,新源县的细粒棘球绦虫序列中有9条为主单倍型（34.6%,9/26）,而石渠县仅有1条（1/14）,显示出新源县在细粒棘球绦虫单倍型结构中的核心位置;石渠县的多房棘球绦虫基因序列中有23条为主单倍型（58.9%,23/39）,而新源县为7条（35.0%,7/20）,表明石渠县在多房棘球绦虫的单倍型结构中更具有主导地位。 结论 新源县和石渠县的棘球绦虫线粒体cox1基因型一致,但遗传多态性存在一定差异,同等空间跨度上细粒棘球绦虫的遗传分化程度要高于多房棘球绦虫,这些差异为探讨棘球绦虫传播方向和动力提供了重要参考依据。
Objective To understand the genetic polymorphisms of cytochrome c oxidase subunit 1 (cox1) gene in mitochondria of Echinococcus spp. in Xinyuan County, Xinjiang and Shiqu County, Sichuan, and discuss Echinococcus transmission directions and dynamics in China. Methods From 2014 to 2018, rodents were captured in Xinyuan County (Xinjiang) and Shiqu County (Sichuan) to collect liver tissues, meanwhile lesioned tissue samples of livestock were collected. DNA was extracted from the livers and lesioned tissues, and underwent PCR to amplify part of Echinococcus mitochondrial cox1 gene. PCR products were sequenced, and the sequencing results were aligned in NCBI database to identify the Echinococcus species. The sequences were edited by Clustal X2 and MAGE 7 softwares, and diversity and neutrality indices were calculated by DnaSP v5 and Arlequin 3.5. Genetic diversities were compared between the two regions. By using E. shiquicus as an outgroup, cox1 sequence-based Bayesian phylogenetic trees were established with MrBayes 3.2.4 software. The haplotype networks were drawn for cox1 genes of Echinococcus spp. using Network 5.0 to analyze haplotype structure. Results One hundred and twenty-two Microtus arvalis were captured and 5 lesioned tissues of sheep were collected in Xinyuan County (Xinjiang); while in Shiqu County (Sichuan), 144 Lasiopodomys fuscus, 44 M. limnophilus and 135 Ochotona curzoniae were captured and 4 lesioned tissues of yaks were collected. A total of 40 sequences of E. granulosus cox1 genes (26 from Xinyuan, and 14 from Shiqu) were amplified from lesioned tissues of 5 sheeps and 4 yaks. Fifty-nine sequences of E. multilocularis mitochondrial cox1 genes (20 from Xinyuan, and 39 from Shiqu) were amplified in 8 M. arvalis, 14 L. fuscus, 5 M. limnophilus and 2 O. curzoniae. All PCR products had a length of 875 bp. E. granulosus showed a higher degree of genetic differentiation (Fst = 0.088 63) between the 2 regions than E. multilocularis (Fst = 0.000 88). The genetic diversity of E. granulosus was lower in Xinyuan (0.002 58 ± 0.000 46) than in Shiqu (0.005 88 ± 0.000 58), while E. multilocularis had higher genetic diversity in Xinyuan (0.002 28 ± 0.000 46) than in Shiqu (0.001 37 ± 0.000 30). The Bayesian phylogenetic tree showed that in both regions E. granulosus had the G1 genotype, and E. multilocularis was of the Asian type. Sequences alignment revealed 25 haplotypes for E. granulosus cox1 gene (15 in Xinyuan, 9 in Shiqu, and 1 shared by both) and 29 haplotypes for E. multilocularis (13 in Xinyuan, 15 in Shiqu, 1 shared by both). The haplotype networks of E. multilocularis and E. granulosus both showed a star-shaped network with a centrally placed main haplotype. There were 9 sequences of E. granulosus being the main haplotype type in Xinyuan (34.6%, 9/26), while only 1 sequence of that type was found in Shiqu (1/14), indicating a central role of Xinyuan County in the E. granulosus haplotype network. On the contrary, there were 23 sequences of E. multilocularis being the main haplotype type in Shiqu (58.9%, 23/39) and 7 of that type in Xinyuan (35.0%, 7/20), indicating that Shiqu County was more predominant in the E. multilocularis haplotype structure. Conclusion There is no apparent difference in the mitochondrial cox1 genotype of Echinococcus found between Xinyuan and Shiqu County, but there are some differences in the genetic polymorphism detected in the two sites. The genetic differentiation level of E. granulosus is higher than that of E. multilocularis over the same spatial span. These findings provide important referential basis for further study on transmission direction and dynamics of Echinococcus.
[1] Eckert J, Gemmell MA, Meslin FX, et al. WHO/OIE manual on echinococcosis in humans and animals: a public health problem of global concern[C]. Paris: World Organisation for Animal Health, 2001.
[2] Wang X, Liu JY, Zuo QQ, et al. Echinococcus multilocularis and Echinococcus shiquicus in a small mammal community on the eastern Tibetan Plateau: host species composition, molecular prevalence, and epidemiological implications[J]. Parasit Vectors, 2018, 11(1): 302.
[3] Wu WP, Wang H, Wang Q, et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 1-14. (in Chinese)
(伍卫平, 王虎, 王谦, 等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 1-14.)
[4] Eckert J, Conraths FJ, Tackmann K.Echinococcosis: an emerging or re-emerging zoonosis?[J]. Int J Parasitol, 2000, 30(12/13): 1283-1294.
[5] Liu HD, Wang HB, Fan HN, et al. Alveolar echinococcosis and immune evasion[J]. Chin J Parasitol Parasit Dis, 2018, 36(6): 655-660. (in Chinese)
(刘寒冬, 王宏宾, 樊海宁, 等. 多房棘球蚴病的免疫逃避机制[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(6): 655-660.)
[6] Shang JY, Zhang GJ, Liao S, et al. A multiplex PCR for differential detection of Echinococcus granulosus sensu stricto, Echinococcus multilocularis and Echinococcus canadensis in China[J]. Infect Dis Poverty, 2019, 8: 68.
[7] Wang ZH, Wang XM, Liu XQ.Echinococcosis in China, a review of the epidemiology of Echinococcus spp.[J]. Ecohealth, 2008, 5(2): 115-126.
[8] Beiromvand M, Akhlaghi L, Fattahi Massom SH, et al. Detection of Echinococcus multilocularis in Carnivores in Razavi Khorasan Province, Iran using mitochondrial DNA[J]. PLoS Negl Trop Dis, 2011, 5(11): e1379.
[9] Smith AT, Xie Y. A Guide to the mammals of China[M]. Changsha: Hunan Education Publishing House, 2009. (in Chinese)
(Smith AT, 解焱. 中国兽类野外手册[M]. 长沙: 湖南教育出版社, 2009.)
[10] He W, Wang Q, Huang Y, et al. Risk factors of echinococcosis in Shiqu County[J]. Chin J Parasitol Parasit Dis, 2019, 37(4): 428-432. (in Chinese)
(何伟, 王谦, 黄燕, 等. 四川省石渠县棘球蚴病流行的影响因素分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 428-432.)
[11] Wumaier M, Osman Y, Simayi A, et al. A survey on Echinococcus infections in animals in Xinjiang Uygur Autonomous Region[J]. Chin J Parasitol Parasit Dis, 2017, 35(2): 145-149. (in Chinese)
(买买提江·吾买尔, 伊斯拉音·乌斯曼, 阿迪力·司马义, 等. 新疆维吾尔自治区动物棘球绦虫感染调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 145-149.)
[12] Nakao M, Sako Y, Yokoyama N, et al. Mitochondrial genetic code in cestodes[J]. Mol Biochem Parasitol, 2000, 111(2): 415-424.
[13] Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0.[J]. Bioinformatics, 2007, 23(21): 2947-2948.
[14] Kumar S, Stecher G, Tamura K.MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Mol Biol Evol, 2016, 33(7): 1870-1874.
[15] Librado P, Rozas J.DnaSP v5: a software for comprehensive analysis of DNA polymorphism data[J]. Bioinformatics, 2009, 25(11): 1451-1452.
[16] Excoffier L, Lischer HE.Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows[J]. Mol Ecol Resour, 2010, 10(3): 564-567.
[17] Xia XH.DAMBE5: a comprehensive software package for data analysis in molecular biology and evolution[J]. Mol Biol Evol, 2013, 30(7): 1720-1728.
[18] Darriba D, Taboada GL, Doallo R, et al. jModelTest 2: more models, new heuristics and parallel computing[J]. Nat Methods, 2012, 9(8): 772.
[19] Ronquist F, Huelsenbeck JP.MrBayes 3: Bayesian phylogenetic inference under mixed models[J]. Bioinformatics, 2003, 19(12): 1572-1574.
[20] Nakao M, McManus DP, Schantz PM, et al. A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes[J]. Parasitology, 2007, 134(Pt 5): 713-722.
[21] Wei YH, Hu Y, Cao JP.Advances in the genetic polymorphism of Echinococcus granulosus[J]. Chin J Parasitol Parasit Dis, 2019, 37(4): 481-485. (in Chinese)
(魏玉环, 胡媛, 曹建平. 细粒棘球绦虫基因多态性的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 481-485.)
[22] Kinkar L, Laurimäe T, Acosta-Jamett G, et al. Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1[J]. Int J Parasitol, 2018, 48(9/10): 729-742.
[23] Nakao M, Li TY, Han XM, et al. Genetic polymorphisms of Echinococcus tapeworms in China as determined by mitochondrial and nuclear DNA sequences[J]. Int J Parasitol, 2010, 40(3): 379-385.
