Sharbatkhori Mitra, Mirhendi Hossein, Jex Aaron R, Pangasa Aradhana, Campbell Bronwyn E, Kia Eshrat B, Eshraghian Mohammad R, Harandi Majid F, Gasser Robin B
Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia.
Electrophoresis. 2009 Aug;30(15):2648-55. doi: 10.1002/elps.200900145.
In the present study, we have extended earlier taxonomic, biochemical and experimental investigations to characterize Echinococcus granulosus from various hosts in Iran utilizing DNA regions (designated pcox1 and pnad1) within the cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 mitochondrial genes, respectively. An emphasis was placed on the characterization of E. granulosus isolates (cyst material) from humans, sheep, goats, cattle and camels, and on assessing their genetic relationships. PCR-based SSCP analysis of pcox1 and pnad1 amplicons derived from individual isolates (n=148) of E. granulosus revealed five (pc1-pc5) and nine (pn1-pn9) electrophoretic profiles, respectively. Sequencing of pcox1 and pnad1 amplicons representing unique SSCP profiles demonstrated that each profile was linked unequivocally to a particular sequence and that single point mutations were readily detectable by SSCP. Phylogenetic analyses of pcox1 and/or pnad1 nucleotide sequence data were conducted using Bayesian inference and maximum likelihood tree-building methods. Following the phylogenetic analyses of concatenated pcox1+pnad1 sequence data, including representatives of all presently recognized Echinococcus species/genotypes as well as Taenia saginata (as the outgroup), the majority of cyst isolates (142 of 148; 95.9%) from humans, ruminants (sheep, goats and cattle) and camels were assigned to the G1-G3 complex of E. granulosus (or E. granulosus sensu stricto), whereas some E. granulosus cysts (6 of 19; 31.6%) from camels were assigned to the G6-G10 complex (or E. canadensis). The present study reinforces the advantages of the mutation scanning-sequencing-phylogenetic approach to explore variation in multiple mitochondrial loci within and among Echinococcus populations, which provides a platform for future, detailed studies of the molecular epidemiology of E. granulosus in Iran and other countries. (Note: The sequences determined in the present study have been deposited in the GenBank database under accession numbers: FJ796203-FJ796207 (pcox1) and FJ796208-FJ796216 (pnad1)).
在本研究中，我们扩展了早期的分类学、生物化学和实验研究，利用细胞色素c氧化酶亚基1和NADH脱氢酶1线粒体基因中的DNA区域（分别命名为pcox1和pnad1）对伊朗不同宿主中的细粒棘球绦虫进行特征描述。重点是对来自人类、绵羊、山羊、牛和骆驼的细粒棘球绦虫分离株（囊液）进行特征描述，并评估它们的遗传关系。对来自细粒棘球绦虫单个分离株（n = 148）的pcox1和pnad1扩增子进行基于PCR的SSCP分析，分别揭示了五种（pc1 - pc5）和九种（pn1 - pn9）电泳图谱。对代表独特SSCP图谱的pcox1和pnad1扩增子进行测序表明，每个图谱都明确与特定序列相关联，并且单点突变可通过SSCP轻松检测到。使用贝叶斯推断和最大似然树构建方法对pcox1和/或pnad1核苷酸序列数据进行系统发育分析。在对串联的pcox1 + pnad1序列数据进行系统发育分析后，包括所有目前公认的棘球绦虫物种/基因型以及牛带绦虫（作为外群）的代表，来自人类、反刍动物（绵羊、山羊和牛）和骆驼中的大多数囊液分离株（148个中的142个；95.9%）被归类为细粒棘球绦虫的G1 - G3复合体（或狭义的细粒棘球绦虫），而来自骆驼的一些细粒棘球绦虫囊肿（19个中的6个；31.6%）被归类为G6 - G10复合体（或加拿大棘球绦虫）。本研究强化了突变扫描 - 测序 - 系统发育方法在探索棘球绦虫种群内部和种群之间多个线粒体基因座变异方面的优势，这为未来在伊朗和其他国家对细粒棘球绦虫分子流行病学进行详细研究提供了一个平台。（注：本研究中确定的序列已存入GenBank数据库，登录号为：FJ796203 - FJ796207（pcox1）和FJ796208 - FJ796216（pnad1））
