Tang Ying, Li Na, Song Mingxin, Roellig Dawn M, Feng Yaoyu, Xiao Lihua
Division of Foodborne, Waterborne and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA; College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, China.
School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China.
Infect Genet Evol. 2016 Nov;45:256-261. doi: 10.1016/j.meegid.2016.09.011. Epub 2016 Sep 12.
Cryptosporidium ubiquitum is an emerging zoonotic pathogen in humans. Recently, a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene was developed for C. ubiquitum, and identified six subtype families (XIIa-XIIf). In this study, we selected five genetic loci known to be polymorphic in C. hominis and C. parvum for the development of a multilocus subtyping tool for C. ubiquitum, including CP47 (cgd6_1590), MSC6-5 (cgd6_4290), cgd6_60, cgd2_3690, and cgd4_370. PCR primers for these targets were designed based on whole genome sequence data from C. ubiquitum. DNA sequence analyses of 24 C. ubiquitum specimens showed the presence of 18, 1, 5, 4, and 5 subtypes at the CP47, MSC6-5, cgd6_60, cgd2_3690, and cgd4_370 loci, respectively. Altogether, 18 multilocus sequence typing (MLST) subtypes were detected among the 19 specimens successfully sequenced at all polymorphic loci. Phylogenetic analyses of the MLST data indicated that the rodent subtype families of XIIe and XIIf were highly divergent from others, and the ruminant XIIa subtype family formed a monophyletic group genetically distant from other rodent subtype families XIIb, XIIc, and XIId. The latter showed no consistent grouping of specimens and formed one large cluster in phylogenetic analysis of concatenated multilocus sequences. This was supported by results of STRUCTURE and F analyses, which further suggested that XIIa originated from one common ancestor whereas XIIb, XIIc, and XIId contained mixed ancestral types, reflecting a close relatedness of the three subtype families and the likely occurrence of genetic recombination among them. Thus, an MLST tool was developed for high-resolution subtyping of C. ubiquitum and results of preliminary characterizations of specimens from humans and animals supported the conclusion on the existence of ruminant and rodent-adapted C. ubiquitum groups.
泛在隐孢子虫是一种新出现的人兽共患病原体。最近，针对泛在隐孢子虫开发了一种靶向60 kDa糖蛋白（gp60）基因的分型工具，并鉴定出6个亚型家族（XIIa - XIIf）。在本研究中，我们选择了已知在人隐孢子虫和微小隐孢子虫中具有多态性的5个基因座，用于开发泛在隐孢子虫的多位点分型工具，包括CP47（cgd6_1590）、MSC6 - 5（cgd6_4290）、cgd6_60、cgd2_3690和cgd4_370。基于泛在隐孢子虫的全基因组序列数据设计了这些靶点的PCR引物。对24份泛在隐孢子虫标本的DNA序列分析显示，在CP47、MSC6 - 5、cgd6_60、cgd2_3690和cgd4_370基因座分别存在18、1、5、4和5个亚型。在所有多态性基因座成功测序的19份标本中，共检测到18种多位点序列分型（MLST）亚型。对MLST数据的系统发育分析表明，XIIe和XIIf的啮齿动物亚型家族与其他家族高度不同，反刍动物XIIa亚型家族形成一个单系群，在遗传上与其他啮齿动物亚型家族XIIb、XIIc和XIId距离较远。后者在串联多位点序列的系统发育分析中没有显示出一致的标本分组，而是形成了一个大的聚类。STRUCTURE和F分析结果支持了这一点，进一步表明XIIa起源于一个共同祖先，而XIIb、XIIc和XIId包含混合的祖先类型，这反映了这三个亚型家族的密切相关性以及它们之间可能发生的基因重组。因此，开发了一种用于泛在隐孢子虫高分辨率分型的MLST工具，对人和动物标本的初步特征分析结果支持了反刍动物和啮齿动物适应的泛在隐孢子虫群体存在的结论。
