Lv Shan, Zhang Yi, Steinmann Peter, Utzinger Jürg, Zhou Xiao-Nong
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, 200025, People's Republic of China.
Swiss Tropical and Public Health Institute, P.O. Box, CH-4002, Basel, Switzerland.
Infect Dis Poverty. 2017 Sep 1;6(1):125. doi: 10.1186/s40249-017-0341-z.
The People's Republic of China (P.R. China) is the presumptive home range of the rat lungworm Angiostrongylus cantonensis, a major aetiological agent of human eosinophilic meningitis. We present a study of the genetic variation of A. cantonensis in P.R. China. Our aim was to deepen the current knowledge pertaining to its origin and global spread from a molecular perspective.
Adult A. cantonensis were collected in the frame of a national survey and identified based on morphological criteria. Polymerase chain reaction (PCR) was employed to amplify the target DNA sequences (cytochrome c oxidase subunit I (cox1), nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) and internal transcribed spacer (ITS)). The PCR product of cox1 was directly submitted to sequencing, while clone sequencing was used for nad1 and ITS. The identity of the samples was verified by comparing the sequences to those of accepted A. cantonensis specimens. The specific composition of substitutions in each gene was analysed, and the genotypes were compared based on the complete cox1, nad1 and ITS genes.
We characterised the complete mitochondrial genes cox1 and nad1 of 130 specimens and obtained 357 nuclear sequences containing two complete ITS (ITS1 and ITS2) and 5.8S rRNA of the same samples. All specimens were genetically confirmed as A. cantonensis. Two major groups (i.e. I and II) were identified according to the phylogeny of cox1 sequences. Group I could be further categorised into six distinct clades. Almost half of the specimens (47.7%) belong to the clade Ia and 22.3% to the group II. The former was widely distributed across the study region. A variable number of repeat units in three microsatellites was observed, resulting in considerable length variation in ITS. Intragenomic variation of ITS sequences was found in a large proportion of the samples. Genotyping showed a striking difference between mitochondrial DNA and ITS.
Our results demonstrate that A. cantonensis is the only rat lungworm species in P.R. China and shows high genetic diversity. Results of diversity and genotyping of A. cantonensis can be impacted by the sequencing strategy and biomarker. Although ITS may be a valuable marker for interspecific identification, it is not suitable for studying the intraspecific variation of A. cantonensis due to its high intragenomic variation and current challenges for direct sequencing.
中华人民共和国是广州管圆线虫的假定栖息地，广州管圆线虫是人类嗜酸性粒细胞性脑膜炎的主要病原体。我们开展了一项关于中国广州管圆线虫基因变异的研究。我们的目的是从分子角度加深对其起源和全球传播的现有认识。
在一项全国性调查中收集成年广州管圆线虫，并根据形态学标准进行鉴定。采用聚合酶链反应（PCR）扩增目标DNA序列（细胞色素c氧化酶亚基I（cox1）、烟酰胺腺嘌呤二核苷酸脱氢酶亚基1（nad1）和内转录间隔区（ITS））。cox1的PCR产物直接进行测序，而nad1和ITS则采用克隆测序。通过将序列与公认的广州管圆线虫标本序列进行比较来验证样本的身份。分析每个基因中替换的具体组成，并基于完整的cox1、nad1和ITS基因比较基因型。
我们对130个标本的完整线粒体基因cox1和nad1进行了特征分析，并获得了357个核序列，其中包含相同样本的两个完整ITS（ITS1和ITS2）以及5.8S rRNA。所有标本在基因上均被确认为广州管圆线虫。根据cox1序列的系统发育确定了两个主要类群（即I和II）。类群I可进一步分为六个不同的分支。几乎一半的标本（47.7%）属于分支Ia，22.3%属于类群II。前者广泛分布于研究区域。在三个微卫星中观察到可变数量的重复单元，导致ITS长度有相当大的差异。在很大一部分样本中发现了ITS序列的基因组内变异。基因分型显示线粒体DNA和ITS之间存在显著差异。
我们的结果表明，广州管圆线虫是中国唯一的鼠肺线虫物种，且具有高度的遗传多样性。广州管圆线虫的多样性和基因分型结果可能受到测序策略和生物标志物的影响。尽管ITS可能是种间鉴定的一个有价值的标记，但由于其高度的基因组内变异以及目前直接测序面临的挑战，它不适合用于研究广州管圆线虫的种内变异。
