Yan Xiaofei, He Sangang, Liu Yiyong, Han Bing, Zhang Ning, Deng Haifeng, Wang Yuqi, Liu Mingjun
College of Life Sciences, Xinjiang Agricultural University, Urumqi, 830052, China.
Institute of Biotechnology, Xinjiang Academy of Animal Science, Key Laboratory of Genetic Breeding and Reproduction of Herbivorous Livestock of Ministry of Agriculture, Xinjiang Key Laboratory of Animal Biotechnology, Urumqi, Xinjiang Province, China.
Exp Parasitol. 2023 Nov;254:108625. doi: 10.1016/j.exppara.2023.108625. Epub 2023 Sep 26.
Gastrointestinal nematode (GIN) infection in sheep has been recognized globally as a major problem challenging animal health and production. The objective of this study is to use a molecular diagnosis of the prevalence for gastrointestinal nematode (GIN) dominant species of Kazakh sheep and its hybrid (Kazakh × Texel). The internal transcribed spacer 2 (ITS-2) sequences of ribosomal DNA (rDNA) were used as the target sequence. In the study, three dominant species of nematodes, namely Haemonchus contortus, Trichostrongylus spp., and Teladorsagia (Ostertagia) circumcincta from the Kazakh sheep and the F1 and F2 generations of Texel × Kazakh sheep hybrids were subjected to molecular identification and phylogenetic analysis. The fecal and single larva genomic DNA were extracted and amplified by PCR using specific primers to determine the infection rate of the three nematode species. In addition, the PCR products were sequenced and analyzed using bioinformatics methods to construct a phylogenetic tree. The results showed that all the three species had their ITS-2 specific amplified. According to the sequence homology analysis of PCR products, the results showed a high homology (above 98.5% homology) with H. contortus, Trichostrongylus spp., T. circumcincta ITS-2 sequences in GenBank. Phylogenetic analysis showed that the ITS-2 sequences of the three species were on the same branch as the ITS-2 sequences of the same species in NCBI. And on different branches from those of the ITS-2 sequences of different families, genera and species. Sequences carried out on three species from different samples showed a close relationship and little genetic difference in phylogenetic tree. The infection rates based on fecal DNA were 35.59, 25.55, and 11.24% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. While the infection rates based on larva DNA, were 24.07, 18.89, and 13.26% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. The seasonal prevalence of the three dominant species in spring was significantly higher than that in autumn and winter. And there was no significant difference between Kazakh, F1 and F2 sheep considering the infection rate of the studied three species of nematodes. This study provides valuable molecular approaches for epidemiological surveillance and for assisting in the control of Nematodirus infection in sheep.
绵羊胃肠道线虫（GIN）感染在全球范围内已被公认为是一个挑战动物健康和生产的主要问题。本研究的目的是利用分子诊断方法检测哈萨克羊及其杂交品种（哈萨克羊×特克塞尔羊）胃肠道线虫优势种的感染率。核糖体DNA（rDNA）的内部转录间隔区2（ITS-2）序列被用作目标序列。在本研究中，对来自哈萨克羊以及特克塞尔羊×哈萨克羊杂交一代和二代的三种优势线虫，即捻转血矛线虫、毛圆线虫属和环形泰勒虫（奥斯特线虫）进行了分子鉴定和系统发育分析。提取粪便和单个幼虫的基因组DNA，使用特异性引物通过PCR进行扩增，以确定这三种线虫的感染率。此外，对PCR产物进行测序，并采用生物信息学方法进行分析，构建系统发育树。结果表明，这三种线虫均有其ITS-2特异性扩增产物。根据PCR产物的序列同源性分析，结果显示与GenBank中捻转血矛线虫、毛圆线虫属、环形泰勒虫的ITS-2序列具有高度同源性（同源性高于98.5%）。系统发育分析表明，这三种线虫的ITS-2序列与NCBI中同物种的ITS-2序列位于同一分支上，且与不同科、属、种的ITS-2序列位于不同分支上。对来自不同样本的这三种线虫进行的序列分析表明，它们在系统发育树中关系密切，遗传差异较小。基于粪便DNA的捻转血矛线虫、毛圆线虫属和环形泰勒虫的感染率分别为35.59%、25.55%和11.24%。而基于幼虫DNA的捻转血矛线虫、毛圆线虫属和环形泰勒虫的感染率分别为24.07%、18.89%和13.26%。这三种优势线虫春季的季节性感染率显著高于秋季和冬季。并且，就所研究的这三种线虫的感染率而言，哈萨克羊、杂交一代和杂交二代羊之间没有显著差异。本研究为绵羊线虫感染的流行病学监测和防控提供了有价值的分子方法。
