Amarante M R V, Bassetto C C, Neves J H, Amarante A F T
Departamento de Parasitologia,Instituto de Biociências, Unesp-Universidade Estadual Paulista,Botucatu,SP,BrazilCEP 18618-970.
J Helminthol. 2014 Dec;88(4):447-52. doi: 10.1017/S0022149X13000412. Epub 2013 May 31.
Agricultural ruminants usually harbour mixed infections of gastrointestinal nematodes. A specific diagnosis is important because distinct species can differ significantly in their fecundity and pathogenicity. Haemonchus spp. and Cooperia spp. are the most important gastrointestinal nematodes infecting ruminants in subtropical/tropical environments. In Brazil, C. punctata is more adapted to cattle than sheep. Additionally, C. spatulata appears to be more adapted to cattle, whereas C. curticei is more adapted to sheep. However, infection of sheep with C. punctata is common when cattle and sheep share the same pasture. Although morphological analyses have been widely used to identify nematodes, molecular methods can overcome technical limitations and help improve species-specific diagnoses. Genetic markers in the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA (rDNA) have been used successfully to detect helminths. In the present study, the ITS-1 region was analysed and used to design a species-specific oligonucleotide primer pair to identify C. curticei. The polymerase chain reaction (PCR) product was sequenced and showed 97% similarity to C. oncophora partial ITS-1 clones and 99% similarity to the C. curticei sequence JF680982. The specificity of this primer pair was corroborated by the analysis of 17 species of helminths, including C. curticei, C. punctata and C. spatulata. Species-specific diagnosis, which has implications for rapid and reliable identification, can support studies on the biology, ecology and epidemiology of trichostrongylid nematodes in a particular geographical location.
农业反刍动物通常感染多种胃肠道线虫。进行特异性诊断很重要，因为不同的线虫种类在繁殖力和致病性上可能有显著差异。血矛线虫属和古柏线虫属是亚热带/热带环境中感染反刍动物的最重要的胃肠道线虫。在巴西，点状古柏线虫对牛的适应性强于对羊的适应性。此外，匙形古柏线虫似乎对牛的适应性更强，而柯氏古柏线虫对羊的适应性更强。然而，当牛和羊共用牧场时，羊感染点状古柏线虫的情况很常见。虽然形态学分析已被广泛用于鉴定线虫，但分子方法可以克服技术限制并有助于改进物种特异性诊断。核糖体DNA（rDNA）的第一和第二内部转录间隔区（分别为ITS-1和ITS-2）中的遗传标记已成功用于检测蠕虫。在本研究中，对ITS-1区域进行了分析，并用于设计一对物种特异性寡核苷酸引物以鉴定柯氏古柏线虫。对聚合酶链反应（PCR）产物进行测序，结果显示其与瘤胃古柏线虫部分ITS-1克隆的相似性为97%，与柯氏古柏线虫序列JF680982的相似性为99%。通过对包括柯氏古柏线虫、点状古柏线虫和匙形古柏线虫在内的17种蠕虫的分析，证实了该引物对的特异性。物种特异性诊断对于快速可靠的鉴定具有重要意义，可为特定地理位置的毛圆科线虫的生物学、生态学和流行病学研究提供支持。
