山羊源3种不同形态阴门盖捻转血矛线虫雌虫的多位点序列分析

旨在解析3种不同形态阴门盖捻转血矛线虫（Haemonchus contortus）的遗传进化差异，本研究从养殖场山羊皱胃中分离到捻转血矛线虫虫体，采用普通光学显微镜观察71条雌虫虫体阴门盖的形态结构特征，并基于捻转血矛线虫ITS-1、ITS-2和nad4基因位点，采用PCR法对不同阴门盖的虫体进行扩增，通过比对多位点序列进行遗传进化分析。经显微镜观察发现，本研究分离到的捻转血矛线虫雌虫阴门盖存在舌瓣形、亚球形和舌-球混合形3种形态，所占比例分别为45.07%（32/71）、50.70%（36/71）和4.23%（3/71）。对43条雌虫全长和阴门至虫体末端长度进行测量和统计分析，结果显示，3种不同形态阴门盖的捻转血矛线虫全长统计学差异不显著（P>0.05），而3种不同形态阴门盖的捻转血矛线虫阴门至虫体末端的长度存在显著性差异（P<0.05）。基于捻转血矛线虫ITS-1、ITS-2和nad4基因进行的序列分析结果显示，不同形态阴门盖虫体的9个样品在3个基因位点上存在不同程度的碱基差异，多态性位点分别为10、13和43个，核苷酸多态性分别为0.85%、1.34%和1.97%。基于ITS-1和ITS-2构建的遗传进化树显示，研究中所用样品与GenBank上H.contortus的参考序列处于同一进化支，表明本研究所分离到的虫体均属捻转血矛线虫。捻转血矛线虫雌虫的阴门盖存在形态学差异，不同形态阴门盖虫体在ITS-1、ITS-2和nad4基因位点上均存在不同程度的碱基差异，此研究结果为捻转血矛线虫的种类鉴定和遗传进化提供了参考数据。

Multi-locus Gene Sequence Analysis of Female Haemonchus contortus with Morphological Differences in Vulval Flap from Goat

To elucidate the genetic and evolutionary differences of Haemonchus contortus with morphological differences in the vulval flap, in this study, fresh worms were isolated from the abomasum of infected goats. The morphological characteristics of the vulval flap of 71 female worms were observed by microscope. Based on the ITS-1, ITS-2 and nad4 gene loci, PCR was used to amplify H. contortus with morphological differences in the vulval flap, and phylogenetic analysis was performed by comparing the sequences of multiple loci. Morphological observation showed there existed three morphologic types of vulval flap for female H. contortus isolated in this study, namely linguiform, spheroid-shaped and ligule-spheroid form, and statistical analysis showed that the proportion of the three types of the vulval flap was 45.07% (32/71), 50.70% (36/71) and 4.23% (3/71), respectively. The body length and the length from the vulva to the posterior of 43 females (20 linguiform, 20 spheroid-shaped and 3 ligule-spheroid form) were measured and analyzed. Statistical analysis showed that there was no significant difference among the body length of the H. contortus with different vulval flap (P>0.05), but a significant difference was found in the length from the vulva to the posterior of the nematode with spheroid-shaped vulval flap compared with the other two types (P<0.05). Sequence analysis of the ITS-1, ITS-2 and nad4 genes of H. contortus showed there existed different levels of base differences at the three gene loci among 9 samples with different vulval flaps, that there were 10, 13 and 43 polymorphic sites, and the nucleotide diversity was 0.85%, 1.34% and 1.97%, respectively. The phylogenetic analysis based on ITS-1 and ITS-2 suggested that the samples here and reference sequence of H. contortus in GenBank were grouped into the same evolutionary branch, indicating that the parasites isolated here belonged to H. contortus. The morphological difference on vulval flap of female H. contortus was observed here and these parasites, on ITS-1, ITS-2 and nad4 gene locus, shown different degrees of base mutation. The results of this study should provide reference data for species identification and molecular epidemiological studies of H. contortus.