Development of a multiplex PCR method for discriminating between Heterakis gallinarum, H. beramporia, and H. indica parasites of poultry
Development of a multiplex PCR method for discriminating between Heterakis gallinarum, H. beramporia, and H. indica parasites of poultry
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DOI：
10.1016/j.vetpar.2021.109463
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发表时间：
2021-05-21
影响因子：
2.6
通讯作者：
Itagaki, Tadashi
中科院分区：
文献类型：
--
作者：
Biswas, Peru Gopal;Ohari, Yuma;Itagaki, Tadashi
鸡异弯线虫、小孢囊线虫和印度线虫是亚洲国家鸡科家禽常见的线虫，其感染有时会导致宿主健康下降。这3种异形藻(Heterakis spp.可以通过雄虫的形态特征来识别；但不能识别雌虫和卵，因为它们没有可靠的形态特征来区分。此外，鸡嗜血杆菌是一种著名的胎儿原生动物组织单胞菌的媒介，这使得区分这三个异种在基础和临床兽医寄生虫学中具有重要意义。我们分析了这三个异种的核糖体18S-ITS1-5.8S-ITS2-28S DNA序列。种间18S、5.8S和28S序列具有很高的同源性，ITS1和ITS2序列相似性为68.5%~93.2%。在ITS1和ITS2连锁的系统发育树上，鸡隐孢子虫、小孢子虫和印度隐翅虫被划分为不同的分支。因此，为了建立一种区分这三种异尖线虫的多重PCR方法，我们在ITS2区域设计了物种特异的反向引物：猪瘟杆菌特异的HgI2-R、伯氏杆菌特异的HbI2-R5和稻瘟病菌特异的HiI2-R。多重聚合酶链式反应分别扩增出鸡嗜血杆菌、小孢囊线虫和印度线虫DNA特异的396、272和482个碱基的片段，而不能扩增其他鸡线虫的DNA片段，如华支睾吸虫、曼氏氧化螺杆菌、鼻咽虫和虎纹夜蛾。这些结果表明，多重聚合酶链式反应可作为一种有用的工具来鉴定和诊断家禽感染鸡嗜血杆菌、小孢子虫和印度细小孢子虫。
Heterakis gallinarum, H. beramporia, and H. indica are common nematodes in gallinaceous poultry in Asian countries, and the infections occasionally lead to declining health of the hosts. These three Heterakis spp. can be identified by the morphological characteristics of the male worms; however, the female worms and eggs cannot be identified because they have no reliable morphological characteristics for discrimination. In addition, H. gallinarum is a well-known vector of fetal protozoan Histomonas meleagridis, making the discrimination between these three Heterakis species important in basic and clinical veterinary parasitology. We analyzed nuclear ribosomal 18S-ITS1-5.8S-ITS2-28S DNA sequences of these three Heterakis species. The 18S, 5.8S, and 28S DNA sequences had very high homology between the species; however, the ITS1 and ITS2 sequence similarity was 68.5 %-93.2 %. H. gallinarum, H. beramporia, and H. indica were divided into separate clades in the ITS1 and ITS2-concatenated phylogenetic tree. Therefore, to develop a multiplex PCR method for discriminating between the three Heterakis species, we designed species-specific reverse primers within the ITS2 region as follows: H. gallinarum-specific HgI2-R, H. beramporia-specific HbI2-R5, and H. indica-specific HiI2-R. The multiplex PCR amplified 396-bp, 272-bp, and 482-bp fragments specific to H. gallinarum, H. beramporia, and H. indica DNA, respectively, and did not amplify the fragments using other chicken nematode DNAs such as Ascaridia galli, Oxyspirura mansoni, Dispharynx nasuta, and Cheilospirura hamulosa. These results suggest that the multiplex PCR would serve as a useful tool for identifying and diagnosing infections of H. gallinarum, H. beramporia, and H. indica in poultry.