Marwaha Sumnil, Brar Basanti, Jain Vinod Kumar, Poonia Rachna, Prasad Minakshi
Veterinary Medicine, Lala Lajpat Rai University of Veterinary and Animal Science, Hisar, India.
Biofertilizer Production Lab, HGIPPCL, CCSHAU, Hisar, India.
Parasitol Res. 2023 May;122(5):1189-1197. doi: 10.1007/s00436-023-07819-1. Epub 2023 Mar 10.
Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 10 and 10 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.
泰勒虫病是一种由蜱传播的疾病，给乳制品行业造成巨大损失。有几种泰勒虫可感染牛。一般来说，在任何地理区域都有不止一种泰勒虫流行；因此，共同感染的几率很高。通过显微镜检查或血清学检测可能无法区分这些物种。因此，在本研究中，对一种多重PCR检测方法进行了标准化和评估，用于快速同时鉴别两种泰勒虫，即环形泰勒虫和东方泰勒虫。设计了物种特异性引物，靶向环形泰勒虫的裂殖子梨形虫体表抗原基因（TAMS1）和东方泰勒虫的主要梨形虫体表蛋白基因，分别产生229 bp和466 bp的特异性扩增子。多重PCR对环形泰勒虫和东方泰勒虫的灵敏度分别为10拷贝和10拷贝。单重和多重PCR均具有特异性，两种引物与其他血液原虫均无交叉反应。为了进行比较评估，对216头牛的血样进行了单重和多重PCR检测这两种泰勒虫。使用多重PCR，发现131只动物感染了泰勒虫病，其中112只感染了环形泰勒虫，5只感染了东方泰勒虫，14只混合感染。这是印度哈里亚纳邦首次报道东方泰勒虫。环形泰勒虫（ON248941）和东方泰勒虫（ON248942）的代表性序列已提交至GenBank。本研究中使用的标准化多重PCR检测方法特异性强、灵敏度高，可用于现场样本的筛查。
