Abstract：
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 102 and 103 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.
摘要：
Theileriosis是一种蜱传疾病，在乳制品行业造成巨大损失。有几种Theileria可以感染牛。一般来说,在任何地理区域普遍存在一种以上的物种；因此，合并感染的可能性很高。通过显微镜检查或血清学测试可能无法区分这些物种。因此,在这项研究中，对多重PCR检测方法进行了标准化和评估，以快速同时鉴别检测两种Theileria。,环流西里虫和东方西里虫。设计了物种特异性引物，以T.arnulata的裂殖子pirosa表面抗原基因（TAMS1）和东方毛虫的主要pirosa表面蛋白基因为目标，产生229bp和466bp的特异性扩增子，分别。多重PCR的灵敏度分别为102和103个拷贝。分别。单纯形和多重PCR是特异性的，并且对于任一引物均未显示与其他嗜血原虫的交叉反应性。对于比较评估，对216头牛的血液样本进行了单纯形和多重PCR检测。使用多重PCR，发现131只动物感染了Theileriosis，其中112人感染了T.annulata，有5人感染了东方毛虫,14人混合感染。这是来自哈里亚纳邦的T.Orientalis的第一份报告，印度。在GenBank中提交了T.annulata（ON248941）和T.Orientalis（ON248942）的代表性序列。本研究中使用的标准化多重PCR检测方法具有特异性，敏感,用于现场样本的筛选。
