Sudan Vikrant, Jaiswal Amit Kumar, Parashar Rahul, Shanker Daya
Department of Parasitology, College of Veterinary Sciences and Animal Husbandry, U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura, 281001, India,
Trop Anim Health Prod. 2015 Jun;47(5):915-9. doi: 10.1007/s11250-015-0808-5. Epub 2015 Apr 7.
Trypanosomosis and bovine tropical theileriosis are important vector-borne protozoan diseases imposing some of the serious constraints on the health and productivity of domestic cattle in tropical and subtropical regions of the world. Following recovery from primary infection of both these conditions, animals become persistent carriers and act as reservoirs of infection thereby playing a critical role in disease epidemiology. The present study describes development and evaluation of duplex polymerase chain reaction (PCR) assays for simultaneous detection of Trypanosoma evansi and Theileria annulata in buffaloes. Following in silico screening for candidate target genes representing each of the pathogens, an optimized duplex PCR assay was established using TBR F/R and TAMS F/R as primer sets encoding for products of 164 and 721 bp for T. evansi and T. annulata, respectively. The results were compared and correlated with conventional Giemsa-stained thin blood smear examination and the single PCR assay. The duplex PCR detected each pathogen with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen. Moreover, single and duplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. evansi and T. annulata, and no evidence of nonspecific amplification from nontarget species was observed. The developed assay may be seen as a good tool for epidemiological studies aiming at assessing the burden of dual infections and improving control of the associated diseases in endemic regions.
锥虫病和牛热带泰勒虫病是重要的媒介传播原生动物疾病，对世界热带和亚热带地区家牛的健康和生产力造成了一些严重限制。从这两种疾病的初次感染中恢复后，动物会成为持续带菌者，并充当感染源，从而在疾病流行病学中发挥关键作用。本研究描述了用于同时检测水牛体内伊氏锥虫和环形泰勒虫的双重聚合酶链反应（PCR）检测方法的开发和评估。通过对代表每种病原体的候选靶基因进行电子筛选，使用TBR F/R和TAMS F/R作为引物对建立了优化的双重PCR检测方法，分别扩增出伊氏锥虫和环形泰勒虫大小为164 bp和721 bp的产物。将结果与传统的吉姆萨染色薄血涂片检查和单重PCR检测进行比较和关联。双重PCR检测每种病原体的灵敏度相同，无论其DNA是单独扩增还是与另一种病原体的DNA一起扩增。此外，单重和双重PCR能够在伊氏锥虫和环形泰勒虫DNA混合物的系列稀释样本中以相同的灵敏度检测到每种病原体，并且未观察到非靶标物种的非特异性扩增迹象。所开发的检测方法可被视为一种良好的工具，用于旨在评估地方病流行区双重感染负担和改善相关疾病控制的流行病学研究。
