Kundave V R, Ram Hira, Banerjee Partha S, Garg Rajat, Mahendran K, Ravikumar G V P P S, Tiwari Ashok K
Division of Parasitology, ICAR-IVRI, Izatnagar-243122 India.
Division of Veterinary Medicine, ICAR-IVRI, Izatnagar-243122 India.
Acta Parasitol. 2018 Dec 19;63(4):759-765. doi: 10.1515/ap-2018-0090.
This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.
本研究描述了一种多重PCR检测方法的开发与评估，该方法用于同时检测牛体内的环形泰勒虫、双芽巴贝斯虫和边缘无浆体感染。该检测方法是利用寄生虫特异性基因组DNA以及分别靶向环形泰勒虫、双芽巴贝斯虫和边缘无浆体的Tams1、18S rRNA和16S rRNA基因的三组PCR引物开发而成。从总共461头疑似血寄生虫感染的牛采集血液样本，进行显微镜检查以记录感染状况，同时，从这些血液样本中提取的基因组DNA用于多重PCR检测方法的优化和验证。血液样本的显微镜检查显示，分别有25.8%和2.4%的样本存在单一和多种血寄生虫。多重PCR结果显示，159例（34.5%）存在单一血寄生虫感染，而82例（17.8%）样本记录为混合感染。本研究中通过多重PCR检测到的单个物种感染发生率分别为：环形泰勒虫26.03%（120/461）、双芽巴贝斯虫3.25%（15/461）和边缘无浆体5.20%（24/461）。多重PCR检测方法的检测限分别为环形泰勒虫、边缘无浆体和双芽巴贝斯虫模板稀释度为10-6、10-6和10-4时，分别对应0.1 pg、0.1 pg和10.0 pg的DNA。基于高诊断敏感性和通量，本研究开发的多重PCR检测方法可作为一种工具，用于对牛蜱传血寄生虫感染进行大规模流行病学调查。
