Faculty of Veterinary Medicine, Department of Parasitology, Adnan Menderes University, Işikli Mevki, 09016 Aydin, Turkey. huseyin
Vet Parasitol. 2010 Dec 15;174(3-4):341-7. doi: 10.1016/j.vetpar.2010.08.025. Epub 2010 Sep 28.
Bovine tropical theileriosis, caused by the tick-borne protozoan Theileria annulata, imposes a serious constraint upon breed improvement programmes and livestock production in tropical and sub-tropical regions of the world. Animals that recover from primary infection serve as carriers and play a critical role in the epidemiology of the disease, acting as reservoirs of infection. However, conclusive identification of carrier animals can be problematic. This study describes assessment of candidate target genes for PCR assay-based detection of T. annulata infected carrier animals. Following in silico screening and rejection of three major multi-copy gene families, an assay based on PCR amplification of a 312 bp segment of the T. annulata gene for cytochrome b (Cytob1 assay) was established. Sensitivity was evaluated using serial dilutions of blood obtained from experimentally infected calves, while specificity was confirmed by testing DNA representing twelve different T. annulata stocks and other Theileria and Babesia species. Direct comparison with other target genes and published data indicated that Cytob1 PCR-based assays provide the greatest level of sensitivity, combined with a high level of specificity and the ability to detect different T. annulata genotypes. It can be concluded that the cytochrome b gene is the optimal target for PCR amplification and its incorporation in a Reverse Line Blot Assay offers the most sensitive method yet devised to detect the parasite in carrier animals. The use of this assay will increase the accuracy of epidemiological studies aimed at improving disease control in endemically unstable regions.
牛环形泰勒虫病由蜱传播的原生动物环形泰勒虫引起，给世界热带和亚热带地区的品种改良计划和畜牧业生产带来了严重的制约。从初次感染中恢复的动物成为携带者，并在疾病的流行病学中发挥关键作用，充当感染的储主。然而，对携带者动物的明确鉴定可能是一个问题。本研究描述了用于基于 PCR 检测环形泰勒虫感染携带者动物的候选靶基因的评估。在对三个主要的多拷贝基因家族进行计算机筛选和拒绝后，建立了一种基于环形泰勒虫细胞色素 b（Cytob1 检测）基因 312 bp 片段 PCR 扩增的检测方法。通过对实验感染小牛的血液进行系列稀释来评估敏感性，同时通过测试代表 12 种不同环形泰勒虫株和其他泰勒虫属和巴贝斯虫属的 DNA 来确认特异性。与其他靶基因和已发表数据的直接比较表明，基于 Cytob1 PCR 的检测方法提供了最高的敏感性，同时具有高特异性和检测不同环形泰勒虫基因型的能力。可以得出结论，细胞色素 b 基因是最适合 PCR 扩增的靶基因，将其纳入反向线印迹检测提供了迄今为止最敏感的方法来检测携带者动物中的寄生虫。该检测方法的应用将提高旨在改善流行不稳定地区疾病控制的流行病学研究的准确性。
