Paliwal Sanjhi, Sudan Vikrant, Shanker Daya
College of Biotechnology, U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura, 281001, India.
Centre for Research Impact and Outcome (CRIO), Chitkara University, Rajpura, Punjab, 14041, India.
Sci Rep. 2025 Jan 2;15(1):431. doi: 10.1038/s41598-024-84892-1.
This study is the first to conduct a sero-surveillance of Bovine Tropical Theileriosis (BTT) caused by the protozoan parasite Theileria annulata (T. annulata) using a recombinant Tams1 protein-based dot-ELISA in cattle, and to compare its efficacy with plate-ELISA, single PCR, nested PCR, and blood microscopy. The goal was to identify the most effective method for the early and accurate detection of theileriosis, which significantly impacts livestock through reduced milk yield and increased mortality. A total of 101 field blood samples were examined using blood smear analysis, single PCR, nested PCR, and dot-ELISA. The recombinant Tams1 protein was successfully cloned and expressed using a pET-30b (+) expression vector in a prokaryotic system. The protein was purified with Ni-NTA chromatography, confirmed for immunoreactivity with T. annulata positive serum via Western blot analysis, and used to optimize both dot-ELISA and plate-ELISA. Both dot-ELISA and plate-ELISA using recombinant Tams1 protein exhibited comparable diagnostic performance, with a kappa value of 0.826 and similar analytical productivity (P = 0.6165). Dot-ELISA revealed a BTT seroprevalence of 58.4% in the cattle population, demonstrating good sensitivity (93.33%) and specificity (90%). The diagnostic performance of dot-ELISA was found to be superior to other molecular techniques, including microscopy, single PCR, and nested PCR. Dot-ELISA is also a sustainable solution in comparison to other laboratory diagnostic techniques with benefits of early diagnosis, reduced waste generation, resource efficiency, cost-effective point of care disease surveillance. With its minimal antigen requirement, Tams1 molecule based dot-ELISA is recommended as an effective tool for epidemiological studies and field surveys of BTT.
本研究首次在牛群中使用基于重组Tams1蛋白的斑点酶联免疫吸附测定（dot-ELISA）对由环形泰勒虫（T. annulata）这种原生动物寄生虫引起的牛热带泰勒虫病（BTT）进行血清学监测，并将其与平板酶联免疫吸附测定（plate-ELISA）、单重聚合酶链反应（single PCR）、巢式聚合酶链反应（nested PCR）及血液显微镜检查的效果进行比较。目标是确定用于早期准确检测泰勒虫病的最有效方法，该病会通过降低产奶量和增加死亡率对家畜产生重大影响。共使用血液涂片分析、单重PCR、巢式PCR及斑点酶联免疫吸附测定对101份野外血液样本进行检测。重组Tams1蛋白在原核系统中使用pET-30b(+)表达载体成功克隆并表达。该蛋白通过镍-氮三乙酸（Ni-NTA）层析法纯化，经蛋白质印迹分析确认其与环形泰勒虫阳性血清具有免疫反应性，并用于优化斑点酶联免疫吸附测定和平板酶联免疫吸附测定。使用重组Tams1蛋白的斑点酶联免疫吸附测定和平板酶联免疫吸附测定均表现出相当的诊断性能，kappa值为0.826且分析效率相似（P = 0.6165）。斑点酶联免疫吸附测定显示牛群中BTT血清阳性率为58.4%，具有良好的敏感性（93.33%）和特异性（90%）。发现斑点酶联免疫吸附测定的诊断性能优于其他分子技术，包括显微镜检查、单重PCR和巢式PCR。与其他实验室诊断技术相比，斑点酶联免疫吸附测定也是一种可持续的解决方案，具有早期诊断、减少废物产生、资源高效利用、具有成本效益的即时护理疾病监测等优点。基于Tams1分子的斑点酶联免疫吸附测定由于其对抗原的需求极少，被推荐作为BTT流行病学研究和现场调查的有效工具。
