Theileria equi is 1 of the emerging and prevailing tick-borne hemoprotozoans adversely affecting the equids worldwide, including Pakistan. The current study aimed to investigate the prevalence and molecular characterization of T. equi in working horses (n = 194), the comparative efficacy of different diagnostic tests, associated risk factors, and hematobiochemical analysis. The blood samples of horses were subjected to microscopic examination, cELISA, and polymerase chain reaction (PCR) and the results revealed a prevalence of 9.79, 21.13, and 13.40%, respectively, for T. equi in working horses. The comparison of microscopy and cELISA results with PCR showed that cELISA had higher sensitivity (84.62%), but lower specificity (88.69%) and accuracy (88.14%) in comparison to microscopy (57.69, 97.62, and 92.27%). Molecular characterization of T. equi by phylogenetic analysis revealed a 61% resemblance of study isolates with each other OL662926, OL662925, and 82% similarity with isolate OL662924 while also showing homology with T. equi isolates of South Africa, South Korea, India, Pakistan, and Brazil. The risk factor analysis revealed a significant association (P < 0.05) of tick control status, previous tick history, tick infestation, house hygiene, deworming/vaccination, and the presence of other livestock species with T. equi infection in horses. The hematobiochemical profile revealed a significant (P < 0.05) decrease in red blood cells (RBCs), hemoglobin (Hb), packed cell volume (PCV), white blood cells (WBCs), platelet (PLT), phosphorus, and an increase in lymphocytes, granulocytes, aspartate aminotransferase (AST), glucose, bilirubin, blood urea nitrogen (BUN), and creatinine in T. equi–infected horses. The current study is the first comprehensive report for comparative evaluation of microscopy, cELISA, and PCR, assessment of epidemiological risk factors as well as hematobiochemical variations due to T. equi infection in Pakistan.

马泰勒虫是一种新兴且普遍存在的蜱传血液原虫，对全世界（包括巴基斯坦）的马科动物产生不利影响。目前的研究旨在调查马毛虫在工作马（n = 194）中的患病率和分子特征、不同诊断测试的比较功效、相关危险因素和血液生化分析。对马的血液样本进行了显微镜检查、cELISA 和聚合酶链反应 (PCR)，结果显示，工作马中马锥虫的患病率分别为 9.79%、21.13% 和 13.40%。显微镜检查和 cELISA 结果与 PCR 的比较表明，与显微镜检查（57.69、97.62 和 92.27%）相比，cELISA 具有较高的灵敏度（84.62%），但特异性（88.69%）和准确性（88.14%）较低。通过系统发育分析对马木犀的分子特征显示，研究分离株与 OL662926、OL662925 之间有 61% 的相似性，与分离株 OL662924 的相似性为 82%，同时还显示出与南非、韩国、印度、巴基斯坦的马木犀分离株的同源性和巴西。危险因素分析显示，蜱虫控制状况、既往蜱虫病史、蜱虫感染、房屋卫生、驱虫/疫苗接种以及其他家畜物种与马匹感染马蜱存在显着相关性（ P < 0.05）。血液生化特征显示红细胞 (RBC)、血红蛋白 (Hb)、红细胞体积 (PCV)、白细胞 (WBC)、血小板 (PLT)、磷显着减少 (P < 0.05)，并且马锥虫感染的马中的淋巴细胞、粒细胞、天冬氨酸转氨酶 (AST)、葡萄糖、胆红素、血尿素氮 (BUN) 和肌酐。目前的研究是第一份对显微镜、cELISA 和 PCR 进行比较评估、流行病学危险因素评估以及巴基斯坦马锥虫感染引起的血液生化变异的综合报告。