Molecular Epidemiology, Hematobiochemical Alterations, and Oxidative Stress-Induced Genotoxicity of Equine Trypanosomiasis in Pakistan

Trypanosoma evansi (T. evansi) infection poses a significant health threat to equines. This study was aimed to assess the prevalence, risk factors, hematobiochemical alterations, and oxidative stress-mediated genotoxicity associated with equine trypanosomiasis in the Rahim Yar Khan District. This cross-sectional study was conducted on 384 equines from October 2024 to September 2025. Blood samples were collected for thin blood film microscopy and PCR assay using RoTat 1.2 primers. Hematological indices were analyzed with an automated hematology analyzer; serum biochemical parameters were quantified via standard assays. Oxidative stress markers, including malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH), were also measured. Genotoxicity was evaluated using the alkaline comet assay. Statistical analyses included the chi-square test, logistic regression, and independent t-tests. T. evansi was detected in 5.99% of samples by microscopy and 10.16% by PCR, with no significant association with species, age, or sex. Infected equines exhibited significant reductions in hemoglobin (5.4 ± 0.6 vs. 10.8 ± 0.5 g/dL; p < 0.0001), total serum protein (2.1 ± 0.3 vs. 5.8 ± 0.2 g/dL; p < 0.0001), albumin, and globulin, alongside elevated hepatic enzymes, blood urea nitrogen, and creatinine (all p < 0.01). Oxidative stress was confirmed by increased MDA (p < 0.0001) and decreased CAT activity (p < 0.001). Genotoxicity was significantly higher in infected animals (genetic damage index; 1.12 ± 0.08 vs. 0.40 ± 0.01; p < 0.01). This study provides the first integrated assessment of molecular epidemiology and oxidative stress-mediated genotoxicity in equines in this region, suggesting the pathogenic impact of the infection and targeted diagnostics for disease management strategies.