Molecular Characterization, Oxidative Stress-Mediated Genotoxicity, and Hemato-Biochemical Changes in Domestic Water Buffaloes Naturally Infected with Trypanosoma evansi Under Field Conditions.

(1) Background: Surra is a debilitating disease of wild and domestic animals caused by Trypanosoma evansi (T. evansi), resulting in significant mortality and production losses in the affected animals. This study is the first to assess the genetic relationships of T. evansi in naturally affected buffaloes from Multan district, Pakistan, using ITS-1 primers and evaluating the effects of parasitemia and oxidative stress on DNA damage and hematobiochemical changes in infected buffaloes. (2) Methods: Blood samples were collected from 167 buffaloes using a multi-stage cluster sampling strategy, and trypomastigote identification was performed through microscopy and PCR targeting RoTat 1.2 and ITS-1 primers. Molecular characterization involved ITS-1 via neighbor-joining analysis. The impact of parasitemia loads was correlated with oxidative stress markers, genotoxicity, and hematobiochemical parameters using Pearson correlation and multivariable regression models. (3) Results: Field-stained thin blood film microscopy and molecular identification revealed 8.98% and 10.18% infection rates, respectively. Phylogenetic analysis based on ITS-1 region sequences of the identified isolates showed close genetic associations with Indian isolates. The mean trypomastigote count observed in the infected buffaloes was 5.15 × 10(6) (±5.3 × 10(2))/µL of blood. The parasitemia loads were significantly correlated with the alterations in oxidative stress markers, DNA damage, and changes in hematobiochemical parameters. Infected animals exhibited significant (p < 0.05) alterations in oxidative stress biomarkers, including catalase, nitric oxide, and malondialdehyde concentrations. Noteworthily, a comet assay revealed a significantly (p < 0.0001) higher mean genetic damage index in the infected buffaloes (0.7 ± 0.04) compared with the healthy ones (0.196 ± 0.004). Alongside significant (p < 0.05) reductions in red cell indices, a marked elevation in leukocyte counts and serum hepatic enzyme levels was recorded in the affected buffaloes. (4) Conclusion: T. evansi isolates of buffaloes from Multan, Pakistan, have genetic similarities to Indian isolates. This study also revealed that higher parasitemia loads induce genotoxicity in the infected animals through oxidative stress and cause hematobiochemical alterations under natural field conditions.