Molecular Identification and Phylogenetic Analysis of Trypanosoma evansi with Assessment of Associated Risk Factors in Camels (Camelus dromedarius) Across Ten Districts of Punjab, Pakistan

Trypanosomiasis significantly impacts camel health and productivity, posing a major challenge to food security in regions with large camel populations. In this study, we investigated the microscopic and molecular prevalence, performed phylogenetic analysis, and explored risk factors associated with Trypanosoma evansi (T. evansi) infection in 400 randomly selected suspected camels (Camelus dromedarius) from 10 districts of Punjab, Pakistan. Blood samples were collected for microscopic examination of Giemsa/Field’s-stained smears, and three PCR primer sets (ITS1CF/BR, pMUTec, RoTat 1.2) were used to detect the presence of T. evansi. PCR-based prevalence was higher (14.8%; CI 11.4–18.6) as compared to the microscopic examination (8.3%; CI 5.7–11.4) of samples. The targeted primers amplified DNA fragments of 210, 205, and 478 base pairs, respectively. Phylogenetic analysis showed 100% homology between local isolates and those from India, Sudan, Malaysia, Egypt, and Kenya. Risk analysis identified female gender (OR 2.1) and being in Southern Punjab (OR: 1.9) as significant factors associated with disease. Significantly (p < 0.05) reduced total protein (5.51 ± 0.05), albumin (2.77 ± 0.04), and globulin (2.57 ± 0.06) levels were found in PCR-positive camels. This study provides new molecular and phylogenetic data on T. evansi in Pakistan.