Development and validation of an agglutination test using in vitro-cultivated Trypanosoma evansi antigens for the diagnosis of surra in cattle.

Trypanosomosis is an important disease in livestock caused by Trypanosoma evansi infections. The CATT/T. evansi is recommended by WOAH for trypanosomosis screening, but its accessibility in Thailand and Southeast Asia remains limited by cold chain transportation and high costs. This study aimed to develop and validate an agglutination test using in vitro-cultivated T. evansi Thai strain antigens. The test was optimized using serum dilutions ranging from 1:2-1:64. Diagnostic performance was evaluated against the commercial CATT/T. evansi test and indirect ELISA. Cross-reactivity was assessed using bovine serum positive for Anaplasma spp., Babesia bigemina, Babesia bovis, Theileria spp., and Leishmania spp. Lyophilized antigen stability was evaluated for 12 months. The developed test showed a sensitivity of 87.07% and a specificity of 96.67% (PPV: 97.12%; NPV: 85.29%) versus CATT/T. evansi, with almost perfect agreement (κ = 0.83). It showed 77.95% sensitivity, 82.86% specificity (PPV: 80.49%; NPV: 80.56%), and substantial agreement (κ = 0.61) against indirect ELISA. No cross-reactivity was observed with five hemoparasites, and lyophilized antigen remained stable for ≥ 12 months at 4-8°C. The newly developed T. evansi agglutination test was applied to 851 cattle serum samples collected from 15 provinces across four regions of Thailand, revealing a seroprevalence of 50.29% (95% CI: 46.94-53.64). This study showed that the newly developed T. evansi agglutination test provides a rapid, field-adaptable alternative for trypanosomosis surveillance.