In vitro cultivation of Trypanosoma equiperdum: Comparison of in vitro and in vivo antigens for an ethical and sustainable serological diagnosis of Dourine.

The protozoan Trypanosoma equiperdum is the causative agent of Dourine, a contagious sexually transmitted disease of equines that can manifest itself in chronic or acute form. Dourine has serious veterinary and economic implications, as there are no vaccines available for prevention and the management of the disease requires the culling of infected animals, together with rigorous sanitary control measures. This study compares the traditional production of T. equiperdum antigen grown in vivo, with an in vitro cultivation methodology developed to eliminate the use of animals. The in vitro and in vivo antigens are evaluated within serological diagnostic methods for Dourine, using Complement Fixation Test and Indirect Fluorescent Antibody Test. These methods are validated according to the principles and procedures outlined the WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, in order to assess their diagnostic performance and ethical sustainability. The results show the sensitivity and specificity of the in vivo antigen is comparable to the in vitro antigen. Precision parameters confirmed the robustness of the assays, with 100% concordance in repeatability and reproducibility. The IFAT showed excellent concordance with the CFT, suggesting its potential as a confirmatory test in eradication programs. The in vitro production system significantly reduces biosafety risks and logistical constraints associated with animal housing and infection. This approach is consistent with the 3Rs (Replacement, Refinement, and Reduction), offering a sustainable alternative to antigen production and supporting more ethical animal management in research, promoting animal welfare and sustainable veterinary diagnostic practices.