A methodological platform for Trypanosoma evansi research and surveillance

Abstract Trypanosoma evansi , the causative agent of surra, infects a wide range of domestic and wild mammals and has a broad geographic distribution across Asia, Africa, and South America. Despite its veterinary importance, experimental tools for its isolation, in vitro maintenance, diagnosis, and structural characterization remain limited. We developed an integrated methodological toolbox comprising in vivo parasite isolation using immunodeficient mice, adaptation to in vitro culture in a modified medium, serological ELISA for multi-host antibody detection, a sensitive TaqMan qPCR assay for parasite detection and species differentiation, and ultrastructural analysis via Ultrastructure Expansion Microscopy applied directly to bloodstream parasites. This platform enabled stable parasite maintenance, accurate diagnosis, epidemiological studies and detailed visualization of cell cycle dynamics, revealing the absence of kinetoplast DNA. Together, this toolbox provides a versatile experimental platform for the study of T. evansi biology and supports future research on this neglected veterinary pathogen.