Development and Preliminary Evaluation of a Chimeric Epitope-Based Antigen (TevISDY) for Serological Detection of...

Trypanosoma evansi remains a neglected yet economically significant pathogen in Latin America, where current serological diagnostic tools suffer from low specificity, cross-reactivity, and limited field applicability. This study presents the design and preliminary evaluation of a novel chimeric antigen derived from epitopes of the TevSTIB805.3.100 protein, a variant surface glycoprotein (VSG) of T. evansi, and explores its potential application as a serological marker for surra. An ISDY-based chimeric antigen was engineered, expressed, purified, and structurally characterized using integrated bioinformatic, molecular, and biochemical analyses. Its immunogenicity and diagnostic performance were assessed through rodent and chicken immunizations, optimized ELISAs, and blinded evaluation of 136 equine field sera, including cross-reactivity panels with babesiosis and anaplosmosis sera. The TevISDY chimera showed high solubility (GRAVY = -1.361), structural stability (instability index = 28.89), and successful purification, with clear immunoreactivity confirmed by Western blot. ELISA assays suggested that the chimeric antigen is immunogenic and may provide good discrimination of T. evansi-positive field sera (AUC 96.23-99.57%), with specificity ranging from 91.74 to 99.70% and accuracy from 95.83 to 99.70%, while preliminary assessment suggested limited cross-reactivity with the Babesia bigemina and Anaplasma marginale samples included in this study. Overall, the TevISDY antigen showed consistent identification of both experimentally infected and field serum samples in the ELISA assays. The observed performance of TevISDY may reflect the use of a single, conserved, pathogen-specific epitope, which could help reduce cross-reactivity and minimize the batch variability commonly associated with whole-cell lysate antigens, suggesting its potential as a more stable serological target for T. evansi. Broader validation in naturally infected animals and integration into multiepitope or rapid diagnostic platforms will be key next steps toward field-deployable surra diagnostics.