DEVELOPMENT OF NOVEL CHIMERIC EPITOPE-BASED DIAGNOSTIC ANTIGENS FOR EHRLICHIOSIS AND ANAPLASMOSIS

Ehrlichiosis and anaplasmosis are increasingly prevalent tick-borne diseases with limitations in current diagnostic methods. This study aimed to develop novel chimeric epitope-based diagnostic antigens with enhanced sensitivity and specificity for detecting Ehrlichia and Anaplasma infection in canines. Potential diagnostic antigens were identified using immunoprecipitation followed by liquid chromatography-mass spectrometry (IP-LC/MS). Chimeric diagnostic constructs were designed incorporating epitopes from identified antigens, epitope localization studies and bioinformatic predictions aided in the selection of antigenic regions. Constructs were evaluated using indirect enzyme-linked immunosorbent assays (ELISA) against well-characterized canine serum panels, including naturally and experimentally infected samples. The results demonstrated the successful development of EDC4, which exhibited 98.46% sensitivity and 96.83% specificity for detecting Ehrlichia spp., including enhanced detection of E. ewingii. Furthermore, ADC1 showed 100% sensitivity and 98% specificity for detecting A. phagocytophilum and A. platys. These novel chimeric constructs outperformed commercially available diagnostic tests, such as SNAP4DX Plus. Future directions include expanding serum panels, investigating additional antigens, conducting time-course infection studies, and exploring the potential for a multiplex assay. This research highlights the potential of chimeric epitope-based antigens for improving the accuracy and breadth of diagnostics for canine ehrlichiosis and anaplasmosis.