Evaluation of the immunodiagnostic potential of recombinant Spm2 antigen using indirect ELISA for detecting Theileria annulata infection in cattle

Bovine tropical theileriosis, caused by Theileria annulata, is an economically important disease that reduces livestock productivity through decreased milk yield, high morbidity, and mortality, particularly in exotic and cross-bred cattle. Timely diagnosis and treatment are crucial for effectively managing susceptible animals. This study aimed to develop a robust serological diagnostic tool targeting the sporozoite and macroschizont 2 antigen (Spm2), a protein expressed across multiple parasite stages including sporozoites, macroschizonts, and piroplasms. The recombinant Spm2 (rSpm2) protein was cloned, expressed in a prokaryotic system, and purified via Ni-NTA chromatography, with its immunoreactivity confirmed through Western blot (WB) analysis using known T. annulata-positive serum samples. An indirect rSpm2-ELISA (Enzyme-linked immunosorbent assay) was developed and standardized using 23 known positive and 30 known negative sera samples. Its diagnostic potential was evaluated by analyzing 360 field serum samples and comparing the results with a previously validated rTaSP-ELISA. The rSpm2-ELISA detected T. annulata infection in 78.9 % of cattle sera, compared to 83.9 % with rTaSP-ELISA. Notably, rSpm2 exhibited no cross-reactivity with reference-positive sera from cattle infected with Babesia bigemina and Anaplasma marginale in WB and ELISA assays. The sensitivity and specificity of the rSpm2-ELISA were determined to be 93.7 % and 98.3 %, respectively, highlighting its strong diagnostic potential. This study is the first comparative serological evaluation of tropical theileriosis using rSpm2-ELISA and rTaSP-ELISA. The high accuracy, specificity, and sensitivity of rSpm2-ELISA make it a promising tool for serological surveillance of tropical theileriosis, particularly in regions where T. parva is absent.