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Utilization of antigenic proteins for the diagnosis of Theileriaa equi infection in equines.

Bassma SM Elsawy, Olfat A Mahdy, Ahmed M Nassar, Mona S Mahmoud

The aim of this study was to characterize Theileriaa equi antigens and detect the most antigenic protein for further use in serological diagnosis (indirect Enzyme-Linked Immunosorbent Assay (iELISA)). One of three donkeys highly infected with Theileriaa equi, as confirmed by nested PCR (nPCR), was splenectomized for antigen preparation. Crude lysate antigens (Ags) (L1 with hemoglobin, L2 without hemoglobin and sonicated crude Ag (S)) were prepared from infected blood with a parasitemia of 15%. The three Ags were electrophoretically analysed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) using Coomassie blue staining and silver staining. The antigenic proteins of these three Ags were detected by immunoblot analysis using serum from a donkey naturally infected with Theileriaa equi. Blood samples obtained from 133 donkeys and 168 horses were examined by iELISA after coating the plates with the most antigenic protein. By Coomassie blue staining, electrophoretic analysis of these three Ags using SDS-PAGE showed 3 common bands for L1, L2 and S at MWt 63, 54 and 31 kDa. However, silver staining showed 5 bands at MWt 70, 54, 31, 28 and 23 kDa. Immunoblot analysis of theL1, L2 and S Ags showed that there was one immune dominant protein at MWt 16 kDa. The common immune reactive band among the three types of Theileriaa equi Ags was the band at 16 kDa. The most antigenic preparation was L2, as shown by immune blot analysis and confirmed by checkerboard titration, and this Ag was utilized in iELISA. iELISA revealed that the overall prevalence of Theileriaa equi was 33.5% (53.4% in donkeys and 17.9% in horses).