Field validation of a competitive ELISA for detection of Theileria annulata infection

Abstract

In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop a recombinant-protein-based indirect enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antibodies in serum of T. annulata-infected animals. To increase the specificity, a competitive ELISA (cELISA) was developed using recombinant TaSP antigen and a monoclonal antibody (1C7) specifically binding to TaSP. Since the cELISA accurately differentiated T. annulata-infected from uninfected animals, a study was performed to analyse the suitability of the cELISA in the field. For this, 230 sera with unknown status from different governorates in the north of Iraq were analysed using both the indirect and competitive ELISA and were compared. There was a significant (p < 0.5 x 10(-19)) correlation (r = 0.556) between the tests, whereby the cELISA detected more sera as negative (44/230) compared to the indirect ELISA (21/270). Accordingly, less sera were determined to be positive in the competitive (186/230) than in the indirect ELISA (209/230). Sensitivity and specificity of the cELISA taking the indirect ELISA as a reference were 84.2% and 52.4%, respectively. Accordingly, the calculated prevalence of T. annulata infection was 90.9%, and the positive predictive value was determined to be 94.6%. Taken together, the cELISA proved its suitability for field application and was found qualified for use in serological surveys to monitor the prevalence of T. annulata infection and to identify carrier animals.