Development and application of a recombinant protein-based indirect ELISA for the detection of serum antibodies against Cystoisospora suis in swine

The apicomplexan parasite Cystoisospora suis which causes neonatal porcine coccidiosis is one of the predominant parasite in suckling piglets. Currently, the immunofluorescence antibody test (IFAT) is the only available serological tool for detecting serum antibodies against C. suis which has several limitations, including bias from interpretation and low throughput. In the present study, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using a previously characterized recombinant merozoite protein for the detection of specific immunoglobulin G (IgG) and IgA against C. suis. The recombinant protein was expressed in Escherichia coli as a N-terminal histidine fusion protein, and its specificity was confirmed in an immunoblot probed with highly positive anti-C. suis sera from experimentally infected piglets. Optimal dilutions of recombinant protein, sera and conjugate were determined by checkerboard titrations, and the serum dilution that gave the greatest ratio between the positive and the negative sera was selected for subsequent analyses. Agreement between the IFAT and the newly developed ELISA was assessed with kappa statistics. The receiver operating characteristic (ROC) curve analysis based on 185 serum samples with known C. suis exposure previously tested in the reference IFAT was used to determine the cut-off value, sensitivity and specificity of the ELISA. For IgG, the ELISA had an estimated cut-off value of 0.82 and sensitivity and specificity values of 94.7% and 98%, respectively, whereas for IgA the estimated cut-off value was 0.41 and sensitivity and specificity values were both100%. According to kappa coefficient, an excellent correlation (κ > 0.8) was found between IFAT and ELISA for both isotypes. The diagnostic accuracy of the test measured as the area under the ROC curve index scaled between 0.98 and 1.0, indicating high discriminatory capacity and its possible application as a serological tool for detecting antibody response in the host following C. suis exposure/immunization and large-scale surveillance studies.