Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers’ recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (Ppos = 0.25–0.96). Negative agreement was very good for all assays (Pneg > 0.94) except NAT (Pneg = 0.66). Sensitivity was ≥0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.