Diagnostic Performance of Four Serological Assays for Bovine Brucellosis and Optimised Cutoff Thresholds in an Endemic Region of Iran

Brucellosis is a severe zoonotic infection impacting dairy cattle, requiring accurate diagnostic assays for efficient control programs. This cross-sectional study was conducted in Alborz Province, Iran, to assess the diagnostic efficacy of four serological tests for Brucella detection. One thousand serum samples were obtained from dairy cattle and analysed over 1 year of age. Furthermore, milk specimens from seropositive cows were cultured for bacteriological analysis. Serological testing detected Brucella antibodies in 33% of samples using RBT, 19.4% by SAT, 17.6% by 2-ME and 33.5% by I-ELISA. Bacterial culture detected Brucella spp. in 16.6% of seropositive milk samples, with all isolates classified as Brucella abortus biovar 3. Statistical methods were used to evaluate the diagnostic accuracy of each test. Bayesian latent class analysis revealed that I-ELISA demonstrated the highest diagnostic accuracy for Brucella infection in dairy cattle, with superior sensitivity and specificity. In contrast, SAT and 2-ME exhibited high specificity but lower sensitivity, while RBT showed moderate sensitivity with low specificity. The receiver operating characteristic (ROC) analysis was performed based on the results obtained from the Bayesian latent class analysis to further evaluate the diagnostic performance of the tests. Furthermore, the ROC curve analysis showed that SAT and 2-ME displayed strong concordance with RBT. The ideal threshold for SAT and 2-ME titers was established at 5.00, optimising sensitivity and specificity. Cohen's kappa analysis assessed agreement levels, revealing that RBT demonstrated the highest concordance with I-ELISA. The results indicate that although RBT offers a simple screening approach, its sensitivity constraints require validation by I-ELISA. A significant portion of infected animals (20%) might be undetected using RBT. These findings underscore the need for various serological assays to identify brucellosis in endemic areas accurately.