Bovine brucellosis, caused mainly by Brucella abortus, is an important cows disease that has created a widespread public health problem in humans. Diagnosis primarily relies on serological testing; however, these assays lack sensitivity and, more importantly, specificity. In this study, we tried to compare the performance of serological tests routinely applied in Iran with antigen detection tests. Also, we examined Brucella species circulating in cows of Fars province, Iran. In addition, the infection rate of Yersinia entrocolitica O9 strain as a probable interfering agent in Brucella spp. serological tests were evaluated. Supramammary lymph nodes were sampled from 98 Brucella spp. reactor cows of Fars province, Iran, analyzed by bacterial culture and molecular tests, including conventional, multiplex, and real-time PCR. Brucella spp. was isolated from 5.1% of cultured samples, while conventional and real-time PCR detected in 15 (15.3%) and 21 (21.4%) samples, respectively. All positive samples were identified as B. abortus. Notably, 78.6% of seropositive cows tested Brucella spp. negative by both molecular tests and culture at the time of slaughtering, which showed a high false-positive rate of serological testing. As Y. enterocolitica O9 was not detected in any lymph node samples, it could be concluded that immunological cross-reaction with this bacterium was not the reason for the few real-time PCR-positive results among Brucella reactor cows. In conclusion, real-time PCR provides valuable information about the Brucella species circulating in the slaughtered cows of each region.

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