Background: Timely diagnosis of brucellosis is the starting point for effective programs to control brucellosis in humans and animals. Aims: This study aimed to detect Brucella infection in milk samples from livestock in Famenin, an endemic region in western Iran, using milk ring test and molecular techniques. Methods: In a cross-sectional study, 738 raw milk samples were randomly collected from cattle, sheep and goats. The milk samples underwent screening using the milk ring test (MRT). In addition, the polymerase chain reaction method was used to detect Brucella spp. applied. DNA in all MRT positive samples. DNA from milk samples was extracted and used for PCR using the BCSP31 gene and IS711 locus. Results: Out of the samples tested using MRT, 46 (6.23%, 95% CI: 2.83-9.63%) yielded positive results. Of the 46 seropositive samples, 42 (91.30%) were from sheep, 4 (8.70%) were from goats, and no bovine sample had positive MRT results. PCR analysis confirmed that 78.26% (36/46) of the MRT positive samples belonged to the genus Brucella. Furthermore, 83.33% (30/36) the confirmed samples were identified as B. melitensis, while 16.66% (6/36) were identified as B. abortus. Conclusion: The results obtained from MRT evaluation of milk samples did not align entirely with the findings of the molecular examinations. The PCR method has minimal biological contamination and high sensitivity and accuracy, especially for determining Brucella species. Assessment of contamination in milk should be routine. This work is necessary to identify hidden infections and also break the chain of transmission of brucellosis.
