BACKGROUND: Brucellosis is one of the most dangerous worldwide infectious zoonotic diseases that are common between ruminants and human. Consumption of infected milk and by-products is the major transmission source to human. In Iran, sheep compared to cow, has a higher rate of contamination with brucellosis. Therefore, early detection and precision could be a starting point for any efficient program to control the disease in human and animals. For brucellosis monitoring, milk ring test (MRT) is recommended but the test is not reliable in sheep herds. Perhaps a more realistic outcome could be achieved by changing the antigen used in MRT. OBJECTIVES: Comparison of two Brucella abortus and Brucella melitensis antigens in MRT for detection of Brucella antibodies in milk, as well as monitoring contamination of  ewe’s milk in Dezful region by detection of B. abortus and B. melitensis genes using PCR. METHODS: In this research, 220 milk samples from 16 different herds were collected from Dezful region’s nomadic at Khuzestan province. As the first step, MRT by two antigens, B. abortus and B. melitensis, were conducted on the samples. Next, the samples were subjected to detect Brucella genes using PCR technique. RESULTS: Results showed that 47 (21/3 %) out of 220 cases were positive by MRT test, in terms of both antigens of B. abortus and B. melitensis. In PCR, out of 220 samples, only 9 (4%) samples were positive for specific genes of B. melitensis which were MRT positive as well. CONCLUSIONS: A significant difference between B. abortus and B. melitensis antigens was not observed in MRT. Although the nature and basis of PCR and MRT methods for the diagnosis of brucellosis is different but a significant difference between the results obtained by PCR and MRT showed that MRT even by changing of antigens is still not authentic. Considering that various methods of identification have their limitations, it is recommended that in ewe’s milk samples, in addition to using a serological method as screening, PCR and culture methods should be used for definitive diagnosis.

BACKGROUND: Causing site direct mutation can be one of the efficient methods to evaluate the characteristics and properties of various genes. Brucellosis is the most common zoonotic infectious disease that would cause great economic losses. Thus, recognition of pathogenic and immunogenic factors in the genus Brucella can lead to control this health problem. Objectives: Considering the importance of site direct mutation in identification of genome structure and numerous ways to achieve this goal, Overlap Extension PCR is introduced as an improved technique for the removal and replacement of the gene target. Methods: For this study, with two-step PCR using specific primers, upstream and downstream fragments from target gene and antibiotic resistance cassette from plasmid pET28a (+), were reproduced and were connected to each other. The resulting fragment was cloned in specific position of pBluescriptIISK(-) plasmid by the restriction enzymes. Then, the construction was transferred into the genome of Brucella abortus by electroporation method. Results: Fusion PCR product was obtained without any change in the nucleotide sequence and then it was cloned into pBluescriptIISK (-) plasmid, finally the construction was replaced and the target gene was deleted. Conclusions: The results of this study show that the Overlap Extension PCR is an optimized and modified technique to create mutations in the bacterial genome structure and can easily be used in the family Brucella.