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Brucellosis remains as a public health concern worldwide. In domestic animals, the disease is characterized by reproductive disorders in male and female. Besides extensive use of serological tests and recent development of molecular biology techniques, microbiological culture of Brucella species is yet considered a “gold standard” method for diagnosis. Here, semen of 335 bovine bulls was subjected simultaneously to microbiological culture in Brucella agar, Farrell media, and CITA media to evaluate comparatively the best selective media for isolation of Brucella sp. Among all 335 samples, B. abortus B19 strain was isolated from semen of five (1.49%) bulls using the three selective media. However, Farrell media was considered the best selective media for microbiological diagnosis, because of allowed isolation of B. abortus B19 strain from bull semen without bacterial commensal or fungal contamination of plates. Key words: Brucella agar, Farrell media, CITA media, bovine bulls, semen.

Brucella melitensis is a Gram negative coccobacillus bacterium from the Brucellaceae family. Brucellosis is an infectious zoonotic disease affecting most species of domestic animals, but sheep and goats, particularly milking breeds, are the most susceptible. Cattle may occasionally be affected and the disease may appear in pigs. vacB gene is one important gene of B. melitensis that encoded a protein (RNase R) and in B. melitensis and Brucella abortus has no impact on bacterial virulence. The aim of the present study was to construct a novel recombinant vector as B. melitensis vacB gene knockouts candidate. B. melitensis were collected from Microbiology laboratory of Islamic Azad University of Shahrekord Branch and cultured into Brucella agar. Genomic deoxyribonucleic acid (DNA) was extracted and the polymerase chain reaction (PCR) were performed using designed primers for amplification of upstream and downstream regions of vacB gene of B. melitensis and kan gene of pET28 vector. Then, amplified fragments were cloned using T/A cloning technique and the construction was transformed into competent Escherichia coli Top10F' strain in LB media. The final construction was confirmed by digestion with XhoI, KpnI, XbaI, and BamHI restriction enzymes. PCR amplified products for flanking regions of vacB gene and kan gene on 1% agarose gel revealed 609, 595, and 870 bp, fragments, respectively. The results showed that vacB gene and kan gene were cloned in E. coli successfully. Sub-cloning of all fragment into pET-32 vector were done successfully and pET-32-UpKan-Down recombinant vector was generated. The findings of this study showed that the designed novel recombinant construct (pET-32-Up-Kan-Down recombinant plasmid) is useful for genetic engineering and for manipulate of vacB gene from B. melitensis.