Brucella abortus is a Gram-negative facultative intracellular pathogen that can affect many species of animals and man, causing brucellosis. LPS of Brucella outer membrane is one of the main virulence factors and has three basic components: O-antigen, core, and Lipid A. Modifications in this structure may alter the morphology of the colonies from smooth to rough. Brucella abortus wbkC gene participates in this LPS biosynthesis.The objective of this work was to obtain a rough vaccine strain, less pathogenic than the available vaccines, that could be differentiated during the screening for vaccinated and infected cattle. The strategies used were: inactivation the wbkC gene by insertion of a kanamycin cassette, transformation of Brucella abortus wild type S2308 by electroporation and molecular characterization of the mutants. Towards to this goal the entire ORF of wbkC gene was amplified by PCR technique using primers designed based on wbkC sequence available in the GenBank. This gene was cloned in pBluescript-KS(+) vector and them disrupted with kanamycin gene (pBlue:wbk-Kan). After electroporation, simple (M24) and double (M5 and M23) recombinants were obtained. These mutants were characterized by PCR, southern and western blot techniques. The mutant colonies M5 and M23 have shown rough morphology when stained with violet crystal. Southern blot confirmed the presence of the disrupted wbkC gene in the mutants M5 and M23 and both, the entire and disrupted gene in the mutat M24. Western blot was performed with mice antibody IgG anti-O LPS and only the strains B. abortus 2308 and B. abortus S19 were positive. The vaccine strain B. abortus RB51 was confirmed to be negative for that antibody, since it is a rough strain. This data confirms that the mutants M5 and M23 have lost part of the O-antigen of the LPS. Mouse model was used to analyze the virulence of the mutants. IRF ^(-/-) mice were infected with 1 x 10⁶ CFU with the following strains: S2308, RB51, S19, M5 and M23.  The mice survival was observed for 30 days post-inoculation and the results indicated that wbkC mutants have showed a decreased virulence compared to the wild type and vaccine strains.
In conclusion, we suggest that modification in the wbkC gene alters the LPS structure, what may result in less virulent strains. Further studies are on going to elucidate the protection aspects of these mutants.