Construction and characterization of a Brucella abortus mutant by inactivation of a gene that codes a 26 kDa protein

One of the principal problems in bovine brucellosis is the difficulty in discriminating infected animals from vaccinated ones with current sere-assays. Strain S19, the vaccine strain, and pathogenic field strains share the same LPS, the main immunogen in Brucella species. As most of the antibodies in infected and vaccinated bovines are anti-LPS, antigenic differences due to different protein composition between strains are masked. The aim of this work is to develop a new vaccine strain with differential immunological response in comparison to pathogenic field strains, and at least the same protective capacity of the actual vaccine. A B. abortus S19 gen that codes for a 26 kDa protein of unknown function was initially cloned in lambda g11. This gene expressed by its own promoter in E. coli. The gene was subcloned in a pBR322 derivative, a suicide plasmid for Brucella because it can not be maintained in these species, and mutagenized in vitro by insertion of a kanamycin resistance gene. The vector bearing the mutated gene was introduced in B. abortus S19 by electroporation. Kanamycin resistant cells that had acquired the resistance gene by homologous recombination with the plasmid, were selected. Gene replacement of the original gene for the mutated one by double recombination, and the lack of expression of the 26 kDa protein were analyzed by Southern and Western blotting respectively. We also studied the kinetics of infection and survival of the mutant strain in BALB/c mice, measuring bacterial replication and weight variation of the spleen. These assays showed no difference between the original and the mutant strain. The great advantage of using the mutant strain as the vaccine strain in that sera from animals vaccinated with this strain can be differenciated from infected animals due to failure to produce antibodies against protein BP26.