IN-VITRO AND IN-VIVO PHENOTYPES RESULTING FROM DELETION OF THE HIGH-TEMPERATURE REQUIREMENT-A (HTRA) GENE FROM THE BOVINE VACCINE STRAIN BRUCELLA-ABORTUS S19

An htrA deletion mutant was created in the bovine vaccine strain, B. a bortus S19, by replacing the majority of the htrA gene with a kanamyci n resistance gene. Antibiotic selection for a double crossover event y ielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient, The B. abort us S19 htrA mutant was significantly more susceptible than the parenta l strain to killing by H2O2 (P < 0.001) and O-2 generated by the redox cycling agent paraquat(P < 0.05) in disk sensitivity assays. Deletion of the htrA gene from S19 produced a bimodal effect on the spleen col onization profile of this strain in BALB/c mice. At one week post-infe ction, the B. abortus S19 htrA mutant colonized the spleens of experim entally infected BALB/c mice at significantly lower levels (P < 0.01) than the parental strain, Enhanced clearance (P < 0.05) was also obser ved at later timepoints, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains coloniz ed the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in BALB/c mice i nfected with the mutant or parental strain were equivalent, These resu lts suggest that the htrA gene product contributes to successful host colonization by S19. However, deletion of this gene does not radically alter the overall, characteristic spleen colonization profile of this vaccine strain in the BALB/c mouse model, nor compromise the capacity of this strain to elicit Brucella specific cellular or humoral immune responses in this experimental host.