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

• Published in: Veterinary microbiology Vol. 49; no. 3; pp. 197 - 207
• Main Authors: Robertson, Gregory T., Elzer, Philip H., Roop, R.Martin
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-04-1996; Elsevier Science
• Subjects: Animals; Bacteriology; Biological and medical sciences; BOVINAE; BRUCELLA ABORTUS; Brucella abortus - drug effects; Brucella abortus - genetics; Brucella abortus - growth & development; Brucella abortus - immunology; Brucella Vaccine; Brucellosis, Bovine - immunology; CALOR; Cattle; CHALEUR; Female; FENOTIPOS; Fundamental and applied biological sciences. Psychology; Gene Deletion; Genes, Bacterial - genetics; Genetics; HEAT; Hydrogen Peroxide - pharmacology; Hypersensitivity, Delayed - immunology; MICE; Mice, Inbred BALB C; Microbial Sensitivity Tests - veterinary; Microbiology; PHENOTYPE; PHENOTYPES; Puromycin - pharmacology; RATON; Reactive oxygen intermediates; SOURIS; Stress response; Time Factors; VACCIN; VACCINES; Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies; VACUNA; Genetics; Brucella abortus; Vaccines; Stress response; Reactive oxygen intermediates; Immune response; Vaccine strain; Virulence; Rodentia; Vaccine; Vertebrata; Mammalia; Brucellaceae; Immunogenicity; Mouse; Infectivity; Deletion; Bacteria; Attenuation; Mutation
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/0378-1135(96)84554-8

An
htrA deletion mutant was created in the bovine vaccine strain,
B. abortus S19, by replacing the majority of the
htrA gene with a kanamycin resistance gene. Antibiotic selection for a double crossover event yielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient. The
B. abortus S19
htrA mutant was significantly more susceptible than the parental strain to killing by H
2O
2 (
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 colonization profile of this strain in
BALB
c
mice. At one week post-infection, the
B. abortus S19
htrA mutant colonized the spleens of experimentally infected
BALB
c
mice at significantly lower levels (
P < 0.01) than the parental strain. Enhanced clearance (
P < 0.05) was also observed at later timepoints, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains colonized the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in
BALB
c
mice infected with the mutant or parental strain were equivalent. These results 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.