Article Figures & Data
Figures
- FIG. 1.
Growth curves in liquid TSB-YE-HS, as determined by the evolution of OD600 with time, of parental B. ovis PA (A and B) and the mutant strains with the genes omp31, omp25, omp25c, omp25d (A), and omp22 (B) inactivated. CFU/ml counts are shown at time points 25 and 46 h for parental and Δomp22 mutant strains (B). Cultures were started at an OD600 of 0.05 and were incubated at 37°C with shaking under a 10% CO2 atmosphere.
- FIG. 2.
Western blot (A and B) and iELISA (C and D) analysis of the B. ovis strains obtained in this work. Western blotting was performed with MAb G02, specific for the B. ovis Omp31 protein, diluted 1/5 (A) or with MAb C09, specific for the Brucella spp. Omp25 protein, diluted 1/6,000 (B). The positions of the molecular mass protein standards are shown by arrows on the left. For iELISA, MAbs specific for Omp31 (G02 and G10, diluted 1/50) (C) or specific for Omp25 (C09, diluted 1/6,400, and F04, diluted 1/800) (D) were used. Black, white, and shaded columns correspond to parental B. ovis PA, mutant strains, and complemented mutant strains, respectively.
- FIG. 3.
Autoagglutination capacity of parental B. ovis PA, its derived strains obtained in this work, and B. abortus RB51. The OD600 values for bacterial suspensions adjusted to an initial OD600 of 0.8 were scored at several time points of static incubation, and the percent OD600 was determined with respect to the OD600 of the initial suspension (100% of OD600). B. ovis PA mutants PNV25dA and PNV22A and the corresponding strains complemented with wild-type omp25d and omp22, respectively, behaved as parental B. ovis PA and are not represented. The SD, which was always lower than 5% of the mean, is not shown.
- FIG. 4.
Kinetics of splenic infection of parental B. ovis PA (A and B), and the mutant strains with the genes omp25, omp25c, omp25d, and omp22 (A) and omp31 (B) inactivated. Mice were inoculated intraperitoneally with 5 × 106 CFU. Results are expressed as the mean ± SD (n = 5) of the log CFU/spleen.
Tables
- TABLE 1.
Brucella strains used in this work
Brucella strain Relevant genotype Relevant characteristic(s) Origin Wild-type strainsa B. abortus RB51 Rough strain obtained under laboratory conditions BCCNb B. ovis PA Parental strain Naturally rough virulent Brucella strain BCCN Mutant strains B. ovis PNV31A Δomp31 Kanr B. ovis PA with a Kan resistance cassette replacing the XcmI-SalI fragment of omp31 This work B. ovis PNV25A omp25::Kanr B. ovis PA with a Kan resistance cassette inserted into the AflII site of omp25 This work B. ovis PNV25cA Δomp25c Kanr B. ovis PA with a Kan resistance cassette replacing the AspI-HinCII fragment of omp25c This work B. ovis PNV25dA Δomp25d Kanr B. ovis PA with a Kan resistance cassette replacing the NheI-ClaI fragment of omp25d This work B. ovis PNV22A Δomp22 Kanr B. ovis PA with a Kan resistance cassette replacing the RsrII-StuI fragment of omp22 This work Complemented mutant strains B. ovis PNV31A-com Δomp31 omp31 Kanr Ampr B. ovis PNV31A complemented with omp31 cloned in pNV31300c This work B. ovis PNV25A-com omp25::Kanromp25 Kanr Ampr B. ovis PNV25A complemented with omp25 cloned in pNV25comc This work B. ovis PNV25cA-com Δomp25c omp25c Kanr Ampr B. ovis PNV25cA complemented with omp25c cloned in pNV25c4c This work B. ovis PNV25dA-com Δomp25d omp25d Kanr Ampr B. ovis PNV25dA complemented with omp25d cloned in pNV25d4c This work B. ovis PNV22A-com Δomp22 omp22 Kanr Ampr B. ovis PNV22A complemented with omp22 cloned in pNV22Dc This work ↵ a Reference strains of the Brucella species (46) were also used for the bacteriological typing of the B. ovis strains obtained in this work.
↵ b BCCN, Brucella Culture Collection of Nouzilly, INRA Centre de Tours, France. B. abortus RB51 and B. ovis PA are strains BCCN V5 and BCCN 76-250, respectively.
↵ c Plasmids were constructed by cloning the corresponding B. ovis PA wild-type omp gene in pBBR1MCS-4.
- TABLE 2.
DNA primers used in this study
Primer use and name Nucleotide sequence Construction of mutant B. ovis PA strains 31MUT-F 5′-AGA ATA AAA CAC ATG CCC-3′ 31MUT-R 5′-GCT GAA TGC GGA GAT GGT-3′ 25MUTZ-F 5′-CGA CCT TAT CCT CCT GAA-3′ 25MUTZ-R 5′-CCA GCA AAA CGT CGC AAA-3′ 25cdMUT-F 5′-TGC GTG GTT CAG ATT TCG-3′ 25cdMUT-R 5′-TTG CCG CTT CCA TCA GGT-3′ 25cMUT-R 5′-AGC CGT AAC CAA CCT GAC-3′ 22MUT-F 5′-GGC AAA GAA GAA GAA TAC-3′ 22MUT-R 5′-CTG CTG GAA TGC CCT GAA-3′ Complementation of mutant B. ovis PA strains 31sd 5′-TGA CAG ACT TTT TCG CCG AA-3′ 31ter 5′-CAT TCA GGA CAA TTC CCG CC-3′ 25A-F 5′-GGA CCG CGC AAA ACG TAA-3′ 25-R 5′-ACC GGA TGC CTG AAA TCC-3′ 25C-F 5′-CTG TGT CCT GTT TGC TAC-3′ 25C-R 5′-TAT TGG GTG AGG ATT GAC-3′ 25D-F 5′-CTA CAT ACA TTC GAC CAG-3′ 25D-R 5′-CTG AAG GGT AAA TGC GGC-3′ 22-F 5′-TCA AGC ATG TTT CCC GCC-3′ 22-R 5′-GTT TGA ATC CCG GCT GTT-3′ - TABLE 3.
Susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum of B. abortus RB51, parental B. ovis PA, and its derived strains obtained in this worka
Brucella strain % Survival after 1-h exposure to: Inhibition zone diam (cm) with hydrogen peroxide % Survival after 4-h exposure to nonimmune ram serum Polymyxin B (1 mg/ml) Na deoxycholate (0.1 mg/ml) B. abortus RB51 0.00 ± 0.00B 88.69 ± 5.45 4.28 ± 0.11C 25.73 ± 4.84B B. ovis PA (parental) 69.59 ± 5.26 89.34 ± 9.30 5.04 ± 0.05 85.52 ± 5.20 B. ovis PNV31A 70.07 ± 13.83 75.51 ± 20.97 6.23 ± 0.11B 64.43 ± 6.97C B. ovis PNV31A-com 69.83 ± 8.63 62.99 ± 22.90D 6.64 ± 0.37B(D) 69.44 ± 3.62D B. ovis PNV25A 75.73 ± 22.13 63.81 ± 3.98D 5.88 ± 0.02B 71.67 ± 2.89D B. ovis PNV25A-com 75.42 ± 4.41 64.74 ± 15.67D 6.08 ± 0.43B 87.21 ± 2.27(D) B. ovis PNV25cA 27.70 ± 11.65B 39.21 ± 7.92B 5.88 ± 0.15B 55.68 ± 7.32B B. ovis PNV25cA-com 7.88 ± 5.18B(D) 32.54 ± 18.15B 7.08 ± 0.04B(B) 17.51 ± 14.17B(B) B. ovis PNV25dA 61.16 ± 8.93 55.44 ± 21.69C 6.05 ± 0.07B 54.05 ± 7.42B B. ovis PNV25dA-com 58.02 ± 12.87 48.70 ± 15.19C 6.29 ± 0.27B 56.13 ± 2.98B B. ovis PNV22A 52.74 ± 3.97 30.68 ± 3.60B 5.90 ± 0.25B 25.38 ± 3.98B B. ovis PNV22A-com 38.52 ± 1.91C 23.31 ± 8.23B 6.09 ± 0.02B 17.41 ± 5.47B ↵ a Assays were performed as detailed in Materials and Methods. Values are means ± SD (n = 3). Statistical comparisons were performed with Fisher's protected least significant differences test. Significant differences between the mutant or complemented strains and parental B. ovis PA or between the complemented strains and the corresponding mutant strain (in parenthesis) are as follows: B, P < 0.0005; C, P < 0.005; D, P < 0.05.
