J Vet Sci.  2015 Dec;16(4):467-473. 10.4142/jvs.2015.16.4.467.

Immunization of BALB/c mice with Brucella abortus 2308DeltawbkA confers protection against wild-type infection

• Li ZQ
• Gui D
• Sun ZH
• Zhang JB
• Zhang WZ
• Zhang H
• Guo F
• Chen CF

• KMID: 2133629
• DOI: http://doi.org/10.4142/jvs.2015.16.4.467

Abstract

Brucellosis is a zoonotic disease that causes animal and human diseases. Vaccination is a major measure for prevention of brucellosis, but it is currently not possible to distinguish vaccinated animals from those that have been naturally infected. Therefore, in this study, we constructed the Brucella (B.) abortus 2380 wbkA mutant (2308DeltawbkA) and evaluated its virulence. The survival of 2308DeltawbkA was attenuated in murine macrophage (RAW 264.7) and BALB/c mice, and it induced high protective immunity in mice. The wbkA mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon. Antibodies to 2308DeltawbkA could be detected in sera from mice, implying the potential for use of this protein as a diagnostic antigen. The WbkA antigen would allow serological differentiation between infected and vaccinated animals. These results suggest that 2308DeltawbkA is a potential attenuated vaccine against 16M. This vaccine will be further evaluated in sheep.

Keyword

2308DeltawbkA; Brucella; vaccine

MeSH Terms

Animals
Antibodies
Brucella abortus*
Brucella*
Brucellosis
Humans
Immunization*
Immunoglobulin G
Interferons
Macrophages
Mice*
Sheep
Staphylococcal Protein A
Vaccination
Virulence
Zoonoses
Antibodies
Immunoglobulin G
Interferons
Staphylococcal Protein A

Figure

• 

  Fig. 1 Intracellular replication of Brucella abortus 2380 wbkA mutant (2308ΔwbkA) within RAW264.7 macrophages. RAW264.7 Macrophages were infected with 2308ΔwbkA, S19, 2308-wbkA and S2308 at a multiplicity of infection of 150. The level of initial infection was the same for these strains. At 4, 8, 24 and 48 h post-infection, infected macrophages were lysed and bacteria were calculated by plating serial dilutions on tryptone soy agar plates. The results demonstrate that the 2308ΔwbkA was unable to achieve the level of colonization reached by the conventional vaccine strain S19, parental strain S2308 and complementary strain 2308-wbkA. Significant differences between the mutant and S2308 or 2308-wbkA are indicated by *p < 0.05 and **p < 0.01.

• 

  Fig. 2 Clearance of 2308ΔwbkA after infection. BALB/c mice were inoculated i.p. with 1 × 106 CFU/mouse of 2308ΔwbkA, S19 and S2308. Control groups were inoculated i.p. with phosphate buffered saline (PBS). Infected mice were sacrificed by CO2 asphyxiation at 3, 7, 14, 21 and 28 days post-infection, after which spleens were removed aseptically and assessed individually for colonization. Values are the means ± SD (n = 5 per time point). Differences in splenic colonization were determined by one-way ANOVA. *p < 0.05, **p < 0.01.

• 

  Fig. 3 Anti-Brucella in serum from mice immunized with 2308ΔwbkA or S19. Mice were inoculated i.p. with 1 × 106 CFU of 2308ΔwbkA or S19. Control groups were inoculated with PBS. Serum samples were collected at 3, 7, 14, 21 and 28 days post-vaccination (n = 5 per time point) and IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA). The results are the means ± SD of the absorbance at 450 nm (OD450). Significant differences between the 2308ΔwbkA and PBS groups are indicated by *p < 0.05.

• 

  Fig. 4 IFN-γ production by spleen cells of BALB/c mice vaccinated with 2308ΔwbkA or S19. Spleens from mice were inoculated i.p. with 1 × 106 CFU of 2308ΔwbkA or S19. Control groups were inoculated with PBS. At 4 weeks post-vaccination, mice were euthanized and splenocytes were recovered and stimulated with either heat-killed S2308, concanavalin A (ConA) or RPMI 1640. Splenocyte culture supernatants were harvested and analysis of IFN-γ secretion (pg/mL) was assayed by ELISA. Significant differences from the same stimulus in PBS-injected mice are indicated by *p < 0.05.

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  Fig. 5 Response of WbkA to 2308ΔwbkA immunization sera. Sera from mice immunized with 2308ΔwbkA, S2308, S19 and PBS were collected. Antibodies to WbkA were detected in these sera by Western blotting. Antibodies against the WbkA protein were not detected in sera from 2308ΔwbkA immunized mice.

• 

  Fig. 6 Humoral immune responses to WbkA were assessed in serum by indirect ELISA. Sera from mice immunized with 2308ΔwbkA, S2308, S19 and PBS were collected. Humoral immune responses against the WbkA protein were not detected in serum from 2308ΔwbkA immunized mice.

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