A novel approach for an immunogen against Corynebacterium pseudotuberculosis infection: An Escherichia coli bacterin expressing phospholipase D.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA) in small ruminants. There is still needed an immunoprophylaxis model, which induces a protective and sustained immune response against the bacteria. In this study, we evaluated a recombinant Escherichia coli bacterin expressing the recombinant phospholipase D (rPLD) protein, the most relevant virulence factor of C. pseudotuberculosis, as a potential vaccine formulation. E. coli BL21 (DE3) Star strain was used for rPLD protein expression and was then inactivated by formaldehyde. Four groups with 10 Balb/c mice each were immunized twice within a 21 days interval: G1-control - 0.9% saline solution; G2- E. coli bacterin/pAE (naked plasmid); G3- E. coli bacterin/pAE/pld; G4-purified recombinant rPLD. Subsequently, the animals were challenged with a C. pseudotuberculosis virulent strain and evaluated for 40 days. The highest survival rate was observed for G3 with 40% protection, followed by 30% in the purified rPLD group (G4). These two groups also showed considerable IgG production when compared with the control group (G1). Also, a higher significant expression of interferon-γ was observed for the experimental groups G2, G3, and G4 when compared with a control group (G1) (p < 0.05). These results represent that a recombinant bacterin can be seen as a promising approach for vaccinal antigens against CLA, being possible to be used in association of different vaccine strategies.

The combination of Brazilian red propolis and recombinant protein rCP01850 in the immunoprophylaxis of Corynebacterium pseudotuberculosis infection in mice.

The immunomodulatory properties of Brazilian red propolis (BRP) have been already described. Also, propolis have been proved to have antibacterial activity on Corynebacterium pseudotuberculosis. An adjuvant effect of red propolis oil was able to induce a significant anti-C. pseudotuberculosis humoral immune response. Here, we demonstrate for the first time the immunostimulant property of BRP hydroalcoholic extract (BRPHE) in a recombinant vaccine against caseous lymphadenitis. Mice BALB/c were allocated in three groups inoculated with: sterile saline solution (G1); BRPHE (G2); or BRPHE combined with the C. pseudotuberculosis rCP01850 recombinant protein (G3) in two doses within a 21-days-interval. Blood samples were collected for the total IgG, IgG1 and IgG2a measurement. Mice were challenged with a virulent C. pseudotuberculosis strain, and other 6 mice were used for IFN-γ and IL-10 levels determination after splenocyte stimulation with the recombinant antigen. G3 showed higher significant levels of antibodies on the 42nd experimental day, with a high IgG2a/IgG1 proportion. G2 and G3 presented significant production of IFN-γ and IL-10, while G3 presented the higher levels of IFN-γ (p < 0.05). After challenge, G2 showed a survival rate of 20%, while 70% of mice from G3 survived the experimental challenge. In conclusion, BRPHE used alone has immunostimulant properties specially on cellular immune response, and when used in combination with the recombinant protein rCP01850 induces cellular and humoral immune responses as well as a significant survival of inoculated mice.