Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis

Abstract

Background: Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge.

Results: The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5).

Conclusions: rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.

Figure 1

Cellular invasion pattern of C. Pseudotuberculosis strain CP09 after 1, 3 and 6 hours of interaction with J774 cells. The results are expressed as the means ± standard deviation of two representative experiments performed in quadruplicate. ***Significant reduction of cellular invasion versus T1 wild-type (wt) strain cellular invasion ; p < 0.5 (ANOVA, Tukey).

Figure 2

Total specific IgG isotype levels in immunised mice with different vaccine formulations. The sera of immunised mice were collected 0, 15, 30, 45, and 60 days post-immunisation and tested by ELISA for the presence of specific IgG1 (A), IgG2a (B) and IgG3 (C). The results are presented as the means and standard deviation (bars) for each experiment with ten animals per group. Significant differences between IgG production in the different groups was calculated employing one-way ANOVA followed by the Tukey post-test. D,d: groups significantly different on day 30 (p < 0.05); E,e: groups significantly different on day 45 (p < 0.05); F,f: groups significantly different on day 60 (p < 0.05). Arrows correspond to the two immunisations (days 0 and 15), and stars indicate the challenge with the virulent strain (Day 30). All bleedings were performed before immunisations and challenge.

Figure 3

Graphical representation of the survival of immunised mice after virulent challenge. The data show the results of an experiment using 10 mice per group. Survival was monitored for 30 days after challenge.