Salmonella Serovars and Vaccination Effect on the Immune Responses of Male and Female Layers.

Salmonella Enteritidis, S. Gallinarum and S. Pullorum are common serovars to infect poultry and cause diseases differently. The antibody production and cellular immune responses of male and female layers were evaluated before and after inoculation. Before inoculation, S. Gallinarum and S. Pullorum could survive and grow in 10% sera from 6-week-old layers, and S. Enteritidis and E. coli were completely eliminated. The weights of the male and female layers were increased the lowest by inoculation with S. Gallinarum, followed by S. Pullorum, and S. Enteritidis. Inoculation with S. Enteritidis, S. Gallinarum and S. Pullorum increased the antibody titer in the males depending on the serovars and maintained same higher antibody level in females. Furthermore, an increased anti-Salmonella IgG titer was associated with bactericidal ability and the level was reduced by serovars and complemente. Despite the vaccination and serovars, the male layers expressed more IgG2a than IgG1, indicating preferential activation of the Th1 pathway. The inoculation number affected the expression level of IFN-γ and IL-12 in the blood not in the secretion of the peripheral blood mononucleated cells (PBMCs) and more inoculations increased the expression of both cytokines. Inoculation increased more reactive oxygen species (ROS) production in polymorphonuclear (PMN) cells, not the PBMCs. ROS production was greater in cells from the males than from the females and greater in the cells treated with S. Enteritidis than S. Gallinarum and S. Pullorum. These three serovars and their vaccinations differed in sera killing and immune responses.

Evaluation of a Salmonella vectored vaccine expressing Mycobacterium avium subsp. paratuberculosis antigens against challenge in a goat model.

Johnes disease (JD), caused by Mycobacterium avium subsp paratuberculosis (MAP), occurs worldwide as chronic granulomatous enteritis of domestic and wild ruminants. To develop a cost effective vaccine, in a previous study we constructed an attenuated Salmonella strain that expressed a fusion product made up of partial fragments of MAP antigens (Ag85A, Ag85B and SOD) that imparted protection against challenge in a mouse model. In the current study we evaluated the differential immune response and protective efficacy of the Sal-Ag vaccine against challenge in a goat model as compared to the live attenuated vaccine MAP316F. PBMCs from goats vaccinated with Sal-Ag and challenged with MAP generated significantly lower levels of IFN-γ, following in vitro stimulation with either Antigen-mix or PPD jhonin, than PBMC from MAP316F vaccinated animals. Flow cytometric analysis showed the increase in IFN-γ correlated with a significantly higher level of proliferation of CD4, CD8 and γδT cells and an increased expression of CD25 and CD45R0 in MAP316F vaccinated animals as compared to control animals. Evaluation of a range of cytokines involved in Th1, Th2, Treg, and Th17 immune responses by quantitative PCR showed low levels of expression of Th1 (IFN-γ, IL-2, IL-12) and proinflammatory cytokines (IL-6, IL-8, IL-18, TNF-α) in the Sal-Ag immunized group. Significant levels of Th2 and anti-inflammatory cytokines transcripts (IL-4, IL-10, IL-13, TGF-ß) were expressed but their level was low and with a pattern similar to the control group. Over all, Sal-Ag vaccine imparted partial protection that limited colonization in tissues of some animals upon challenge with wild type MAP but not to the level achieved with MAP316F. In conclusion, the data indicates that Sal-Ag vaccine induced only a low level of protective immunity that failed to limit the colonization of MAP in infected animals. Hence the Sal-Ag vaccine needs further refinement to increase its efficacy.

Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD mutant as a vaccine candidate against challenge in a caprine model.

Johne's disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor ß (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.

Immune response and protective efficacy of live attenuated Salmonella vaccine expressing antigens of Mycobacterium avium subsp. paratuberculosis against challenge in mice.

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants that leads to diarrhea and eventually death. Existing vaccines have proven useful in limiting disease progression but have not been effective in preventing infection. To address this problem we constructed an attenuated Salmonella (ΔyejE; ΔssaV) strain harboring a plasmid that expressed a fusion protein comprised of the Salmonella Type III secretion system (T3SS) effector SopE and MAP antigens (85A, 85B, SOD, 74F) and evaluated its potential as vaccine candidate against MAP infection in mice. Of various SopE-MAP fusion proteins analyzed, only SopE104-Ag85A C-terminal(202-347)-SOD N-terminal(1-72)-Ag85B C-terminal(173-330) and SopE104-74F(1-148+669-786)were successfully expressed and secreted into culture media as revealed by western blot analysis. Mice immunized with attenuated Salmonella (ΔyejE; ΔssaV) harboring the SopE104-Ag85A C-terminal(202-347)-SOD N-terminal(1-72)-Ag85B C-terminal(173-330) and SopE104-74F(1-148+669-786)plasmid generated a potent and long lasting Th1 response characterized by production of IFN-γ. The cytokine profile varied at various time points after immunization and challenge, which showed down regulation of Th2 cytokines (IL-4, IL-10) and up-regulation of proinflammatory cytokines (IL-12 and IL-17). Further, the immune response correlated with protection as revealed by reduced bacterial load and improved histopathology of spleen and liver, which showed fewer granulomas and lower numbers of acid-fast bacilli as compared to PBS controls. Interestingly, vaccination with antigens mixed with Ribi adjuvant (Agmix+Ribi) imparted better protection than the attenuated salmonella vectored vaccine. Thus, priming with a live recombinant Salmonella strain that secretes MAP antigens represents a promising approach that could lead to development of an efficacious and cost effective vaccine for Johne's disease.