1) At 9 dpi, groups B and C, both vaccinated only with LV showed

1). At 9 dpi, groups B and C, both vaccinated only with LV showed the highest levels of CD8+ T cells in caecal tonsils (Fig. 4). Before challenge, low levels of CD8+ T cells were detected (Fig. 4), suggesting that levels of CD8+ cells returned to basal levels during the interval between vaccination and challenge, as seen before [36] and [42]. After challenge, the population of CD8+ T cells constantly increased in groups B and C. This may suggest a controlled clonal expansion of memory CD8+ cells in these vaccinated birds. Furthermore, high numbers of CD8+ T cells persisted for longer periods, in birds that were vaccinated only with the LV (groups B and

C). Otherwise, the combination of LV and KV (group E), generated lower levels of CD8+ T cells, similarly to the PFI-2 solubility dmso KV (group D), whereas unvaccinated birds had rapid influx of cytotoxic T cells in the liver, possibly attracted by invasive bacteria in this organ. Birds which received one dose of LV (group

B) showed the highest levels of IFN-γ in spleen before challenge. This cytokine is important for macrophage activation [42] and [43], however after challenge of vaccinated birds, the levels of this Smad inhibitor cytokine decreased. This may be related to the development of acquired immunity mechanisms, obviously different from the innate immune response that is triggered in unvaccinated birds after primary infection (Fig. 3). Paratyphoid salmonellosis is frequently limited to the gastrointestinal tract; thus the control of bacterial invasion must occur primarily at the intestinal mucosae and gut associated lymphoid tissue (GALT), specifically the caecal tonsils. Considering this, the highest production of IFN-γ in the caecal tonsils was seen in groups C and E (Fig. 4). At 6 dpi, the expression of IFN-γ was significantly higher in group E, which could be associated with the

ability of birds in this group to control the first phase of SE infection; colonization and invasion. As shown in Fig. 1, control of SE in caecal contents was clearly faster in groups C and E than in the control groups A and D. The association these of IFN-γ production and clearance of primary Salmonella infection was suggested previously [35], [42] and [44]. However, in this study, IFN-γ levels decreased after challenge (1 dpi) of vaccinated birds, reaching similar levels to the unvaccinated group A, suggesting that the development of acquired immunity in vaccinated birds is not solely dependant on IFN-γ. IL-12 has an important role in stimulating the production of IFN-γ, recruiting naïve CD8+ T cells and CTLs and developing the CD8+ memory cells [45] and [46]. The present study detected high expression of this cytokine in vaccinated birds before challenge (Fig. 3). At 1 dbi IL-12 levels in caecal tonsils were elevated in all vaccinated groups in comparison with unvaccinated birds (group A).

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