Bacterial loads in cecum content (A), mLN (B), spleen (C) and liver (D)

were assessed by plating at day 4 p.i.. n.s., statistically not significant; *, statistically significant (p < 0.05, Two-way ANOVA). MT4 protects wild-type C57BL/6 mice when challenged with wild-type S. Typhimurium The immunogenic potential of MT4 in wild-type C57BL/6 mice was analyzed by previously established BAY 57-1293 molecular weight vaccination and challenge protocol using TTSS-2 deficient S. Typhimurium strain [34]. Three groups of wild-type C57BL/6 mice were vaccinated with MT4 (n = 10), MT5 (n = 10) and PBS (negative control; n = 10). The fecal shedding was analyzed as a measure of cecal colonization during vaccination period. Both, MT5 and MT4 strains reached a bacterial load of ~109 CFU/g (of cecal content) in the gut lumen at the day 1 p.v.; however, the bacterial loads slightly declined at day 14 and day 28 p.v. (Figure 2A). Half the number of vaccinated mice (MT5, n = 5; MT4, n = 5; PBS, n = 5) were sacrificed Doxorubicin order to analyze cecal inflammation and the colonization levels in different systemic sites at day 30 p.i. With both the strains, cecum colonization was maintained up to ~107-9 CFU/g. The bacterial load

in mLN was lower as compared to the acute infection experiments (compare Figure 1B to 2B) whereas cecal mucosa did not show any sign of disease (Figure 2C). The remaining mice were analyzed for protection against a challenge with wild-type S. Typhimurium. At day 30 p.v., the remaining vaccinated mice (MT4, n = 5; MT5, n = 5; PBS, n = 5) were treated with 20 mg of ampicillin to remove regrown gut flora and any residual vaccine strain. Mice groups were then challenged with wild-type S. Typhimurium at day 31st (200 CFU by gavage). The wild-type S. Typhimurium was able to colonize the lumen efficiently and reached the carrying capacity by day 3 p.c. in all three immunized groups (Figure 3A). Mice in the PBS treated control group suffered from severe enteropathy (Figure 3B). In contrast, Reverse transcriptase the mice immunized with MT5 and MT4 strains did not show any signs of mucosal inflammation (Figure 3B). Furthermore, spleen and liver colonization by wild-type S. Typhimurium was significantly

reduced in both the vaccinated groups (p < 0.05; Figure 3A). Thus, the data indicates that MT4 strain conferred equivalent level of protection from Salmonella inflicted disease as MT5 strain. Figure 2 Vaccination experiment analyzing the attenuation of MT4 at day 30 p.v. For vaccination, C57BL/6 mice were treated with PBS (n = 10; grey solid circles), MT5 (5x107CFU; n = 10; black solid circle) and MT4 (5×107 CFU; n = 10; open circle). (A) Fecal shedding as analyzed by plating. PBS-controls: below detection limit (stripped line); (B) Colonization by the vaccine strains (MT5, n = 5 and MT4, n = 5) in cecal content, mLN, spleen and liver; (C) Cecal pathology at day 30 p.v.. n.s., not significant; *, statistically significant (p < 0.05).