## 3 Results
### Prophylactic Effect of the \(\Delta\)fruR Mutant against Virulent Salmonella Infection in Mice  
The prophylactic efficacy of pre-infection with the three mutant strains was investigated in BALB/c mice by challenging them with virulent _Salmonella_ (ST2173) infection at 21 dpi (Figure 3A). Mice that were pre-infected with _Salmonella_ strains or PBS showed comparable body weights up to 28 dpi; however, additional ST2173 infection at a dose of LD\({}_{50}\) caused alterations in body weight that were dependent on the pre-infection composition (Figure 4A). Mice that were not immunized with any of the _Salmonella_ strains but were administered the virulent ST2173 strain (P control) lost 23% of their weight compared with healthy mice (N control, administered PBS alone) at 35 dpi (14 days post-challenge; 14 dpc). However, mice that were pre-infected with the _Salmonella_ strains, with the exception of the \(\Delta\)_ffu_ mutant, did not lose weight, even after lethal ST2173 infection. Notably, mice that were immunized with the \(\Delta\)_fruR_ mutant gained weight for 14 days after ST2173 infection, showing body weights equivalent to those of the N control (Figure 4A). Pathological signs such as hepatomegaly and splenomegaly are typically observed in mice suffering from systemic infection with _Salmonella_, as shown in the P control (Figs 4B and S2). The liver and spleen also became enlarged in the challenged mice after pre-infection with WT or \(\Delta\)_ssrAB_ and \(\Delta\)_ffu_ mutant strains (Figure 4B). By contrast, mice immunized with the \(\Delta\)_fruR_ mutant strain exhibited no signs of hepatosplenomegaly, showing liver and spleen weights that were similar to those of healthy mice (Figure 4B). The bacterial burden of the liver and spleen exhibited a tendency that was reminiscent of the phases of hepatosplenomegaly. Moreover, immunization with the \(\Delta\)_fruR_ mutant strain resulted in the lowest bacterial burden in the organ among the tested _Salmonella_ strains, showing a 78.9-fold (liver) and 692.7-fold (spleen) lower number of _Salmonella_ compared with the P control challenged without immunization (Figure 4C). Immunization with the \(\Delta\)_ssrAB_ and \(\Delta\)_ffu_ mutant strains moderately reduced the bacterial counts in the liver and spleen compared with the P control cases. The monitoring of mouse survival for 35 days after the challenge with ST2173 revealed that P control mice not immunized with any _Salmonella_ strains showed severe morbidity, including a ruffled hair coat, dull eyes, shivering, and weight loss, and all succumbed to death within 7 dpc. However, mice immunized with the \(\Delta\)_fruR_ mutant strain exhibited a survival of 100% against lethal ST2173 infection (Figure 4D), indicating the potency of the \(\Delta\)_fruR_ mutant strain as a live vaccine. Interestingly, WT _Salmonella_ and the \(\Delta\)_ssrAB_ mutant displayed substantial protection activity (75% survival with WT _Salmonella_; 66.7% survival with the \(\Delta\)_ssrAB_ strain) against ST2173 infection (Figure 4D). Taken together, these findings suggest that the \(\Delta\)_fruR_ mutant strain, as a potential candidate vaccine, afforded a successful protective activity against _Salmonella_ infection. Figure 4: Comparison of prophylactic effects between _Salmonella_ mutant strains in mice. As depicted in Figure 3A, groups of mice were immunized with wild-type (WT) or mutant _Salmonella_ strains and then challenged with the ST2173 strain (\(10^{8}\) CFU/mouse for the protection assay; \(10^{10}\) CFU/mouse for the survival assay). The N control received PBS in both inoculations, whereas the P control received ST2173 inoculation at challenge tests after PBS administration. The body weight was measured at 28 and 35 dpi (**A**). All mice tested in the protection assay were euthanized at 14 days post-challenge (14 dpc; 35 dpi), and the livers and spleens were weighed (**B**) and used to count the ST2173 cells residing in these organs (**C**).