Salmonella Typhimurium lacking the Znuabc transporter is attenuated and immunogenic in pigs

Abstract

Meat contamination by Salmonella spp. is emerging as a major cause of human enteric infections in industrialized countries. The attempts to reduce human cases of salmonellosis encompass pre- and post-harvest interventions. In this context, vaccination of pigs may represent an effective instrument in eliminating/reducing Salmonella burden through the food chain. We have previously demonstrated that Salmonella Typhimurium lacking the ZnuABC transporter (S. Typhimurium ΔznuABC) is a promising candidate live vaccine in different mouse models of Salmonella Typhimurium infection. In this study, we confirmed in pigs the attenuation of S. Typhimurium ΔznuABC. Moreover, we evaluated the safety and immunogenicity of S. Typhimurium ΔznuABC administered to pigs by the oral route. We monitored clinical conditions of animals and we conducted a microbiological culture and a quantification of the humoral and cellular immune response, respectively, on fecal and blood samples of pigs. After vaccination with attenuated S. Typhimurium ΔznuABC, pigs showed a modest degree of hyperthermia. In addition, fecal shedding of S. Typhimurium ΔznuABC could not be detected 28 days after the inoculum. Furthermore, vaccination with S. Typhimurium ΔznuABC elicited a distinct production of anti-Salmonella antibodies and IFN-γ. Taken together, these results suggest that S. Typhimurium ΔznuABC is attenuated and immunogenic in pigs. Although the vaccine dosages do not guarantee complete safety there is ample margin to set up better conditions of use, suggesting that S. Typhimurium ΔznuABC could be a promising attenuated strain to be used as live mucosal vaccine for oral delivery.

Fig 1. S. Typhimurium ΔznuABC shows lower virulence in pigs compared to wild type S. Typhimurium ATCC 14028

(Fig. 1A). Mean values and SD bars of body temperature of groups (A–C) at different time points. Results of group A or B were compared with results of group C. Differences were estimated using 2 ways analysis of variance with a Tuckey’s multiple comparison test and considered significant when* P-value ≤0.05. (Fig. 1B) Mean values and SD bars of CFU/g of S. Typhimurium ΔznuABC (group A) and S. Typhimurium ATCC14028 (group B) shed in faeces. Results of group A were compared with results of group B. Differences were estimated using non parametric Mann Whitney test and were considered significant when * P-value ≤0.05.

Fig 2. Group A and group B display a rise of body temperature after vaccination with S. Typhimurium ΔznuABC

Mean values and SD bars of body temperature of each group at different time points. Two-way ANOVA and Bonferroni corrections were applied. * P-value ≤0.05.

Fig 3. The weight gain of pigs of vaccinated groups is heterogeneous

Weight gain of animals of different groups. Each symbol represents one animal and bars represent mean value of the group.

Fig 4. Faecal shedding of S. Typhimurium ΔznuABC

Faecal elimination of S. Typhimurium ΔznuABC by group A and group B after oral administration respectively with a dose of 5×10⁸ and 5×10⁷ CFU/pig. Mean values with SD bars of CFU/g of each group at different time points.

Fig 5. S. Typhimurium ΔznuABC has a limited survival in faecal material

Survival of S. Typhimurium ΔznuABC and S. Typhimurium ATCC 14028 in artificial contaminated faeces maintained at room temperature.

Fig 6. S. Typhimurium ΔznuABC and inactivated S. Typhimurium ATCC14028 induce a similar pattern of humoral immune response

After vaccination, serum of pigs was collected at several time points; a commercially available ELISA test was used to detect antibodies against Salmonella. The X-axis (time) of figure is divided in two segments. Each segment has different intervals coinciding with time points. This solution was adopted to ameliorate the graphical illustration of data. Mean values with SD bars of antibodies titres of each group at different time points. Groups with asterisk are statistically different from group D at the time point considered *P-value ≤0.05.