Outer membrane vesicles (OMVs) are membranous structures frequently observed in Gram-negative bacteria that contain bioactive substances. These vesicles are rich in bacterial antigens that can activate the host\'s immune system, making them a promising candidate vaccine to prevent and manage bacterial infections. The aim of this study was to assess the immunogenicity and protective efficacy of OMVs derived from Salmonella enterica serovar Typhimurium and S. Choleraesuis, while also focusing on enhancing OMV production. Initial experiments showed that OMVs from wild-type strains did not provide complete protection against homologous Salmonella challenge, possible due to the presence of flagella in the purified OMVs samples, which may elicit an unnecessary immune response. To address this, flagellin-deficient mutants of S. Typhimurium and S. Choleraesuis were constructed, designated rSC0196 and rSC0199, respectively. These mutants exhibited reduced cell motility and their OMVs were found to be flagellin-free. Immunization with non-flagellin OMVs derived from rSC0196 induced robust antibody responses and improved survival rates in mice, as compared to the OMVs derived from the wild-type UK-1. In order to enhance OMV production, deletions of ompA or tolR were introduced into rSC0196. The deletion of tolR not only increase the yield of OMVs, but also conferred complete protection against homologous S. Typhimurium challenge in mice. Collectively, these findings indicate that the flagellin-deficient OMVs with a tolR mutation have the potential to serve as a versatile vaccine platform, capable of inducing broad-spectrum protection against significant pathogens.

Salmonella is one of the most spread foodborne pathogens worldwide, and Salmonella infections in humans still represent a global health burden. The main source of Salmonella infections in humans is represented by contaminated animal-derived foodstuffs, with pork products being one of the most important players. Salmonella infection in swine is critical not only because it is one of the main causes of economic losses in the pork industry, but also because pigs can be infected by several Salmonella serovars, potentially contaminating the pig meat production chain and thus posing a significant threat to public health globally. As of now, in Europe and in the United States, swine-related Salmonella serovars, e.g., Salmonella Typhimurium and its monophasic variant Salmonella enterica subsp. enterica 1,4,[5],12:i:-, are also frequently associated with human salmonellosis cases. Moreover, multiple outbreaks have been reported in the last few decades which were triggered by the consumption of Salmonella-contaminated pig meat. Throughout the years, changes and evolution across the pork industry may have acted as triggers for new issues and obstacles hindering Salmonella control along the food chain. Gathered evidence reinforces the importance of coordinating control measures and harmonizing monitoring programs for the efficient control of Salmonella in swine. This is necessary in order to manage outbreaks of clinical disease in pigs and also to protect pork consumers by controlling Salmonella subclinical carriage and shedding. This review provides an update on Salmonella infection in pigs, with insights on Salmonella ecology, focusing mainly on Salmonella Choleraesuis, S. Typhimurium, and S. 1,4,[5],12:i:-, and their correlation to human salmonellosis cases. An update on surveillance methods for epidemiological purposes of Salmonella infection in pigs and humans, in a \"One Health\" approach, will also be reported.

Salmonella is a common foodborne and zoonotic pathogen. Only a few serovars carry a virulence plasmid (pSV), which enhances the pathogenicity of the host. Here, we investigated the pathogenicity roles of the pSVs among wild-type, plasmid-less, and complemented S. Typhimurium, S. Enteritidis S. Choleraesuis in invasion, phagocytosis, and intracellular bacterial survival in human THP-1 cells and cell death patterns by flow cytometry and difference in cell death patterns between pig and human S. Choleraesuis isolates with large pSCVs. Virulence plasmid (pSTV) led to slightly increasing cellular apoptosis for S. Typhimurium; virulence plasmid (pSEV) enhanced apoptosis and necrosis significantly for S. Enteritidis; and pSCV reduced apoptosis significantly for S. Choleraesuis. After complementation, pSTV increased the intracellular survival of pSCV-less Choleraesuis and the cytotoxicity against human THP-1 cells. Using the Cytochalasin D to differentiate the invasion of S. Choleraaesuis and phagocytosis of THP-1 cells determined that pSCV were responsible for invasion and phagocytosis at 0 h and inhibited intracellular replication in THP-1 cells, and pSTV were responsible for invasion and increased intracellular survival for S. Choleraesuis in THP-1 cells. The human isolates with large pSCV induced more cellular apoptosis and necrosis than the pig isolates. In conclusion, human S. Choleraesuis isolates carrying large pSCVs were more adapted to human THP-1 cells for more cell death than pig isolates with large pSCV. The role of pSVs in invasion, phagocytosis, intracellular survival and apoptosis differed among hosted serovars.

Regulated delayed attenuation is a well-studied strategy for retaining the immunogenicity of Salmonella-vectored vaccines. In this study, this strategy was used to optimize two previously constructed recombinant Salmonella enterica serovar Typhimurium vaccines expressing S. Choleraesuis O-polysaccharides (OPS). The novel vaccine strains SLT31 (Δasd ΔrmlB-rfbP ΔPcrp::T araC PBAD) and SLT33 (Δasd ΔrfbP ΔpagL::T araC PBADrfbP ΔPcrp::T araC PBAD) were constructed by replacement of the native crp promoter with the arabinose-dependent araC PBAD promoter. As controls, two vaccine strains with direct crp mutations were also constructed, namely, SLT30 (Δasd ΔrmlB-rfbP Δcrp) and SLT32 (Δasd ΔrfbP ΔpagL::T araC PBADrfbP Δcrp). Then, the ability to deliver the heterologous S. Choleraesuis OPS on the Asd+ plasmid pCZ1 to the mouse immune system was evaluated in the strains with or without regulated delayed attenuation. The SLT30 (pCZ1) and SLT31 (pCZ1) strains expressed only the heterologous OPS, while the SLT32 (pCZ1) and SLT33 (pCZ1) strains co-expressed the homologous and heterologous OPS. The strain SLT31 (pCZ1) or SLT33 (pCZ1), which exhibited regulated delayed attenuation, colonized mouse tissues significantly better and stimulated stronger antibody responses against S. Choleraesuis LPS post immunization than the SLT30 (pCZ1) or SLT32 (pCZ1) strain. Immunization with SLT31 (pCZ1) or SLT33 (pCZ1) resulted in a significant reduction in bacterial loads in mouse tissues and a greater degree of protection against a lethal S. Choleraesuis dose compared with the effects observed after SLT30 (pCZ1) or SLT32 (pCZ1) immunization (100% vs. 80% or 70% vs. 50%, respectively). In addition, all four vaccines conferred complete protection against S. Typhimurium challenge. Overall, our study demonstrates that regulated delayed attenuation via an araC PBAD-regulated crp gene can enhance the cross-protection by Salmonella-vectored vaccines expressing heterologous OPS, and strain SLT31 (pCZ1) is a good candidate vaccine for preventing both S. Typhimurium and S. Choleraesuis infections.

Salmonella infections remain a big problem worldwide, causing enteric fever by Salmonella Typhi (or Paratyphi) or self-limiting gastroenteritis by non-typhoidal Salmonella (NTS) in healthy individuals. NTS may become invasive and cause septicemia in elderly or immuno-compromised individuals, leading to high mortality and morbidity. No vaccines are currently available for preventing NTS infection in human. As these invasive NTS are restricted to several O-antigen serogroups including B1, D1, C1, and C2, O-antigen polysaccharide is believed to be a good target for vaccine development. In this study, a strategy of O-serotype conversion was investigated to develop live attenuated S. Typhimurium vaccines against the major serovars of NTS infections. The immunodominant O4 serotype of S. Typhimurium was converted into O9, O7, and O8 serotypes through unmarked chromosomal deletion-insertion mutations. O-serotype conversion was confirmed by LPS silver staining and western blotting. All O-serotype conversion mutations were successfully introduced into the live attenuated S. Typhimurium vaccine S738 (Δcrp Δcya) to evaluate their immunogenicity in mice model. The vaccine candidates induced high amounts of heterologous O-polysaccharide-specific functional IgG responses. Vaccinated mice survived a challenge of 100 times the 50% lethality dose (LD50) of wild-type S. Typhimurium. Protective efficacy against heterologous virulent Salmonella challenges was highly O-serotype related. Furthermore, broad-spectrum protection against S. Typhimurium, S. Enteritidis, and S. Choleraesuis was observed by co-vaccination of O9 and O7 O-serotype-converted vaccine candidates. This study highlights the strategy of expressing heterologous O-polysaccharides via genetic engineering in developing live attenuated S. Typhimurium vaccines against NTS infections.