HtpG contributes to Salmonella Typhimurium intestinal persistence in pigs

Salmonella enterica subspecies enterica serovar Typhimurium ( Salmonella Typhimurium) contamination of pork, is one of the major sources of human salmonellosis. The bacterium is able to persist and hide in asymptomatic carrier animals, generating a reservoir for Salmonella transmission to other animals and humans. Mechanisms involved in Salmonella persistence in pigs remain poorly understood. In the present study, we demonstrate that the Salmonella htpG gene, encoding a homologue of the eukaryotic heat shock protein 90, contributes to Salmonella Typhimurium persistence in intestine-associated tissues of pigs, but not in the tonsils. HtpG does not seem to play an important role during the acute phase of infection. The contribution to persistence was shown to be associated with htpG-dependent Salmonella invasion and survival in porcine enterocytes and macrophages. These results reveal the role of HtpG as a virulence factor contributing to Salmonella persistence in pigs

Application of the DIVA principle to Salmonella Typhimurium vaccines in pigs avoids interference with serosurveillance programmes

Salmonellosis is one of the most important bacterial zoonotic diseases in humans and Salmonella infections are often linked with the consumption of contaminated pork. In order to reduce Salmonella Typhimurium infections in humans, minimization of the Salmonella intake into the food chain is important. Vaccination has been proposed to control Salmonella infections in pigs. However, pigs vaccinated with the current vaccines cannot be discriminated from infected pigs with the lipopolysaccharide (LPS) -based serological tests used in European serosurveillance programmes. We therefore examined which LPS encoding genes of Salmonella Typhimurium can be deleted to allow differentiation of infected and vaccinated pigs, without affecting the vaccine strain’s protective capacity. For this purpose, deletion mutants in Salmonella strain 112910a, used as vaccine strain, were constructed in the LPS encoding genes: ∆rfbA, ∆rfaL, ∆rfaJ, ∆rfaI, ∆rfaG and ∆rfaF. Inoculation of BALB/c mice with the parent strain, ∆rfaL, ∆rfbA or ∆rfaJ strains but not the ∆rfaG, ∆rfaF or ∆rfaI strains protected significantly against subsequent infection with the virulent Salmonella Typhimurium strain NCTC12023. Immunization of piglets with the ∆rfaJ or ∆rfaL mutants resulted in the induction of a serological response lacking detectable antibodies against LPS. This allowed a differentiation between sera from pigs immunized with the ∆rfaJ or ∆rfaL strains and sera from pigs infected with their isogenic wild type strain

An update on pathogenesis and control of Salmonella infections in pigs

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Tissue-specific Salmonella Typhimurium gene expression during persistence in pigs

Salmonellosis caused by Salmonella Typhimurium is one of the most important bacterial zoonotic diseases. The bacterium persists in pigs resulting in asymptomatic 'carrier pigs', generating a major source for Salmonella contamination of pork. Until now, very little is known concerning the mechanisms used by Salmonella Typhimurium during persistence in pigs. Using in vivo expression technology (IVET), a promoter-trap method based on Delta purA attenuation of the parent strain, we identified 37 Salmonella Typhimurium genes that were expressed 3 weeks post oral inoculation in the tonsils, ileum and ileocaecal lymph nodes of pigs. Several genes were expressed in all three analyzed organs, while other genes were only expressed in one or two organs. Subsequently, the identified IVET transformants were pooled and reintroduced in pigs to detect tissue-specific gene expression patterns. We found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes during Salmonella peristence in pigs. Furthermore, we compared the persistence ability of substitution mutants for the IVET-identified genes sifB and STM4067 to that of the wild type in a mixed infection model. The Delta STM4067::kanR was significantly attenuated in the ileum contents, caecum and caecum contents and faeces of pigs 3 weeks post inoculation, while deletion of the SPI-2 effector gene sifB did not affect Salmonella Typhimurium persistence. Although our list of identified genes is not exhaustive, we found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes of pigs and we identified STM4067 as a factor involved in Salmonella persistence in pigs. To our knowledge, our study is the first to identify Salmonella Typhimurium genes expressed during persistence in pigs

Salmonella Typhimurium resides largely as an extracellular pathogen in porcine tonsils, independently of biofilm-associated genes csgA, csgD and adrA

In European countries, Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) is the serovar most frequently isolated from slaughter pigs1. Porcine carcass contamination with Salmonella Typhimurium can largely be attributed to persistently infected pigs. Even though tonsils are a predilection site for Salmonella persistence in pigs, virulence mechanisms necessary for cell invasion and intracellular survival do not contribute to tonsillar colonization2, suggesting that Salmonella Typhimurium resides mainly extracellularly in porcine tonsils. Biofilm formation is a mechanism used by several bacteria to survive in an extracellular context or in hostile environments3. The role of biofilm formation in Salmonella Typhimurium persistence in pigs is still unknown. It was the aim of the present study to determine whether Salmonella Typhimurium persists intracellularly or extracellularly in tonsils of pigs. Additionally, the role of biofilm formation in persistence of Salmonella Typhimurium in porcine tonsils was determined

Salmonella Typhimurium interferes with the humoral immune response in pigs

Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from slaughter pigs in Europe. Circumvention of the host’s immune system by Salmonella might contribute to persistent infection of pigs. We found that Salmonella Typhimurium strain 112910a, which is able to persist in pigs, was capable of downregulating the expression of major histocompatibility class II (MHC II) molecules on porcine alveolar macrophages (PAM) in a Salmonella pathogenicity island 2 (SPI-2) dependent way and that MHC II downregulation was Salmonella strain dependent. The MHC II downregulation capacity was abolished when bacteria were opsonized with Salmonella-specific antibodies. Furthermore, intracellular proliferation of Salmonella Typhimurium opsonized with Salmonella positive pig serum was significantly impaired compared to that of the bacteria opsonized with negative pig serum. In a subsequent in vivo experiment, Salmonella Typhimurium strain MB2216 that did not induce MHC II downregulation in vitro, was shed less and persisted less but induced earlier seroconversion in pigs than strain 112910a. From the in vitro data, it is proposed that Salmonella Typhimurium downregulates the humoral immune response to promote intracellular survival inside porcine macrophages, contributing to long-term Salmonella persistence in pigs. The fact that the less persistent strain MB2216 induced earlier seroconversion than strain 112910a is of major interest for Salmonella-monitoring programs primarily based on serology, since this indicates that more persisting strains are more likely to escape serological detection

HtpG and STM4067 contribute to long-term Salmonella Typhimurium persistence in pigs

Persistent Salmonella Typhimurium infections in pigs often result in asymptomatic carrier pigs and are a major concern for food safety and human health. Tonsils and lymph nodes play a key role in the persistence of Salmonella Typhimurium in pigs, but very little is known about the underlying mechanisms. After bacterial invasion in pigs, the porcine immune system will respond to clear the Salmonella infection and bacterial survival strategies for (long-)term persistence will become important. For the identification of Salmonella Typhimurium genes specifically induced in tonsils and lymph nodes at 3 weeks post inoculation, a genome-wide screening method was performed using in vivo expression technology (IVET). Using IVET we were able to identify different Salmonella Typhimurium genes that play a role in long-term persistence in pigs. Furthermore, we were able to show a role for Salmonella Typhimurium genes htpG (encoding a heat-shock protein) and STM4067 (encoding a protein with an unknown function) in long-term persistence in the porcine intestines and lymph nodes, although their exact role remains to be clarified

Application of the DIVA principle to Salmonella Typhimurium vaccines in pigs avoids interference with serosurveillance programmes

Salmonellosis is one of the most important bacterial zoonotic diseases in humans and Salmonella infections are often linked with the consumption of contaminated pork. In order to reduce Salmonella Typhimurium infections in humans, minimization of the Salmonella intake into the food chain is important

How and why Salmonella Typhimurium circumvents seroconversion in pigs

Salmonellosis is one of the most important bacterial zoonotic diseases and represents a considerable burden to humans in both developing and developed countries1. The consumption of contaminated pork is a major source of Salmonella Typhimurium infections in humans. Porcine carcass contamination is correlated with long-term Salmonella persistence in pigs, resulting in so called asymptomatic ‘carrier pigs’. Since interference of several pathogens with the host’s immune system has already been shown to be of major importance for successful long-term infection2, Salmonella Typhimurium persistence in pigs could be the result of the bacterium being able to circumvent the host’s cellular and/or humoral immune responses. It was the aim of the present study to examine if, how and why Salmonella Typhimurium escapes the porcine humoral immune system