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

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