Identification and characterisation of Salmonella enterica serovar Typhimurium factors playing a role in the colonisation of the porcine gut

Salmonella is an important food borne pathogen. Over 100,000 cases of human Salmonella infection are reported in the European Union each year, resulting in an economic burden estimated to be around 3 billion Euros per year (EFSA, 2012). In a European Food Safety Authority (EFSA) survey between 2006 and 2007 S. Typhimurium was the most common serovar of Salmonella isolated from pig carcasses (EFSA, 2008a). Pigs can be asymptomatic carriers of S. Typhimurium (Berends et al., 1996) and contaminated pork contributes significantly to the number of human infections. It has been estimated that the porcine Salmonella reservoir contributes between 10-20% of human salmonellosis cases per year (VLA, 2010). In addition to improvements in biosecurity and husbandry practices, immune-prophylaxis is an important method to reduce the prevalence of food borne pathogens such as Salmonella in reservoir species. An understanding of the molecular basis of bacterial colonisation and persistence in the reservoir host is crucial to rational vaccine design and targeting relevant species. S. Typhimurium expresses multiple surface factors involved in adherence and colonisation of gut epithelium in several host species. The aim of this project was to identify factors involved in S. Typhimurium colonisation of the porcine gut. The work presented here specifically focuses on the role of flagella in the colonisation of porcine gut epithelium. Flagella are motility organelles possessed by many bacterial species. Flagella can also function as surface adhesins, shown in Escherichia coli O157:H7 (Mahajan et al., 2009), and Pseudomonas (De Bentzmann et al., 1996, Lillehoj et al., 2002). Flagellin is the major flagellar filament structural protein approximately 50kDa in size. Salmonella enterica has the ability to switch between two alternate, antigenic forms of its flagellin filament protein, expressing either FliC or FljB (Macnab, 1996). The biological relevance of these two types of flagella filament protein is still not understood. It has been postulated that the presence of a second phase type of flagella may offer an advantage to the bacteria by avoiding recognition by the immune system. However, studies have shown that both FliC and FljB flagella activate Toll-like receptor-5 (TLR-5) mediated by nuclear factor (NF)-κB signalling (Simon and Samuel, 2007b). One specific objective of this research was to compare the role of flagellar phase types in S. Typhimurium adherence and colonisation of porcine gut. To this end a porcine colonic primary epithelial cell culture and ex vivo tissue explants were developed as in vitro infection models. Primary colonic cell cultures were phenotypically characterised using specific markers for epithelial and M cells. In addition to primary epithelial cell culture, porcine intestinal epithelial cell line, IPEC-J2, was also used for specific flagellar interaction studies. The role of flagella in interaction of S. Typhimurium to porcine intestinal epithelium was tested using S. Typhimurium strain SL1344 and flagella mutant derivative strains. Flagella mutant strains exhibited reduced binding to porcine intestinal epithelial cells. Purified flagella proteins were also shown to bind porcine intestinal epithelial cells. Moreover, flagella specific anti-sera suppressed S. Typhimurium adherence to both porcine intestinal epithelial cells as well as porcine colonic explants. The immuno-protective role of flagella as a potential S. Typhimurium vaccine candidate was tested during vaccine efficacy studies in pigs. Parenteral immunisation of pigs with purified FliC and FljB flagella proteins induced production of both IgG and IgA antibodies. The vaccination of pigs with Salmonella flagella provided some protection against challenge as fewer ileum tissue samples from the pigs in the vaccinated group tested positive for Salmonella. The intestinal contents from the vaccinated pigs tested for Salmonella post mortem appeared to also have lower levels of Salmonella compared to un-vaccinated controls, though these were not significantly different between groups. This project has identified flagella as one potential subunit of a multivalent subunit vaccine to help control salmonellosis in the porcine reservoir