Induction of IL-6 production A macrophage invasion assay was conducted with J774A.1. After 1 hour and 4 hours of incubation, the last 3 hours with gentamicin present in the medium, supernatants were removed and assayed for the presence of cytokine IL-6 using a commercially available kit (Promokine, mouse
IL-6 ELISA kit). Positive controls consisted of purified IL-6 supplied with the kit, and negative controls consisted of wells not infected with bacteria. Animal challenge experiments Per oral and intraperitoneal virulence were assessed using competitive challenge assays with five C57BL/6 female mice (Taconic Black6 mice) of 6–8 weeks of age per group. The protocol followed the instructions of Jelsbak et al.[48] for intra peritoneal SB431542 cell line challenge, while a challenge dose of 8 × 106 CFU was used for per oral challenges. In all experiments, S. Dublin was given a 10 times reduced dose compared to S. Typhimurium. The ratio between the wild type and the mutant strain in the broth used for challenge as well as the ratio in the spleen 4–5 days post challenge was determined by patching of 100 colonies from the broth and from the spleen of each mice onto LB agar without antibiotic GSK2126458 datasheet and 100 colonies onto LB agar with the relevant antibiotic. For statistical analysis of the difference between input and output ratios, an estimate of the variation on the input ratio was needed. This was obtained
by combining the results from the patching of all input pools into one distribution and using this as an average input ratio. The animal experimentation was conducted with permission from Florfenicol the Animal Experiments Inspectorate (http://www.foedevarestyrelsen.dk/Dyr/Dyrevelfaerd/Dyreforsoegstilsynet/Sider/forside.aspx) in accordance
with Danish law (license number: 2009/561–1675). Statistical analysis Statistical analyses were made using the statistical software package GraphPath Prism 5. Mean CFU of bacterial strains in cell assays and cytotoxicity levels were compared using Bonferroni’s multiple comparison test. Comparison of mean competitive index between wild type and mutant strains and oxidative responses were done using unpaired YM155 mw T-test. P<0.05 was considered significant. Acknowledgments Tony Bønnelycke and Gitte Pedersen are thanked for skillful technical assistance. Kelly T. Hughes, Washington University, Seattle, WA is thanked for providing the plasmid pPR2 with S. Typhimurium fliC. José Breschiani is thanked for help with the electron-microscopy pictures. References 1. Joys TM: The covalent structure of the phase-1 filament protein of Salmonella Typhimurium and its comparison with other flagellins. J Biol Chem 1985, 260:15758–15761.PubMed 2. Popoff MY: LL: Antigenic formulas of the Salmonella serovars. Paris: WHO collaboration Centre for reference and research on Salmonella; 2007. 3. McQuiston JR, Fields PI, Tauxe RV, Logsdon JMJ: Do Salmonella carry spare tyres. Trends Microbiol 2008, 16:142–148.PubMedCrossRef 4.