The strains were propagated in LB broth or LB agar at 37°C. Table 3 List of strains used in this study. strain strain ID SPI present SPI absent reference S. Enteritidis 147 Nal wild EPZ015666 price type 7F4 1, 2, 3, 4, 5 none [28] S. Enteritidis 147 Nal ΔSPI1 4A10 2,3,4,5 1 [30] S. Enteritidis 147 Nal ΔSPI2 5D10 1,3,4,5 2 [30] S. Enteritidis 147

Nal ΔSPI3 6A9 1,2,4,5 3 [30] S. Enteritidis 147 Nal ΔSPI4 4B10 1,2,3,5 4 [30] S. Enteritidis 147 Nal ΔSPI5 4J1 1,2,3,4 5 [30] S. Enteritidis 147 Nal ΔSPI1-5 5E9 none 1,2,3,4,5 [30] S. Enteritidis 147 Nal SPI1o 5G10 1 2,3,4,5 [30] S. Enteritidis 147 Nal SPI2o 5H9 2 1,3,4,5 [30] S. Enteritidis 147 Nal SPI3o 5J10 3 1,2,4,5 [30] S. Enteritidis 147 Nal SPI4o 5D9 4 1,2,3,5 [30] S. Enteritidis 147 Nal SPI5o 5H10 5 1,2,3,4 [30] S. Enteritidis 147 Nal Δlon 16H2 1, 2, 3, 4, 5 none [33] S. Enteritidis 147 Nal ΔrfaL 14E5 1, 2, 3, 4, 5 none [33] Experimental infection of mice In all the experiments, six-week-old Balb/C mice were orally infected with 104 CFU (equivalent to 100 × LD50 of the wild type strain) of the wild type strain or each of the mutants in a SBI-0206965 supplier volume of 0.1 ml using a gastric gavage without any neutralisation of gastric acid prior the

infection. In the first animal infection, 12 groups of 10 mice each were infected with all the SPI mutants and wild type S. Enteritidis. A negative control group consisted of 3 uninfected animals. On day 5 post-infection, 3 mice from each group including Ferroptosis inhibitor all non-infected control mice were sacrificed and used for the determination of bacterial counts in liver, spleen and caecum, two-color flow cytometry of splenic lymphocytes, histology in liver and caecum, and lymphocyte proliferation assay. The remaining 7 mice were left for monitoring of feacal shedding and mortalities until day 21 post infection when the experiment was terminated. Faecal shedding was monitored on a daily basis by transferring the mice into a clean plastic box and collecting pooled fresh droppings 30 minutes later. Bacterial counts in liver, spleen, caecal content and faecal droppings

were determined using a standard plating method described previously [31]. For the purposes of statistical analysis, a viable count of log10 < 2.5 (limit for direct plate detection) obtained Rucaparib from a sample positive only after enrichment was rated as log10 = 1.0 whereas samples negative for S. Enteritidis after enrichment were rated as log10 = 0. During the post mortem analysis, liver and caecal samples were also taken for histological examinations. The samples were fixed in 10% neutral buffered formalin for 24 h, embedded in paraffin wax, sectioned at 5 μm, and stained with haematoxylin-eosin. In the second animal infection, 3 mice per group, including 3 non-infected mice, were infected with the wild-type S. Enteritidis, or with ΔSPI2, lon or rfaL mutants. In this experiment, four-colour flow cytometry detecting CD3, CD19, CD14 and CD16 in splenic lymphocytes was performed.