Third, we used click here mutants in which the entire SPI1 and/or the entire structural operon of SPI2 are deleted (Figure 1). This inactivates all the genes involved in both SPI1 and SPI2 T3SS apparatus synthesis and prevents the action of SPI1 regulators on SPI2 gene expression. Using this approach, we compared the colonization of the wild-type to that of each of the mutants. We report here that SPI1 contributes to the colonization of both the cecum and spleen of the chicken.

In contrast, SPI2 contributes to colonization of the spleen but not the cecum and, in the absence of SPI1, inhibits cecal colonization. Additionally, we show that the contribution of SPI1 in the spleen is greater than that of SPI2. IWR-1 cost These results differ from those observed during the infection of mice by Typhimurium, where SPI2 plays a major role during selleck inhibitor systemic colonization. Results To assess the roles of SPI1 and SPI2 in the colonization of the gut and internal organs of the chicken, we used a mixed infection approach [30]. We orally infected one-week old chickens with mixtures of two strains. Each strain carried different antibiotic resistance markers providing a simple means of identification. At days three, seven, and fourteen post-infection, groups of chickens were euthanized. The spleen and a sample

of cecum from each bird were recovered, processed and plated for enumeration of colonies as described in the Methods section. The ratio of the two strains recovered from each organ was determined and compared to the input ratio to determine the competitive index (CI, ratio of the two strains from an

organ divided by the ratio in the suspension used for the infection). In Vitro Testing of SPI1 and SPI2 Mutants All strains containing SPI1 and SPI2 mutations were assayed for in vitro growth and invasion of the chicken macrophage cell line MQ-NSCU [31]. All mutants strains grew at approximately the same rate at the parent strain χ4138 (data not shown). Additionally, all mutants containing the Δspi1 mutation were approximately thirty times less invasive than those with Afatinib solubility dmso an intact SPI1 (data not shown) SPI1 contributes to the colonization of the cecum and of the spleen in chicken In chickens infected with the wild type strain and its isogenic mutant lacking the entire SPI1 (Δspi1), the Δspi1 cells were significantly reduced in the ceca at days three (P < 0.0001) and fourteen (P < 0.0001) post-infection (Figure 2A). At day seven post-infection the difference between the two strains was not significant (Figure 2A). Figure 2 Contribution of SPI1 to the colonization of chicken cecum (A) and spleen (B) by Typhimurium. Competitive indexes are from mixed oral infections in chickens with the wild type and the Δspi1 (deletion of SPI1) strains.