phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens

Abstract

Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the interactions between chickens and S. enterica is therefore important for vaccine design and subsequent decrease in the incidence of human salmonellosis. In this study we therefore characterized the interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested the response of HD11 chicken macrophage-like cell line to S. Enteritidis infection monitoring the transcription of 36 genes related to immune response. All the mutants and the wild type strain induced inflammatory signaling in the HD11 cell line though the response to SPI1 mutant infection was different from the rest of the mutants. When newly hatched chickens were inoculated, the phoP as well as the SPI1 mutant did not induce an expression of any of the tested genes in the cecum. Despite this, such chickens were protected against challenge with wild-type S. Enteritidis. On the other hand, inoculation of chickens with the aroA or SPI2 mutant induced expression of 27 and 18 genes, respectively, including genes encoding immunoglobulins. Challenge of chickens inoculated with these two mutants resulted in repeated induction of 11 and 13 tested genes, respectively, including the genes encoding immunoglobulins. In conclusion, SPI1 and phoP mutants induced protective immunity without inducing an inflammatory response and antibody production. Inoculation of chickens with the SPI2 and aroA mutants also led to protective immunity but was associated with inflammation and antibody production. The differences in interaction between the mutants and chicken host can be used for a more detailed understanding of the chicken immune system.

Figure 1

Invasion and intracellular survival of the wild-type S. Enteritidis and its mutants in HD11 cells. HD11 cells were inoculated with approx. 10⁵ CFU S. Enteritidis wild-type or the SPI1, SPI2, aroA or phoP mutant (MOI = 1). Four and 24 h post infection, intracellular bacterial counts were determined. Bars represent mean and SD.

Figure 2

Expression of genes in HD11 cells inoculated with wild-type S. Enteritidis and its mutants. The SPI1 mutant stimulated a different response in HD11 cell line than the remaining strains or mutants. Panels in green, genes suppressed by S. Enteritidis infection 24 hours post infection (hpi). Panels in red, genes induced by S. Enteritidis infection. Bars represent expression normalized to the average expression of 3 house-keeping genes. * - statistically significant difference from the expression in the non-infected cells at P ≤ 0.05.

Figure 3

Salmonella counts in organs of chickens inoculated with wild-type S. Enteritidis and its mutants. Chickens were inoculated on day of hatch with SPI1, SPI2, phoP and aroA S. Enteritidis mutants and challenged on day 22 of life. The data represent individual values and median CFU/g tissue. Asterisks indicate statistically significant differences from chickens inoculated with wild-type S. Enteritidis at P ≤ 0.05 (*). Non-vaccinated chickens infected on day 22 of life are designated as “nv”. Some values from the chicken cecum are missing due to an overgrowth of plates with non-Salmonella microbiota resistant to nalidixic acid.

Figure 4

Gene expression in chickens inoculated with wild-type S. Enteritidis and its mutants. Chickens were infected on day of hatch with wild-type S. Enteritidis or SPI1, SPI2, phoP and aroA mutants, and euthanized 4 days later. Heat map shows average gene expression in groups of chickens. Asterisks indicate statistically significant difference from the expression in the non-infected chickens at P ≤ 0.05 (*), P ≤ 0.01 (**), or P ≤ 0.001 (***). Maximal fold increase is shown to highlight the differences between the green and red color range for each of the genes. Minimal and maximal expression levels are shown to remind that higher fold inductions are more frequent for genes with a lower basal expression.

Figure 5

Salmonella counts in organs of chickens infected with wild-type S. Enteritidis at different ages. Chickens were infected with wild-type S. Enteritidis on day 1, 22 or 43 of life and euthanized 4 days later. The data represent individual values and median CFU/g tissue. Asterisks indicate statistically significant difference from Salmonella counts in 5-day-old chickens 4 days post infection at P ≤ 0.05 (*).

Figure 6

Gene expression in inoculated chickens 4 days after challenge with wild-type S . Enteritidis. Chickens were inoculated on day of hatch with SPI1, SPI2, phoP and aroA S. Enteritidis mutants, challenged on day 22 of life and euthanized 4 days later. Heat map shows average gene expression in groups of chickens. Group I of genes represents genes belonging mainly to innate immune response, group II genes belonging to Th1 cell mediated immune response and group III is mainly associated with B-lymphocyte development and antibody production. Asterisks indicate statistically significant difference from the non-infected chickens at P ≤ 0.05 (*), P ≤ 0.01 (**) or P ≤ 0.001 (***). Maximal fold increase is shown to highlight the differences between the green and red color range for each of the gene. Minimal and maximal expression levels are shown to remind that higher fold inductions are more frequent for genes with a lower basal expression.