Chicken innate immune response to oral infection with Salmonella enterica serovar Enteritidis

Abstract

The characterization of the immune response of chickens to Salmonella infection is usually limited to the quantification of expression of genes coding for cytokines, chemokines or antimicrobial peptides. However, processes occurring in the cecum of infected chickens are likely to be much more diverse. In this study we have therefore characterized the transcriptome and proteome in the chicken cecum after infection with Salmonella Enteritidis. Using a combination of 454 pyrosequencing, protein mass spectrometry and quantitative real-time PCR, we identified 48 down- and 56 up-regulated chicken genes after Salmonella Enteritidis infection. The most inducible gene was that coding for MMP7, exhibiting a 5952 fold induction 9 days post-infection. An induction of greater than 100 fold was observed for IgG, IRG1, SAA, ExFABP, IL-22, TRAP6, MRP126, IFNγ, iNOS, ES1, IL-1β, LYG2, IFIT5, IL-17, AVD, AH221 and SERPIN B. Since prostaglandin D2 synthase was upregulated and degrading hydroxyprostaglandin dehydrogenase was downregulated after the infection, prostaglandin must accumulate in the cecum of chickens infected with Salmonella Enteritidis. Finally, above mentioned signaling was dependent on the presence of a SPI1-encoded type III secretion system in Salmonella Enteritidis. The inflammation lasted for 2 weeks after which time the expression of the "inflammatory" genes returned back to basal levels and, instead, the expression of IgA and IgG increased. This points to an important role for immunoglobulins in the restoration of homeostasis in the cecum after infection.

Figure 1

Northern blot analysis for 4 selected genes responding to Salmonella Enteritidis infection in the cecum. MMP7 and ExFABP were identified by real-time PCR as upregulated after the infection and AQP8 and ADH1 were identified as downregulated after the infection. GAPDH was used as a loading control. NI1, NI2 and NI3, mRNA purified from the cecum of non-infected chickens. INF1, INF2 and INF3, mRNA purified from the cecum of infected chickens.

Figure 2

Salmonella Enteritidis colonization of the cecum, liver and spleen after oral infection at the day of hatching. The cecum (triangles) was colonized as early as at the first sampling time, 24 h after the infection. Colonization of the liver (circles) and spleen (squares) required an additional 24 h and these organs became highly colonized from 48 h after the infection. The liver and spleen remained colonized at constant counts until day 12 of life and thereafter a gradual decrease in Salmonella Enteritidis counts was observed.

Figure 3

Heat map of gene expression in the chicken cecum, with or without Salmonella Enteritidis infection. The heat map for each gene was calculated based on its minimal and maximal recorded expressions. The color coding (light green, the lowest expression, light red, the highest expression) thus characterizes each gene profile and the same shade of red or green color for two different genes does not necessarily correspond to the same fold increase.

Figure 4

PCA of the chickens clustered according to their gene expression in the cecum. Sixty-eight percent of variability explained by coordinate 1 is likely to be associated with the infection status. Coordinate 2 explaining an additional 8% of variability seems to be chicken age. Green and red color, clustering of the non-infected and infected chickens, respectively. Numbers indicate age of each particular chicken in days.