Gene Expression Profiles of Chicken Embryo Fibroblasts in Response to Salmonella Enteritidis Infection

The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria

Gene expression in the chicken caecum in response to infections with non-typhoid Salmonella

International audienceChickens can be infected with Salmonella enterica at any time during their life. However, infections within the first hours and days of their life are epidemiologically the most important, as newly hatched chickens are highly sensitive to Salmonella infection. Salmonella is initially recognized in the chicken caecum by TLR receptors and this recognition is followed by induction of chemokines, cytokines and many effector genes. This results in infiltration of heterophils, macrophages, B- and T-lymphocytes and changes in total gene expression in the caecal lamina propria. The highest induction in expression is observed for matrix metalloproteinase 7 (MMP7). Expression of this gene is increased in the chicken caecum over 4000 fold during the first 10 days after the infection of newly hatched chickens. Additional highly inducible genes in the caecum following S. Enteritidis infection include immune responsive gene 1 (IRG1), serum amyloid A (SAA), extracellular fatty acid binding protein (ExFABP), serine protease inhibitor (SERPINB10), trappin 6-like (TRAP6), calprotectin (MRP126), mitochondrial ES1 protein homolog (ES1), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), avidin (AVD) and transglutaminase 4 (TGM4). The induction of expression of these proteins exceeds a factor of 50. Similar induction rates are also observed for chemokines and cytokines such as IL1β, IL6, IL8, IL17, IL18, IL22, IFNγ, AH221 or iNOS. Once the infection is under control, which happens approx. 2 weeks after infection, expression of IgY and IgA increases to facilitate Salmonella elimination from the gut lumen. This review outlines the function of individual proteins expressed in chickens after infection with non-typhoid Salmonella serovars

Functional classification of genes induced in CEFs after infection with wild-type <i>S</i>. Enteritidis.

<p>Genes are ranked in descending order of their expression fold change. Functional annotation of genes was performed with the STRING database v9.1. and is represented by gene ontology (GO) terms for biological process (BP).</p

Interaction analysis of genes inducible in CEFs infected with <i>S</i>. Enteritidis.

<p>Figure presents a confidence view of protein interactions in chicken (<i>Gallus gallus</i>) generated by the STRING Database v9.1 for genes significantly upregulated more than threefold in CEFs in response to both <i>Salmonella</i> strains. Lines represent associations based on experimental data, co-expression, databases and/or homology.</p

Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium's Adaptive Mechanisms of Intramacrophage Survival and Replication.

Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria

The Early Innate Response of Chickens to <i>Salmonella enterica</i> Is Dependent on the Presence of O-Antigen but Not on Serovar Classification

<div><p><i>Salmonella</i> vaccines used in poultry in the EU are based on attenuated strains of either <i>Salmonella</i> serovar Enteritidis or Typhimurium which results in a decrease in <i>S</i>. Enteritidis and <i>S</i>. Typhimurium but may allow other <i>Salmonella</i> serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with <i>S</i>. Infantis compared to <i>S</i>. Enteritidis and <i>S</i>. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of <i>Salmonella</i> serovars Enteritidis, Typhimurium and Infantis as well as with their <i>rfaL</i> mutants and characterized the early <i>Salmonella</i>-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the <i>rfaL</i> mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and <i>Salmonella</i> was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.</p></div

Additional file 1: of phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens

List of primers used in real-time PCR. Primers used for the quantification of chicken gene expression by real time PCR are listed in this file

PCA plot of the chickens clustered according to their gene expression in the cecum and heat map correlation coefficients between factor 1 and individual gene expression.

<p>Open black circles, <i>S</i>. Enteritidis 147; Open blue circles, <i>S.</i> Enteritidis G1481; open black squares, <i>S</i>. Typhimurium LT2; open blue squares, <i>S</i>. Typhimurium 2002; open red squares, <i>S</i>. Typhimurium 2454; open black triangles, <i>S</i>. Infantis 1516; open blue triangles, <i>S</i>. Infantis 514; closed black circles, <i>S</i>. Enteritidis 147 <i>rfaL</i> mutant; closed black squares, <i>S</i>. Typhimurium LT2 <i>rfaL</i> mutant; closed black triangles, <i>S</i>. Infantis 1516 <i>rfaL</i> (I) mutant; closed blue triangles, <i>S</i>. Infantis 1516 <i>rfaL</i> (II) mutant; closed red triangles, <i>S</i>. Infantis 1516 <i>rfaL</i> (III) mutant. symbol “plus”, non-infected chickens. PCA also showed that a single factor explained nearly 80% of the variation in the chicken response. This factor was the scope of inflammation itself as high and significant correlations were observed between the expression of individual genes and the positioning of corresponding chickens along X axis. Genes are arranged from the most positively correlated to the most negatively correlated ones.</p

Cytokine gene expression in the cecum of orally infected chickens.

<p>Columns represent geometric means of the relative expressions of respective genes. Vertical bars represent 95% confidence intervals regarding the geometric means. Superscripts above columns denote statistically significant differences among groups (columns sharing the same superscript are not significantly different from each other, columns that have no superscript in common are significantly different from each other). NI, expression in the non-infected chickens. SE, expression in the chickens infected with <i>S</i>. Enteritidis 147. STM, expression in the chickens infected with <i>S</i>. Typhimurium LT2. SI, expression in the chickens infected with <i>S</i>. Infantis 1516. SE <i>rfaL</i>, expression in the chickens infected with <i>S</i>. Enteritidis <i>rfaL</i> mutant. STM <i>rfaL</i>, expression in the chickens infected with <i>S</i>. Typhimurium <i>rfaL</i> mutant. SI <i>rfaL</i>, expression in the chickens infected with <i>S</i>. Infantis <i>rfaL</i> (I) mutant. Mind logarithmic scaling of Y-axis.</p