Virulence potential of five major pathogenicity islands (SPI-1 to SPI-5) of Salmonella enterica serovar Enteritidis for chickens

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the <it>Salmonella </it>genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of <it>Salmonella </it>to infect chickens. In this study, we therefore constructed <it>Salmonella </it>Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host.</p> <p>Results</p> <p>The mutant lacking all 5 major SPIs was still capable of colonising the caecum while colonisation of the liver and spleen was dependent on the presence of both SPI-1 and SPI-2. In contrast, the absence of SPI-3, SPI-4 or SPI-5 individually did not influence virulence of <it>S</it>. Enteritidis for chickens, but collectively they contributed to the colonisation of the spleen. Proinflammatory signalling and heterophil infiltration was dependent on intact SPI-1 only and not on other SPIs.</p> <p>Conclusions</p> <p>SPI-1 and SPI-2 are the two most important pathogenicity islands of <it>Salmonella </it>Enteritidis required for the colonisation of systemic sites in chickens.</p

aro Mutations in Salmonella enterica Cause Defects in Cell Wall and Outer Membrane Integrity▿

In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response

Contact with adult hen affects development of caecal microbiota in newly hatched chicks.

Chickens in commercial production are hatched in a clean hatchery environment in the absence of any contact with adult hens. However, Gallus gallus evolved to be hatched in a nest in contact with an adult hen which may act as a donor of gut microbiota. In this study, we therefore addressed the issue of microbiota development in newly hatched chickens with or without contact with an adult hen. We found that a mere 24-hour-long contact between a hen and newly hatched chickens was long enough for transfer of hen gut microbiota to chickens. Hens were efficient donors of Bacteroidetes and Actinobacteria. However, except for genus Faecalibacterium and bacterial species belonging to class Negativicutes, hens did not act as an important source of Gram-positive Firmicutes. Though common to the chicken intestinal tract, Lactobacilli and isolates from families Erysipelotrichaceae, Lachnospiraceae and Ruminococcaceae therefore originated from environmental sources instead of from the hens. These observation may have considerable consequences for the evidence-based design of the new generation of probiotics for poultry

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

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

<i>Salmonella</i> secreted proteins.

<p>Panel A, strains included in the initial part of this study. 1, <i>S</i>. Enteritidis 147. 2, <i>S</i>. Enteritidis 147 <i>rfaL</i> mutant. 3, <i>S</i>. Typhimurium LT2. 4, <i>S</i>. Typhimurium LT2 <i>rfaL</i> mutant. 5, <i>S</i>. Infantis 1516. 6, <i>S</i>. Infantis 1516 <i>rfaL</i> (I) mutant. Lane M, molecular weight standard. This analysis was repeated 3 times for each strain or mutant with similar results in each of the replicates. Panel B, strains included in the second part of this study. 1, <i>S</i>. Enteritidis G1481. 2, <i>S</i>. Typhimurium 2002. 3, <i>S</i>. Typhimurium 2454. 4, <i>S</i>. Infantis 514. 5, <i>S</i>. Infantis 1516 <i>rfaL</i> (II) mutant. 6, <i>S</i>. Infantis 1516 <i>rfaL</i> (III) mutant. Lane M, molecular weight standard. This analysis was repeated 3 times for each strain or mutant with similar results in each of the replicates.</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

Correlation between gene expression and <i>Salmonella</i> counts in the liver (log CFU/g), and correlation between upregulated and downregulated genes.

<p>Each dot represents a single chicken characterized by <i>Salmonella</i> counts in the liver, average expression calculated from expression of all genes which positively respond to <i>Salmonella</i> infection or average expression calculated from expression of all genes which negatively respond to <i>Salmonella</i> infection. A, correlation between average expression of upregulated genes and <i>Salmonella</i> counts in the liver. B, correlation between average expression of downregulated genes and <i>Salmonella</i> counts in the liver. C, correlation between average expression of upregulated and downregulated genes.</p