The ability of Salmonella typhimurium to invade the intestinal mucosal cells is an important step in pathogenesis. This invasion process requires genes encoded on the Salmonella pathogenicity island 1 (SPI1). Two transcriptional activators, HilA and InvF, encoded in SPII regulate the expression of invasion genes in response to environmental stimuli such as osmolarity, oxygen tension, and pH. During its pathogenic life cycle, Salmonella typhimurium is also exposed to short-chain fatty acids (SCFA), especially acetate, propionate, and butyrate, in the intestinal lumen, as well as the SCFA used as food preservatives. The effects of SCFA on the expression of hilA and invF-lacZY transcriptional fusions were examined to determine the potential role of SCFA in the pathogenesis of Salmonella typhimurium. Growth rates were reduced by increasing SCFA concentrations at pH 6 but not at pH 7. At pH 7, hilA and invF expression was induced by acetate but not by propionate or butyrate, while at pH 6, all SCFA induced hilA and invF expression at 1 h. In general, hilA and invF expression levels when compared to respective control responses were higher at 1 h than at 4 and 8 h in the presence of most SCFA concentrations at pH 6. However, expression levels at 4 and 8 h were either similar or higher than the 1-h responses for the hilA-lacZY fusion strain in the presence of acetate while exposure to 20 mM propionate yielded similar levels of expression at 1, 4, and 8 h. The pH-dependent manner of induction suggests that entry of SCFA into the cell was necessary for induction. We speculate that SCFA may serve as an environmental signal that triggers the expression of invasion genes in the gastrointestinal tract.

Leghorn hens over 50 weeks of age were assigned to two treatment groups designated as either unmolted controls or molted. A forced molt was induced by a 9-day feed withdrawal, and each hen was challenged orally with 105 Salmonella enteritidisorganisms on day 4 of feed withdrawal. On days 4 and 9 of molt, the numbers of lactobacilli and the concentrations of lactate, acetate, propionate, and butyrate, and total volatile fatty acids in the crops decreased while crop pH increased significantly (P < 0.05) in the molted hens compared to the controls. S. enteritidis crop and cecal colonization, in addition to spleen and liver invasion, increased significantly (P < 0.05) in the molted hens compared to the controls. The invasive phenotype of Salmonella spp. is complex and requires several virulence genes which are regulated by the transcriptional activator HilA. Samples of the crop contents from the molted and unmolted birds were pooled separately, centrifuged, and filter sterilized. The sterile crop contents were then used to measure the expression of hilA. By using a lacZYtranscriptional fusion to the hilA gene in S. enteritidis, we found that hilA expression was 1.6- to 2.1-fold higher in the crop contents from molted birds than in those from control birds in vitro. The results of the study suggest that the changes in the microenvironment of the crop caused by feed deprivation are important regulators of S. enteritidis survival and influence the susceptibility of molted hens to S. enteritidis infections. Furthermore, our in vitro results on the expression of hilA suggest that the change in crop environment during feed withdrawal has the potential to significantly affect virulence by increasing the expression of genes necessary for intestinal invasion.

Limiting Salmonella Enteritidis from table eggs can involve intervention approaches at several levels of the production cycle, beginning at the hatchery and ending at the processing or table egg production facilities. Likewise, interventions that limit Salmonella Enteritidis dissemination can be implemented at various stages during the life cycle of infection of Salmonella in the laying hen. However, achieving complete elimination of Salmonella infestation in egg products has remained elusive. There is a multitude of reasons for this, including adaptability of the organism, virulence properties, and persistence. Likewise, environmental factors in the layer house such as transmission routes, reservoirs, and feed sources can influence the exposure of susceptible laying hens to Salmonella Enteritidis. Consequently, successful applications of control measures depend not only on the timing of when they are applied but also on effective surveillance to detect frequency and level of infection of Salmonella. Several studies demonstrated that molt induction by feed withdrawal altered the immune system and the gastrointestinal tract of hens, making them susceptible to Salmonella Enteritidis colonization of the gastrointestinal tract. To alleviate this, the development of alternative methods to induce a molt became necessary. The use of several fiber-containing diets was shown to effectively induce a molt with alfalfa-based diets being the most extensively studied. Further reduction of Salmonella Enteritidis levels in eggs will probably require application of multiple interventions at several steps during egg production and processing as well as a better understanding of the mechanisms used by Salmonella Enteritidis to persist in laying flocks.

Pathogens express virulence genes in response to the combination of environmental conditions present in the host environment. The crop is the first gastrointestinal environment encountered in birds. However, feed withdrawal alters the crop environment resulting in an increased pH, and decreased concentrations of lactate, glucose and amino acids compared with unmoulted birds. Salmonella enteritidis infections increase significantly in hens that have been force moulted by feed withdrawal. The present study examined the effects of pH, carbohydrate sources, amino acids and lactate on expression of Salm. enteritidis virulence by measuring expression of hilA. The hilA gene encodes a transcriptional activator that regulates expression of Salmonella virulence genes in response to environmental stimuli. HilA expression was determined using a poultry isolate of Salm. enteritidis carrying a hilA‐lacZY transcriptional fusion from Salm. typhimurium. The media used were Luria Bertani (LB) broth and LB broth diluted 1 : 5 (DLB). The expression of hilA was 2·9‐fold higher in DLB broth compared with LB broth which suggested that there is a nutritional component to the regulation of hilA. Addition of 0·2% glucose, fructose or mannose to LB and DLB reduced hilA expression 1·5 to twofold. Addition of 0·2% Casaminoacids, arabinose, fucose, or lactose had little effect on hilA expression. Lactate (25 and 50 mmmol l−1) reduced hilA expression at pH 6, 5 and 4, with the lowest expression occurring at pH 4. Based on these results it appears that the composition of the crop lumen could potentially influence Salm. enteritidis virulence expression.

This study demonstrates that the growth of S. typhimurium in Luria Bertani broth supplemented with acetate, propionate, butyrate, or a mixture of the three SCFA, affected cell-association and the ability to invade cultured HEp-2 cells. Cell-association and invasion was determined after growth for 4 h of growth in the presence of the SCFA at pH 6 and 7. The results suggest that the growth rate of the culture may have affected cell-association and invasion since accompanying the significant decrease in growth rate in the presence of SCFA at pH 6 was a decrease in cell-association and invasion. However, the results also suggest that the individual SCFA may play a role in modulating cell-association and the invasion phenotype and the regulation of cell-association and invasion by the SCFA was dependent on the concentration and the pH of the medium. Although the growth rates were similar for S. typhimurium in the SCFA mixture, butyrate (100 mM) and propionate (50 mM) at pH 6, differences in cell-association and invasion were observed among these cultures. Also, at pH 7, differences were observed among the SCFA treatments even though the growth rates were similar.