Conflicting results have been published concerning the effect of different stressors on the shedding of Salmonella Typhimurium in pigs [33–38]. However, our findings elucidate that a natural stress stimulus like feed withdrawal causes recrudescence of a Salmonella Typhimurium infection in carrier pigs. Feed withdrawal before transport to the slaughterhouse is a common practice to reduce the risk of carcass and environmental contamination because a decrease of the gastrointestinal tract weight results in a lower risk of lacerations during evisceration . However, we showed that feed withdrawal practices could result in an increased risk of contamination. Martín-Peláez et al. hypothesized that the increased faecal excretion of Salmonella Typhimurium after feed withdrawal could be the result of a lower short-chain fatty acids concentration, an increased pH or an increased number of lactic acid bacteria such as lactobacilli .
Until now, the mechanism of stress related recrudescence of Salmonella is not well understood and the investigation of this phenomenon is hindered by the lack of appropriate animal models [40, 41]. The higher Salmonella Typhimurium numbers in pigs subjected to feed withdrawal stress, suggest that this model is a valuable tool for the study of stress related Salmonella recrudescence. We hypothesized that cortisol plays a role in the stress related recrudescence of Salmonella Typhimurium by pigs. During a stress reaction, the sympathetic nervous system and hypothalamic-pituitary-adrenal axis become activated, resulting in the release of catecholamines and glucocorticoids, respectively . These stress hormones can affect the host immune response, but the pathogenesis of an infection can also be altered by direct effects of these stress mediators on the bacteria .
We showed that social stress and starvation result in elevated serum cortisol levels. Starvation can result in hypoglycaemia, which causes an increased secretion of cortisol to stimulate the gluconeogenesis . Müller et al. showed that a starvation period up to 5 days in miniature pigs, results in a slight, but insignificant elevation of plasma cortisol levels . Therefore, the elevated serum cortisol levels, seen in the carrier pigs that were subjected to feed withdrawal is probably the result of a combination between the feed withdrawal itself and the stress that is involved.
We revealed that a short-term treatment of carrier pigs with a high dose of dexamethasone results in the recrudescence of Salmonella Typhimurium. This confirms that the release of corticosteroids in the bloodstream itself could alter the outcome of a Salmonella Typhimurium infection in pigs, resulting in recrudescence of the infection. Smyth et al. showed that long-term treatment of mice with dexamethasone promotes a dose-dependent increase in Salmonella Typhimurium growth within mouse livers and spleens . The increased numbers of bacteria described by Smyth et al. are probably the result of the immunosuppressive activity of glucocorticoids. Pigs are remarkably resistant to immunosuppression of dexamethasone, even at a high dose of 2 mg/kg body weight [27, 45–47]. Therefore, the dexamethasone induced recrudescense of Salmonella Typhimurium in pigs is probably not the direct consequence of the immunosuppressive activity of dexamethasone.
We also demonstrated that this glucocorticoid mediated effect was not the result of a direct effect on the bacterium. Earlier research has shown that norepinephrine in vitro promotes the growth and the motility of Salmonella enterica[48, 49]. However, we provide evidence that cortisol does not cause an increase in growth in LB and DMEM medium, or any significant changes in the gene expression of Salmonella Typhimurium when grown in a complex medium, at a physiological stress concentration of 1 μM . In contrast to the absence of a direct effect on the bacterium, we showed that cortisol and dexamethasone promote intracellular proliferation of Salmonella Typhimurium in porcine macrophages, in a dose-dependent manner at concentrations (0.1 to 100 μM) that do not exert a notable effect on cell viability. Nevertheless, this increased survival was not observed 3D4/31 and IPEC-J2 cells. Although Salmonella is an extensively studied bacterium, still many questions remain about the intracellular environment of Salmonella within different host cells. After invasion, Salmonella resides within Salmonella containing vacuoles (SCV) which serves a unique intracellular compartment where it resides and eventually replicates. Maturation of the SCV has been studied in different cell types and these studies indicate that the SCV biogenesis may not be generalized . Possibly, cortisol affects the SCV biogenesis in primary macrophages and not in other cell types, which results in an increased survival of the bacterium in these primary macrophages.
Although we showed that catecholamines did neither affect the intracellular proliferation nor the invasion of Salmonella Typhimurium in primary macrophages and IPEC-J2 cells, catecholamines have been shown to promote the growth and motility of Salmonella[48, 49, 51]. Concentrations of the catecholamines were not determined in the in vivo trial since they have a half-life of approximately 3 min and because their serum levels change in matter of seconds [52, 53]. However, it is commonly known that a stress reaction also results in the release of catecholamines. Recently, Pullinger et al. demonstrated that the release of norepinephrine in pigs by administration of 6-hydroxydopamine, enhances the faecal extretion of Salmonella Typhimurium . Therefore, it is possible that catecholamines and glucocorticoids act in a synergistic way to cause a sudden increase of Salmonella Typhimurium shedding in stressed animals. Since stress is very common in food producing animals and since these stress hormones and derivatives are frequently used in human and animal medicine, their effects need further examination [55, 56]. The elucidation of the mechanisms through which stress and its hormones alter the susceptibility to an infection could help to improve the prevention and treatment of Salmonella Typhimurium infections in pigs, and as a consequence help to reduce the number of cases of human salmonellosis.
In conclusion, we showed that the glucocorticoid cortisol is involved in a stress induced recrudescence of Salmonella Typhimurium in carrier pigs. In addition to this, we pointed out that cortisol promotes the intracellular proliferation of Salmonella Typhimurium in porcine macrophages which is caused by an indirect effect through the cell.