Stress induced Salmonella Typhimurium re-excretion by pigs is associated with cortisol induced increased intracellular proliferation in porcine macrophages

Infections of pigs with Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) often result in the development of carriers that intermittently excrete Salmonella in very low numbers. During periods of stress, recrudescence of Salmonella may occur. The mechanism of stress related re-excretion of Salmonella by pigs is poorly understood and the aim of the presented study was to determine the role of the stress hormone cortisol on Salmonella re-excretion by pigs. We showed that a 24 hour feed withdrawal increases the Salmonella Typhimurium load in pigs, which is correlated with increased cortisol blood levels. A second in vivo trial showed that the stress related re-excretion of Salmonella Typhimurium in pigs can be induced by intramuscular injection of dexamethasone. Furthermore we demonstrated that cortisol promotes intracellular proliferation of Salmonella Typhimurium in porcine alveolar macrophages, but not in intestinal epithelial cells, at a concentration (1 μM) that did not exert a notable effect on porcine cell viability and gene expression of Salmonella Typhimurium. This implies that the enhanced survival of Salmonella is probably caused by an indirect effect of cortisol on the cell. Introduction Pigs infected with Salmonella Typhimurium can carry this bacterium asymptomatically in their tonsils, gut and gut-associated lymphoid tissue for months resulting in so called Salmonella carriers. During periods of stress recrudescence of Salmonella may occur (Berends et al., 1996). Until now, the mechanism of stress related re-excretion of Salmonella in pigs is not well known. We hypothesized that cortisol plays a role in the stress related recrudescence of Salmonella Typhimurium in pigs. Material and Methods Salmonella strain: Salmonella Typhimurium strain 112910a, isolated from a pig stool sample and characterized previously by Boyen et al. (2008), was used. In vivo trials: In a first in vivo trial, we investigated the effect of different types (feed withdrawal, isolation and overcrowding) of stress on the re-excretion of Salmonella Typhimurium by carrier pigs. In a second in vivo trial, we intramuscularly injected carrier pigs with 2 mg dexamethasone per kg body weight to test our hypothesis that cortisol plays a role in the recrudescence of Salmonella Typhimurium in pigs. Cytotoxicity assays: The cytotoxic effect of cortisol on porcine alveolar macrophages and IPEC-J2 cells was determined using the lactate dehydrogenase cytotoxicity detection kit (Roche Applied Science, Bazel, Switzerland), in accordance to the manufacturer’s instructions. Effect of cortisol on the growth of Salmonella Typhimurium: The effect of cortisol on the growth of Salmonella Typhimurium in LB broth was examined during 24 hours. Effect of cortisol on the gene expression of Salmonella Typhimurium: RNA was isolated from Salmonella Typhimurium using the SV Total RNA purification kit (Promega, Leiden, the Netherlands). Gene expression was measured using a Salmonella microarray constructed at the Institute of Food Research, Norwich, UK. Invasion and intracellular survival assays: The ability of Salmonella Typhimurium to invade and proliferate in PAM and IPEC-J2 cells after exposure to cortisol was performed as described by Boyen et al., 2009. Macrophage chemiluminiscence: The effect of cortisol was examined on the reactive oxygen species production of porcine alveolar macrophages, as described by Boyen et al., 2006.