6-hydroxydopamine-mediated release of norepinephrine increases faecal excretion of Salmonella enterica serovar Typhimurium in pigs

Abstract

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. In pigs, transport and social stress are associated with reactivation and spread of Salmonella Typhimurium infection. The stress-related catecholamine norepinephrine (NE) has been reported to activate growth and virulence factor expression in Salmonella; however the extent to which NE contributes to stress-associated salmonellosis is unclear. We studied the impact of releasing NE from endogenous stores during Salmonella Typhimurium infection of pigs by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals. Treatment of pigs with 6-OHDA 7 or 16 days post-oral inoculation with Salmonella Typhimurium produced elevated plasma NE levels and transiently, but significantly, increased faecal excretion of the challenge strain. Oral administration of NE to Salmonella Typhimurium-infected pigs also transiently and significantly increased shedding; however pre-culture of the bacteria with NE did not alter the outcome of infection. Salmonella has been proposed to sense and respond to NE via a homologue of the adrenergic sensor kinase QseC. A DeltaqseC mutant of Salmonella Typhimurium was consistently excreted in lower numbers than the parent strain post-oral inoculation of pigs, though not significantly so. 6-OHDA treatment of pigs infected with the DeltaqseC mutant also increased faecal excretion of the mutant strain, albeit to a lesser extent than observed upon 6-OHDA treatment of pigs infected with the parent strain. Our data support the notion that stress-related catecholamines modulate the interaction of enteric bacterial pathogens with their hosts.

Figure 1.

Intravenous administration of 6-OHDA to pigs increases plasma NE levels and faecal excretion of Salmonella Typhimurium. Plasma NE (ng/mL) was quantified by ELISA immediately before and 1 h after treatment with a total of 40 mg/kg 6-OHDA or diluent 7 days post-inoculation with ST4/74 nal^R (panel A) or 16 days post-inoculation (panel C). The impact of treatment on the course of faecal excretion is shown for pigs treated at 7 and 16 days post-inoculation in panels B and D, respectively. Values shown are the least square means (LSM) ± standard error (SE) of the LSM. p values ≤ 0.05 are marked with an asterisk. † denotes significant differences in the 6-OHDA group relative to pre-treatment samples.

Figure 2.

Oral administration of NE, but not pre-culture with NE, alters the course of Salmonella Typhimurium infection in pigs. Panel A shows the impact of oral administration of c. 200 mg/kg NE or diluent to groups of 2 pigs 17 days post-inoculation with ST4/74 nal^R. Values are the fold change in mean log₁₀ CFU Salmonella/g relative to samples collected immediately prior to treatment. Panel B shows the magnitude of faecal excretion of Salmonella Typhimurium after oral dosing of groups of 3 pigs with c. 1 × 10⁸ CFU ST4/74 nal^R amplified in LB medium for 2 h in the presence of 5 mM NE or diluent. Values shown are the least square means (LSM) ± standard error (SE) of the LSM. p values ≤ 0.05 are marked with an asterisk.

Figure 3.

The putative adrenergic sensor kinase QseC does not significantly influence intestinal colonisation of pigs. 6-week-old pigs were inoculated with 1.50 × 10⁹ CFU ST4/74 nal^R (n = 5) or 1.53 × 10⁹ CFU ST4/74 nal^R ΔqseC (n = 6) and the course of faecal excretion of the bacteria followed daily for 16 days. Values shown are the least square means (LSM) ± standard error (SE) of the LSM. † denotes p values ≤ 0.1.

Figure 4.

QseC influences, but is not essential for, increased faecal excretion of Salmonella Typhimurium in response to 6-OHDA treatment. Plasma NE (ng/mL) was quantified by ELISA immediately before and 1 h after treatment with 40 mg/kg 6-OHDA or diluent 16 days post-inoculation with CFU ST4/74 nal^R ΔqseC (panel A). The impact of 6-OHDA treatment on the course of faecal excretion of ST4/74 nal^R ΔqseC is shown in panel B. Values shown are the least square means (LSM) ± standard error (SE) of the LSM. p values ≤ 0.05 are marked with an asterisk.