Salmonella pathogenicity island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in calves

Abstract

We have used signature-tagged mutagenesis to identify mutants of the host-specific Salmonella enterica serotype Dublin which were avirulent in calves and/or BALB/c mice. A mutant with a transposon insertion in the sseD gene of Salmonella pathogenicity island 2 (SPI-2), which encodes a putative secreted effector protein, was identified. This mutant was recovered from the bovine host but not from the murine host following infection with a pool of serotype Dublin mutants. However, a pure inoculum of the sseD mutant was subsequently shown to be attenuated in calves following infection either by the intravenous route or by the oral route. The sseD mutant was fully invasive for bovine intestinal mucosa but was subsequently unable to proliferate to the same numbers as the parental strain in vivo. Both the sseD mutant and a second SPI-2 mutant, with a transposon insertion in the ssaT gene, induced significantly weaker secretory and inflammatory responses in bovine ligated ileal loops than did the parental strain. These results demonstrate that SPI-2 is required by serotype Dublin for the induction of both systemic and enteric salmonellosis in calves.

FIG. 1

Recovery of bacteria from the spleens (▪) and livers (□) of BALB/c mice 4 days after intravenous infection with SD3246 Nal^r or the SPI-2 (sseD and ssaT) mutant strains. Each bar represents the mean recovery of bacteria from three mice and is presented with the standard error of the mean.

FIG. 2

Intravenous infection of calves with SD3246 Nal^r (four calves) or the sseD mutant strain (three calves). (a) Mean rectal temperature responses of calves following infection with either the parental strain (●) or the sseD mutant strain (○) for 7 days. Each datum point is presented with the standard error of the mean. (b) Recovery of bacteria from systemic sites and intestinal sites of calves 7 days after infection with the parental (▪) or the sseD mutant strain (□). Each bar represents the mean recovery of bacteria and is presented with the standard error of the mean. Samples which were positive only after enrichment culture are indicated with circles.

FIG. 3

Bovine secretory (a) and inflammatory (b) responses in ileal loops 12 h after infection with SD3246 Nal^r or the SPI-1 (sipD) and SPI-2 (sseD and ssaT) mutant strains. Negative control loops (-ve) were injected with LB medium. The secretory response is expressed as the volume of fluid within a loop divided by the length of the loop. The PMN influx ratio is expressed as the PMN influx within a test loop divided by the PMN influx in the control loops. Each bar is derived from the mean secretory or inflammatory response from three loops and is presented with the standard error of the mean. The SPI-1 mutant strain was tested in two calves, and all other strains were tested in three calves. The results from one representative calf are presented.

FIG. 4

Oral infection of calves with SD3246 Nal^r (two calves) or the sseD mutant strain (two calves). (a) Mean rectal temperature responses, mean bacterial recovery per gram of feces, and daily scour scores of calves following infection with either the parental strain (● and ▪) or the sseD mutant strain (○ and □) for 6 days. Each bar or datum point is presented with the standard error of the mean. Each scour score represents the daily sum of scores from two animals. Points where data are taken from one animal only are indicated with triangles. (b) Recovery of bacteria from systemic sites and intestinal sites of calves 5 or 6 days after infection with the parental strain (▪) or the sseD mutant strain (□). Each bar represents the mean recovery of bacteria and is presented with the standard error of the mean. Samples that were positive only after enrichment culture are indicated with circles.

FIG. 5

Recovery of gentamicin-protected bacteria from Int407 epithelial cells over 24 h following infection with SD3246 Nal^r (▪), the sipD mutant (□), the sseD mutant (●), or the ssaT mutant (▵). Each datum point represents the mean recovery of bacteria from three wells and is presented with the standard error of the mean.

FIG. 6

Recovery of gentamicin-protected bacteria from Int407 epithelial cells 12 h after infection with SD3246 Nal^r or the SPI-2 (sseD and ssaT) mutant strains. Each bar represents the mean recovery of bacteria from six wells and is presented on a linear scale with the standard error of the mean.

FIG. 7

Recovery of gentamicin-protected bacteria from bovine ileal loops 2 h (a) and 8 h (b) after infection with SD3246 Nal^r or the SPI-1 (sipD and sipB) and SPI-2 (sseD and ssaT) mutant strains. Each bar is derived from the mean secretory or inflammatory response from six loops (two calves) with the exception of the sipD bar, which is derived from the mean of three loops (one calf). Each bar is presented with the standard error of the mean.