Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model

Abstract

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.

FIG. 1.

Virulence of different S. enterica serovars in streptomycin pretreated mice. Six (or five) streptomycin-pretreated mice were infected for 3 days with 5 × 10⁷ CFU of the indicated serovar (Typhimurium SL1344, Dublin SARB13, Paratyphi A SARB42, Pullorum X3543, Gallinarum X3796, and Enteritidis 125109). (A to E) Bacterial loads in the cecal content (A), the liver (B), the spleen (C), and the mLN (D). The dotted line indicates the limits of detection, and the horizontal bars indicate the medians. (E) Histopathological analysis. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars); PMN infiltration (black dotted bars); reduction in the number of goblet cells (white dotted bars); and desquamation, erosion, and ulceration of the epithelial layer (white bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. Differences in colonization or the total pathological score (sum of the separate scores) were statistically analyzed by using the exact Mann-Whitney U test (in comparison to SL1344).

FIG. 2.

Cecal inflammation at 3 days p.i. HE-stained cecal tissue sections from six representative animals of the experiment shown in Fig. 1. The cecal tissues were obtained from mice infected with serovar Typhimurium SL1344 (A and D), serovar Dublin SARB13 (B and E), serovar Paratyphi A SARB42 (C and F), serovar Pullorum X3543 (G and J), serovar Gallinarum X3796 (H and K), or serovar Enteritidis 125109 (I and L). Boxes in panels A, B, C, G, H, and I indicate the area shown at the higher magnification. L, intestinal lumen; e, edema; g, goblet cell; sa, submucosa. Magnifications are indicated by the black bars. Scale bars: A, B, C, G, H, and I, 200 μm; D, E, F, J, K, and L, 100 μm.

FIG. 3.

Serovar Dublin colitis in the streptomycin-pretreated mice. Five streptomycin-pretreated mice were infected for 3 days with 5 × 10⁷ CFU of serovar Dublin strains SARB13, SD2229, or SD3246. (A to E) Bacterial loads in the cecal content (A), the liver (B), the spleen (C), and the mLN (D). The dotted line indicates the limit of detection, and the horizontal bars indicate the medians. (E) Histopathological analysis. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars); PMN infiltration (black dotted bars); reduction in the number of goblet cells (white dotted bars); and desquamation, erosion, and ulceration of the epithelial layer (white bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. Differences in colonization or the total pathological score (sum of the separate scores) were statistically analyzed by using the exact Mann-Whitney U test.

FIG. 4.

Virulence of Gallinarum and Pullorum strains in streptomycin-pretreated mice. Five streptomycin pretreated mice were infected for 3 days with 5 × 10⁷ CFU of serovar Gallinarum X3796, Gallinarum 287/91, Pullorum X3543, or Pullorum 449/87. (A to E) Bacterial loads in the cecal content (A), the liver (B), the spleen (C), and the mLN (D). The dotted line indicates the limit of detection, and the horizontal bars indicate the medians. (E) Histopathological analysis. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars), PMN infiltration (black dotted bars), reduction in the number of goblet cells (white dotted bars), and desquamation, erosion, and ulceration of the epithelial layer (white bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. Differences in colonization or the total pathological score (sum of the separate scores) were statistically analyzed by using the exact Mann-Whitney U test.

FIG. 5.

Histopathological changes in mice infected with serovar Gallinarum 287/91 at 3 days p. i. A representative image of the cecal tissue from the experiment shown in Fig. 5 is shown. The 5-μm cecal tissue section was stained with HE. (A and B) Cecum of streptomycin-pretreated mouse infected with serovar Gallinarum strain SG287/91. (C) Enlarged sections showing PMNs. The images are representative for all animals from each group. Box in panels A indicates the area shown at the higher magnification. L, intestinal lumen; e, edema; p, PMN; sa, submucosa. Magnifications are indicated by the black bars. Scale bars: A, 200 μm; B, 100 μm; C, 20 μm.

FIG. 6.

Virulence of serovar Enteritidis strains in streptomycin-pretreated mice. Five streptomycin-pretreated mice were infected for 3 days with 5 × 10⁷ CFU of serovar Enteritidis strain 125109, 5496/98, or 832/99. (A to E) Bacterial loads in the cecal content (A), the liver (B), the spleen (C), and the mLN (D). The dotted line indicates the limits of detection, and the horizontal bars indicate the medians. (E) Histopathological analysis. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars); PMN infiltration (black dotted bars); reduction in the number of goblet cells (white dotted bars); and desquamation, erosion, and ulceration of the epithelial layer (white bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. Differences in colonization or the total pathological score (sum of the separate scores) were statistically analyzed by using the exact Mann-Whitney U test.

FIG. 7.

Virulence of serovar Paratyphi A strains in streptomycin-pretreated mice. Five streptomycin-pretreated mice were infected for 3 days with 5 × 10⁷ CFU of serovar Paratyphi A strain SARB42, 2804/96, or 5314/98. (A to E) Bacterial loads in the cecal content (A), the liver (B), the spleen (C), and the mLN (D). The dotted line indicates the limits of detection, and the horizontal bars indicate the medians. (E) Histopathological analysis. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars); PMN infiltration (black dotted bars); reduction in the number of goblet cells (white dotted bars); and desquamation, erosion, and ulceration of the epithelial layer (white bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. Differences in colonization or the total pathological score (sum of the separate scores) were statistically analyzed by using the exact Mann-Whitney U test.

FIG. 8.

Invasion assay. Invasion of polarized m-IC_cl2 cells by different strains of serovar Dublin, Paratyphi A, Gallinarum, Pullorum, and Enteritidis. Typhimurium SL1344 and SB161 (SL1344, ΔinvG) served as a control. The data are normalized to the number of CFU recovered from the wells infected with Typhimurium SL1344. The data are presented as the mean (± the standard deviation) of triplicate results obtained in three independent experiments.