Analysis of the contribution of Salmonella pathogenicity islands 1 and 2 to enteric disease progression using a novel bovine ileal loop model and a murine model of infectious enterocolitis

Abstract

We have developed a novel ileal loop model for use in calves to analyze the contribution of Salmonella enterica serovar Typhimurium type III secretion systems to disease processes in vivo. Our model involves constructing ileal loops with end-to-end anastamoses to restore the patency of the small intestine, thereby allowing experimental animals to convalesce following surgery for the desired number of days. This model overcomes the time constraint imposed by ligated ileal loop models that have precluded investigation of Salmonella virulence factors during later stages of the infection process. Here, we have used this model to examine the enteric disease process at 24 h and 5 days following infection with wild-type Salmonella and mutants lacking the virulence-associated Salmonella pathogenicity island 1 (SPI-1) or SPI-2 type III secretion systems. We show that SPI-2 mutants are dramatically attenuated at 5 days following infection and report a new phenotype for SPI-1 mutants, which induce intestinal pathology in calves similar to wild-type Salmonella in the 5-day ileal loop model. Both of these temporal phenotypes for SPI-1 and SPI-2 mutants were corroborated in a second animal model of enteric disease using streptomycin-pretreated mice. These data delineate novel phenotypes for SPI-1 and SPI-2 mutants in the intestinal phase of bovine and murine salmonellosis and provide working models to further investigate the effector contribution to these pathologies.

FIG. 1.

(A) Pathological scores in streptomycin-pretreated mice 48 h postinfection with wild-type S. enterica serovar Typhimurium or strains lacking functional SPI-1, SPI-2 or both type III secretion systems. (B) Pathological scores from mice infected for 120 h (5 days). Pathological scores are stacked averages from 8 mice per group, error bars indicate the standard deviation of the total score. Bacterial loads in the colon (C) and spleen (D) of streptomycin-treated mice for above strains at 48 h and 120 h postinfection. Each black circle represents data from one mouse. Bars represent the geometric mean for the group. The type III secretion status of each strain is given below each panel next to the respective locus. All P values are derived from comparisons to wild-type Salmonella (SPI-1⁺, SPI-2⁺).

FIG. 2.

Histopathological changes at 48 h (A, C, E, and G) and 120 h (B, D, F, and H) postinfection in streptomycin-pretreated mice infected with wild-type S. enterica serovar Typhimurium (A and B) or strains lacking functional SPI-1 (SPI-1⁻) (C and D) or SPI-2 (SPI-2⁻) (E and F) type III secretion systems or both (SPI-1⁻, SPI-2⁻) (G and H). Tissues were paraffin embedded, section and stained with hematoxylin and eosin. Photographs are of representative tissues for each group.

FIG. 3.

(A) Secretory responses in ileal loops 24 h after infection with wild-type S. enterica serovar Typhimurium, or mutants lacking SPI-2 (SPI-2⁻) or SPI-1 (SPI-1⁻) type III secretion. The secretory response in control loops inoculated with physiological saline was also determined (saline). The secretory response is expressed as the volume of fluid divided by the length of the loop. Data are the means with standard deviation from two duplicate loops. (B) Bacterial loads in calf ileal loops at day 5 after infection. Loop contents were collected and the number of Salmonella contained in this fluid was enumerated by selective plating. Values are expressed as the mean number of CFU per ml of loop fluid from two to four infected loops with standard errors.

FIG. 4.

Pathological changes in calf ileal loops at 24 h postinfection. (A) Pathology scores from calf ileal loops at 24 h after infection with either saline alone, wild-type S. enterica serovar Typhimurium or mutants lacking SPI-2 or SPI-1 type III secretion (SPI-2⁻, SPI-1⁻, respectively). White bars and black bars indicate initial infectious doses of 1 × 10³ and 1 × 10⁶ CFU per loop, respectively. Saline groups are similarly colored but are not differentially dosed. Pathology scores for loops infected with wild-type bacteria are from 2 animals while scores for the SPI-2 and SPI-1 mutants are from one animal each. Pathology was scored on a scale from 1 to 4 according to the criteria defined in the Materials and Methods. (B) Pathology scores from calf ileal loops at 5 days after inoculation with either saline alone, wild-type S. enterica serovar Typhimurium or mutants lacking SPI-2 or SPI-1 type III secretion (SPI-2⁻ and SPI-1⁻, respectively). White bars and black bars indicate initial infectious doses of 1 × 10³ and 1 × 10⁶ CFU per loop, respectively. Pathology scores for wild-type-infected loops are from four individual loops from two animals. Pathology scores for loops infected with SPI-1 and SPI-2 mutants are from two separate loops from two animals each. Villus height from matched tissue sections was also determined from loops infected with 1 × 10³ (solid squares) and 1 × 10⁶ (solid circles) CFU at 24 h (C) and 5 days (D) after infection.

FIG. 5.

Histopathological changes in calf ileal loops at 24 h and 5 days after inoculation with either saline alone (A, B, and B′), wild-type S. enterica serovar Typhimurium (C, D, and D′), or mutants lacking SPI-1 (E, F, and F′) or SPI-2 (G, H, and H′) type III secretion systems. Shown are hematoxylin- and eosin-stained sections of the intestinal epithelium. White bars, 0.250 mm.