A piglet model of acute gastroenteritis induced by Shigella dysenteriae Type 1

Abstract

Background: The lack of a standardized laboratory animal model that mimics key aspects of human shigellosis remains a major obstacle to addressing questions about pathogenesis, screening therapeutics, and evaluation of vaccines.

Methods: We characterized a piglet model for Shigella dysenteriae type 1.

Results: Piglets developed acute diarrhea, anorexia, and dehydration, which could often be fatal, with symptom severity depending on age and dose. Bacteria were apparent in the lumen and on the surface epithelium throughout the gut initially, but severe mucosal damage and bacterial cellular invasion were most profound in the colon. Detached necrotic colonocytes were present in the lumen, with inflammatory cells outpouring from damaged mucosa. High levels of interleukin (IL)-8 and IL-12 were followed by high levels of other proinflammatory cytokines. Elevated levels of tumor necrosis factor-alpha, IL-1beta, IL-6, and IL-10 were detected in feces and in gut segments from infected animals. Bacteria were present inside epithelial cells and within colonic lamina propria. In contrast, an isogenic strain lacking Shiga toxin induced similar but milder symptoms, with moderate mucosal damage and lower cytokine levels.

Conclusion: We conclude that piglets are highly susceptible to shigellosis, providing a useful tool with which to compare vaccine candidates for immunogenicity, reactogenicity, and response to challenge; investigate the role of virulence factors; and test the efficacy of microbial agents.

Fig. 1

Bacterial excretion in feces (A) and diarrheal rates (B) over 7 days after an oral challenge with 5×10⁹ CFU/animal of wild type SD1 strain 1617 (total 35 animals) and its isogenic Stx minus strain (total 25 animals). 1A shows that significant differences (p<0.05) in the rate of bacterial excretion occurred on days 5-7 after challenge. Each bar in 1B indicates the daily percentage of piglets with diarrhea with #representing significantly higher values between the 2 bacterial strains at each time points (p<0.05).

Fig. 2

Bacterial colonization in the gastrointestinal (GI) tract. Luminal contents were collected from stomach, jejunum, ileum, and colon from piglets euthanized 8 hr and 1, 2, 3, and 5 days after challenge. The error bars represent standard errors of the means of data (n=5 ∼ 18) at each time-points. #, represents significantly higher values than other GI segments (p<0.05).

Fig. 3

Cytokine excretion in the feces. Seven cytokine profiles were measured by ELISA over 8 days after challenge. The error bars represent standard errors of the means of data (n=5 ∼ 18) time-points. Cytokine concentrations (pg/ml) in rectal swabs were calculated by regression based on the standard curves of respective standard recombinant cytokines. # represents statistically significantly higher values (p<0.05).

Fig. 4

Cytokine responses measured by ELISA in the luminal contents of GI tissues (stomach, jejunum, ileum, and colon). The error bars represent standard errors of the means of data (n=3 ∼ 5) time-points. Cytokine concentrations (pg/ml) in the GI contents were calculated by regression based on the standard curves of respective standard recombinant cytokines. # represent statistically significantly higher values (p<0.05).

Fig. 4

Cytokine responses measured by ELISA in the luminal contents of GI tissues (stomach, jejunum, ileum, and colon). The error bars represent standard errors of the means of data (n=3 ∼ 5) time-points. Cytokine concentrations (pg/ml) in the GI contents were calculated by regression based on the standard curves of respective standard recombinant cytokines. # represent statistically significantly higher values (p<0.05).

Fig. 5

Micrographs of gut sections of a piglet euthanized one day after challenge: Upper panel shows H&E stained sections, while the lower panel shows bacterial antigen localization by IHC in the corresponding section. Areas marked by the rectangles in the upper panels are presented at higher magnification in the lower panels.

Fig. 6

Micrographs of colon sections taken from a piglet euthanized on day 2 after bacterial challenge. Serial sections of colon were stained with H&E (A), and with IHC (B-D) to illustrate the location of bacterial antigen in the mucosa. The areas marked by the 2 rectangles in B are presented at higher magnification in C (luminal regions) and D (crypt-submucosal junction).

Fig. 7

Electron micrographs of sections taken from the colon one day after bacterial challenge; A showing organisms (arrowed) closely adherent to colonocyte surfaces or inside cells; and B showing deeply embedded in the lamina propria (B) surrounded by inflammatory cells (arrowed bacteria is enclosed by dotted line).