Intimin, tir, and shiga toxin 1 do not influence enteropathogenic responses to shiga toxin-producing Escherichia coli in bovine ligated intestinal loops

Abstract

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle.

FIG. 1.

Enteropathogenic responses to STEC strains 84-289 (O157:H7) and PMK5 (O103:H2) and sterile brain heart infusion (BHI) broth in the intestines of ca. 28-day-old conventional calves. (A) Fluid accumulation. Each strain was tested in triplicate mid-ileal loops, and the mean V/L ratio (+ SEM) from the three independent experiments is shown. (B) Neutrophil infiltration. The total ¹¹¹In activity in the loop contents and mucosa was corrected for loop length, and the mean of three measurements in the mid-ileum was derived. Neutrophil influx is the ratio of the mean ¹¹¹In activity in the test loops to ¹¹¹In activity in the negative control loops. The mean neutrophil influx (+ SEM) from the three calves is shown.

FIG. 2.

Histological analysis of mid-ileal mucosa from ligated loops inoculated with E. coli PMK5 (A) or sterile medium (B). Hematoxylin and eosin staining was used. Magnification, ×400.

FIG.3.

Confocal laser scanning microscopy showing PMK5 and isogenic eae, tir, and stx mutants interacting with HeLa cells or intestinal mucosa. (A) FAS of HeLa cells incubated with PMK5 and the isogenic mutants indicates that the eae and tir mutants are incapable of nucleating F-actin under the sites of bacterial adhesion. Magnification, ×1,000. (B and C) Sections of mid-ileal mucosa from inoculated loops stained for F-actin and bacteria. The images in column B show rare microcolonies formed by the wild type and the stx mutant. Microcolonies were not detected with the eae and tir mutants; however, small numbers of bacteria could occasionally be seen on the ileal mucosa. Column C contains typical fields showing an almost complete absence of bacteria on the intestinal epithelium and numerous bacteria and neutrophils in the gut lumen. Magnification (B and C), ×630. Green, F-actin stained with Oregon green 514-phalloidin; red, bacteria detected with rabbit anti-O103 typing serum and anti-rabbit immunoglobulin-Alexa⁵⁶⁸.

FIG. 4.

Enteropathogenic responses to E. coli PMK5 and isogenic eae, tir, and stx1 mutants in the mid-ileum of ca. 28-day-old conventional calves. (A) Fluid accumulation. To correct for interanimal variation, the mean V/L ratio for each mutant strain was expressed as a percentage of the mean V/L ratio for the wild type in the same calf. The mean percentage plus SEM from three independent experiments is shown. (B) Neutrophil influx. The total ¹¹¹In activity in the contents and mucosa of test loops was corrected for loop length and expressed as a ratio to the activity in negative control loops. Neutrophil influx for the mutant strains was then calculated as a percentage of the influx for the wild type, and the mean of the three experiments plus SEM was derived.