C57BL/6 and congenic interleukin-10-deficient mice can serve as models of Campylobacter jejuni colonization and enteritis

Abstract

Campylobacter jejuni is a globally distributed cause of human food-borne enteritis and has been linked to chronic joint and neurological diseases. We hypothesized that C. jejuni 11168 colonizes the gastrointestinal tract of both C57BL/6 mice and congenic C57BL/6 interleukin-10-deficient (IL-10(-/-)) mice and that C57BL/6 IL-10(-/-) mice experience C. jejuni 11168-mediated clinical signs and pathology. Individually housed mice were challenged orally with C. jejuni 11168, and the course of infection was monitored by clinical examination, bacterial culture, C. jejuni-specific PCR, gross pathology, histopathology, immunohistochemistry, and anti-C. jejuni-specific serology. Ceca of C. jejuni 11168-infected mice were colonized at high rates: ceca of 50/50 wild-type mice and 168/170 IL-10(-/-) mice were colonized. In a range from 2 to 35 days after infection with C. jejuni 11168, C57BL/6 IL-10(-/-) mice developed severe typhlocolitis best evaluated at the ileocecocolic junction. Rates of colonization and enteritis did not differ between male and female mice. A dose-response experiment showed that as little as 10(6) CFU produced significant disease and pathological lesions similar to responses seen in humans. Immunohistochemical staining demonstrated C. jejuni antigens within gastrointestinal tissues of infected mice. Significant anti-C. jejuni plasma immunoglobulin levels developed by day 28 after infection in both wild-type and IL-10-deficient animals; antibodies were predominantly T-helper-cell 1 (Th1)-associated subtypes. These results indicate that the colonization of the mouse gastrointestinal tract by C. jejuni 11168 is necessary but not sufficient for the development of enteritis and that C57BL/6 IL-10(-/-) mice can serve as models for the study of C. jejuni enteritis in humans.

FIG. 1.

Colon gross pathology in a C57BL/6 IL-10^−/− mouse given oral Campylobacter jejuni and sacrificed 30 days after infection. (A) Normal colon from a congenic uninfected control mouse 30 days after gavage with trypticase soy broth. (B) Colon from the Campylobacter jejuni-infected mouse is enlarged and pale, with a thickened wall and watery contents.

FIG. 2.

Presence of Campylobacter jejuni in four segments of the GI tract and feces in mice inoculated with C. jejuni 11168. Colonization rates were ranked according to numbers of Campylobacter colonies on the plate as 0 (none), 1 (approximately 1 to 20 CFU), 2 (approximately 20 to 200 CFU), 3 (over 200 CFU), and 4 (confluent growth). All cultures from the uninoculated control mice were negative for Campylobacter jejuni. (A to E) Short-term and long-term time course experiments. Open diamonds, male C57BL/6 IL-10^−/− mice; filled squares, female C57BL/6 IL-10^−/− mice. (F to J) Dose-response experiment. Filled diamonds, male C57BL/6 IL-10^−/− mice; filled squares, female C57BL/6 IL-10^−/− mice; open diamonds, male C57BL/6 IL-10^+/+ mice; open squares, female C57BL/6 IL-10^+/+ mice.

FIG. 3.

Representative sections of the ileocecocolic junction of C57BL/6 IL-10^−/− mice given oral Campylobacter jejuni 11168 and sacrificed 30 days after infection. Sections are stained with either hematoxylin and eosin (A, B, C, E, G, and H) or anti-Campylobacter jejuni-specific immunohistochemical stain (D, F, I, and J). Bars represent 100 μm. (A) Low-power view (magnification, ×2) of the ileocecocolic junction of an uninfected C57BL/6 IL-10^−/− control mouse. PC, proximal colon; C, cecum; I, ileum. (B) Section (similar to A) from a C57BL/6 IL-10^−/− mouse given oral Campylobacter jejuni. Note the extensive inflammatory response in all segments. (C) Cross section of the proximal colon from a C57BL/6 IL-10^−/− mouse given oral Campylobacter jejuni (magnification, ×10). Note crypt hyperplasia, extensive mononuclear cell infiltrates, crypt abscesses (small arrow), and neutrophilic exudates. Arrowheads show the extent of mucosal thickening compared to panel E. (D) Serial section adjacent to the section in C that was stained with the immunohistochemical stain. Campylobacter jejuni (brown stain) can be seen in crypts (D1 inset) and crypt abscesses (D2 inset). (E) Cross section of the proximal colon from an uninfected control mouse (magnification, ×20). (F) Same cross section of the proximal colon from an uninfected control mouse stained for Campylobacter jejuni using the immunohistochemical stain, with no positive staining seen. (G) Mucosa from the ileocecocolic junction in an infected mouse showing a lesion with sloughing epithelium associated with Campylobacter jejuni infection (arrows). (H) View of mesenteries underlying the ileocecocolic junction from an infected C57BL/6 IL-10^−/− mouse showing extensive mononuclear cell infiltrate, edema, and fibrosis. Arrowheads show the extent of perivascular cellular infiltrate for one vessel. (I) Low-power view (magnification, ×20) of an individual colon villus tip stained for Campylobacter jejuni. Arrows indicate the borders of the villus tip. Insert I1 shows Campylobacter jejuni within vacuoles of a phagocytic cell. (J) High-power view (magnification, ×100) of the boxed area in I. Campylobacter jejuni was associated with epithelial cells (arrows) and among and apparently inside of inflammatory cells underlying the epithelium.

FIG. 4.

Histopathology scores of sections of ileocecocolic junctions from C57BL/6J IL-10^−/− mice inoculated with Campylobacter jejuni 11168. Histopathology was scored as described in Materials and Methods. The bar labeled “TSB” shows data for all 32 control mice (inoculated with tryptose soya broth); the control mice were analyzed as a single group regardless of the time of sacrifice. Asterisks indicate statistically significant differences (P ≤ 0.05) between Campylobacter jejuni-infected animals and grouped control animals from the same experiment by Fisher's exact test with Bonferroni correction for multiple comparisons as described in Materials and Methods. Boxes enclose data falling within the first and third quartiles of the distributions; the heavy bar within each box indicates the median of the distribution, and whisker bars indicate the maximum and minimum values observed. (A) Time course experiments. (B) Dose-response experiment.

FIG. 5.

Representative cross sections from the ileocecocolic junctions of C57BL/6 IL-10^−/− mice given oral Campylobacter jejuni 11168 and sacrificed 30 days after infection. Sections are stained with hematoxylin and eosin; bars represent 100 μm. (A) Low-power view (magnification, ×10) of sloughing colon villus tip from an infected mouse. Note extensive largely neutrophilic inflammatory exudates. The arrowhead shows a crypt abscess. Small arrows show the edge of the sloughing epithelium. (B) High-power view of neutrophilic inflammatory exudates from the infected mouse shown in A (magnification, ×100). Note the many neutrophils present in the exudate. Arrows show degenerating epithelial cells. (C) Colon cross section from an infected mouse showing extensive mononuclear cell infiltrates (magnification, ×10). Arrows point to the colon villus tip. (D) High-power view of mononuclear inflammatory exudates from the infected mouse shown in C (magnification, ×100). Arrows point to three of the many mononuclear cells in this field.

FIG. 6.

Antibody response as measured by ELISA. Each column indicates the mean absorbance value/μg plasma protein; error bars are standard errors. Panels A through F show results for the long-term time course experiment for total IgG, IgA, and IgM (A); IgG1 (B); IgG2b (C); IgG2c (D); IgG3 (E); and IgA (F). Panel G shows results for IgM for both short- and long-term time course experiments. Panels H through M show results for the dose-response experiment for total IgG, IgA, and IgM (H); IgG1 (I); IgG2b (J); IgG2c (K); IgG3 (L); and IgA (M). Gray bars represent values from C57BL/6 IL-10^−/− mice, and white bars represent values from C57BL/6 mice.