Responses of cattle to gastrointestinal colonization by Escherichia coli O157:H7

Abstract

Recent research has established that the terminal rectum is the predominant colonization site of enterohemorrhagic Escherichia coli O157:H7 in cattle. The main aim of the present work was to investigate pathological changes and associated immune responses at this site in animals colonized with E. coli O157:H7. Tissue and gastrointestinal samples from a total of 22 weaned Holstein-cross calves challenged with E. coli O157:H7 were analyzed for bacterial colonization and pathology. Five unexposed age-matched calves were used as comparative negative controls. E. coli O157:H7 bacteria induced histopathological alterations of the rectal mucosa with enterocyte remodeling. This was often associated with removal of the colonized epithelial layer. Immunogold labeling and transmission electron microscopy (TEM) showed E. coli O157 bacteria on pedestals, as part of attaching and effacing lesions. These pathological changes induced a local infiltration of neutrophils that was quantified as larger in infected animals. Rectal mucosal immunoglobulin A responses were detected against the E. coli O157:H7 antigen. This work presents evidence that E. coli O157:H7 is not a commensal bacteria in the bovine host and that the mucosal damage produced by E. coli O157:H7 colonization of the terminal rectum induces a quantifiable innate immune response and production of specific mucosal antibodies.

FIG. 1.

Histopathological changes induced by E. coli O157:H7 colonization at the terminal rectum of cattle. (A) Apical surface of the enterocyte partly eroded by an E. coli O157 microcolony (arrow). (B) A larger E. coli O157 microcolony causing further erosion of the cytoplasm. An E. coli O157 colony is illustrated (arrow). (C) Enterocytes (arrows) heavily colonized with E. coli O157 are shown sloughing off from the basal membrane. Bacterial aggregates surround the enterocyte. (D) A larger E. coli O157 microcolony is shown on the lamina propria, following the shedding of the epithelial layer. Histopathology was carried out as described in Materials and Methods.

FIG. 2.

Ultrastructure analysis of E. coli O157 interaction with the bovine terminal rectum. (A) Immunogold staining of E. coli O157 organisms. The image shows a single bacterium intimately attached through a pedestal to the host enterocyte, as part of an A/E lesion. Immunolabeling and TEM were carried out as described in Materials and Methods. (B) Cross-section of an E. coli O157 microcolony at the bovine terminal rectum. The bacteria are all intimately attached to the damaged epithelium (black arrows), inducing effacement of the microvilli. Unaffected brush border is visible on the neighboring cell (white arrow). (C) Aggregates of bacteria eroding the apical surface of colonized enterocytes (black arrows). Bacteria are intimately attached to the apical surface of the enterocyte (white arrows). (D) Extravasated polymorphic mononuclear leukocyte (N) adjacent to a bacterial cluster (black arrow).

FIG. 3.

Histological granulocyte quantification. (A) Hematoxylin- and-eosin-stained bovine rectal mucosa colonized with E. coli O157:H7. The dashed outline highlights colonized epithelium. Arrows indicate infiltration of granulocytes. (B) Box plots show the granulocyte counts in the terminal rectums of exposed and unexposed animals. The boxes contain 50% of the data, and the median count is illustrated by the black triangle. ⧫, 1st quartile; ▪, minimum; ▴, median; ○, mean; ×, maximum; •, 3rd quartile. Samples were prepared and granulocytes identified as described in Materials and Methods.

FIG. 4.

Detection of mucosal antibody responses to protein antigens of E. coli O157. Mucosal antibody was obtained from homogenates of rectal mucosa sampled from three animals that had histopathological lesions detectable by microscopy. The homogenates were used to blot whole-cell preparations of E. coli strains O157 and K-12 and, in one case, O26. Multiple E. coli O157 immunoreactive bands were detected in comparison to either E. coli K-12 or E. coli O26. Trypsin digestion of the bacterial preparations removed most of the immunoreactive material. This is demonstrated with a blot using the homogenate from animal 3 (lanes labeled “Trypsinized”). Lanes marked “no primary” are controls containing E. coli O157 and K-12 preparations incubated with all the reagents, except for the rectal homogenate. Mucosal homogenates and Western blots were prepared as described in Materials and Methods.