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Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli-induced diarrhea and intestinal inflammation in infant rabbits - PubMed

Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli-induced diarrhea and intestinal inflammation in infant rabbits

Jennifer M Ritchie et al. Infect Immun. 2003 Dec.

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a group of food-borne pathogens that can cause diarrhea, colitis, and the hemolytic uremic syndrome (HUS). The importance of several of the proposed EHEC virulence factors lacks experimental verification in animal models. The limitations of current animal models led us to reexamine the infant rabbit model for the study of EHEC pathogenicity. Here, we report that intragastric inoculation of a Shiga toxin 2 (Stx2)-producing E. coli O157:H7 clinical isolate into infant rabbits led to severe diarrhea and intestinal inflammation but no signs of HUS. We constructed a set of isogenic derivatives of this isolate with deletions in several putative virulence genes, including stx(2), eae, tir, and ehxA, to investigate the contribution of individual virulence factors to EHEC pathogenicity. stx(2) increased the severity and duration of EHEC-induced diarrhea. Furthermore, although stx(2) had no role in EHEC intestinal colonization nor was it required for EHEC-induced inflammation, stx(2) altered how the host responded to EHEC infection by promoting heterophilic infiltration of the colonic epithelium and lamina propria. Intragastric inoculation of purified Stx2 also induced inflammation and diarrhea in this model. Diarrhea and intestinal inflammation were also dependent on EHEC colonization, as EHEC derivatives with deletions in eae and tir did not colonize, form attaching and effacing lesions, or develop clinical signs of disease. Our studies indicate that infant rabbits are a useful model for investigation of the intestinal stage of EHEC pathogenesis and suggest that Shiga toxin may play a critical role in causing diarrhea and inflammation in patients infected with EHEC.

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Figures

FIG. 1.
FIG. 1.

Gross and histologic findings in infant rabbits infected with EHEC strain 905 or its stx2 derivative, 905Δstx2. Feces-contaminated area of rabbits inoculated with 905 at 7 days postinoculation (A) versus the lack of diarrhea in rabbits inoculated with 905Δstx2 (B) are shown, as well as lack of formed pellets and edematous nature of the colon of rabbits infected with 905 (C) versus normal appearance of colon containing formed ingesta in PBS-treated rabbits (D). (E to I) Representative H&E-stained sections from the mid-colon of infant rabbits infected with 905, 905Δstx2, PBS, or purified Stx2. (E) Suppurative colitis in a rabbit infected with 905 (magnification, ×20). (F) Cluster of heterophils (black arrow) shown at a higher magnification in a 905-infected rabbit (magnification, ×40). (G) No inflammation in the colon of PBS-treated rabbit (magnification, ×20). (H) Lymphocytic colitis with fewer heterophils in rabbit infected with 905Δstx2. (I) Crypt abscess (black arrow) in the distal colon of rabbit inoculated with purified Stx2 (magnification, ×40).

FIG. 2.
FIG. 2.

Weight gains in infant rabbits inoculated with PBS, 905, or one of its derivatives. Rabbits were weighed prior to inoculation and at 7 days postinoculation, and the difference in weight was expressed as a percentage of the initial rabbit weight. Rabbits infected with 905 gained significantly (P < 0.05) less weight than those infected with 905Δstx2. Error bars represent the standard deviations of the means.

FIG. 3.
FIG. 3.

Recovery of 905 or one of its derivatives (CFU g−1) from intestinal segments or stool from infected rabbits. Numbers of bacterial CFU were determined in sections taken from the ilea (A), ceca (B), mid-colons (C), and stools (D) of rabbits 7 days postinoculation. Open symbols represent samples below detection limits. Bars represent the mean value for each treatment.

FIG. 4.
FIG. 4.

Stx2 concentrations (ng g−1) in stools of rabbits inoculated with PBS, 905, or one of its derivatives. Stool samples were removed from the large intestines of rabbits 7 days postinoculation, and Stx2 concentrations were determined by enzyme-linked immunosorbent assay. Bars represent the mean values.

FIG. 5.
FIG. 5.

Colitis scores from the distal colons of infant rabbits inoculated with PBS, 905, or one of its derivatives. H&E-stained sections were scored for heterophils (A), mononuclear cells (B), and edema or congestion (C). Bars represent the median values.

FIG. 6.
FIG. 6.

Transmission electron micrographs of tissue sections from the colons of rabbits infected with 905 (A) or 905Δeae (B). Note the loss of microvilli from the epithelial cells in panel A. The colons of rabbits inoculated with 905Δeae contained no adherent bacteria, although bacteria could be seen in the intestinal lumen adjacent to rows of intact microvilli. [magnification, ×4,900 (A) or ×2,200 (B); scale bars, 1 μm].

FIG. 7.
FIG. 7.

Colitis scores from the distal colons of infant rabbits inoculated with purified Stx2 or HI-Stx2. Rabbits were sacrificed 2 days postinoculation. H&E-stained sections were scored as described in Materials and Methods and also assessed for crypt abscesses. Shown are data for heterophils (A) and crypt distortion (B). Bars represent the median values.

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