Immunology and Infection
Published: February 19th, 2013
Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.
This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.
Infection by enteric bacterial pathogens triggers gastrointestinal (GI) inflammation, as well as intestinal pathology and pathophysiology, including diarrhea and intestinal epithelial barrier dysfunction. The virulence mechanisms by which bacterial pathogens colonize the GI tract of their hosts, as well as specific host responses that defend against such infections are poorly understood, however recent advances in the modeling of enteric bacterial infections have begun to aid our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). The IBDs Crohn's Disease (CD) and UC are complex diseases of unknown e....
1. Preparation of Citrobacter rodentium Inoculum and Oral Gavage of Mice
During a standard infection experiment, mice are infected with approximately 2.5 x 108 CFU through gavage of 100 μl overnight C. rodentium culture. Infection of C57BL/6 mice with C. rodentium results in modest and transient weight loss and diarrhea. Although a rare occurrence with C57BL/6 mice, animals may become ill and require euthanization. Therefore, mice should be monitored for degree of weight loss and symptoms of distress such as piloerect fur and hunched posture, to determine.......
Citrobacter rodentium infection provides a valuable model for the study of both infectious disease and colitis in mice. This unique model allows for the characterization of both host responses, as well as the pathogenic properties of bacteria. The steps outlined in this protocol detail the successful use of this model.
There are several critical steps in this protocol to keep in mind when inducing colitis and analyzing responses. First, the preparation of a fresh overnight .......
This work was supported by operating grants to BAV from the Crohn's and Colitis Foundation of Canada (CCFC) and the Canadian Institutes for Health Research (CIHR). GB was funded by a graduate studentship from CIHR. BAV is the Children with Intestinal and Liver Disorders (CHILD) Foundation Chair of Pediatric IBD Research and the Canada Research Chair in Pediatric Gastroenterology.....
|Name of Reagent/Material
|Add 25 g of LB powder to 1L of water. Autoclave before using.
|Square bottom plate with grid
|Falcon culture tube
|Bulb tipped gastric gavage needle
|Fine Science Tools
|1 ml syringe
|4 kDa FITC-dextran
|Acid citrate dextrose
|20 mM ctiric acid, 110 mM sodium citrate, 5 mM dextrose
|Black 96-well plate
|Metal beads (5 mm)
|5 ml vial
|Coplin staining jar
|Sodium citrate buffer
|10 mM sodium citrate, 0.05% Tween 20, pH 6.0
|Bovine Serum Albumin (BSA)
|2% goat serum, 1% BSA, 0.1% triton X-100, 0.05% Tween 20, 0.05% sodium azide, 0.01 M PBS, pH 7.2, mix & store at 4 °C.
|Antibody dilution buffer
|0.1% triton X-100, 0.1% BSA, 0.05% sodium azide, 0.04% EDTA
|Blocking buffer & Antibody dilution buffer for tir
|Same recipes as above, but without addition of detergents (triton X-100 and tween 20)
|Prolong Gold Antifade Reagent with DAPI
|Benchtop incubation shaker
|Barnstead Lab Line
|Black & Decker
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