The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Preparation of Citrobacter rodentium Inoculum and Oral Gavage of Mice

1. Prepare and autoclave Luria Bertani broth (LB).
2. Obtain viable C. rodentium from a frozen glycerol stock and streak onto LB agar plate using a sterile inoculating loop or pipette tip. Incubate at 37 °C overnight. Inoculate 3 ml of sterile LB broth in a falcon culture tube with colonies from the LB plate using an inoculating loop or pipette tip. Preparation of the inoculum should be done using aseptic techni.......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
Luria Broth	ABM	G247	Add 25 g of LB powder to 1L of water. Autoclave before using.
Square bottom plate with grid	Fisher	08-757-11A
Falcon culture tube	Sarstedt	62.515.006
Bulb tipped gastric gavage needle	Fine Science Tools	18060-20
1 ml syringe	BD Biosciences	309659
4 kDa FITC-dextran	Sigma	FD-4
Citric acid	Sigma	C7129
Sodium citrate	Fisher	S279-500
Dextrose	Fisher	D16.1
Acid citrate dextrose			20 mM ctiric acid, 110 mM sodium citrate, 5 mM dextrose
Black 96-well plate	Fisher	07-200-762
Metal beads (5 mm)	Qiagen	69989
10% formalin	Fisher	5F93-4
5 ml vial	DiaMed	STK3205
Hematoxylin	Fisher	H345-23
Eosin	Fisher	E511-100
Xylene	Fisher	HC700-1GAL
Tween 20	Sigma	P5927
Coplin staining jar	VWR	47751-792
Sodium citrate buffer			10 mM sodium citrate, 0.05% Tween 20, pH 6.0
Pap pen	Cedarlane	Mu22
Goat serum	Sigma	G902-3
Bovine Serum Albumin (BSA)	Fisher	BP1600-100
Triton X-100	Sigma	T8532
Sodium azide	Sigma	SZ002
Blocking buffer			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	Invitrogen	P-36931
Coverslips	Fisher	12.54SE
Benchtop incubation shaker	Barnstead Lab Line	Max Q4000
Fluorometer	Perkin Elmer	Victor2D
Refrigerated centrifuge	Beckman Coulter	Microfuge 22R
Steamer	Black & Decker
Fluorescence microscope	Zeiss	Axio Image.Z1