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.

1. Preparation of Citrobacter rodentium Inoculum and Oral Gavage of Mice
<ol>
 <li>Prepare and autoclave Luria Bertani broth (LB).</li>
 <li>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 &deg;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 techniques.</li>
 <li>Incubate the C. rodentium culture aerobically at 37 &deg;C overnight in a benchtop incubation shaker at 200 rpm. The inoculated LB broth should appear cloudy after overnight culture.</li>
 <li>Use a bulb tipped gastric gavage needle attached to a 1 ml syringe to gavage each mouse with 100 &mu;l of the overnight C. rodentium culture. Gently scruff the mouse, by firmly grasping the loose skin over its neck and back with thumb and fingers. Pull back the animal's head with your index finger to immobilize the head and straighten the esophagus for insertion of the gavage needle.</li>
 <li>Maintain the mouse in an upright position and direct the bulb tip of the gavage needle along the side of its mouth and over its tongue. Gently pass the needle along the roof of the mouth and advance it down the esophagus. If there is any resistance felt during this procedure, remove the gavage needle and re-insert it.</li>
 <li>Slowly inject 100 &mu;l of the bacterial inoculum and gently remove the gavage needle. Monitor the breathing rate and behavior of the mouse after returning it to its cage.</li>
</ol>
Notes:
<ul>
 <li>In steps 2 and 3, also prepare a falcon culture tube using aseptic techniques with 3 ml of sterile LB broth to be cultured overnight to ensure that there is no bacterial contamination of the broth itself. The LB broth should appear clear after overnight culture.</li>
 <li>Gently agitate the culture within the falcon tube prior to loading syringes for gavage so that an equal dose of bacteria is delivered to each mouse, in step 4.</li>
 <li>Overnight culture should be discarded if it has been inoculated for over 24 hr.</li>
 <li>All C. rodentium infections and housing of infected animals should be carried out in a Biosafety Level 2 facility.</li>
</ul>
2. Measuring Colonic Epithelial Barrier Permeability in C. rodentium-infected Mice
<ol>
 <li>Measure barrier integrity at a desired time point post-infection. </li>
 <li>On the day of the assay, prepare enough 4 kDa fluorescein isothiocyanate (FITC)-dextran dissolved in phosphate buffered saline (PBS) to a concentration of 80 mg ml-1 to gavage each mouse with 150 &mu;l and to prepare the standard curve.</li>
 <li>Scruff the mouse and gavage with 150 &mu;l of FITC-dextran. Withdraw food from the cage at this time.</li>
 <li>Anesthetize mice 4 hr after gavaging and collect as much blood as possible (~450 &mu;l) through cardiac puncture. Add the blood to a final concentration of 3% acid-citrate dextrose in a microcentrifuge tube to deter coagulation. Euthanize the mice and collect colonic tissues in PBS for bacterial counts (steps 3.1 - 3.5) and in 10% formalin for tissue fixation and ultimately immunofluorescence staining (steps 4.1 - 4.8).</li>
 <li>Spin the blood samples at 1,000 x g for 12 min in a centrifuge at 4 &deg;C. Collect the serum and dilute to 1/10 and 1/100 in PBS. Add 100 &mu;l of each sample at these two dilutions to a 96 well plate in replicates.</li>
 <li>To prepare the standard curve, dilute the original 80 mg ml-1 FITC-dextran used to gavage the mice in PBS to the following concentrations: 800, 400, 200, 100, 50, 25, 12.5, 6.25, and 0 &mu;g/ml. Add 100 &mu;l of each concentration to a 96 well plate in triplicate.</li>
 <li>Quantify the fluorescence of each sample using a fluorometer at an excitation wavelength of 485 nm and 535 nm emission wavelength.</li>
 <li>Analyze the raw data by plotting the standard curve. Input the raw data for each sample into the equation of the line generated by the standard curve to determine the concentration of FITC-dextran in each sample.</li>
</ol>
Notes:
<ul>
 <li>From step 4 onward, keep blood samples on ice and minimize exposure to light.</li>
 <li>When measuring epithelial barrier integrity a time point at, or before, 7 days post-infection is optimal, as severe tissue damage occurs after this point. Measuring barrier integrity prior to severe damage occurring allows you to determine maximal differences in permeability during C. rodentium infection between mouse strains.</li>
 <li>Ensure that uninfected control mice are also tested for epithelial barrier integrity.</li>
</ul>
3. Measurement of Bacterial Load in Tissues of C. rodentium-infected Mice
<ol>
 <li>Add 1 ml sterile PBS and an autoclaved metal bead to a round bottomed 2 ml centrifuge tube using aseptic technique. Individual tissues collected from each animal will be placed in separate PBS tubes. Weigh the tubes prior to addition of the tissue.</li>
 <li>Remove the cecum and colon from the mouse. Separate the cecum from the colon and push out the luminal contents using forceps. Add the contents to a PBS tube. After separating 0.5 cm sections from the distal colon and cecum for 10% formalin fixation, cut open the remaining colon and cecum longitudinally and wash with sterile PBS before placing tissues in tubes.</li>
 <li>Weigh the PBS tubes with the tissue and then homogenize the samples using a bead beater for 6 min at 30 Hz.</li>
 <li>Using aseptic techniques, add 180 &mu;l sterile PBS per well to a 96 well plate. Add 20 &mu;l of each homogenized sample to the first well in each row. Mix well and serially dilute, adding 20 &mu;l from each previous well to obtain dilutions from 10-1 (first well) to 10-6. Plate 10 &mu;l of each dilution from each sample in triplicate on a square bottom LB plate with a grid. Incubate overnight at 37 &deg;C.</li>
 <li>Count colony forming units (CFU), then average the 3 counts, and record the dilution at which each sample was counted. Multiply average CFU by 50, as 20 &mu;l of the original 1 ml sample was used, and by the dilution factor at which the sample was counted resulting in CFU/ml. Finally, divide by the tissue weight to determine CFU/g.</li>
</ol>
4. Histological Assessment and Immunofluorescence Staining of Infected Colon Tissues
<ol>
 <li>Collect tissues as in step 3.2. Store tissues in formalin overnight, then wash with 70% ethanol. Embed tissues in paraffin and cut 5 &mu;m sections for staining. Stain with hematoxylin &amp; eosin for histological assessment and proceed to the following steps for immunofluorescence staining.</li>
 <li>To deparaffinize tissues prior to staining, place slides in a coplin staining jar and put in 65 &deg;C water bath for 10 min. Next, place slides in xylene for four washes of 2 min each. Slides should then be rehydrated with two 5-minute washes in 100% ethanol, followed by one 5 min wash in each of the following: 95% ethanol, 75% ethanol and dH20. These washes should be done in a fume hood to avoid exposure to harmful vapors.</li>
 <li>Place slides in the coplin jar with pre-warmed sodium citrate buffer and then place into a steamer for 30 min, then let sit for 30 min. This process breaks the protein cross-links formed by formalin fixation retrieving potential antigenic sites.</li>
 <li>Wash with PBS azide for 5 min, three times. Dry slides and mark area around tissue with a PAP pen to create a hydrophobic barrier, keeping the staining reagents localized on the tissue.</li>
 <li>Block tissue for 1 hr at room temperature with blocking buffer (e.g. &alpha;-goat serum) in an immunostaining moisture chamber.</li>
 <li>Dilute primary antibody to desired concentration using antibody dilution buffer. Pour off blocking buffer and add 50-100 &mu;l of primary antibody to tissues. Incubate at 4 &deg;C overnight or at room temperature for 2 hr.</li>
 <li>Wash with PBS azide for 5 min, three times. Add 50-100 &mu;l of secondary antibody diluted in antibody dilution buffer to the tissues and incubate at room temperature for 1 hr in the dark. Wash with dH2O for 5 min, three times.</li>
 <li>Dehydrate slides, add DAPI Prolong Gold mounting medium and apply a coverslip. View slides under a fluorescence microscope.</li>
</ol>
Notes:
<ul>
 <li>PBS azide can be substituted with freshly prepared PBS as an alternative to avoid bacterial growth in the wash medium from step 4 onward.</li>
</ul>

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The authors declare that they have no competing financial interests.

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 C. rodentium inoculum in LB broth is required for successful infection of mice. As a dose of 108-109 organisms is required to infect most strains of mice, if the inoculum is left in the shaker or on the bench top at room temperature for extended periods, there will not be enough viable organisms remaining in the culture to induce colitis upon infection. It is also important to agitate the bacterial inoculum before loading the syringe for infection, to ensure that each mouse receives an equal dose of bacteria. When collecting tissues upon terminus of the infection, complete submersion of the colonic tissues in an ample volume of 10% formalin is required for proper tissue fixation to occur. It is suggested that tissues be added to formalin in a 5 ml vial, rather than into microcentrifuge tubes as a 10:1 ratio of fixative to tissue is recommended. Proper fixation is essential for later histological analysis, as well as immunofluorescence staining of these tissues. It is also important to keep in mind that different knockout strains will have varying responses upon induction of C. rodentium colitis. When infecting a new strain for the first time, mice should be closely monitored to determine a humane end-point for the experiment, if needed. Time points for analysis of epithelial barrier integrity, bacterial loads, and histology can be defined based on the response of the mouse strain of interest to the infection.
As plating of tissue homogenates on LB plates to determine bacterial loads is not an entirely selective method, performing PCR for a C. rodentium specific gene on bacterial colonies can be done to differentiate C. rodentium from other bacterialcolonies on the plate. Another option is to plate tissue homogenates on MacConkey agar, as this medium is more selective for C. rodentium than LB agar. An alternative approach is to use a streptomycin resistant wild-type C. rodentium strain instead.Substitution with a streptomycin resistant strain will result in the induction of the same colitis model described in this protocol. The benefit of using the streptomycin resistant strain is for easier analysis of bacterial loads, as tissue homogenates from these infections can be plated on LB agar supplemented with streptomycin rather than LB agar alone. This avoids potential growth of commensal microbes on the plate, making the CFU counting process easier and more accurate. Another alternative while measuring bacterial loads, is to incubate plates after plating dilutions of tissue homogenates on LB agar at either 37 &deg;C overnight or at room temperature for 2-3 days, resulting in slower bacterial growth, before counting.
This protocol outlines the steps required to produce a robust model of infectious colitis, as well as the methods used to characterize C. rodentium infection in mice. Aside from using this model to examine pathogen-host interactions and immune responses, it can also be used to study how a bacterial infection can increase the risk of colon cancer. This can be done by exposing C. rodentium infected mice to the carcinogen azoxymethane, or by studying how infection impacts on epithelial cell proliferation, or on genes involved in epithelial cell differentiation or tumor development15. Using the infectious colitis model outlined in this protocol, bacterial numbers can also be assessed at extra-intestinal sites, such as the mesenteric lymph nodes, spleen and liver. Higher numbers in these organs may indicate that the mouse strain being tested is highly susceptible to the infection. Using the immunofluorescence staining technique described, a virtually endless number of mechanisms in response to infection can be explored. Once familiar with the techniques detailed here, the protocol can be modified to collect tissues to measure other endpoints, such as for RNA extraction and assessment of gene expression levels, to more thoroughly characterize responses to C. rodentium infection.

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 etiology, characterized by chronic intestinal inflammation and tissue damage. Many mouse models of intestinal inflammation exist, from spontaneous inflammation in genetically modified strains, such as IL10 -/- mice, to chemical challenges with compounds, such as dextran sodium sulfate (DSS) and dinitrobenzene sulfonic acid (DNBS)1. It has been hypothesized that the maladaptive chronic inflammatory responses present in IBD patients develop in genetically susceptible individuals upon abnormal exposure of the intestinal mucosal immune system to enteric bacteria2, therefore the study of models of infectious colitis also offers significant potential for defining potentially pathogenic host responses to enteric bacteria. Citrobacter rodentium induced colitis is one of the rare models of infectious colitis that has been well characterized1,3, allowing for the analysis of host responses to enteric bacteria and further understanding of potential mechanisms of IBD pathogenesis; an essential step in developing novel preventative and therapeutic treatments.
C. rodentium is a gram negative attaching and effacing (A/E), murine specific bacterial pathogen that is closely related to the important human pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)3-8. The family of A/E pathogens intimately attach to the apical host cell membrane of the cecal and colonic epithelium, forming a non-invasive pedestal-like structure on the host cell. Oral challenge with C. rodentium of 108-109 organisms produces a robust model of infectious colitis characterized by colonic hyperplasia or elongation of the crypts, mononuclear immune cell infiltration and goblet cell depletion3,4. The initial site of colonization, a few hours after challenge, is at the cecal patch, followed by progression to the distal colon 2 to 3 days after infection3. In immunocompetent mouse strains, clearance of the pathogen is achieved 3 to 4 weeks after infection1,3,4. However, many genetically modified strains, i.e. gene deficient or knockout (-/-) mice, have been found to display increased susceptibility to infection resulting in exaggerated damage and/or chronic infection and inflammation9-14. Use of this infectious colitis model in these knockout strains, many lacking innate signaling proteins, has been indispensible in revealing several host proteins integral to resolution of intestinal infection and inflammation.

<table><tbody><tr><td>Luria Broth</td><td>ABM</td><td>G247</td><td>Add 25 g of LB powder to 1L of water. Autoclave before using.</td></tr><tr><td>Square bottom plate with grid</td><td>Fisher</td><td>08-757-11A</td><td></td></tr><tr><td>Falcon culture tube</td><td>Sarstedt</td><td>62.515.006</td><td></td></tr><tr><td>Bulb tipped gastric gavage needle</td><td>Fine Science Tools</td><td>18060-20</td><td></td></tr><tr><td>1 ml syringe</td><td>BD Biosciences</td><td>309659</td><td></td></tr><tr><td>4 kDa FITC-dextran</td><td>Sigma</td><td>FD-4</td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C7129</td><td></td></tr><tr><td>Sodium citrate</td><td>Fisher</td><td>S279-500</td><td></td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D16.1</td><td></td></tr><tr><td>Acid citrate dextrose</td><td></td><td></td><td>20 mM ctiric acid, 110 mM sodium citrate, 5 mM dextrose</td></tr><tr><td>Black 96-well plate </td><td>Fisher</td><td>07-200-762</td><td></td></tr><tr><td>Metal beads (5 mm)</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>10% formalin</td><td>Fisher</td><td>5F93-4</td><td></td></tr><tr><td>5 ml vial</td><td>DiaMed</td><td>STK3205</td><td></td></tr><tr><td>Hematoxylin</td><td>Fisher</td><td>H345-23</td><td></td></tr><tr><td>Eosin</td><td>Fisher</td><td>E511-100</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>HC700-1GAL</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P5927</td><td></td></tr><tr><td>Coplin staining jar</td><td>VWR</td><td>47751-792</td><td></td></tr><tr><td>Sodium citrate buffer</td><td></td><td></td><td>10 mM sodium citrate, 0.05% Tween 20, pH 6.0</td></tr><tr><td>Pap pen</td><td>Cedarlane</td><td>Mu22</td><td></td></tr><tr><td>Goat serum</td><td>Sigma</td><td>G902-3</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher</td><td>BP1600-100</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8532</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>SZ002</td><td></td></tr><tr><td>Blocking buffer</td><td></td><td></td><td>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 &amp;amp; store at 4 &amp;deg;C.</td></tr><tr><td>Antibody dilution buffer</td><td></td><td></td><td>0.1% triton X-100, 0.1% BSA, 0.05% sodium azide, 0.04% EDTA</td></tr><tr><td>Blocking buffer &amp;amp; Antibody dilution buffer for tir</td><td></td><td></td><td>Same recipes as above, but without addition of detergents (triton X-100 and tween 20)</td></tr><tr><td>Prolong Gold Antifade Reagent with DAPI</td><td>Invitrogen</td><td>P-36931</td><td></td></tr><tr><td>Coverslips</td><td>Fisher</td><td>12.54SE</td><td></td></tr><tr><td>Benchtop incubation shaker</td><td>Barnstead Lab Line</td><td>Max Q4000</td><td></td></tr><tr><td>Fluorometer</td><td>Perkin Elmer</td><td>Victor2D</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Beckman Coulter</td><td>Microfuge 22R</td><td></td></tr><tr><td>Steamer</td><td>Black Decker</td><td></td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axio Image.Z1</td><td></td></tr></tbody></table>

the citrobacter rodentium mouse model: studying pathogen and host contributions to 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.

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 the extent to which different strains are affected by the infection.
Results presented in Figures 1 through 4 are representative of an infection done using an overnight culture prepared from a frozen glycerol stock. At 7 days post-infection, mice were anesthetized and blood was collected through cardiac puncture. Figure 1 shows FITC-dextran measured in the serum of C57BL/6 mice, as well as from Toll like receptor 2 (TLR2) knockout mice, which have previously been found to exhibit impaired epithelial barrier integrity during infection9. In the presence of an intact intestinal epithelial barrier, 4 kDa FITC-dextran is poorly permeable through this layer. Therefore, increased levels of FITC-dextran in serum suggest an impairment of intestinal epithelial barrier integrity during infection allowing this molecule to leak across. As a control, serum levels in uninfected mice gavaged with FITC-dextran are also presented.
By one week post-infection, bacterial loads in the colon have been found to peak around 109 CFU/g3,4. Bacterial loads can be measured at desired time points post-infection to confirm infection, as well as to assess if different knockout mice suffer from increased or decreased bacterial burdens. As C. rodentium is a luminal pathogen that can intimately adhere to intestinal epithelial cells, bacterial loads in the luminal contents, cecal, and colonic tissues can be measured. Figure 2 shows typical bacterial loads measured at day 7 post-infection cecal and colonic tissues as well as within the intestinal lumen of C57BL/6 mice.
Most of the pathology observed during C. rodentium infection in C57BL/6 mice occurs in the distal 2 cm of the colon11. Macroscopically by day 7 post-infection, a thickening of the colonic mucosa as well as a shortening in length of the colon is observed, with little overt damage seen in C57BL/6 mice. Normally during infection, C57BL/6 mice suffer only moderate inflammation and pathology characterized histologically by immune cell infiltration, elongation of colonic crypts and goblet cell depletion (Figure 3).
As seen in the H&amp;E sections in Figure 3, several host responses are mounted during infection with C. rodentium. To further characterize these changes, immunofluorescence staining can be utilized to examine changes in proteins of interest, such as markers of proliferation, cell death, or innate and adaptive immune responses. Immunofluorescence staining is also valuable in examining aspects of the bacterial response, such as its localization within the infected tissue. An example of this staining technique, examining a host protein ki67 (red), a marker for cell proliferation and the C. rodentium protein tir, green, to examine bacterial localization at day 12 post-infection is presented in Figure 4.
<img src="/files/ftp_upload/50222/50222fig1.jpg" alt="FITC dextran assay bar graph comparing WT and TLR2 -/- at baseline and D7 p.i., ug/ml results." fo:content-width="3.5in" fo:src="/files/ftp_upload/50222/50222fig1highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
 Figure 1. Measurement of serum FITC-dextran to assess epithelial barrier integrity. Mice were gavaged with 4 kDa FITC-dextran under uninfected conditions and at 7 days post-infection, following which serum FITC-dextran levels were determined. C57BL/6 (WT) mice exhibit a negligible increase in FITC-dextran serum levels at 7 days post-infection compared to uninfected conditions. In contrast, TLR2 -/- mice have significantly increased levels of FTIC-dextran in serum compared to baseline suggesting severely impaired barrier integrity in this strain during C. rodentium infection, as has previously been shown.
<img src="/files/ftp_upload/50222/50222fig2.jpg" alt="Bacterial count graph; CFU/g vs lumen, cecum, colon; microbial analysis, data plot, comparative study." fo:content-width="4in" fo:src="/files/ftp_upload/50222/50222fig2highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
 Figure 2. C. rodentium load in lumen, cecum, and colon of infected C57BL/6 mice at 7 days post-infection. Lumen, cecal, and colonic tissues were collected, homogenized, and plated in serial-dilution on LB agar. Each circle represents a single sample collected from individual animals. Solid lines indicate the geometric mean while vertical error bars indicate SEM.
<img src="/files/ftp_upload/50222/50222fig3.jpg" alt="Histological diagram showing intestinal hyperplasia post-infection; microscopy comparison at 100 μm." fo:content-width="5in" fo:src="/files/ftp_upload/50222/50222fig3highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
 Figure 3. Histological analysis of damage caused by C. rodentium infection. By day 7 post-infection moderate immune cell infiltration, as well as elongation of crypts, goblet cell depletion and mild edema is observed compared to control uninfected tissue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50222/50222fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/50222/50222fig4.jpg" alt="Fluorescence microscopy, intestinal tissue, uninfected vs. day 12 post infection comparison." data-altupdatedat="1747911167000" data-alt="Figure 4"> 
 Figure 4. Immunofluorescence staining on uninfected and day 12 post-infection tissues of C57BL/6 mice. Distal colonic tissue is stained for the host protein ki67 (red) and the C. rodentium protein tir (green).

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

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.

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

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

26.4K Views.  BC Children's Hospital. Citrobacter rodentium infectie biedt een waardevol model voor enterische bacteriële infecties te bestuderen, evenals het hosten immuunreacties en colitis bij muizen. Dit protocol beschrijft de meting van de barrière integriteit, pathogeen belasting en histologische schade waardoor de grondige karakterisering van pathogeen en gastheer bijdragen aan murine infectieuze colitis.

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

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Medicine

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Biology

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.

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

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