Name: the citrobacter rodentium mouse model: studying pathogen and host contributions to infectious colitis
Uploaded: 2013-02-19T13:00:00
Description: Watch this Scientific Journal Video about The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis at JoVE.com

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 techni...

<ol>
	<li>Nell, S., Seurbaum, S., Josenhans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+microbiota+on+the+pathogenesis+of+IBD:+lessons+from+mouse+infection+models.">The impact of microbiota on the pathogenesis of IBD: lessons from mouse infection models.</a> Nature Reviews Microbiology. 8, 564-577 (2010).</li><li>Cario, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+Receptors+in+Inflammatory+Bowel+Diseases:+A+Decade+Later.">Toll-like Receptors in Inflammatory Bowel Diseases: A Decade Later.</a> Inflammatory Bowel Diseases. 16 (9), 1583-1597 (2010).</li><li>Eckmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Inflammatory+Bowel+Disease.">Animal Models of Inflammatory Bowel Disease.</a> Annals New York Academy of Sciences. , 1027-38 (2006).</li><li>Mundy, R., MacDonald, T. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+host+responses+to+enteric+bacterial+pathogens:+a+balancing+act+between+resistance+and+tolerance.">Innate host responses to enteric bacterial pathogens: a balancing act between resistance and tolerance.</a> Cellular Microbiology. 14, 475-484 (2012).</li><li>MacDonald, T. T., Frankel, G., Dougan, G., Goncalves, N. S., Simmons, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+defences+to+Citrobacter+rodentium.">Host defences to Citrobacter rodentium.</a> International Journal of Medical Microbiology. 293, 87-93 (2003).</li><li>Borenshtein, D., McBee, M. E., Schauer, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+the+Citrobacter+rodentium+infection+model+in+laboratory+mice.">Utility of the Citrobacter rodentium infection model in laboratory mice.</a> Current Opinion in Gastroenterology. 24, 32-37 (2008).</li><li>Gibson, D. L., Ma, C., Rosenberger, C. M., Bergstrom, K. S. B., Valdez, Y., Huang, J. T., Khan, M. A., Vallance, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+2+plays+a+critical+role+in+maintaining+mucosal+integrity+during+Citrobacter+rodentium-induced+colitis.">Toll-like receptor 2 plays a critical role in maintaining mucosal integrity during Citrobacter rodentium-induced colitis.</a> Cellular Microbiology. 10, 388-403 (2008).</li><li>Dennis, A., Kudo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p50+subunit+of+NF-&#954;B+is+critical+for+in+vivo+clearance+of+the+non-invasive+enteric+pathogen+Citrobacter+rodentium.">The p50 subunit of NF-&#954;B is critical for in vivo clearance of the non-invasive enteric pathogen Citrobacter rodentium.</a> Infection &amp; Immunity. 76 (11), 4978-4988 (2008).</li><li>Gibson, D. L., Ma, C., Bergstrom, K. S. B., Huang, J. T., Man, C., Vallance, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyD88+signalling+plays+a+critical+role+in+host+defence+by+controlling+pathogen+burden+and+promoting+epithelial+cell+homeostasis+during+Citrobacter+rodentium-induced+colitis.">MyD88 signalling plays a critical role in host defence by controlling pathogen burden and promoting epithelial cell homeostasis during Citrobacter rodentium-induced colitis.</a> Cellular Microbiology. 10 (3), 618-631 (2008).</li><li>Lebeis, S. L., Bommarius, B., Parkos, C. A., Sherman, M. A., Kalman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+signaling+mediated+by+MyD88+is+required+for+a+protective+innate+immune+response+by+neutrophils+to+Citrobacter+rodentium.">TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium.</a> Journal of Immunology. 179 (1), 566-577 (2007).</li><li>Bry, L., Brenner, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+T+cell+dependent+serum+antibody,+but+not+the+gut-associated+lymphoid+tissue,+for+surviving+acute+mucosal+infection+with+Citrobacter+rodentium,+an+attaching+and+effacing+pathogen.">Critical role of T cell dependent serum antibody, but not the gut-associated lymphoid tissue, for surviving acute mucosal infection with Citrobacter rodentium, an attaching and effacing pathogen.</a> Journal of Immunology. 172 (1), 433-441 (2004).</li><li>Simmons, C. P., Clare, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+for+B+lymphocytes+and+CD4++T+cells+in+immunity+to+infection+by+the+attaching+and+effacing+pathogen+Citrobacter+rodentium.">Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium.</a> Infection &amp; Immunity. 71 (9), 5077-5086 (2003).</li><li>Ahmed, I., Chandrakesan, P., Tawfik, O., Xia, L., Anant, S., Umar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Roles+of+Notch+and+Wnt/&#946;-Catenin+Pathways+in+the+Regulation+of+Hyperplasia+and/or+Colitis+in+Response+to+Bacterial+Infection.">Critical Roles of Notch and Wnt/&#946;-Catenin Pathways in the Regulation of Hyperplasia and/or Colitis in Response to Bacterial Infection.</a> Infection &amp; Immunity. 80 (9), 3107-3121 (2012).</li></ol>

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 ...

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 border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <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; store at 4 &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; 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 &amp; 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 &mu;l 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...

Watch this Scientific Journal Video about The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis at JoVE.com

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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