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>

<ol>
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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 ...

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...

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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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JoVE Journal

Immunology and Infection

26.3K 次观看.  BC Children's Hospital. 枸橼酸rodentium感染提供了一个有价值的模型来研究肠道细菌感染以及宿主的免疫反应和结肠炎的小鼠。该协议概述了屏障的完整性测量，病原体负荷和病理损害，从而为深入鉴定病原体和宿主小鼠的感染性肠炎的贡献。

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

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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 ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...