Establishing a Mouse Model of Clostridium difficile Infection

Abstract

Source: Winston, J. A., et al. Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291. J. Vis. Exp. (2016)

This video demonstrates the procedure for inoculating antibiotic-pretreated mice with Clostridium difficile spores to establish a murine model of infection for studying pathogenesis and host responses.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of the Clostridium difficile Spore Inoculum and the Oral Gavage of the Mice

NOTE: Mice challenged with C. difficile spores should be housed in a Biosafety Level 2 animal facility.

1. Obtain a C. difficile spore stock of the desired strain (in this case, R20291) that was prepared and stored at 4 °C in sterile water. Determine the enumeration of this spore stock prior to use.
2. Based on the known concentration (spores/ml) of the spore stock, calculate the desired dilution to obtain 10⁵ spores per 25 µl. Ensure the inoculum volume is adequate to inoculate all mice in the experiment (25 µl per mouse), to perform the enumeration of the inoculum (approximately 30 µl), and to account for pipetting error.
3. Vortex the original C. difficile spore stock. Then, resuspend the calculated volume (from step 1.2) of the original C. difficile spore stock in sterile water within a screw-capped microcentrifuge tube. Perform this aerobically in a laminar flow hood. Identify this diluted mixture as the "spore inoculum."
4. Place the spore inoculum into a 65 °C water bath and heat it for 20 min.
   NOTE: This step will ensure that only active C. difficile spores remain after the heat treatment.
5. Within the laminar flow hood, take 50 µl of heated spore inoculum, place it in a sterile microcentrifuge tube, and pass it into the anaerobic chamber. Use this sample for spore enumeration.
6. Within the laminar flow hood, load the remaining heated spore inoculum into a 1ml syringe attached to a sterile bulb-tipped gastric gavage needle. Utilize a manual syringe stepper to ensure accurate and rapid repeat dosing between mice. Ensure that the gavage needle is filled with the heated spore inoculum prior to use.
   NOTE: A new sterile gavage needle for each mouse is not required. However, if various doses of C. difficile spores are administered, a new gavage needle is recommended for each dose.
   NOTE: C. difficile spores are highly resistant to traditional disinfectants. Therefore, the use of a sporicidal disinfectant, such as #62 Perisept Sporicidal Disinfectant, is necessary. The manufacturer recommends a 2 min contact time to destroy C. difficile spores.
7. Gavage each mouse with 25 µl of the spore inoculum. Restrain each mouse gently by grasping the animal by the loose skin of the neck and back in order to immobilize the head. Using the index finger further immobilizes the animal's head and to elongate the esophagus. Ensure that the animal does not vocalize or show other signs of distress during restraint.
   NOTE: The total volume given to mice by gavage should not exceed 10 ml/kg bodyweight (0.1 ml/10 g).
8. Maintain the mouse in an upright, vertical position and gently pass the gavage needle into the side of the mouth. Direct the gavage needle along the roof of the mouth and slowly advance the gavage needle into the esophagus.
   NOTE: If any resistance is encountered, the gavage needle may be entering into the trachea and thus should NOT be advanced, but rather removed and repositioned immediately.
9. After the gavage needle is approximately halfway into the esophagus, inject 25 µl of the heated spore inoculum and gently withdraw the gavage needle from the esophagus.
   NOTE: Forceful advancement of the gavage needle may lead to the rupture of the esophagus or stomach. Therefore, if resistance is encountered when advancing the gavage needle, it is best to immediately remove and reposition the gavage needle.
10. Return the animal to its cage and observe it for any coughing or signs of respiratory distress, as this may indicate inadvertent tracheal administration or aspiration of the heated spore inoculum.
    NOTE: Mice are extremely susceptible to C. difficile following antibiotic treatment; therefore, precautions to prevent cross-contamination between cages is essential. This is especially important when managing antibiotic-treated but unchallenged mice as controls.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#62 Perisept Sporidicial Disinfectant Cleaner	SSS Navigator	48027	This product will require dilution as recommended by the manufacturer
C57BL/6J Mice	The Jackson Laboratory	664	Mice should be 5 - 8 weeks of age
2 ml Micro Centrifuge Screw Cap	Corning	430917