Virulence Studies of Clostridium difficile    

Materials and Reagents

1. Institutional Animal Care and Use Committee (IACUC)-approved animal use protocol
   
2. C. difficile spores
   
3. Female Syrian golden hamsters (80 g–120 g)
   
4. Clindamycin Injection, USP (150 mg/ml) (Hospira, catalog number: 0409-4052 )
   
5. Dulbecco’s Modified Eagle Medium (DMEM)
   

Equipment

1. Animal feeding needles (gavage needles)
   
2. 1 ml syringe
   
3. Dedicated BSL2 animal facility
   
4. Scale
   

Procedure

1. Weigh each hamster.
   
2. Gavage 100 μl clindamycin to each hamster (30 mg clindamycin / kg body weight).
   Note: Dilute the stock clindamycin to the appropriate concentration using DMEM.
   
3. After 5 days, gavage 100 μl C. difficile spores.
   Notes:
   
   1. Depending on the strain of C. difficile used, the infectious dose can vary. However, in several strains, inoculation with approximately 100 spores will result in lethal disease.
      
   2. There are several methods to ensure reproducible infectious doses [e.g. suspending spores in phosphate buffered saline (PBS)]. Our laboratory uses Teflon-coated tubes to reduce spore adherence. Other laboratories can get reproducible infectious doses in water.
      
4. Closely monitor animals for signs of infection including: wet tail, poor fur coat, lethargy, loss of 15% body weight.
   Note: Signs of disease are commonly observed in 2 – 4 days post infection. Be sure to closely monitor the animals for the above symptoms. Moribund animals can succumb to disease quickly.
   
5. Humanly euthanize moribund animals, in accordance with your protocol that was approved by your Institutional Care and Animal Use Committee (IACUC). Score the time required to reach a moribund state.
   

Acknowledgments

We would like to thank Charlotte Allen and Ritu Shrestha for aiding in the development of this protocol. This protocol is based upon previously published C. difficile virulence studies by Sambol et al. (2001), wherein the authors analyzed the infection of hamsters by epidemiologically important C. difficile isolates. This work was supported by the American Heart Association National Scientist Development grant to JAS (No. 11SDG7160013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

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1. Francis, M. B. and Sorg, J. A. (2013). Virulence Studies of Clostridium difficile. Bio-protocol 3(24): e1002. DOI: 10.21769/BioProtoc.1002.
   
2. Francis, M. B., Allen, C. A., Shrestha, R. and Sorg, J. A. (2013). Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5): e1003356.