Materials and Reagents

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

Equipment

Animal feeding needles (gavage needles)
1 ml syringe
Dedicated BSL2 animal facility
Scale

Procedure

Weigh each hamster.
Gavage 100 μl clindamycin to each hamster (30 mg clindamycin / kg body weight).
Note: Dilute the stock clindamycin to the appropriate concentration using DMEM.
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.
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.
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

Antonopoulos, D. A., Huse, S. M., Morrison, H. G., Schmidt, T. M., Sogin, M. L. and Young, V. B. (2009). Reproducible community dynamics of the gastrointestinal microbiota following antibiotic perturbation. Infect Immun 77(6): 2367-2375.
Aubry, A., Hussack, G., Chen, W., KuoLee, R., Twine, S. M., Fulton, K. M., Foote, S., Carrillo, C. D., Tanha, J. and Logan, S. M. (2012). Modulation of toxin production by the flagellar regulon in Clostridium difficile. Infect Immun 80(10): 3521-3532.
Bartlett, J. G., Chang, T. W., Moon, N. and Onderdonk, A. B. (1978). Antibiotic-induced lethal enterocolitis in hamsters: studies with eleven agents and evidence to support the pathogenic role of toxin-producing Clostridia. Am J Vet Res 39(9): 1525-1530.
Carter, G. P., Douce, G. R., Govind, R., Howarth, P. M., Mackin, K. E., Spencer, J., Buckley, A. M., Antunes, A., Kotsanas, D., Jenkin, G. A., Dupuy, B., Rood, J. I. and Lyras, D. (2011). The anti-sigma factor TcdC modulates hypervirulence in an epidemic BI/NAP1/027 clinical isolate of Clostridium difficile. PLoS Pathog 7(10): e1002317.
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.
Kuehne, S. A., Cartman, S. T., Heap, J. T., Kelly, M. L., Cockayne, A. and Minton, N. P. (2010). The role of toxin A and toxin B in Clostridium difficile infection. Nature 467(7316): 711-713.
Lyras, D., O'Connor, J. R., Howarth, P. M., Sambol, S. P., Carter, G. P., Phumoonna, T., Poon, R., Adams, V., Vedantam, G., Johnson, S., Gerding, D. N. and Rood, J. I. (2009). Toxin B is essential for virulence of Clostridium difficile. Nature 458(7242): 1176-1179.
McFarland, L. V. (2008). Update on the changing epidemiology of Clostridium difficile-associated disease. Nat Clin Pract Gastroenterol Hepatol 5(1): 40-48.
Sambol, S. P., Tang, J. K., Merrigan, M. M., Johnson, S. and Gerding, D. N. (2001). Infection of hamsters with epidemiologically important strains of Clostridium difficile. J Infect Dis 183(12): 1760-1766.
Voth, D. E. and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 18(2): 247-263.
Wilson, K. H. and Perini, F. (1988). Role of competition for nutrients in suppression of Clostridium difficile by the colonic microflora. Infect Immun 56(10): 2610-2614.