C. difficile cytotoxin assay:
A cytotoxicity assay was performed as previously described with the following modifications (25). Briefly, ATCC CCL-81 Vero cells were grown to confluence in Dulbecco modified Eagle medium (Gibco catalog number 11965) supplemented with 10% fetal bovine serum (catalog number 16140) and 1% penicillin-streptomycin (Gibco catalog number 15070). Cells were plated to a density of 10 5 cells/well. Mouse cecal content was diluted 1:10 in sterile PBS, passed through a 0.22- m filter, and serially diluted to 10:6. Filtered samples were tested in duplicate with a corresponding control to which both antitoxin (Techlab catalog number T5000) and sample were added. A positive control of C. difficile TcdA (List Biologicals catalog number 152C) was used. Samples were incubated overnight at 37°C, and the cytotoxic titer was determined as the reciprocal of the highest dilution that produced 80% cell rounding.