the toxin a- and toxin b - specific recombination vectors pjir3051 and pjir3050 were constructed by cloning a 566 bp tcda ( accession m30307 ) pcr product ( nt 10711637 ) and the equivalent tcdb ( accession x53138 ) region , respectively , into the shuttle vector pjir1456 ( accession u90554 ) . these plasmids were transferred by conjugation to c. difficile strain jir8094 and the resultant chromosomal mutants analyzed by pcr , southern blotting and plasmid rescue13 . filtered supernatants of 2ty broth cultures ( 72 h ) were used for western blots , cytotoxicity and toxin neutralization assays . serial two - fold dilutions of these supernatants were used in vero and ht-29 cell cytotoxicity assays , with the end - point scored as the last dilution yielding complete cpe . toxins were neutralized with affinity purified polyclonal antibodies specific for toxin a and toxin b ; neutralization was carried out at the last dilution giving complete cpe . partially purified ammonium sulphate - precipitated toxin proteins were western blotted using toxin a - specific affinity purified polyclonal goat antibodies . clindamycin was administered orogastrically to render the hamsters susceptible to infection ( day zero ) . on day fecal pellets were collected daily for 12 days , then weekly until day 30 and inoculated onto selective tccfa plates to monitor colonization . fecal pellets were analyzed for the presence of toxin a using a toxin a - specific elisa and for the presence of toxin b using a human foreskin fibroblast cytotoxicity assay . full methods and any associated references are available in the online version of the paper at www.nature.com/nature .