Herein we present evidence for the therapeutic potential of colonization factor (CF)-specific egg yolk antibodies (IgY) for potentially treating acute and recurring Clostridium difficile infection (CDI) in humans. The study involved cloning, expressing as 6¾His-tagged proteins in Escherichia coli, and Ni-affinity purifying three previously identified CFs (FliC, FliD and Cwp84) from C. difficile. The recombinant CF antigens were then used to immunize Leghorn chickens and CF-specific IgY antibodies were prepared from their eggs. The specificity and titre of the resulting C. difficile CF-specific IgY antibodies were assessed by ELISA and Western immunoblotting techniques. The antibodies were also screened for their ability to inhibit C. difficile adherence to human colon-derived T84 cells, and, based on these findings, one of them (FliD-specific IgY) was evaluated for its potential to prevent C. difficile-mediated morbidity and mortality in Syrian hamsters. The results revealed that purified FliD-specific IgY significantly protected hamsters from C. difficile strain 630 infection relative to control animals treated with carbonate buffer alone or IgY produced from unimmunized chicken eggs. The results suggest that egg yolk preparations obtained from chickens immunized with recombinant C. difficile CFs may represent another safe and cost-effective treatment option in humans suffering from acute or recurring CDI. INTRODUCTIONConventional antibiotics alter both the numbers and complexity of the normal gastrointestinal (GI) microflora, thereby reducing the ability of these protective organisms to competitively exclude opportunists from colonizing the GI tract. This disruption may render hospitalized subjects susceptible to colonization by C. difficile (Job & Jacobs, 1997). Once established in the colon, C. difficile expresses two very large exotoxins, toxin A (TcdA) and toxin B (TcdB), which are primarily responsible for the clinical symptoms of C. difficile infection (CDI) (Voth & Ballard, 2005). These range from diarrhoea to pseudomembranous colitis, toxic megacolon and multi-system organ failure, which may be fatal.The accepted approach to treating CDI is to replace the antibiotic responsible for initiating the condition with one, typically metronidazole, to which C. difficile is sensitive. In about 80 % of cases, this approach leads to complete recovery without further complications. In the remaining cases, however, recurring CDI (rCDI) can occur, due, presumably, to a failure of the normal GI microflora to become re-established after the metronidazole is discontinued. This leaves the colon of these individuals susceptible to C. difficile relapse or reinfection. Multiple recurrences are not unusual, resulting in a significant reduction in the quality of life in these individuals.In response to the increasing risk of CDI to human health, much research has focused on new and improved treatment regimens, particularly in subjects suffering from rCDI. These studies have concentrated on novel toxin neutralizing agents, an...

Transgenic C57BL/6 mice expressing human serum amyloid P component (HuSAP) are resistant to Shiga toxin 2 (Stx2) at dosages that are lethal in HuSAP-negative wild-type mice. However, it is well established that Stx2 initiates extra-intestinal complications such as the haemolytic-uremic syndrome despite the presence of HuSAP in human sera. We now demonstrate that co-administering purified Escherichia coli O55 lipopolysaccharide (LPS), at a dosage of 300 ng/g body weight, to HuSAP-transgenic mice increases their susceptibility to the lethal effects of Stx2. The enhanced susceptibility to Stx2 correlated with an increased expression of genes encoding the pro-inflammatory cytokine TNFα and chemokines of the CXC and CC families in the kidneys of LPS-treated mice, 48 hours after the Stx2/LPS challenge. Co-administering the glucocorticoid dexamethasone, but not the LPS neutralizing cationic peptide LL-37, protected LPS-sensitized HuSAP-transgenic mice from lethal doses of Stx2. Dexamethasone protection was specifically associated with decreased expression of the same inflammatory mediators (CXC and CC-type chemokines and TNFα) linked to enhanced susceptibility caused by LPS. The studies reveal further details about the complex cascade of host-related events that are initiated by Stx2 as well as establish a new animal model system in which to investigate strategies for diminishing serious Stx2-mediated complications in humans infected with enterohemorrhagic E. coli strains.