Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.
Animals ; Antibodies, Bacterial/blood/*immunology/therapeutic use ; Bacterial Proteins/immunology/therapeutic use ; Bacterial Toxins/immunology/therapeutic use ; Clostridium Infections/microbiology/prevention & control/*veterinary ; Clostridium difficile/*immunology ; Enterotoxins/immunology/therapeutic use ; Female ; Horse Diseases/microbiology/*prevention & control ; Horses ; Immune Sera/*immunology ; Immunization, Passive/*veterinary ; Mice ; Mice, Inbred C57BL ; Spores, Bacterial/immunology

BACKGROUND: Enzyme immunoassay (EIA) capable of detecting both toxin A and toxin B is strongly recommended for the diagnosis of Clostridium difficile associated disease. Therefore, we evaluated two different EIAs for the detection of C. difficile toxin A/B. METHODS: For a total of 228 stool specimens we performed bacteriologic cultures for C. difficile and examined for toxin A and toxin B using enzyme linked fluorescent immunoassay (ELFA; VIDAS CDAB, Bio-Merieux sa, France) and ELISA (C.DIFFICILE TOX A/B II, TECHLAB, USA). We also performed PCR assays for toxin A and B genes in 117 C. difficile isolates that grew from the stool cultures and compared the results with those obtained with the two different EIAs. RESULTS: The concordance rate between ELFA and ELISA was 85.5% (195/228). Using the culture and PCR results as the standard, the sensitivity/specificity of the ELFA and ELISA were 65.0%/72.1% and 71.8%/70.3%, and for positive/negative predictive values were 78.4%/69.6% and 71.8%/70.3%, respectively (P value >0.05). No differences were observed between the results of ELFA and ELISA with toxin A- toxin B+ strains of C. difficile. CONCLUSIONS: The sensitivity of the ELISA was slightly higher than that of ELFA for toxin A and toxin B, but the specificity and positive predictive value of the ELFA were rather higher than those of the ELISA, although no statistical differences were observed. A bacteriologic culture and PCR assay for toxin genes are recommended in case the both EIAs are negative.
Bacterial Proteins/*analysis/genetics/immunology ; Bacterial Toxins/*analysis/genetics/immunology ; Clostridium difficile/genetics/isolation & purification/*metabolism ; Enterotoxins/*analysis/genetics/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Feces/microbiology ; Fluorescent Dyes/chemistry ; Humans ; Reagent Kits, Diagnostic

The incidence and severity of Clostridium difficile infection (CDI) has increased over the past decades. It is related to the emergence of hypervirulent strains and increased use of antibiotics. The incidence of refractory CDI to standard therapies and the risk for recurrent CDI are also increasing. Current guidelines recommend the first recurrence to be treated with the same agent used for the initial episode. However, data are lacking to support any particular treatment strategy for severe refractory CDI or cases with multiple recurrence. Treatments currently available for CDI are inadequate to prevent recurrence. Widely used method for managing a subsequent recurrence involves tapering followed by pulsed doses of vancomycin. Other potentially effective strategies for recurrent CDI are use of other antibiotics such as fidaxomicin, nitazoxanide, rifaximin, tigecycline, and teicoplanin. There are efforts to recover gut microflora and to optimize immune response to CDI. These include use of probiotics, fecal microbiota transplantation, intravenous immunoglobulin, monoclonal antibodies directed against C. difficile toxins, and active vaccination. However treatment of patients with refractory CDI and those with multiple CDI recurrences is based on limited clinical evidence, and there is an ongoing need for continued research to improve the outcomes these patients.
Anti-Bacterial Agents/therapeutic use ; Antibodies, Monoclonal/immunology/therapeutic use ; Clostridium difficile/drug effects/pathogenicity ; Enterocolitis, Pseudomembranous/*drug therapy ; Humans ; Immunoglobulins, Intravenous/therapeutic use ; Probiotics/therapeutic use ; Recurrence ; Vancomycin/therapeutic use