Survival of Clostridium Difficile and Its Toxins in Equine Feces: Implications for Diagnostic Test Selection and Interpretation

Abstract

Although Clostridium difficile is recognized as a cause of enterocolitis in horses and humans, there has been little work published regarding the lability of C. difficile and its toxins in feces. A significant decrease in recovery of C. difficile from inoculated equine fecal samples occurred during storage. Recovery after storage in air at 4 degrees C decreased from 76% (37/49) after 24 hours to 67% (33/49) at 48 hours and 29% (14/ 49) after 72 hours. In contrast to aerobic storage, 25 of 26 samples stored anaerobically at 4 degrees C yielded growth of C. difficile for 30 days, whereas the organism was only detected for 2.5 +/- 2.52 days (x +/- SD) in paired samples stored aerobically. The use of an anaerobic transport medium was effective in maintaining viability of C. difficile. These findings indicate that poor aerotolerance is the reason for the rapid decrease in culture yield. In contrast to C. difficile organisms stored aerobically at 4 degrees C, C. difficile toxins were considerably more stable and could be detected by enzyme-linked immunosorbent assay in both broth and inoculated fecal samples for at least 30 days. The poor survival of C. difficile but the stability of its toxins when feces are stored aerobically must be considered when submitting samples for diagnosis of C. difficile-associated enterocolitis in horses and when interpreting laboratory results.

A comparison of Clostridium difficile diagnostic methods for identification of local strains in a South African centre

“…However, the assays in the current study were conducted according to the manufacturers' specifications. In addition, previous studies examining stool samples from animals observed no toxin degradation during storage at 220 uC (Keessen et al, 2011;Weese et al, 2000). Another possibility is that the toxins were diluted by the increased volume of stool produced during diarrhoea.…”

Diagnosis and treatment of non-infectious and infectious foal diarrhoea

“…Exclusion of enrolment of the organism is equally challenging as sample handling requires meticulous attention. Survival of C. difficile rapidly declines within 48-72h if samples, stored at 4°C, are exposed to air (Weese et al 2000). Although the toxins are more resistant and can be recovered even from samples stored aerobically at 4°C for at least 30d, a single toxin negative sample, particularly if taken early in the disease process may not be sufficient to rule involvement of toxigenic C. difficile out (Arroyo et al 2004, Weese et al 2000.…”

“…Survival of C. difficile rapidly declines within 48-72h if samples, stored at 4°C, are exposed to air (Weese et al 2000). Although the toxins are more resistant and can be recovered even from samples stored aerobically at 4°C for at least 30d, a single toxin negative sample, particularly if taken early in the disease process may not be sufficient to rule involvement of toxigenic C. difficile out (Arroyo et al 2004, Weese et al 2000. Equally, culture and toxin may rapidly become negative within 48-72h after initiation of treatment with metronidazole (Arroyo et al 2004).…”

Free fecal water in the horse – an unsolved problem

“…Die bakterielle Kultivierung des C. difficile gestaltet sich schwieriger und Untersuchungen mehrerer Kotproben können hierfür nötig werden (Jones 1989, Marler et al 1992). Die Detektion der Toxine A und B über Immunassays, zellzytotoxische Assays oder eine PCR, zum Nachweis der für die Toxinbildung verantwortlichen Gene des Bakteriums, stellen sensitivere Methoden dar (Jones 2000, Weese et al 2000, Jones 1989). (Mackenthun et al 2013).…”