Freezing or adding trypsin inhibitor to equine intestinal contents extends the lifespan of Clostridium perfringens beta toxin for diagnostic purposes

摘要

Clostridium perfringens type C causes necrotizing enteritis mostly in neonatal animals of several species, including horses. The virulence of C. perfringens type C is mostly mediated by beta toxin (CPB). This toxin is highly sensitive to the action of trypsin and other proteases, which explains the increased susceptibility of neonatal animals to type C infections. Final confirmation of type C disease diagnosis should be based on detection of CPB in the intestinal content of affected animals. However, because CPB is so sensitive to the action of proteases, it is believed that this toxin persists for only a limited period of time in specimens of intestinal content of animals collected for diagnostic purposes. This study was therefore performed to determine the stability of CPB in intestinal content of horses stored at different temperatures and to evaluate the use of trypsin inhibitor to extend the lifespan of CPB in intestinal content of horses. When the intestinal content of horses that had been spiked with different amounts of CPB was tested by a capture ELISA technique to detect CPB, 319 LD 50 of CPB per milliliter was the lowest amount that could be detected. When equine intestinal content spiked with 319 LD 50/ml was stored at 4 °C, CPB was detected by ELISA until day 8 after spiking. Samples spiked with the same amount of CPB and stored at -20 °C were positive for at least 5 weeks after spiking. When intestinal samples spiked with 319 LD 50/ml of CPB were mixed with 0.1 mg/ml or 1.0 mg/ml of trypsin inhibitor and stored at 4 °C, all the samples were positive for at least 5 weeks after spiking. This study demonstrates that C. perfringens CPB present in equine intestinal samples stored at 4 °C cannot be detected by ELISA for more than 8 days. Freezing the samples at -20 °C or adding trypsin inhibitor before storage at 4 °C preserves the lifespan of CPB for at least 5 weeks.