A New Purification Method for Beta-Toxin of Clostridium perfringens Type C Vaccinal Strain

Abstract

Background: Clostridium perfringens type C strains cause severe necrotizing enteritis in sheep, calves, goats, pigs and humans. Beta-toxin is introduced to be the essential virulence factor of this microorganism. In the present study, a new method was established for the purification of beta toxin from culture supernatant fluid of C. perfringens type C vaccinal strain. Methods: The four steps of the purification scheme involved ammonium sulfate precipitation, cation exchange chromatography (CM- Sepharose), anoion exchange chromatography (DEAECellulose) and gel filtration (Sephadex G-100). Results: Beta toxin was purified about 78-fold from the Sephadex G-100 column with a yield of about 16.7% in terms of lethality of the toxin. The molecular weight of the beta toxin as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 37 KD. The LD50 for adult mice was 2.21 μg/kg. Human umbilical vein endothelial cells (HUVEC) exposed to CPB showed a cell border retraction, cytoplasmic blebbing, cell shrinkage and cell rounding. The toxin was heat labile and was inactivated by trypsin. Conclusions: In conclusion, the results of this study showed the new protocol is suitable for purification of beta toxin of C. perfringens type C regarding good purity, good yields and high activity of beta toxin.

Keywords

• Clostridium
• perfringens
• type C
• Beta-toxin
• Enterotoxemias
• chromatography