IDENTIFICATION AND PARTIAL CHARACTERIZATION OF AN ACTINOMYCES-PYOGENES HEMOLYSIN

Twenty-two Actinomyces pyogenes isolates were recovered from hepatic a bscesses in cattle and evaluated for hemolysin production. Hemolysin w as collected from supernatant of cultures grown in 6% CO2 in brain hea rt infusion (BHI) broth. The effect of oxidizing and reducing agents, enzymes, temperatures and pH on hemolytic activity were studied using sheep erythrocytes as the target cells. Our study showed that A. pyoge nes hemolysin is oxygen stable; sensitive to treatment by protease, tr ypsin, and amylase; and destroyed by treatment at extreme temperatures (56 and 100 degrees C) and pH (pH 3 and 11). Production of hemolysin was studied in BHI, RPMI-1640, and a defined serum-free A. pyogenes me dium under aerobic and anaerobic conditions. Maximum hemolysin was pro duced in BHI incubated aerobically in 6% CO2 and to a lesser degree an aerobically in RPMI-1640. No hemolysin was produced in the defined A. pyogenes medium. Differential filtration, isoelectric focusing and sod ium dodecyl sulfate polyacrylamide gel electrophoresis identified two hemolysin proteins with pI values of 3.40 and 9.45 and estimated molec ular masses of 62 and 58 kDa, respectively. Cell-free supernatant samp les positive for hemolysin activity also were screened for leukotoxin activity. Significant levels of leukotoxin were detected in all sample s screened.