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Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenesswine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS‐polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L‐cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coliby means of ZAP Express vector. Analysis by SDS‐polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coliis the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenesswine isolate differed only slightly (97.6% identity) from the sequence of plogene from A. pyogenesstrain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100–6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol‐activated cytolysins (cholesterol‐binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenesseems to be a novel member of the thiol‐activated cytolysin family.

Two strains of Staphylococcus aureuswere examined for behavior in the cutaneous tissue of mice by the fluorescent antibody technique, hematoxylin and eosin staining. When about 108viable cells of an α‐hemolysin‐producing strain (Wood 46) were inoculated subcutaneously into a mouse, they multiplied in the subcutaneous tissue of the mouse and gradually entered the corium to produce α‐hemolysin and nuclease. Edematous and necrotic lesions were observed in the cutaneous tissue where the organisms had multiplied. When 108viable cells of a β‐hemolysin‐producing strain (Kitami 3–9D) were inoculated into a mouse, they multiplied within a narrow extent surrounded mainly by infiltrating leukocytes and produced mainly β‐hemolysin. The changes of cutaneous tissue were weaker in mice inoculated with Kitami 3‐9D strain than in mice inoculated with strain Wood 46. When 106viable cells of both strains were inoculated into mice, they were phagocytized by leukocytes. Neither multiplication of organisms nor production of any active extracellular substance was observed in these mice. Edema, degeneration, and necrosis were also noticed in the cutaneous tissue of mice inoculated with α‐ and β‐hemolysin. In addition, the infiltration of leukocytes was inhibited mainly by α‐hemolysin.