Summary A haemolysin produced by Actinomyces pyogenes ATCC 8164 was purified from culture supernatant by ammonium sulphate and polyethylene glycol precipitation, ion‐exchange chromatography on DEAE‐Sephacel, and fast‐protein‐liquid‐chromatography on Superose 12 prep grade. The purified haemolysin, designated as pyolysin, displayed a single band on poly‐acrylamide gel electrophoresis, indicating a molecular weight of 55000. Additionally, using gel filtration, the same molecular weight was estimated. Further studies of the eluate of ion‐exchange chromatography using isoelectric focusing also revealed a single protein band at pH 9.38 with haemolytic activity. A specific antiserum produced against pyolysin inhibited the haemolytic activity. The purity of the isolated protein was also determined by Western Blot analysis with antiserum obtained from a cow inoculated with culture supernatant from A. pyogenes and Peptococcus indolicus. The isolated pyolysin appeared to be heat‐labile and displayed cytotoxic effects on poly‐morphonuclear leucocytes and on pTK2 kidney cells.

The present study was designed to evaluate the accuracy of the API Coryne test system for identification of Actinomyces pyogenes. The test system correctly identified 36 of 42 A.pyogenes and 4 of 5 comparatively studied Arcunobacterzum huemolyticum-cultures. The biochemical profiles of the remaining 6 A.pyogenes-and 1 A. huemo[yticum-cultures were not included in the analytical profile index. None of the cultures were misidentified. According to the API database (ATB Plus V 1.5.4.) the unidentified cultures could be correctly identified as A.pyogenes and A. huemolyticurn respectively. A greater repertoire of A.pyogenes specific biochemical profiles incorporated into the analytical profile index would improve the applicability of this test system for veterinary diagnostics. 22. SPIRENSEN, G. H., 1978: Studies on the occurrence of Peptococcus indolicus and Corynebacterium pyogenes in abscesses in swine, and on the occurrence of Pc. indolicus in apparently normal skin and mucous membranes of piglets. Nord. Veterinaermed. 30, 282-285. 23. VEGA, L. E., and T. L. GAVAN, 1970: Corynebacteriumpyogenesa pathogen in man. Report of a case. Cleve. Clin. Q. 37, 207-214. 37, 593-600.

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This study was designed to further characterize the fibrinogen-binding properties of Actinornyces pyogenes. The fibrinogen-binding capacities of a selected A. pyogenes culture could be significantly enhanced by heat pretreatment (60 "C, 1 h) of the bacteria. The fibrinogen-binding site seemed to be a protein which was specific for fibrinogen. In phagocytosis studies, binding of fibrinogen to A. pyogenes significantly increased the phagocytic capacity of bovine polyrnorphonuclear leucocytes. ;i. Corresponding author U.S. Copyright Clearance Center Code Statement: 0931 -1793/94/4109 -0588$10.50/0 Fibrinogen-binding Properties of Actinornyces Pyogenes 589 Material and Methods Bacterial cultureThe A . pyogenes culture used in this study (A. pyogenes 103) has been described previously (LAMMLER, 1990). This culture, originally isolated from porcine brain has been characterized biochemically and serologically (LAMMLER and BLOBEL, 1988;DING and LAMMLER, 1992). The A. pyogenes culture was grown microaerobically on Columbia blood-agar plates (Oxoid, Wesel, Germany) containing 5 % sheep blood, or in Todd Hewitt Broth (THB, Gibco, Karlsruhe, Germany) for 48 h at 37 "C in a candle jar. For comparative purposes, the fibrinogen-binding group A streptococcal culture Streptococcus pyogenes 45986 was used (CHHATWAL et al., 1985). Binding assaysRadiolabelling: Human fibrinogen (Deutsche Kabi, Miinchen, Germany) was radiolabelled using the chloramin-T method. (HUNTER and GREENWOOD 1962). The binding assays were performed with 20 pl (40 ng; 20 000-40 000 ~pm)'~'I-fibrinogen as described recently (CHHATWAL et a]., 1985).Heat treatment: For heat treatment, the cells, suspended in 0.25 mol/1 phosphate buffered saline (PBS), p H 7.5, were exposed to 60 or 95 "C for different time intervals. Heat-treated cells were subsequently used in the binding assays.Guanidinium extraction: Following the method described by RUSSEL and FACKLAM (1975), the bacteria were cultivated, resuspended in 6 M guanidinium hydrochloride (Merck, Darmstadt, Germany) (10 % of the original cultivation volume) and incubated for 1 h at room temperature with shaking. After washing (3 X ) with PBS, the bacteria were adjusted photometrically to approximately lo9 bacteria/ml (10 % transmission at 620 nm, Bausch and Lomb, Rochester, NY, USA) and used in the binding assay (CHHATWAL et al., 1984).Ultrasonication: For ultrasonication, the bacteria were cultivated, resuspended in PBS and sonicated (20 kHz, Sonifier B12, Branson Sonic Power, Danbury, USA) five times for 10 s. After washing (twice with PBS), the bacteria were photometrically adjusted and used in the binding assay. Pronase, and trypsin treatment:For enzyme treatment, the photometrically adjusted bacteria (lo9 bacteria/ml) were suspended in PBS, incubated with 50 pg of pronase (Merck) and trypsin (Merck), and incubated for 1 h at 37 "C. The bacteria were washed with PBS and subsequently used in the binding assay (LAMMLER et al., 1983).

Cell surface hydrophobicities of Actinomyces pyogenes were determined by measuring the adherence of the bacteria to hexadecane droplets and by salt aggregation tests. Among 42 A.pyogenes cultures tested 25 (60 Oh) adhered strongly (adherence 3 75 YO) and 17 (40 YO) less pronounced (adherence between 25-75 Yo) to the hexadecane droplets. Pre-treatment of the bacteria with proteolytic enzymes completely eliminated the adherence properties whereas heat treatment had no effect. The salt aggregation studies revealed that 4 (10 %) cultures aggregated in ammonium sulfate solutions of a molarity of 0.05 mol/l, 5 (12 YO), 14 (33 %) and 3 (7 YO) cultures in ammonium sulfate solutions with molarities of 3 1.5 mol/l, 3 3 mol/l and 3 4.5 mol/l, respectively. No aggregation at all could be observed with 16 (38 %) of the cultures. Pronase treatment completely eliminated the salt aggregation reactions, trypsin-and heat treatment had no effect. The results from hexadecane adherence and salt aggregation did not correspond. The differences in surface hydrophobicities, possibly related to adherence properties of A . pyogenes, could be used for epidemiological typing of individual cultures of this bacterial species.