Mutants of Actinobacilluspleuropneumoniue strain HK 361 (serotype 2) were isolated which were deficient in type I1 (Ca2+-dependent) haemolysin activity (Hly-). Some of the Hly-mutants secreted a potent, heat-labile extracellular cytotoxic activity against porcine alveolar macrophages. Comparison of cell-free culture supernatant from the parent strain and some Hly-mutants by SDS-PAGE and immunoblotting revealed the loss of a major extracellular polypeptide of 109 kDa. Two Hly-mutants which in addition failed to secrete a 120 kDa polypeptide produced no extracellular cytotoxic activity, suggesting that the 120 kDa protein was the cytotoxin. Antiserum raised to the culture supernatant from a Hly-mutant lacking the 109 kDa polypeptide recognized the 120 kDa band, but not the 109 kDa band, in immunoblots and neutralized the cytotoxic activity, but not the haemolytic activity, of A. pleuropneumoniue. The 120 kDa polypeptide and extracellular cytotoxic activity were widespread among A. pleuropneumoniue strains, but absent from related bacterial pathogens of the pig: Actinobacillus suis, Huemophilus purusuis and Pusteurellu multocidu. A clear correlation was found between the presence of the 120 kDa pdypeptide'and cytotoxic activity in culture supernatants. The cytotoxic activity of all the strains tested was neutralized by antibody to the Hly-extracellular material and by convalescent pig serum. It is proposed that the 120 kDa polypeptide represents the cytotoxin of A. pleuropneumonia?, that it is distinct from the haemolysin, and that it be termed pleurotoxin.

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