The three different pore-forming RTX-toxins of Actinobacillus pleuropneumoniae are reviewed, and new and uniform designations for these toxins and their genes are proposed. The designation ApxI (for &tinobacillus pZeuropneumoniae RTX-toxin I) is proposed for the RTX-toxin produced by the reference strains for serotypes 1, 5a, 5b, 9,lO and 11, which was previously named haemolysin I (HlyI) or cytolysin I (ClyI). This protein is strongly haemolytic and shows strong cytotoxic activity towards pig alveolar macrophages and neutrophils; it has an apparent molecular mass in the range 105 to 110 kDa. The genes of the apxZ operon will have the designations apxZC, apxZA, apxZB, and apxZD for the activator, the structural gene and the two secretion genes respectively. The designation ApxII is proposed for the RTX-toxin which is produced by all serotype reference strains except serotype 10 and which was previously named App, HlyII, ClyII or Cyt. This protein is weakly haemolytic and moderately cytotoxic and has an apparent molecular m a s between 103 and 105 kDa. The genes of the apxZZ operon will have the designations apxZZC for the activator gene and apxZZA for the structural toxin gene. In the apxZZ operon, no genes for secretion proteins have been found. Secretion of ApxII seems to occur via the products of the secretion genes apxZB and apxZD of the apxZ operon. The designation ApxIII is proposed for the nonhaemolytic RTX-toxin of the reference strains for serotypes 2, 3, 4, 6 and 8, which was previously named cytolysin 111 (ClyIII), pleurotoxin (Ptx), or macrophage toxin (Mat). This protein is strongly cytotoxic and has an apparent molecular mass of 120 kDa. The genes of the apxZZZ operon have the designations apxZZZC, apxZZZA, apxZZZB and apxZZZD for the activator gene, the structural gene and the two secretion genes respectively.

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.

The ability of pig alveolar macrophages to phagocytose Actinobacillus pleuropneumoniae HK 361, which produces both haemolysin II (Apxll) and pleurotoxin (Apxlll), has been studied. Macrophages incubated w i t h HK 361 in the presence of normal pig serum were rapidly killed. Incubation of the macrophages with a haemolysin-def icient mutant (HK 361 e), which possesses only cytotoxic activity (Apxlll), also caused gross damage to the macrophages. A mutant (HK 361 h) which produces neither ApxlI nor Apxlll in i t s culture supernatant allowed longer survival of the macrophages than did either the parent strain or mutant e when incubated w i t h normal pig serum. Prolonged incubation with mutant h resulted in an increase in the number of damaged macrophages, but not to the same extent as w i t h either HK 361 or mutant e. The number of mutant h cells phagocytosed in the presence of normal pig serum was low. The addition of either hyperimmune rabbit serum, raised against whole formalin-treated HK 361 cells, or convalescent pig serum from a pig recovering from a serotype 3 infection, which contained antibody against both ApxlI and Apxlll, did not increase the survival of macrophages incubated with either HK 361 or mutant e. However, incubation of mutant h with convalescent pig serum did result in damage-free macrophages. This serum, which possessed neutralizing capabilities against the toxic activities of Apxll and Apxlll, enhanced the number of mutant h cells phagocytosed compared to the numbers phagocytosed in normal pig serum. Killed bacteria were rapidly phagocytosed and did not damage macrophages. The number of phagocytosed killed bacteria appeared to be similar to that seen with live mutant h cells when incubated in the presence of convalescent pig serum. The presence of a toxic activity associated with the haemolysin-and cytotoxin-negative mutant may represent an additional cell-associated toxin which is not present in its culture supernatant. It appears therefore, that in the absence of extracellular ApxlI and Apxlll and in the presence of convalescent pig serum A. pleuropneumoniae is readily phagocytosed.