Phagocytosis by pig alveolar macrophages of Actinobacillus pleuropneumoniae serotype 2 mutant strains defective in haemolysin II (Apxll) and pleurotoxin (ApxIII)

Abstract

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.

Association ofActinobacillus pleuropneumoniaeCapsular Polysaccharide with Virulence in Pigs

“…These toxins, referred to as ApxI, ApxII, ApxIII, and ApxIV (12,43), are thought to be the primary factors responsible for the hemorrhagic lesions characteristic of swine pleuropneumonia. A. pleuropneumoniae mutants that lack one or more of their normal exotoxins completely or partially lose virulence and the ability to induce lesions in lungs (8,22,33,41,53). ApxI has the greatest hemolytic and cytotoxic activity and is produced by serotypes 1, 5, 9, 10, and 11.…”

Identification of theActinobacillus pleuropneumoniaeLeucine-Responsive Regulatory Protein and Its Involvement in the Regulation of In Vivo-Induced Genes

“…However, the methods by which A. pleuropneumoniae infects and causes disease in swine are still not fully understood. While a variety of virulence factors have been reported to contribute to the pathogenesis of A. pleuropneumoniae (1,3,4,9,13,39,48,56,62,63), little is known about what signals induce expression of these virulence factors during infection. Certain environmental cues, such as iron limitation, heat shock, oxidative stress, and osmotic stress, have been shown to play a part in the regulation of virulence genes in other organisms.…”

[Cu,Zn]-Superoxide Dismutase Mutants of the Swine PathogenActinobacillus pleuropneumoniaeAre Unattenuated in Infections of the Natural Host

“…Although infection elicits a vigorous neutrophil and alveolar macrophage response in vitro with a substantial respiratory burst of oxygen free-radical production (7), these cells are rapidly killed by the bacteria. This toxicity is principally mediated by the secreted bacterial cytolytic toxins ApxI and ApxII (3,15,39), toxins which also inhibit neutrophil chemotaxis and phagocytosis (36). Only in the absence of Apx toxins or in the presence of convalescent-stage pig serum are significant numbers of A. pleuropneumoniae internalized by neutrophils, and evidence suggests that once internalized the bacteria are rapidly killed (2,3).…”

“…This toxicity is principally mediated by the secreted bacterial cytolytic toxins ApxI and ApxII (3,15,39), toxins which also inhibit neutrophil chemotaxis and phagocytosis (36). Only in the absence of Apx toxins or in the presence of convalescent-stage pig serum are significant numbers of A. pleuropneumoniae internalized by neutrophils, and evidence suggests that once internalized the bacteria are rapidly killed (2,3). Thus, while our in vitro data demonstrate the potential for [Cu,Zn]-SOD to facilitate bacterial survival in an oxygen free radical-rich environment, this survival mechanism appears to be redundant (at least during acute experimental infection) in the presence of the potent Apx toxins.…”

“…pleuropneumoniae causes disease predominantly as an extracellular pathogen, with little evidence for a significant intracellular phase (3,6). Pulmonary defense against A. pleuropneumoniae is mediated by resident alveolar macrophages and by migratory neutrophils that are recruited to the lung alveoli as early as 3 h after the onset of infection (20,34).…”