Interaction between heat-stable hemolytic substance from Haemophilus pleuropneumoniae and porcine pulmonary macrophages in vitro

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Abstract
Cytocidal and anti-phagocytic effects of the heat-stable hemolytic substance produced in culture supernatant by Haemophilus pleuropneumoniae serotype 2 on porcine pulmonary macrophages (PMP) were investigated in vitro. The substance was partially purified by sucrose density gradient ultracentrifugation, and it seems to be a carbohydrate. The partially purified preparation of the heat-stable carbohydrate with hemolytic activity was used as H. pleuropneumoniae hemolysin throughout this study. Viability of the PMP cells exposed to hemolysin with a hemolytic unit of 32 decreased by about 35% during the first 30-min exposure. The response was hemolysin dose- and exposure time-dependent and was neutralized by the anti-hemolysin antiserum. The PMP cells were found to be the most sensitive to hemolysin among the pulmonary, peritoneal, and peripheral blood macrophages obtained from the same animal. Heat treatment of the hemolytic substance in the culture supernatant at 121 degrees C for 2 h did not significantly (P greater than 0.05) alter the cytocidal and hemolytic activities. Pretreatment of the PMP cells with hemolysin at a sublethal dose significantly (P less than 0.01) reduced their phagocytic abilities. Thus, the heat-stable carbohydrate H. pleuropneumoniae hemolysin appears to possess the potential for both cytocidal and antiphagocytic effects on the PMP cells. Death and impaired phagocytosis of the PMP cells by the hemolysin, if it occurs in vivo, would enhance the chances of survival for an invading H. pleuropneumoniae organism. These findings promote understanding of the pathogenesis of pleuropneumonia caused by H. pleuropneumoniae in swine.