Effects of Actinobacillus pleuropneumoniae hemolysin on porcine neutrophil function

In an attempt to gain insight into the events that take place during Actinobacillus pleuropneumoniae infection, the present study was designed to ascertain the effects of bacterial toxicity on porcine neutrophil functions and viability. Incubation of phagocytes (2 x 10(6)) with opsonized A. pleuropneumoniae 4074 (2 x 10(7) CFU) resulted in phagocytic uptake of less than or equal to 4%. At the same bacterium-to-phagocyte ratio, levels of lactate dehydrogenase activity of 74 and 81% were detected in the extracellular medium after 1.5 and 3 h of incubation, respectively. Furthermore, the ingested bacteria were not killed by the phagocytes. These effects were ascribed to hemolysin produced by the bacteria, because the presence of hemolysin-neutralizing antibody prevented overt cellular damage, significantly increased phagocytic uptake (P less than 0.001), and resulted in an approximately 10-fold decrease in the number of CFU of the ingested bacteria. Cytolytic doses of isolated hemolysin caused dose-related loss of cell viability, diminished bactericidal activity of toxin-treated phagocytes for Escherichia coli, and decreased the ability of the phagocytes to undergo a respiratory burst upon stimulation with phorbol myristic acetate. In contrast, sublytic doses of the hemolysin activated the phagocytes and caused them to respond to phorbol myristic acetate with increased generation of superoxide anion. Because heated (100 degrees C, 5 min) hemolysin preparations did not produce similar effects, we contend that the observed effects were not due to contaminating endotoxin. The data presented herein indicate that A. pleuropneumoniae hemolysin is a potent antiphagocytic virulence factor by virtue of its leukocidal activity. Sublytic doses of the toxin may have important effects on the oxidative metabolism of phagocytic cells.