BINDING OF PASTEURELLA-HAEMOLYTICA LEUKOTOXIN TO BOVINE LEUKOCYTES

Pasteurella haemolytica is the principal bacterial pathogen in the bov ine respiratory disease complex. This organism produces an exotoxin (r eferred to as leukotoxin) during logarithmic-phase growth that is a po tent leukocyte-modulating agent. At low concentrations, it activates n eutrophils and mononuclear phagocytes to release inflammatory mediator s, while at the same time making these cells destined to undergo apopt otic cell death. At higher concentrations, the toxin causes rapid swel ling and loss of cell viability. In this study, we demonstrated that t oxin binding can be directly evaluated by flow cytometry with biologic ally active biotinylated leukotoxin. Leukotoxin binding was blocked by the addition of a neutralizing anti-leukotoxin monoclonal antibody an d was not detected when bovine leukocytes were incubated with culture filtrates from a mutant strain of P. haemolytica that does not produce biologically active leukotoxin. In addition, treatment of bovine leuk ocytes with protease K eliminated subsequent binding of leukotoxin, su ggesting that there is a protein on the leukocyte surface that is eith er a leukotoxin binding site or is required for stabilization of leuko toxin binding. We did not detect binding of biotinylated leukotoxin to porcine or human leukocytes, which have been reported previously to b e resistant to the lytic effects of the leukotoxin. These findings sug gest that there may be a specific binding site for P. haemolytica leuk otoxin on bovine hut not on porcine or human leukocytes and that it mi ght be involved in the activation and lytic activities of the leukotox in.