Ultrastructural characterization of apoptosis in bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin

Objective-To characterize ultrastructural changes of bovine lymphocytes exp osed to Pasteurella haemolytica leukotoxin (LKT). Sample population-Partially purified LKT from a wild type P haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant P haemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves. Procedure-Isolated bovine lymphocytes were incubated with various concentra tions of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmi ssion electron microscopy. A cytochemical Klenow DNA fragmentation assay wa s used to examine lymphocytes for DNA fragmentation. Results-Lymphocytes incubated with LKT at a high concentration (1.0 toxic U /ml) had ultrastructural evidence of cytoplasmic and nuclear membrane ruptu re and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastru cturally, these cells had nuclear membrane blebbing, cytoplasmic vaculation , chromatin condensation, nuclear fragmentation, and membrane-bound apoptot ic bodies. Incubation of lymphocytes with LKT at extremely low concentratio ns (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. I nclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastru ctural changes in bovine lymphocytes. Conclusions and Clinical Relevance-low concentrations of LKT induce apoptos is and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocyt es may allow bacteria to escape host immune surveillance and colonize the h ost.