Ultra-structure and localisation of formazan formed by human neutrophils and amoebae phagocytosing virulent and avirulent Legionella pneumophila

Legionella pneumophila (LP) strains of differing virulence were incubated with a solution of nitroblue-tetrazolium (NBT) at a concentration of 1 mg.ml-1 in the presence of Acanthamoeba polyphaga or human polymorphonuclear neutrophils (PMN). Reduction of NBT to formazan occurred at a faster rate in the presence of virulent strains. Reduction appeared to be temperature dependent; at 37 degrees C the reaction rate was higher than at 20 degrees C. On microscopic examination, deposits of formazan around Legionella cells were observed inside amoebae similar to those deposited in human neutrophils. Electron microscopy revealed electron-dense particles surrounding virulent legionellae, which appeared to be associated with formazan formation. Formazan formation inside amoebae may suggest the presence of a respiratory burst against LP, which is more intense with virulent strains.