The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the
antigens of bound organisms were susceptible to
pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1%
paraformaldehyde for 30 min or 0.25%
trypsin for 15 min. These results indicate that the ehrlichial
ligand and host cell receptor are likely
surface proteins. Following incubation at 37 degrees C, bound E. risticii and/or its
antigens were removed with
pronase and indirect immunofluorescent staining in the presence of
saponin was used to examine intracellular ehrlichiae. Our results indicate that E. risticii was internalized into P388D1 cells within 3 h and proliferated by 48 h of incubation. The microfilament-disrupting agent
cytochalasin D and the
transglutaminase inhibitor
monodansylcadaverine were used to differentiate between phagocytosis (sensitive to cytochalasin) and receptor-mediated endocytosis (sensitive to
monodansylcadaverine) of E. risticii by P388D1 cells. In concentrations that produced distinctive morphological changes and inhibited phagocytosis of
polystyrene latex beads,
cytochalasin D did not suppress the infectivity of E. risticii. Binding, internalization, or proliferation of E. risticii was not affected by
cytochalasin D. However,
monodansylcadaverine inhibited
infection of E. risticii in a dose-dependent manner. The agent did not affect the attachment of ehrlichiae to host cells, but it did suppress internalization and proliferation. These results suggest that E. risticii is internalized by receptor-mediated endocytosis and that productive
infection by E. risticii does not depend on phagocytosis by the P388D1 cells. Although E. risticii did not bind to the surface of equine PMN leukocytes at 4 degrees C, organisms were taken up by this cell at 37 degrees C. E. risticii, however, failed to survive in equine PMN leukocytes.