Ovine neutrophils spontaneously underwent apoptosis during culture in vitro, as assessed by morphological changes and exposure of annexin V binding sites on their cell surfaces. The addition of conditioned medium from concanavalin A-treated ovine peripheral blood mononuclear cells (PBMC) could partially protect against this progression into apoptosis, but dexamethasone and sodium butyrate could not. Actinomycin D accelerated the rate at which ovine neutrophils underwent apoptosis. Neutrophils isolated from sheep experimentally infected with Anaplasma phagocytophilum showed significantly delayed apoptosis during culture ex vivo, and the addition of conditioned medium from PBMC to these cells could not delay apoptosis above the protective effects observed after in vivo infection. The ability of neutrophils from A. phagocytophilum-infected sheep to activate a respiratory burst was increased compared to the activity measured in neutrophils from uninfected sheep, but chemotaxis was decreased in neutrophils from infected sheep. These data are the first demonstration that in vivo infection with A. phagocytophilum results in changes in rates of apoptosis of infected immune cells. This may help explain how these bacteria replicate in these normally short-lived cells.