lei and B. thailandensis in 20% HI serum had no effect on internalization compared to unopsonized bacteria. These data suggest that serum opsonization increases the ability of neutrophils to internalize both B. pseudomallei and B. thailandensis in a complement-dependent manner. Additionally, bacterial opsonization in $5% serum elicits a rapid reduction in numbers of viable bacteria, which is associated with the respiratory burst. Interestingly, B. pseudomallei opsonized with 1% NHS demonstrated increased internalization by neutrophils but did not stimulate bactericidal 21990348 activity, whereas B. thailandensis showed increased uptake and rapid killing at this serum concentration, suggesting there are differences in the amount of reactive oxygen species produced in response to these species. The ability 19668186 of B. pseudomallei and B. thailandensis to subsequently survive after internalization was measured in the absence and presence of DPI for 2 h post-infection. The numbers of unopsonized B. pseudomallei and B. thailandensis within neutrophils were not reduced during the 2 h assessment period. Opsonization of B. pseudomallei and B. thailandensis in 1% serum produced some reduction in bacterial numbers, but these values were not significant. Opsonization of both species in 5% and 10% serum Piceatannol site elicited a significant reduction in intracellular numbers, and increasing the serum concentration to 20% produced a further significant reduction in bacterial numbers compared to the lower Neutrophil Killing of Opsonized B. Pseudomallei serum concentrations. Notably, opsonization of both species in 20% HI serum produced no reduction in bacterial numbers, and neutrophils treated with DPI were unable to reduce the numbers of either B. pseudomallei or B. thailandensis regardless of the serum concentration used for opsonization. The results demonstrate that opsonization of both B. pseudomallei and B. thailandensis with $5% NHS elicits significant activation and bactericidal activity by human neutrophils, and this is dependent on induction of a respiratory burst. Bacterial opsonization with serum is required for rapid induction of the neutrophil respiratory burst To further delineate the affects of serum opsonization on eliciting neutrophil killing, a luminol-based chemiluminescence assay was utilized to quantify the kinetics and magnitude of the neutrophil respiratory burst induced by B. pseudomallei and B. thailandensis. Neutrophils co-cultured with unopsonized bacteria did not induce a respiratory burst and appeared similar to uninfected cells. Neutrophils co-cultured with B. pseudomallei and B. thailandensis opsonized in 5%, 10%, or 20% NHS induced a rapid and substantial respiratory burst compared to unopsonized bacteria, whereas bacteria opsonized in 1% serum produced a more intermediate response. The maximum respiratory burst for B. pseudomallei and B. thailandensis opsonized with $5% serum was reached within 23 min after inoculation. These values were all significantly greater than neutrophils exposed to unopsonized bacteria, which produced little to no ROS. Neutrophils infected with either bacteria opsonized with 1% serum did show some increase in ROS activity, but these levels were not significantly different from background values. It is noteworthy that we 
also did not observe significant neutrophil killing of B. pseudomallei or B. thailandensis opsonized with 1% NHS. Neutrophils infected with 20% HI opsonized B. pseudomallei or B. thailandensis produced bas