Figure 3
Killing assay. A Standard curve with 8 dilutions DNA from live Map (red boxes) 4 × 107 to 4 copies. Blue square data points represent DNA from two sets of controls; the lower set of squares are from Map DNA (2 × 107 Map) prepared from mixtures of 100% live, 50% live/50% dead, and 100% dead bacteria after PMA treatment. Upper four squares are from DNA isolated from Map after 3 h incubation with MoMΦ. Squares represent mixtures of Map used to infect MoMΦ, prepared at 2 × 107 Map: 100% live, 75% live 25% dead, 25% live 75% dead and 100% dead Map. CT values represent average of duplicate preparations of DNA. B Confocal microscopy showing abundance of K10GFP taken up during 3 h incubation with MoMΦ. C Killing by effector cells plotted on standard curve from A. CT values of mdPBMC from vaccinated steer with MoDC-pulsed ΔMap/relA (a: gold box) or MMP (b: green box) and mdPBMC from one control steer stimulated with MoDC pulsed with ΔMap/relA (c: yellow box) or MMP (d: gray box). D Summary of 6 replications of killing assay comparing killing activity mdPBMC from vaccinated and control steers. Mean and standard deviation for each treatment effect (n = 6 independent experiments). Two-way ANOVA was significant (F = 88.3205; P < 0.0001) and included significant interaction effect between treatments and steer-status (F = 46.1411; P < 0.0001). Within unvaccinated steer S1 and vaccinated steer S3, mean effects of T6-∆Map/relA and T6-MMP were significantly greater than T6-control (each, P < 0.0001; ***S1 not shown). Mean effects within unvaccinated steer S2 either smaller (T6-∆Map/relA, P = 0.0027; *not shown) or not different (T6-MMP; P = 0.3668) than T6-control. Effect of T6-∆Map/relA or T6-MMP for vaccinated steer S3 was significantly greater than effect for either unvaccinated steer (each, P < 0.0001).