FIG. 5.

Elimination of extracellular H. somnus does not alter endothelial cell apoptosis. Volumes containing 5 × 10⁴ endothelial cells were adhered to glass coverslips and incubated with H. sonnus pathogenic isolates 8025, 649, and 2336 and asymptomatic carrier isolates 127P and 129Pt at an MOI of 10:1 for 1 h at 37°C with 5% CO₂. After 1 h, each experimental group was further divided into two subgroups. For the first subgroup (■), H. somnus organisms were not removed from the endothelial cells. For the second subgroup (□), nonadherent H. somnus organisms were washed away and gentamicin was added (5 μg/ml) to kill residual extracellular bacteria. The two groups were incubated for an additional 5 h at 37°C with 5% CO₂, and apoptosis was detected by Hoechst 33342 staining. Controls consisted of untreated cells (UT) and staurosporine (200 nM; Stauro)-treated endothelial cells as a positive control. These data illustrate the mean ± SEM of three separate experiments. Statistical analysis was performed by ANOVA followed by the Tukey-Kramer significant difference test. Significant levels of endothelial cell apoptosis were detected whether H. somnus was removed (P < 0.05) compared to untreated endothelial cells.