Ficoll paque density gradient

Calves were bled at birth (before colostrum feeding), 6 h after birth (following second feeding of colostrum), and on d 1, 3, 7, 14, 21 and 28. Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll-Paque density gradient (1.077 g/mL; BD Falcon, Franklin Lakes, NJ), from whole blood collected using 40 mM EDTA (10% vol/vol) as described previously (Shafer-Weaver et al., 1999) . Following initial centrifugation, plasma was removed, aliquoted, and stored at -20°C until further analysis. Mononuclear cells were resuspended at 1 × 10 7 cells/mL in PBS containing 0.05% sodium azide.

Each calf was bled before each vaccination series and once a month for 3 mo subsequent to their respective vaccination month for each series. Specifically, blood samples were taken the month before vaccination (mo -1), within 4 wk after vaccination (mo 0), 4 to 8 wk after vaccination (mo 1), and 9 to 12 wk after vaccination (mo 2). Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll-Paque density gradient (1.077 g/mL; BD Falcon, Franklin Lakes, NJ) from whole blood collected using 40 mM EDTA (10% vol/vol), as described previously (Shafer-Weaver et al., 1999) .