Vaccination and challenge experiments.
Sixteen Zebu cattle and 16 Holstein-Friesian cattle were vaccinated withc 1x106 BCG SSI cfu/animal at week 0. Eight weeks after vaccination, 16 control and 16 vaccinated Zebu cattle, as well as 15 control and 16 vaccinated Holstein-Friesian cattle were challenged intranodally with BCG Tokyo, as indicated previously (Villarreal-Ramos et al., 2014), with 1x107 cfu each. The number of animals per group was determined based on previously published (Villarreal-Ramos et al., 2014) and non-published experimental data, which indicated that a comparisons between vaccinated and non-vaccinated HF cattle required 12 animals to reach a statistical power of 70.7%, whilst another experiment, using 17 animals, reached a statistical power of 96.7%.
Determination of bacterial load in lymph nodes .
Left and right prescapular lymph nodes (LN) were dissected from each animal at post-mortem. One of these LNs was used for evaluating bacterial load as previously described (Villarreal-Ramos et al., 2014). Briefly, LNs were trimmed and submerged briefly in 70% ethanol prior to weighing and slicing for processing in a stomacher (Seward, U.K.) for 2 min with 7 ml of PBS. One hundred µl of LN macerate was spread in each of 2 plates, as well as preparing serial dilutions for plating on 7H11 agar plates (Gallagher & Horwill, 1977). Results are presented as counts per organ. The limit of detection of this assay in each individual plate is 70 cfu/organ, since we plated two plates, the limit of detection for this assay could be considered to be 35 cfu/organ; the discontinuous line in the graph in figure 1 indicates the limit of detection. Animals for which no cfu were detected in any of the two plates were placed below the line indicating the limit of detection.