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.