208 B.M. Buddle et al.
the same day (Dean et al., 2015), but not with
the combination of subcutaneously and orally
administered BCG (Buddle et al., 2008). Pasteur
and Danish strains of BCG induced similar pro-
tection, although the kinetics of the cellular
immune response varied with the two strains
(Wedlock et al., 2007; Hope et al., 2011). Neona-
tal or very young calves were protected at least
as well as older calves (Buddle et al., 2003; Hope
et al., 2005). Vaccination of cattle with BCG 3
weeks after an experimental challenge with
M. bovis did not produce a beneficial effect, nor
increased tuberculous pathology (Buddle et al.,
2016). Protection against experimental chal-
lenge was shown to be effective at ≤12 months
post-vaccination, but had waned by 24 months
post-vaccination (Thom et al., 2012).
Two studies were undertaken to determine
the effect of revaccination with BCG. In the first
study, calves vaccinated within 8 hours of birth
or at 6 weeks of age showed a high level of pro-
tection against M. bovis, while those vaccinated
within 8 hours of birth and revaccinated at
6 weeks of age had a reduced level of protection
(Buddle et al., 2003). The revaccinated calves
with the lowest level of protection had the
strongest antigen-specific IFN-γ responses, sug-
gesting that revaccination had induced an inap-
propriate immune response. In neonatal calves,
antigen-specific IFN-γ responses remained at
elevated levels for longer than those seen in older
calves, possibly due to a more active BCG infec-
tion and BCG revaccination when immune
responses were at high levels may be contra-
indicated. In contrast, calves vaccinated with
BCG at 2 to 4 weeks of age and revaccinated at
2 years of age when immunity had waned,
showed a significant level of protection when
challenged 6 months later, while those receiving
only the initial vaccine dose were not protected
(Parlane et al., 2014).
Trials in Ethiopia and Mexico that were
undertaken in field conditions with exposure of
vaccinated and non-vaccinated calves to in-
contact, tuberculin-reactor cows, demonstrated
a significant level of protection in the vaccinated
calves (Ameni et al., 2010; Lopez-Valencia et al.,
2010). In a large-scale field trial in New
Zealand, cattle vaccinated orally with BCG and
exposed to tuberculin-reactor cattle and a wild-
life reservoir of infection had a significant level
of protection compared to non-vaccinated
cattle, with an estimated vaccine efficacy of 67%
for preventing infection (Nugent et al. 2017).14.2.2 New generation TB vaccinesIn the past two decades, large amounts of fund-
ing have been provided to develop human TB
vaccines and efforts to develop and evaluate
cattle TB vaccines have greatly benefited from
this research. The different types of TB vaccines
that have recently been tested in cattle include
live attenuated mycobacteria, which could
replace BCG, and subunit TB vaccines such as
DNA, protein, and virus-vectored vaccines,
which could be used to boost immunity induced
by BCG (Parlane and Buddle, 2015, summarized
in Table 14.1).
Published reports evaluating live attenu-
ated mycobacterial vaccines in cattle have
included modified BCG strains, a M. bovis auxo-
troph and deletion mutants of M. tuberculosis
and M. bovis. Significant protection against chal-
lenge with a virulent M. bovis strain combined
with lower pulmonary histological scores was
obtained by a BCG expressing Ag85B, in com-
parison with vaccination using BCG alone (Rizzi
et al., 2012). Additional modifications have been
used, including a zmp1 deletion, with the ratio-
nale that the Zmp1 protein in mycobacteria pre-
vents activation of the inflammasome, thereby
inhibiting maturation of the phagolysosome and
MHCI and II dependent mycobacterial antigen
presentation. Improved T-cell memory responses
in comparison with BCG was obtained by vacci-
nation involving either of two BCG zmp1 dele-
tants (Khatri et al., 2014). Recent experiments to
assess its protective efficacy have demonstrated a
trend towards better protection against lesions in
the thoracic lymph nodes (B. Khatri, unpub-
lished data). Vaccination with a cocktail of four
BCG Danish mutants (BCG∆leuCD, BCG∆fdr8,
BCG∆mmA4, BCG∆pks16) induced significant
protection in cattle against M. bovis challenge to
a level comparable with wild-type BCG Danish
(Waters et al., 2015).
Vaccination with either of two attenuated
M. bovis strains derived by UV irradiation, with
deletions not defined, produced significant pro-
tection against challenge with M. bovis in
calves naturally pre-sensitized to environmental