Vaccination of Domestic and Wild Animals Against Tuberculosis 211
(Thom et al., 2012). The T-cell subset involved in
this response was almost exclusively CD4+, par-
ticularly CD45RO+CD62Lhigh ‘central memory’-
like phenotype. In contrast, the effector/ effector
memory T-cell phenotype (CD45RO+CD62Llow)
was the main contributor to ex vivo ELISPOT
responses, not necessarily a predictor of vaccine
protection (Blunt et al., 2015).
A number of immune parameters have
been associated with pathology post-challenge,
which indirectly correlates with vaccine efficacy.
These include ex vivo ESAT-6-induced produc-
tion of IFN-γ (Vordermeier et al., 2002) as well as
IL-17A and IL-22 (Aranday-Cortes et al., 2012).
In addition, a chemokine, IP-10 (CXCL10), was
shown to be associated with up-regulation in
infected versus non-infected animals (Aranday-
Cortes et al., 2012). Micro-RNAs (miRNA) that
serve as important regulators of gene expression
and are known to play a role in both innate and
adaptive immunity have been investigated as
markers of pathology. The expression of mi155
following PPD-stimulation of PBMCs was shown
to be associated with disease severity in M. bovis-
infected cattle (Golby et al., 2014).
14.2.4 DIVA testsVaccination with TB vaccines can compromise
the interpretation of the tuberculin skin test,
which serves as the primary screening test for
‘test-and-slaughter’ bovine TB control strate-
gies. Eighty per cent of BCG-vaccinated calves
have been shown to react in the tuberculin skin
test at 6 months post-vaccination, decreasing to
10–20% by 9 months post-vaccination (Whelan
et al., 2011b). Alternative DIVA (differentiating
infected from vaccinated animals) tests will be
required for countries intending to use vaccina-
tion alongside conventional test-and-slaughter
control strategies. DIVA tests have now been
developed using antigens from the M. tuberculo-
sis complex that are not expressed or secreted by
BCG and can be used instead of bovine PPD in
the whole-blood IFN-γ or skin tests. The first two
antigens used in the DIVA IFN-γ test were the
early secreted antigen target 6 kDa protein
(ESAT-6) and culture filtrate protein 10 (CFP10),
which are encoded in the RD1 region of
M. tuberculosis and M. bovis, but not in BCG. This
test had the added benefit of differentiating
M. bovis-infected cattle from those infected with
environmental mycobacteria or M. avium subsp.
paratuberculosis (Buddle et al., 1999; Vordermeier
et al., 2001). The sensitivity of this test was still
lower than that for the IFN-γ test using avian
and bovine PPDs and further antigen mining
was required to identify additional antigens.
Following comparative transcriptome analysis
of Mycobacterium species, Rv3615c was added
to the antigen-specific IFN-γ test to enhance
sensitivity (Sidders et al., 2008). Although, this
protein is not located in the RD1 region, its
secretion is dependent on the esx-1 secretion
system located in the RD1 region. A recent
evaluation of the whole-blood IFN-γ test
incorporating ESAT-6, CFP10 and Rv3615c in
75 BCG-vaccinated, M. bovis-infected cattle
and 179 BCG-vaccinated, non-infected animals
revealed estimates of 96% sensitivity and 95.5%
specificity (Chambers et al., 2014).
The most efficient means of using a DIVA
whole-blood IFN-γ test would be for re-testing
tuberculin-positive cattle; however, a more cost-
effective method would be to use the DIVA anti-
gens in the primary screening skin test. Use of
ESAT-6, CFP10 and Rv3615c in the DIVA skin
test in cattle has now been shown to have a high
sensitivity for M. bovis-infected cattle, while not
compromised by vaccination with BCG or with
vaccines against Johne’s disease (Whelan et al.,
2010; Jones et al., 2012). The cost of the
reagents used in the test could be reduced by
expression as a fusion protein and lowering the
concentration of the antigens by display on
nanoparticles such as polyester beads produced
in bacteria (Parlane et al., 2016).14.3 Vaccination of GoatsTB infection of goats is caused by M. bovis or
M. caprae and in the natural disease, lesions are
predominantly found in the lungs and associ-
ated lymph nodes, indicating an aerosol route of
infection (Pesciaroli et al., 2014). The disease is
responsible for economic losses in endemic areas
and infected goats may be a source of TB for cat-
tle or humans. Caprine TB is present in a num-
ber of European countries, but currently there
are no caprine TB control campaigns in the