174 R. Waters and M. Vordermeier
12.2 Immunopathogenesis as Related
to Development of Diagnostic Tests
Bovine peripheral blood CD4, CD8 and γd T cells
from M. bovis-infected cattle proliferate and dis-
play an overt activated phenotype (i.e. increased
CD25, CD26, CD44, CD45RO and CD69 expres-
sion) upon stimulation with mycobacterial anti-
gens (Rhodes et al., 2000; Waters et al., 2003;
Maue et al., 2005; El-Naggar et al., 2015).
Antigen- specific activation is also accompanied
by a robust and diverse cytokine response rang-
ing from pro-inflammatory (e.g. IFN-γ, IL-17,
and IL-1) to immunosuppressive, regulatory and
tissue remodeling responses (e.g. IL-10 and
TGF-β) (Rhodes et al., 2000; Vordermeier et al.,
2002; Jones et al., 2010a; Aranday-Cortes et al.,
2012; McGill et al., 2014; Shu et al., 2014;
Palmer et al., 2015; Waters et al., 2015a). To add
to the complexity, polyfunctional T cells express-
ing various combinations of IFN-γ, IL-2 and
TNF-α are also elicited by M. bovis infection/
bacillus Calmette–Guérin (BCG) vaccination
(Whelan et al., 2011; Dean et al., 2015; Maggioli
et al., 2015a); yet, the nature of protective ver-
sus detrimental polyfunctional T-cell responses
to infection and vaccination remains unclear.
With that said, initial studies indicate that
increasing responses by CD4+ cells co-producing
IFN-γ+ and TNFα+ (particularly the CD45RO+,
CCR7+, CD62Lhi subset) are associated with the
degree of pathology (disease severity), possibly
correlating with antigen load (Maggioli and
Waters, unpublished observations). Studies with
M. tuberculosis infection in HIV-infected humans
suggest that CD4 T cells secreting IL-2 alone or
with other cytokines correlate with beneficial
responses (Day et al., 2008; Wilkinson and
Wilkinson, 2010), whereas a high proportion of
IFN-γ+TNFα+ cells is detected in patients with
active but not latent disease (Chiacchio et al.,
2014; Salgame et al., 2015). Thus, the complex-
ity and diversity of the immune response to TB
provides many opportunities for the discovery
and development of diagnostically useful bio-
markers of infection.
An essential component of the response to
M. tuberculosis infection in mice and humans is
the production of IFN-γ by T helper 1 (Th1) CD4
T cells (Cooper and Torrado, 2012). Immune
deficiencies affecting CD4 T cells and IL-12/
IFN-γ/STAT1 signaling pathways result in more
severe disease in TB-infected individuals (Cooper
et al., 2007; Diedrich and Flynn, 2011). As such,
it is not surprising that IFN-γ and delayed-type
hypersensitivity (DTH) responses are useful cor-
relates of infection for bovine TB (reviewed by
Schiller et al., 2010 for cattle and Walzl et al.,
2011 for humans). For diagnostic purposes in
cattle, IFN-γ responses are generally measured
in whole blood cultures for convenience and
after overnight (i.e. 16–24 hour) stimulation
with antigen (i.e. IFN-γ release assay, IGRA).
Measure of IFN-γ by peripheral blood mononu-
clear cells (PBMCs) in long-term cultures (i.e.
~14 days) is also useful as a measure of vaccine-
elicited protection (Whelan et al., 2008b). For
instance, BCG alone or in combination with
viral-vectored subunit vaccines elicit long-term
cultured IFN-γ responses that correlate with
reduced colonization and severity of tubercu-
lous lesions upon subsequent challenge with
virulent M. bovis (Vordermeier et al., 2009;
Waters et al., 2009). Recent studies have demon-
strated that the responding cells within these
long-term cultures are primarily CD4+ central
memory T cells (Blunt et al., 2015; Maggioli
et al., 2015b). Thus, contingent on the type of
assay performed, a measure of IFN-γ responses is
valuable both as a surrogate of infection and
protection. Additional host biomarkers beyond
IFN-γ have also emerged as potential candidates
for use in blood-based TB tests for humans
(reviewed by Walzl et al., 2011; Salgame et al.,
2015) and cattle (e.g. IL-1β, IL-2, TNF-α, nitric
oxide, IP-10, IL-17 and IL-22) (Waters et al.,
2003, 2012; Vordermeier et al., 2009; Jones
et al., 2010a; Blanco et al., 2011, 2013; Bhuju
et al., 2012; Rhodes et al., 2014; Goosen et al.,
2015).12.3 Current Ante-mortem Testing
SchemesA major impediment for the control of bovine
TB is the relatively poor accuracy of current
ante-mortem tests compounded by difficulties
in the ability to reliably detect tuberculous
lesions and the agent (primarily M. bovis) in all
infected animals due, in part, to the focal nature
of the disease. For instance, the accuracy
of tuberculin skin test (TST) ranges from