Immunological Diagnosis 183
12.7 Antibody-Based AssaysAntibody-based assays are appealing due to ease
and convenience of sample collection, storage
and analysis. Until recently, the poor sensitivity
of antibody-based tests has prevented wide-
spread development and use of these assays for
the diagnosis of TB in cattle (Pollock et al.,
2001). Several serologic tests designed to detect
antibodies to sero-dominant M. bovis antigens
(e.g. MPB83, MPB70, ESAT-6 and CFP10) have
recently emerged for field validation studies in
cattle (Lyashchenko et al., 2000; Whelan et al.,
2008a, 2010b; Green et al., 2009; Waters et al.,
2011). Indeed, a commercial ELISA to MPB83/
MPB70 (M. bovis Ab Test, IDEXX Laboratories,
Westbrook, Maine; Waters et al., 2011) is
approved for use in cattle for bovine TB control
programs by the Office International des Epizo-
oties and US Department of Agriculture; how-
ever, applications of this test are currently
limited to ancillary applications such as confir-
mation of infection and potentially to detect
M. bovis-infected cattle anergic to TST. A com-
mercial immunochromatographic test (Dual-
Path Platform VetTB Assay, Chembio Diagnostic
Systems, Medford, New York; Lyashchenko et al.,
2013) is also approved for use in deer and ele-
phants in several countries and may be applied
for TB diagnosis in multiple other zoo and alter-
native livestock species. With serologic tests for
bovine TB, injection of PPDs for skin tests signifi-
cantly boosts antibody responses to specific
antigens in M. bovis-infected cattle, including
animals without detectable antibody responses
prior to skin test(s) (Lightbody et al., 1998,
2000; Waters et al., 2006b, 2011; Casal et al.,
2014). PPD-boosted antibody responses are
targeted to specific antigens (e.g. MPB83 and
MPB70) and accompanied by an increase in
avidity of antibodies to MPB83/70 (Waters
et al., 2015b). Thus, it is generally recommended
that serologic tests for bovine TB in cattle be
applied after skin test. Recently, Casal et al.
(2014) demonstrated that use of serology
applied after skin test in combination with TST
increases the number of tuberculosis-positive
animals detected within TB-affected cattle herds
as compared to that of skin test alone. Currently,
the best promise for developing an improved
antibody-based test is the discovery of antigens
that are recognized early after infection and pref-
erably without the requirement for injection of
PPD for skin test to achieve detectable levels.
It should also be noted that proteome-wide
definition of the M. bovis B-cell antigenome in
cattle still awaits elucidation to a similar degree
as the use of protein arrays has been undertaken
for M. tuberculosis in humans or non-human
primates (Kunnath-Velayudhan et al., 2010,
2012). Furthermore, the apparent sero-
dominance of antigens such as MPB70 and
MPB83 may be due to a bias introduced into
antigen screening by using sera from skin-test
positive reactors, whose antibody responses are
boosted by an application of a prior tuberculin
test (as alluded to in the previous paragraph). As
MPB83 is the main intact protein that can be
demonstrated in bovine PPD by SDS-PAGE and
immune-blotting (Whelan and Vordermeier,
unpublished data), it is perhaps not surprising
that this protein, together with responses to its
homologue MPB70, are dominant in bovine TB.
Recent data using sera from skin-test negative
cattle with confirmed TB (but IGRA-positive)
suggest that this is the case. While MPB83
responses were still very frequent, they were less
so compared to sera from skin-test positive cattle.
By contrast, recognition of less dominant anti-
gens observed in skin-test positive cattle became
more frequent using sera from skin-test negative
TB cattle (Waters et al., 2017). Thus, we hypoth-
esize that proteome-wide antigen mining for
sero-dominant antigens using sera from skin-
test negative TB animals could yield additional
relevant targets for sero-diagnosis that could
increase the sensitivity of serology to detect
tuberculous cattle.12.8 Host Markers in Sera, Urine,
Saliva and Other Bodily FluidsHistorically, much of the work on evaluation
of pre-formed biomarkers in serum has been
limited to evaluation of traditional markers
of inflammation such as C-reactive protein,
mannose- binding lectin, alpha-1-acid glycopro-
tein, serum adenosine deaminase, complement
components, fibronectin, erythrocyte sedimen-
tation rate, prolidase activity, various cytokines
and other commonly measured hematologic