Immunological Diagnosis 175
52–100% (sensitivity) and 55–99% (specific-
ity) contingent on the type of TST applied,
interpretation criteria, study population, prev-
alence and other factors (de la Rua-Domenech
et al., 2006; Schiller et al., 2010; Bezos et al.,
2014). Particularly in countries with a low
prevalence of bovine TB, traditional slaughter
inspection has a low sensitivity for detection of
tuberculous cattle (e.g. <20% in Australia in
the 1980s [Corner et al., 1990], 31.4% in Cata-
lonia, Spain [Garcia-Saenz et al., 2015] and
28.5% in Texas, USA [Chioino, 2003; APHIS,
2009]). Even with enhanced inspection, visual
detection of tuberculous lesions at post mortem
is seldom greater than 60% (Corner et al.,
1990; Buddle et al., 2015). For these reasons,
the determination of bovine TB test accuracy is
difficult, especially in countries with a low prev-
alence of disease in which TB-affected herds are
scarce.
Specific applications for ante-mortem
bovine TB testing currently include routine
surveillance to identify TB-affected herds, move-
ment tests, epidemiologic trace-back testing
resulting from detection of a tuberculous car-
cass at slaughter, and in TB-affected herds to
delineate animals going to a slaughter plant (test
negative) versus being condemned for rendering
(test positive). While a few new tests have
emerged (Bezos et al., 2014), TST and IGRAs
remain as the principal tests used in bovine TB
control programmes. The intradermal TST may
be applied as a single injection of M. bovis puri-
fied protein derivative (PPD) in the caudal fold or
at the base of the tail (i.e. caudal fold test, CFT)
or in the mid-cervical region (i.e. single intrader-
mal test, SIT). A major confounding variable for
use of M. bovis PPD alone for skin test is that test
specificity is often jeopardized by prior exposure
to ubiquitous non-tuberculous Mycobacteria
species. Thus, a comparative test may be applied
in which Mycobacterium avium-derived PPD and
M. bovis-derived PPD are delivered at adjacent
sites on the neck (designated as either compara-
tive cervical test [CCT] or single intradermal
comparative cervical test [SICCT]). The CFT is
currently used as a primary test for cattle in
the southern hemisphere and North America,
whereas the SICCT is used as the primary test in
the UK and Ireland. The SIT is used in many
countries in continental Europe. To improve
specificity, CCT and/or IGRAs may be used as
secondary tests (i.e. as a follow-up to CFT or SIT),
thereby reducing the number of animals falsely
identified as TB-infected by the primary test. In
known-infected herds, IGRAs may also be used
in parallel with a skin test for prioritization of
animal removal and for decision making on
which animals may be sent to slaughter as likely
non-infected, thereby salvaging value for the
farmer and reducing indemnity costs for regula-
tory agencies. While approved by the OIE for use
as a primary test, IGRAs are currently not
routinely used as a primary test (Bezos et al.,
2014). Further details on TST and IGRAs and
their specific applications in bovine TB control
programmes are provided in recent reviews of
the subject (de la Rua-Domenech et al., 2006;
Schiller et al., 2010; Bezos et al., 2014; Buddle
et al., 2015).
Tuberculins, including PPDs, are a poorly
defined and complex mix of proteins, lipids and
carbohydrates of inherently poor specificity as
many of the compounds within PPDs are anti-
genically cross-reactive amongst the various
mycobacterial species. Given the complex
nature of PPDs, standardization and potency
evaluation of various lots for use in TST and
IGRAs is difficult (Bakker et al., 2005; Good
et al., 2011). PPD potency is routinely evaluated
in guinea pigs by comparison to an interna-
tional standard using a multiple comparative
skin test; however, these results may not corre-
late to a similar potency in cattle (Dobbelaer
et al., 1983; Good et al., 2011). Tuberculin
activity may also be evaluated with blood sam-
ples from M. bovis-infected or sensitized cattle
by comparison of activity in serial dilutions of
PPDs using IGRAs to determine the relative
potency 30 (RP30) defined as ‘the protein con-
centration (μg/ml) or as activity (iu/ml) of a
given PPD needed to obtain 30 per cent of the
response (RP30) of the peak value of a refer-
ence PPD (ODmax)’ (Schiller et al., 2010). Poten-
cies of PPDs may also be evaluated in DTH
assays (in vivo) in cattle, although this approach
is cumbersome due to the requirement of costly
experimental infection studies or access to natu-
rally infected cattle. While a combination of
guinea pig and cattle testing for PPD potency in
comparison to international standards is opti-
mal (OIE, 2009), PPD potency testing in cattle is
rarely conducted due to costs and logistical
demands (Bezos et al., 2014).