Bovine tuberculosis

Immunological Diagnosis 181

immediate antigen stimulation partially resolved
this limitation with the third generation of the
Quantiferon assay (Mahomed et al., 2006). Simi-
lar approaches, while in development, are not
currently available for use with samples from
cattle. Particularly in large countries with
diverse environmental conditions and shipping
networks, improved methods to assure sample
viability are critical for the continued use of
CMI-based tests requiring live and fully func-
tional leukocytes. The variability in sample
conditions associated with shipment in many
countries (e.g. hot or cold conditions within
aeroplane cargo holds, physical turbulence of
the container, and time to antigen stimulation)
adversely impacts the reliability of the Bovigam
assay. An ‘in-tube’ approach including use of
portable field-ready incubators could remedy
this pitfall. For instance, the stimulation phase
could be initiated in the field and samples sent to
satellite laboratories for plasma harvest after the
necessary incubation time. Then, stimulated
plasma samples could be either sent to regional
laboratories or simply analysed at the satellite
laboratory for IFN-γ or other biomarkers within
the samples. Once the logistical and technical
hurdles are overcome, an ‘in-tube’ approach for
immediate antigen stimulation may prove par-
ticularly useful for assays that are less robust
than IGRAs and/or assays that utilize multi-
parameter readouts.
While whole blood assays have greatly
improved the convenience and technical com-
patibility of CMI-based tests for diagnostic labo-
ratories, detection of biomarkers within the
sample continues to require laboratory-based
assays such as ELISA or ELISPOT. Thus, develop-
ment of point-of-care assays for use in the field
could prove useful for animal/patient-side appli-
cations and for remote locales. Indeed, user-
friendly lateral flow assays for the detection of
IP-10 and CCL4 in antigen-stimulated whole
blood samples from humans, including ambient
shipping/storage of reagents and lightweight
stand-alone readers, are in development for
use in remote and resource-limited settings
(Corstjens et al., 2016). In addition, biomarker
detection from dried blood spots on filter paper
is another technology applicable for field use
(reviewed by Chegou et al., 2014), as demon-
strated by Skogstrand et al. (2012) in proof of
concept studies using a Luminex-based assay.

Once host biomarker signatures of TB infection

are validated, similar approaches could be used

for humans and cattle.

12.6.3 Alternative approaches including

flow cytometry and gene-expression

profiling

Novel methods for detecting CMI responses are

also being explored for diagnostic purposes. For

instance, flow cytometric-based tests are in

development for the diagnosis of TB in humans

(reviewed by Rovina et al., 2013). Specifically,

flow cytometry approaches may be used to

determine cytokine production, phenotype/

activation marker status of responding

cells, polyfunctionality and immunosuppressive

markers – all of which are useful for clinical and

diagnostic applications. With cattle, El-Naggar

et al. (2015) recently demonstrated the utility of

a flow cytometric-based assay to detect M. bovis

PPD- and ESAT-6/CFP10-specific intracellular

IFN-γ responses by naturally infected cattle. For

this assay, whole blood stimulation also included

use of monoclonal antibodies specific for bovine

CD28 and CD49d as co-stimulatory molecules to

improve the capacity of lymphocytes to respond

to specific antigen. Use of co-stimulatory mole-

cules and measurement of intracellular IFN-γ

reduced the stimulation phase of the assay from

18 hours to 6 hours. Downsides to this approach

are the requirement for flow cytometry instru-

mentation and expertise, as well as the necessity

of immediate delivery of samples to a laboratory

for cell viability assurance. Use of an ‘in-tube’

approach in which both antigens and co-

stimulatory molecules are contained in the

blood collection vessel may improve the conve-

nience of the assay; however, samples would still

need to be delivered within 2 hours to the labora-

tory for initiation of the intracellular cytokine

staining protocol. Clinical use of flow cytometry

is rapidly increasing in hospitals for use in infec-

tious, neoplastic, hematologic and immune defi-

ciency disorders; thus, improvements in this

approach are on the horizon.

Another tactic for measure of CMI

responses for diagnostic applications is to evalu-

ate expression (i.e. mRNA typically using RT-

qPCR) of cytokines and chemokines in response