Biomarkers in the Diagnosis of Mycobacterium tuberculosis Complex Infections 197
13.4 Circulating microRNABiomarker research has also pointed to circulat-
ing microRNA (miRNA) as a potential prognos-
tic and diagnostic biomarker (Farrell et al.,
2015). MiRNAs are short (~22 nt), single-
stranded, non-coding RNAs that regulate mRNA
expression. MiRNAs are important regulators of
gene expression and play a key role in regulating
both the innate and adaptive immune responses.
Recent work has shown that expression of miR-
155 was more than 40 times higher in naturally
infected cattle with visible pathology compared
with infected animals that presented without
visible pathology (Golby et al., 2014). This sug-
gests that miR-155 could distinguish active from
latent infection, and could be used as a diagnos-
tic and prognostic biomarker to identify infected
animals as well as be used for Differentiating
Infected from Vaccinated Animals (DIVA) test-
ing, although more research is still needed in
this area (Abd-el-Fattah et al., 2013; Golby et al.,
2014). What makes miRNAs particularly
appealing as potential biomarkers is that they
have tissue-specific expression patterns that can
serve as a fingerprint for disease and also can be
detected by RT-PCR assays and microarray tech-
niques (Williams et al., 2013). In addition, it is
hypothesized that different stages of mycobacte-
rial infection have distinct miRNA signatures
(Farrell et al., 2015). Another fascinating fact
about them is that serum miRNA is stable to
repeated freeze–thaw cycles as well as to heat,
acidic and alkaline conditions.
13.5 Serum CytokinesAlveolar macrophages and pulmonary dendritic
cells represent the first line of defence against
invading mycobacterial pathogens (Kaufmann,
2004). This process releases chemokines, which
attract monocytes and other inflammatory cells
to the lungs (Kleinnijenhuis et al., 2011). Che-
mokines are a form of cytokines – a group of
mainly soluble proteins and glycoproteins that
modulate the immune system. Examples include
interleukins (ILs), interferons (IFNs), growth
factors, colony stimulating factors, the tumour
necrosis factor (TNF) family, and chemokines
(Choi et al., 2016).
Phagocytic cells trigger the adaptive
immune response by presentation of mycobacte-
rial antigens to T cells. Once a macrophage is
infected with mycobacteria, it releases interleu-
kins 12 and 18 (IL-12 and IL-18). The released
cytokines stimulate CD4, CD8 and natural killer
cells to produce interferon gamma (IFN-γ) and
TNF alpha (Villarreal-Ramos et al., 2003). T cells
respond to the released IFN-γ in a positive feed-
back loop leading to the production of more IFN-
γ. The IFN-γ activates macrophages to kill the
invading mycobacteria by activating nitric oxide
synthase, which produces nitric oxide while the
TNF-α is critical for the initiation of the immune
response against infection with mycobacteria
(Kaufmann, 2004; Das et al., 2016). In a study
published by Thacker et al. (2007), expression of
IFN-γ, TNF-α, iNOS and IL-4 by peripheral blood
mononuclear cells (PBMC) was increased in
response to infection, whereas IL-10 expression
decreased. There was also a positive association
between Th1 responses and disease severity but
as infection progressed, the differences in gene
expression between the low and high pathology
groups were indistinguishable, implying a possi-
ble influence of early Th1 response on pathology
(Thacker et al., 2007). Characterization of the
bovine immune response has been done by sev-
eral research teams; examples include use of
real-time PCR and bovine immune microarrays
to characterize bovine cytokine/chemokine/
transcription factor etc. responses to bovine TB
(Schiller et al., 2010). Other cellular immunity-
based tests include the development of bovine
cytokine and chemokine multiplex systems
detecting several parameters in a single sample,
as well as the use of monoclonal antibodies that
recognize bovine cytokines (Coad et al., 2010;
Schiller et al., 2010).
A granuloma is a compact, organized col-
lection of mature macrophages, which arises in
response to persistent stimuli (Ramakrishnan,
2012). Necrotic areas called caesium occur
within granulomas as a result of dying cells.
Cells that make up the granuloma include neu-
trophils, dendritic cells, B and T cells, natural
killer cells, fibroblasts and cells that secrete
extracellular matrix components (Harding and
Boom, 2010). After formation of granulomas,
several scenarios may occur: cessation of
infection or dormancy, progression of infection
with dissemination to other organs, as well as