The Pathology and Pathogenesis of Mycobacterium bovis Infection 127
both diseases have improved our understanding
of the mechanisms involved in mammalian
infection with pathogenic mycobacteria (Waters
et al., 2014; Waters and Palmer, 2015). Follow-
ing inhalation, the bacilli are deposited within
the terminal respiratory bronchioles and alveo-
lar lumina, where they are phagocytized by resi-
dent alveolar macrophages (Palmer et al., 2016).
Infected macrophages will start producing cyto-
kines, chemokines and enzymes (Aranday-
Cortés et al., 2013; Palmer et al., 2015, 2016;
Salguero et al., 2017) (see Chapter 10). Macro-
phages produce both pro- and anti- inflammatory
cytokines, inducing the activation of innate
immune response with the involvement of neu-
trophils, monocytes, macrophages and dendritic
cells (Etna et al., 2014). After the innate immune
response, the adaptive response is initiated as
dendritic cells containing bacilli may migrate
from the primary site of infection within the
lung to local lymph nodes, activating naïve T
lymphocytes through cytokine production and
antigen presentation (Palmer et al., 2016) (see
Chapter 10). After activation and expansion, T
lymphocytes migrate to the lesion in the lung,
giving shape to the granuloma together with
epithelioid macrophages and MNGCs (Etna et al.,
2014).
The granuloma is a very dynamic structure
where cells can migrate from and to the lesion.
Studies using zebra fish (Danio rerio) as a model
of tuberculosis have shown that mycobacteria
can leave and enter granulomas using infected
macrophages as vehicles, and this process can
contribute to the dissemination of bacilli to
other tissues and organs (Bold and Ernst, 2009;
Volkman et al., 2010). Granulomas develop indi-
vidually and the disease is controlled at the level
of the granuloma (Lin et al., 2014).
Modern techniques have been used recently
to study the local immune response in-depth
within tuberculous granulomas. Among them,
a combination of laser-capture micro-dissection
(LCMD) and quantitative polymerase chain
reaction (qPCR), has proven to be very useful to
quantify the expression of cytokine and chemo-
kine mRNA within individual lesions (Aranday-
Cortés et al., 2013). Classic IHC and a novel
chromogenic ISH technique (RNAScope) linked
to digital image analysis has also been used
recently to quantify the expression of cytokine/
chemokine mRNA and protein by different cell
population within the lesions (Aranday-Cortés
et al., 2013; Palmer et al., 2015, 2016; Salguero
et al., 2017).
The early stages of the granuloma (stages I
and II) express large amounts of IL-17A
(Aranday- Cortés et al., 2013), a cytokine that
has been proposed as a possible biomarker for
bovine tuberculosis and playing a very impor-
tant role in the maturation of the granuloma
(Blanco et al., 2011; Waters et al., 2015). This
upregulation in early stage granulomas is also
observed for CXCL9 and CXCL10. These high
levels of CXCL9 and CXCL10 are related to the
recruitment of more inflammatory cells at
the initial stages of the disease to assist in the
destruction and control of the pathogen in the
site of infection (Aranday-Cortés et al., 2013).
The level of these chemokines decrease in the
late granuloma stages when the response has
been overwhelmed and the recruitment of cells
is less important than the production of a physi-
cal barrier, through walling the bacteria within
a fibrous capsule, to control the pathogen
(Algood et al., 2003; Widdison et al., 2009).
There is a consistent high level of expres-
sion of IFN-γ in all stages of granuloma forma-
tion (Fig. 9.7), as the host mounts a typical Th1
response against M. bovis (Pollock et al., 2001;Fig. 9.6. CD79a+ staining in stage I, II and IV
granulomas in the lung of a cow experimentally
infected with M. bovis. Scattered CD79a+ cells
can be observed within the rim of inflammatory
cells surrounding the necrotic core of the stage IV
granuloma and interspersed within the stage I and
II granulomas. The formation of a nest of B cells
can be observed in the lesion with a high number
of CD79a+ cells. (IHC, 100×)