194 S.I. Wanzala and S. Sreevatsan
microRNA and small RNAs) in plasma are prom-
ising diagnostic tools, requiring only a limited
blood sample. Recently completed human and
animal genomes have brought refinements in
technology including nucleotide and protein
sequencing, mass spectrometry and microar-
rays (nucleic acid and protein arrays), and these
have allowed researchers to elucidate funda-
mental biological processes of chronic diseases
such as cancers, neurological disorders, cardio-
vascular disease and several infectious diseases
(Maruvada et al., 2005; Scaros and Fisler, 2005;
Jacobsen et al., 2008).
13.3.1 Circulating nucleic acid approachCirculating nucleic acids (CNAs) are segments of
DNA and RNA that are devoid of cellular mate-
rial and are detected in biological fluids. They are
present in small amounts in the plasma of
healthy individuals. However, their increased
levels are associated with disease conditions.
Mandel and Metais first reported CNA in 1948
in human plasma (Mandel, 1947; Anker, 2000).
Later studies on CNA were mainly focused on
autoimmune diseases like lupus erythematosus
(Tan et al., 1966) and rheumatoid arthritis
(Ayala et al., 1951). Thirty years later, the diag-
nostic implications of CNA were recognized by
Leon et al. in 1977, when he reported high levels
of CNAs in patients with pancreatic cancer
and demonstrated that levels of plasma CNA
decreased after chemotherapy (Leon et al.,
1977). Since then, elevated levels of CNA have
been reported in chronic illness (Lui et al., 2002;
Schutz et al., 2005), trauma (Lo et al., 2000),
acute stroke (Rainer et al., 2003), myocardial
infarction (Chang et al., 2003), prenatal diagno-
sis (Lo et al., 1997; Chim et al., 2005) and vari-
ous cancerous diseases (Sorenson et al., 1994;
Vasioukhin et al., 1994; Hibi et al., 1998; Capone
et al., 2000; Shao et al., 2002).
In the last decade, CNA has gained more
attention because of its potential application as
a non-invasive, rapid and sensitive tool for
molecular diagnosis and monitoring of acute
pathologies. Most CANA-based laboratory diag-
nosis involves amplification of either RNA or
DNA with primers designed for single-copy cod-
ing regions. These CNA tests primarily detect the
functional genes associated with exogenous
nucleic acid (Lui et al., 2002), for example, those
belonging to West Nile virus, parvovirus B-19,
human immunodeficiency virus, hepatitis B
virus, hepatitis A virus, etc. In addition to detect-
ing single-copy exogenous nucleic acids, CNA
diagnostics are being developed that detect
endogenous, repetitive sequences (Stroun et al.,
2001). Often chronic diseases that lead to cell
stress and the release of nucleic acids into the
blood show a consistent pattern of endogenous
CNAs in serum.
Although most of the diagnostic CNA sig-
natures are associated with human disease, a
few researchers have detected CNA in cattle dis-
eases (Brenig et al., 2002; Schutz et al., 2005;
Shaughnessy et al., 2015). Researchers have
studied repetitive sequences including short Alu
repeat sequences (SINE-like sequence of pri-
mates) in bovine spongiform encephalopathy
(BSE) where they identified the 3′ region of Bov-
tA fragments in PCR products derived from the
serum of confirmed BSE cases or BSE-exposed
cohorts (Schutz et al., 2005). These repetitive
elements identified in BSE and other infectious
disease lead us to believe that a similar pat-
tern may exist in a chronic infection such as
bovine TB.Mechanism for release of CNA
into circulationVarious hypotheses have been proposed as to the
mechanism of release of CNA in biological flu-
ids. However, there are controversies related to
these hypotheses and the actual origin of CNA
still remains ambiguous. On the one hand,
necrosis and apoptosis has been considered as
major pathway for CNA release (Lo et al., 2000;
Lichtenstein et al., 2001); in contrast, it has been
reported that cellular necrosis may not be an
important pathway as plasma DNA levels fall
rather than rise following radiation therapy
(Tan et al., 1966). Researchers have considered
cellular apoptosis as a source of plasma CNA
based on the fact that the electrophoretic pat-
tern produced by plasma CNA is similar to that
found with DNA extracted from apoptotic cells
(Kamm and Smith, 1975). Apoptosis-induced
increased CNA level has been demonstrated in
the plasma of patients with lung cancer ( Fournie
et al., 1995). Several in vivo experiments in mice