Mycobacterium bovis Molecular Typing and Surveillance 63
helped understand the population structure and
evolutionary history of the pathogen across the
British Isles, Western Europe and beyond. MLVA
and spoligotyping confirmed that M. bovis
recovered from European badgers (Meles meles)
exhibits a strong geographic association with
cattle herds sharing the same molecular type
( Goodchild et al., 2012).
Irish efforts began in concert with North-
ern Ireland, specifically with regard to spoligo-
typing and RFLP technologies (Roring et al.,
1998) applied to bovine, cervine, ovine, caprine,
porcine and meline samples (Costello et al.,
1999). Distribution of most common molecular
types was seen across all species and indicated
that transmission between multiple hosts was a
feature of M. bovis epidemiology (Costello et al.,
1999). RFLP techniques were subsequently
deployed in the analysis of M. bovis isolates from
badgers and cattle in four areas subjected to
badger culling (Olea-Popelka et al., 2005).
Despite good evidence of cattle and badgers
sharing the same molecular type, badger isolates
exhibited low spatial clustering that may consti-
tute evidence of increased badger mobility over
time (Olea-Popelka et al., 2005). Surveillance
data indicated that combined spoligotype and
MLVA methods had enhanced resolution
(McLernon et al., 2010), and when applied to
badgers with differing disease prevalence across
a wide geographic area (Furphy et al., 2012),
indicated that two distinct MLVA types were
associated with high and low prevalence popu-
lations and that multiple strain types could be
found in the same host (Furphy et al., 2012).
IS611O RFLP was used by Spanish
researchers to investigate M. bovis isolates from
cattle and goat hosts (Gutierrez et al., 1995) but
lacked resolution. Subsequently, spoligotyping
provided superior resolution (Aranaz et al.,
1996), proving useful in ruling out epidemio-
logical association between cattle and human
cases of M. bovis (Romero and Aranaz, 2006).
Combined MLVA and spoligotyping has since
been used to infer M. bovis transmission between
wild boar and domestic pigs (Parra et al., 2003),
to survey M. bovis diversity in wildlife including
deer and wild boar (Parra et al., 2005; Menta-
berre et al., 2014), to confirm a role for wildlife
in transmission to domestic animals and vice
versa (Romero et al., 2008) and to investigate
M. bovis diversity in widely distributed bovine
hosts (Rodriguez-Campos et al., 2013). With the
advent of MLVA (Navarro et al., 2014), the ease
with which molecular epidemiological informa-
tion could be gathered has increased the utility
of these data and a MLVA and spoligotype data-
base has been developed to aid epidemiological
surveillance in Spain (Rodriguez-Campos et al.,
2012a). More recent application of MLVA and
spoligotyping in a high prevalence area indi-
cated that persistence of the same molecular
types within small spatial localities was a driver
of ongoing infection rather than re-introduction
from external sources (de la Cruz et al., 2014),
and that multiple molecular types were found in
single hosts as a result of super-infection
(Navarro et al., 2015).
Italian researchers have applied spoligo-,
MLVA and IS 6110 RFLP typing to human
M. bovis isolates (Lari et al., 2006, 2011). A
study in Tuscany identified that 2.3% of 829
cases were caused by M. bovis infection (Lari
et al., 2007). Subsequently, optimization of an
MLVA panel for the Italian M. bovis population
was undertaken using isolates collected from
cattle between 2000 and 2006 in northern Italy
(Boniotti et al., 2009). In a recent specific epide-
miological application, MLVA and spoligotyping
data helped identify a stable source of M. bovis
infection in a joint enterprise cattle and goat
farm (Zanardi et al., 2013) where the presence
of a strain identical to that isolated in a previous
breakdown suggested on-farm persistence,
potentially in goats, which were not being tested
under the statutory TB eradication scheme
(Zanardi et al., 2013).
Some of the earliest work on characteriza-
tion and standardization of MLVA methodolo-
gies to the MTC was undertaken in France in the
mid-2000s (Le Fleche et al., 2006). Application
of these techniques to human patients con-
firmed that M. bovis was responsible for 2% of
human TB cases during a 5-year retrospective
study (Mignard et al., 2006). The lack of clonal
evolution and relatedness between these human
cases was useful in excluding transmission
between human cases (Mignard et al., 2006).
More recently, large-scale spoligotyping and
MLVA of 4654 cattle and wildlife isolates col-
lected since 1978 has increased the knowledge
of the local M. bovis population structure, which
consists of several clonal groups, some exhibit-
ing geographic restriction (Hauer et al., 2015).