Zoonoses and Public Health
REVIEW ARTICLE
Molecular Epidemiology of Mycobacterium bovis in Humans
and Cattle
~ eda-Vazquez3 and H. Castan
~ eda-Vazquez3
A. El-Sayed1, S. El-Shannat1, M. Kamel1,2, M. A. Castan
1
2
3
Laboratory of Molecular Epidemiology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt
Faculty of Veterinary Medicine, Freie Universit€
at Berlin, Berlin, Germany
Laboratory of Mastitis and Molecular Diagnostic, Department of Veterinary Medicine, Division of Veterinary Sciences, University of Guadalajara,
Guadalajara, Mexico
Impacts
• Tuberculosis is a serious re-emerging disease worldwide. The problem of
•
•
antibiotic resistance is becoming increasingly common. The rate of
childhood TB increases annually.
Mycobacterium bovis is responsible for great economic losses and represents
a severe threat to public health.
The purpose of this review was to highlight the seriousness of M. bovis for
both humans and animals, and to focus on the importance of molecular epidemiological studies of M. bovis and the recent tools used in such studies.
Keywords:
Molecular Epidemiology; Mycobacterium
bovis; spoligotyping; tuberculosis
Correspondence:
A. El-Sayed. LME, Faculty of Veterinary
Medicine, Giza Square, 11451 Giza, Egypt.
Tel.: +201097770926; Fax: +2035725240;
E-mail: aaelsayed@cu.edu.eg
Received for publication June 30, 2015
doi: 10.1111/zph.12242
Summary
Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is a serious
re-emerging disease in both animals and humans. The evolution of the Multiand Extensively drug-resistant M. bovis strains (MDR-TB and XDR-TB) represents a global threat to public health. Worldwide, the disease is responsible for
great economic losses in the veterinary field, serious threat to the ecosystem, and
about 3.1% of human TB cases, up to 16% in Tanzania. Only thorough investigation to understand the pathogen’s epidemiology can help in controlling the disease and minimizing its threat. For this purpose, various tools have been
developed for use in advanced molecular epidemiological studies of bTB, either
alone or in combination with standard conventional epidemiological approaches.
These techniques enable the analysis of the intra- and inter-species transmission
dynamics of bTB. The delivered data can reveal detailed insights into the source
of infection, correlations among human and bovine isolates, strain diversity and
evolution, spread, geographical localization, host preference, tracing of certain
virulence factors such as antibiotic resistance genes, and finally the risk factors for
the maintenance and spread of M. bovis. They also allow for the determination of
epidemic and endemic strains. This, in turn, has a significant diagnostic impact
and helps in vaccine development for bTB eradication programs. The present
review discusses many topics including the aetiology, epidemiology and importance of M. bovis, the prevalence of bTB in humans and animals in various countries, the molecular epidemiology of M. bovis, and finally applied molecular
epidemiological techniques.
Although many researchers use the expression ‘molecular
epidemiology’, its actual meaning is sometimes unclear.
The most accepted definitions of molecular epidemiology
in simple terms are the use of molecular techniques (tools)
to define disease and its pre-clinical state by identifying the
presence of susceptible genes (Shpilberg et al., 1997), or the
science that utilizes molecular biology to define the distribution of disease in a population (descriptive epidemiology) and relies heavily on integration of traditional (or
analytical) epidemiological approaches to identify the
© 2015 Blackwell Verlag GmbH Zoonoses and Public Health, 2016, 63, 251–264
251
Introduction
Molecular Epidemiology of Mycobacterium bovis
aetiological determinants of this distribution (Snow, 2011).
It can also be defined as the combination of standard classical epidemiological approaches with molecular techniques
to track specific strains of pathogens in order to understand
the distribution of disease in populations (Skuce and Neill,
2001).
Molecular epidemiological studies aim in general to provide full characterization of infectious agents in order to
determine the physical sources, route of transmission, virulence genes, vaccine-relevant antigens and drug resistance
(Levin et al., 1999). In practice, in TB specialized laboratories, molecular epidemiology helps to determine the source
of infection and outbreaks, recognize the relationship
between different outbreaks and detect wild animal reservoirs of M. bovis. In addition, they can provide insight into
the risk factors for bTB transmission by allowing the identification of the disease’s dynamics (Garnier et al., 2003;
Zumarraga et al., 2005; Ramos et al., 2014). This can be
achieved through the application of various advanced
molecular-based techniques such as multilocus VNTR analysis (MLVA), Spacer Oligonucleotide typing (Spoligotyping) and Restriction Fragment Length Polymorphism
(RFLP) analysis in strain typing. These techniques provide
important epidemiological information about isolates of
M. bovis that may help in the control of bovine TB by indicating possible links between diseased animals, detecting
and sampling outbreaks, and even demonstrating cases of
laboratory cross-contamination between samples, as
reported by Mendez et al. (2006), who solved the problem
of laboratory cross-contamination by spoligotyping. In
addition, these techniques can provide clear data about the
identity of single epidemic strains and distinguish them
from epidemiologically unrelated isolates. Additional data
about the role and distribution of certain incriminated
genotypes can only be obtained through the genetic characterization of pathogens isolated from outbreaks. Genotyping is increasingly becoming a standard tool for disease
control and eradication programs, as it provides significant
insight into the sources of infection, identification of
ecosystem, transmission routes between livestock and wildlife or humans, and transmission routes of bTB within
migrating livestock (Medeiros Ldos et al., 2010; Schiller
et al., 2010; Ramos et al., 2014).
A. El-Sayed et al.
Mycobacterium bovis is one of the members of the Mycobacterium tuberculosis complex (MTBC) and is the causative
agent of bovine tuberculosis (bTB) (Smith et al., 2006; Palmer, 2007; Hershberg et al., 2008; Wirth et al., 2008; Miller
and Sweeney, 2013; Kamel et al., 2014). To date, the known
members of the Mycobacterium tuberculosis complex consist
of: M. tuberculosis, M. canettii, M. africanum subtypes I
and II), M. bovis, M. caprae, M. microti, M. pinnipedii and
the attenuated M. bovis BCG (Bacillus Calmette-Guerin)
vaccine strain (OIE, 2014), in addition to M. orygis (van
Ingen et al., 2012), M. mungi (Alexander et al., 2010), Dassie bacillus (Parsons et al., 2008) and Chimpanzee bacillus
(Coscolla et al., 2013). All members of the MTBC exhibit a
99.9% sequence similarity and their 16SrRNA are also conserved, with the exception of M. canetti (Dos Vultos et al.,
2008) and are capable of causing a serious disease with similar pathology (Rodriguez-Campos et al., 2014). Like other
mycobacteria, M. bovis is very robust in the environment
and can survive under extreme environmental conditions
(Courtenay et al., 2006; Fine et al., 2011). It can be isolated
for about 6 weeks from carcasses and 4 weeks from faecal
matter, depending on weather conditions, but its survival
can be prolonged up to 58 days in contaminated water, up
to 2 years in covered dungs and up to 5 months under diffuse sunlight conditions. Mycobacterium bovis bacilli in
game parks can also be spread by rains to contaminate
grazing areas, making cattle more susceptible to infection
from wildlife. However, the amount required for indirect
transmission of the disease is clearly higher than that
needed for direct or aerosol transmission (Cosivi et al.,
1995; Tanner and Michel, 1999; Sweeney et al., 2007; Fine
et al., 2011). Once bTB is established in a herd, it spreads
via aerosols, suckling, direct contact between animals and
sharing of water and feed. The bTB infection in maintenance hosts can persist through horizontal transfer in the
absence of any other source of M. bovis and can be transmitted to other susceptible hosts (Biet et al., 2005).
Although M. bovis is the main causative agent of bovine
tuberculosis, it has the broadest host range among all
MTBC members. Although it was observed in 1898 that the
mycobacteria isolated from humans and cows vary in their
infectious power in different animal species, the two species
were first separated in 1970 (Karlson and Lessel, 1970).
Mycobacterium bovis can infect farm animals such as cattle,
farmed buffalo, goats and farmed dear. While goats are
considered reservoir hosts of M. bovis (Kamel et al., 2014),
other animal species such as pigs, cats, dogs, horses and
sheep are considered spillover hosts. In addition, bovine
tuberculosis has been reported worldwide in wild ruminants, with very similar symptoms to those of domestic
ruminants (Biet et al., 2005). At present, cases of M. bovis
infections have been reported in more than 40 free-ranging
wild animal species, such as equines, camels, wild boars,
deer, antelopes, foxes, mink, ferrets, rats, primates, llamas,
kudus, elands, tapirs, elks, elephants, sitatungas, oryxes,
addaxes, rhinoceroses, possums, ground squirrels, otters,
seals, hares, moles, raccoons, coyotes and several predatory
felines including lions, tigers, leopards and lynx (OIE,
2014). Other wildlife species are major reservoirs of
M. bovis infection in different countries including
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Aetiology, Epidemiology and Zoonotic Importance
A. El-Sayed et al.
Molecular Epidemiology of Mycobacterium bovis
White-tailed deer (USA), wild boar (Spain), brush tail possums (New Zealand), African buffalo (South Africa), in
addition to badgers (Sweeney et al., 2007; Fitzgerald and
Kaneene, 2012; Hardstaff et al., 2014; Pesciaroli et al.,
2014).
The pathogenesis of bovine tuberculosis is not as well
understood as that of human tuberculosis. Advances in the
field of human tuberculosis have been made using several
small animal models of M. tuberculosis infection. Extrapolations of findings from small animal models have been
applied to bovine tuberculosis, but these extrapolations
may not always be relevant as there are clear differences in
host susceptibility, host immune response, environment,
host ecology and behaviour (Palmer and Waters, 2006).
Despite significant variations in size, appearance and distribution of the tuberculous lesions in different species, the
majority of lesions in affected wildlife species closely resemble those of domestic cattle (Zanella et al., 2008; Drewe
et al., 2009). The severity of the disease and the nature of
the clinical symptoms depend on the species susceptibility,
invading genotype and on the prevalence of bTB (Waters
et al., 2014). It is even believed that some breeds of cattle
and deer could be resistant to M. bovis infections (Delahay
et al., 2001; Michel, 2002; Brotherstone et al., 2010;
Rodrıguez Campos, 2013; Bermingham et al., 2014).
The aerosol transmission of M. bovis is the most frequent form of transmission between herd members, and
spillover to other species occurs via different means of
transmission. Although humans, dogs and cats can contract
the infection through direct contact with infected herds,
they are more susceptible to the digestive form of the disease. Drinking infected milk, feeding on infected carcasses
or even coming into contact with pus secreted from draining fistulae of lymph nodes can be the means of disease
transmission (Michel et al., 2006, 2007). However, it was
suggested that dogs are more likely to be infected with
M. tuberculosis following exposure to infected humans
(Birn et al., 1965; Greene and Gunn-Moore, 2012;
Macpherson, 2012).
Not only is M. bovis the principal cause of re-emerging
TB in free-living or captive wildlife, domestic livestock and
non-human primates, but it also has zoonotic potential to
cause human infections that are indistinguishable from
those produced by the human type (M. tuberculosis).
Therefore, identification of the TB causative agent at the
subspecies level is important for public health. Mycobacterium bovis strains are, in contrast to M. tuberculosis, not
transmitted effectively between humans (Runyon, 1959,
CDC, 2011; Gonzalo-Asensio et al., 2014) and are usually
associated with extrapulmonary TB in humans (Malama
et al., 2014; Han et al., 2015). However, the isolation of
M. bovis from human sputum in some reports may indicate its potential for human-to-human infection, especially
The exact prevalence of M. bovis infections in humans is
unknown and underdiagnosed, especially in various African countries (Cosivi et al., 1998). It is commonly assumed
that M. bovis is less virulent and has less transmission
potential among human populations than M. tuberculosis.
This could be attributed to the presence of three mutations
affecting the virulence regulation system PhoPR in M. bovis
and the closely related M. africanum lineage 6 (GonzaloAsensio et al., 2014). Nevertheless, M. bovis remains a serious opportunistic infection in HIV-infected persons.
Therefore, it is important to public health policy makers to
be able to differentiate human infections caused by M. bovis from those due to M. tuberculosis (Cosivi et al., 1998;
Wedlock et al., 2002). The free movement of cattle within
and between countries and even continents certainly facilitated the worldwide distribution of bovine tuberculosis and
the spread of newly evolved antibiotic-resistant strains
(Sechi et al., 2001). For this reason, great efforts are being
made to understand the population structure of M. bovis
through the use of recent molecular techniques that allow
for the identification of the dominant M. bovis strain in larger geographic locations and for the tracing of the source
of introduction (Kamerbeek et al., 1997; Lindstedt, 2005;
M€
uller et al., 2009). The re-emergence of bovine tuberculosis among animals and humans is a serious worldwide concern, especially in developing countries. Mycobacterium
bovis infections in cattle herds were detected in 109 countries worldwide between 2005 and 2010 (Ramos et al.,
2014). People living in close contact with infected cattle or
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253
in closed populations such as prisons (Romero et al., 2006;
Gumi et al., 2012). The human-to-human transmission
can be differentiated from animal-to-human transmission
by the application of fingerprinting tools (Perez-Lago et al.,
2014).
For a long time, due to the close relationship between
Mycobacterium bovis and mycobacterium tuberculosis, it was
believed that M. bovis was the evolutionary precursor to
M. tuberculosis. Then, recent molecular epidemiological
studies suggested that M. bovis evolved in parallel with
M. tuberculosis, possibly through the independent evolution of both types from another precursor mycobacteria
likely related to M. canetti (Smith, 2003). However, the
M. bovis genome was sequenced in 2003, and its size was
clearly found to be smaller than that of M. tuberculosis,
which indicates that its evolution from an M. tuberculosislike common ancestor most likely originated from an RD9
deleted strain such as M. africanum subtype I, in contrast
to M. tuberculosis, which is more closely related to
M. africanum subtype II (Mostowy et al., 2004, 2005;
Huard et al., 2006).
Epidemiological Prevalence of Bovine Tuberculosis
Molecular Epidemiology of Mycobacterium bovis
A. El-Sayed et al.
children who drink unpasteurized milk are at risk of
infection. On the other hand, tuberculosis caused by M. tuberculosis was also reported in animals living in close contact with humans (Cadmus et al., 2006; Srivastava et al.,
2008). It was estimated that about 3.1% of human TB cases
worldwide are caused by M. bovis. While <1% of human
infections are caused by M. bovis in Spain, the prevalence
in Africa ranges from 3.9% in Nigeria, up to 7% in Uganda,
and even 16% in Tanzania (Romero et al., 2006; Malama
et al., 2014). It was reported that in some developing countries, M. bovis is responsible for 5–10% of all human TB
cases and 30% of all TB cases in children (Wedlock et al.,
2002). Mycobacterium bovis could be isolated from approximately 3.9–5% of human tuberculosis patients in Nigeria
(Idigbe et al., 1986; Cadmus et al., 2006; Mawak et al.,
2006) and 0.6–1.85% of patients with pulmonary tuberculosis in Burkina Faso. The pathogen was also detected in
26.5% of unpasteurized milk samples (Sanou et al., 2014).
In Zambia, the spoligotyping of 49 M. bovis strains with
bovine and human origin revealed a high homogeneity
among the existing genotypes and even identical MIRUVNTR genetic profiles. The same genetic profile characterized by the absence of spacers 3, 9, 16 and 39–43, SB 0120/
SIT 482 was reported in tuberculous cattle in Algeria, Brazil, France and Zambia, and in human patients from Italy
and Germany. This profile differs from that observed in
East African and Southern African countries (Malama
et al., 2014); however, similar observations were reported
in Ethiopia and in Spain, where M. bovis isolates from
human and cattle shared the same genetic profile even in
multidrug-resistant M. bovis (MDR) strains. Nevertheless,
some spacers and certain MIRU-VNTR, which are not used
in the current spoligotyping system, could differentiate
between M. bovis of both human and cattle origin. They
differ in a copy at ETR-A and at QUB-3232 (Romero et al.,
2006; Gumi et al., 2012).
Of 55 African and 36 Asian countries, only seven African
and seven Asian countries apply adequate disease control
measures; the remaining 77 countries control the disease
inadequately or not at all. About 94% of cattle and 99% of
buffalo populations in Asia are either partly controlled for
bTB or not controlled at all. Therefore, about 85% of African cattle and 82% of the human population in Africa, and
94% of the human population in Asia live at high bTB risk
(Cosivi et al., 1998). In Latin America, about 24% of cattle
and 60% of the human population live in areas where there
is no control or only limited control for bovine TB (Cosivi
et al., 1995; Muller et al., 2013). This is thought to be a
result of wildlife reservoirs that spread the infection to
farmed animals, especially cattle. Such reservoirs make
eradication more complicated and extremely difficult, and
necessitate special programs to control the disease (Cosivi
et al., 1998; Renwick et al., 2007; More et al., 2015).
Molecular epidemiological tools are now applied to
provide novel information about the spread of the disease
and to analyse its transmission dynamics (Small and Van
Embden, 1994; Savine et al., 2002). In Africa, epidemiological studies of TB are difficult to perform due to cost and
the lack of information and trained teams. However, some
primary data are available from certain African countries.
In Nigeria and Cameroon, the spoligotyping profiles of
some isolated M. bovis strains offer some information
about their distribution among human and animal populations in West Africa (M€
uller et al., 2009). Molecular epidemiological typing of the isolates based on spoligotyping,
RDAf1 deletion and MIRU-VNTR typing was performed
and resulted in detection of 12 spoligotypes, and the
obtained data grouped the 33 investigated M. bovis isolates
into seven clusters, including isolates exclusively from cattle
(5) or humans (1) or from both (1). These isolates were
shown to belong mainly to the African 1 (Af1) clonal complex (81.8%) and, to a lesser extent, to the putative African
5 (Af5) clonal complex (18.2%) according to (Sanou et al.,
2014).With the aid of spoligotyping, M. bovis strains of
bovine origin isolated from Mali could be divided into two
groups. The first group is marked by the distinct loss of
spacer 30 and accounts for 65% of the strains detected and
resembles strains detected in Chad, Cameroon and Nigeria.
This spoligotype is closely related to the BCG-like type
which dominates in France, suggesting that M. bovis could
have been imported to this region during the French colonial period, with 12/13 isolates lacking spacer 6 in addition
to spacer 30. The second group (6/7 isolates), lacking spacers 4 and 5, resembles spoligotype patterns previously identified in France, Spain and North Africa (Muller et al.,
2008). However, a strong relationship between North African (mainly Algerian) and French isolates was expected
due to the importation of live animals from Europe to
Algeria during the French colonial period (1830–1962) and
later (Sahraoui et al., 2009). In West Africa, African 1
(Af1) M. bovis clone spoligotype pattern SB0944 was found
to dominate only in Chad, Nigeria, Mali and Cameroon,
indicating a separate ancestor for this clone in the aforementioned countries. They are closely related and characterized by specific chromosomal deletion (RDAf1) and the
absence of spacer 30 in the spoligotype patterns. On the
other hand, a different clone (African 2; Af2) was detected
only in East Africa (Ethiopia, Burundi, Uganda and Tanzania), which might be less virulent than Af1 due to the
entire lack of mce2 operon as a consequence of the RDAf2
deletion in Af2 clone, in addition to the mce3 operon
which is already absent in all M. bovis strains. However,
the Af2 strains are capable of inducing typical tubercle
lesions in cattle. This clone is defined by a specific chromosomal deletion (RDAf2) with the absence of spacers 3 to 7
in their patterns. It is possible that all Af2 spoligotype
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A. El-Sayed et al.
patterns were derived from an ancestral spoligotype pattern
equivalent to that of the vaccine strain BCG (SB0120, missing spacers 3, 9, 16 and 39 to 43), with the additional deletion of spacers 4 to 7 (SB0133). While the strains of the Af2
clonal complex were detected only in cattle reared in East
Africa, the Af1 clonal complex is reported only in cattle
from West-Central Africa (Berg et al., 2011). In Mozambique and South Africa, non-Af1–non-Af2 clones are
reported, most likely the European clone (European 1;
Eu1) characterized by a specific deletion called RDEu1 and
the loss of spoligotype spacer 11 (spacers 4 to 7 are usually
present) according to (Berg et al., 2011). The Eu1 clone is
rarely reported in Africa with the exception of South
Africa. Nearly 99% of bTB found in England and Ireland
belong to the Eu1 characterized by the deletion of chromosomal region RDEu1 and absence of spacer 11 in their pattern. This clone also dominates in locations of former
trading partners and English speaking colonies of the UK.
This may be attributed to the massive export of Hereford
beef cattle in the 18th century from the UK to its former
colonies, which would be the vehicle for the spread of this
clone. However, the Eu1 is present in 14% of French, Portuguese and Spanish isolates belonging to this profile and is
rare in other European countries. Meanwhile, with the
exception of Brazil, the Eu1 clonal complex dominates in
both North and South America, especially in Argentina,
Chile, Ecuador, Mexico and North America. The Eu1 clone
was even reported in Korea and Kazakhstan but not in
Africa except South Africa (Ameni et al., 2010; Berg et al.,
2011; Mwakapuja et al., 2013; Rodrıguez Campos, 2013).
The use of modern molecular epidemiological tools
enabled the identification of three clonal lineages of M. bovis, namely African 1, African 2 and European 1. Members
of each group have a distinct spoligotype signature and are
characterized by a unique deletion present in each member
of the complex. The African 1 and African 2 groups are
restricted to central-west Africa and eEast Africa, respectively, and were never detected in cattle outside of these
regions, while the European 1 members are globally distributed. It is thought that this global distribution resulted
from the exportation of modern cattle breeds, such as Herefords bred in the UK in the 18th Century, to the former British colonies. The cows acted as a vehicle for the global
spread of this closely related group of strains (Smith, 2012).
Molecular Epidemiology of Mycobacterium bovis
ment of effective and standardized genotyping systems of
M. bovis to create widely accepted standard genotyping
protocols (Durr et al., 2000b). Thus, different techniques
have been developed for the genotyping of M. bovis,
including whole genome techniques (e.g. restriction
endonuclease analysis [REA], the restriction fragment
length polymorphism /RFLP, pulsed-field gel electrophoresis/PFGE, whole genome sequencing and whole genome
microarray) and the partial genome techniques such as
specific repeated sequences (e.g. tandom-repeated
sequences and non-tandom repeated sequences); random
sequences (e.g. random amplified polymorphic deoxyribonucleic acid /RAPD analysis); house-keeping genes (e.g.
multilocus sequence typing /MLST); regions of difference
(direct repeats /DR typing) and single nucleotide polymorphism (SNP typing). The Tandom Repeated Sequences
include the VNTR typing and IS6110 amplification while
the Non-tandom-repeated sequences include the IS6110RFLP, IS1081-RFLP, PGRS-RFLP, DR-RFLP and the spacer
oligonucleotide typing (Spoliogotyping) (Kremer et al.,
1999; Durr et al., 2000a,b; Gormley et al., 2014; Jagielski
et al., 2014).
RAPD, MTPR and PGRS techniques
Overview of the most commonly used molecular
epidemiological techniques
Although different molecular epidemiological studies have
been performed to enable better traceability of M. bovis
infections and to detect the source of outbreaks (Haddad
et al., 2001), there are still several problems in the develop-
Not all of these techniques are in use for various reasons,
mainly the lack of reproducibility, high cost and complexity. Among the first PCR-based genotyping techniques was
the Random Amplified Polymorphic Deoxyribonucleic
Acid Analysis (RAPD). This technique is rarely used now
due to its lack of reproducibility and poor discrimination
(Jagielski et al., 2014). Similarly, there is Ampliprinting of
the IS6110 targeting the Major Polymorphic Tandem
Repeat (MPTR). The MPTR region resembles the DR
region and consists of 10-bp direct repeats separated by
5-bp spacers. The MPTR also lacks reproducibility and
therefore is not used as a routine genotyping method
(Kremer et al., 1999; Collins, 2011). Because other regions
in the genome of Mycobacteria contain up to 80% GC
nucleotides, these Polymorphic GC Rich Sequences (PGRS)
can be used for molecular typing; however, they achieve
only high discriminatory power in strains with a high copy
number of IS6110. For this reason, IS6110-RFLP became
the gold standard for both human and bovine tuberculosis
over many years. However, its use in bovine tuberculosis is
less common due to the lower copy number of IS6110 in
M. bovis (Collins, 2011). Although Single Nucleotide Polymorphism (SNP) typing has a clear discriminatory power,
it is not commonly used because it is a two-step technique
(PCR then sequencing, or PCR then restriction enzyme),
which makes it expensive and time-consuming (Abadia
et al., 2010; Schurch et al., 2011; Rodrıguez Campos, 2013;
Jagielski et al., 2014).
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Molecular Epidemiology of Mycobacterium bovis
Molecular Epidemiology of Mycobacterium bovis
A. El-Sayed et al.
The introduction of ‘spacer oligonucleotide’ or ‘spoligotyping’ for molecular typing of MTBC started in 1997 (Kamerbeek et al., 1997). It is a PCR-based method like other
techniques, but it avoids the technical difficulties facing
IS6110 RFLP typing. At the beginning, spoligotyping
showed limited value for epidemiological transmission
when used alone, so it was combined with IS6110 RFLP
analysis (Kremer et al., 1999). Spoligotyping is now
increasingly used in molecular epidemiology of MTC as
well as in species and strain differentiation. This technique
depends on the genetic differences between strains due to
loss of spacers at the direct repeat (DR) region in MTC
(Kamerbeek et al., 1997). Spoligotyping is more discriminating than IS61110 RFLP, but equally as discriminating as
DR-RFLP, which identifies genome polymorphism in the
DR region. Nevertheless, spoligotyping remains the preferred technique as it is easy to do and to interpret (Durr
et al., 2000b).
As the introduction of any mutation (e.g. deletion) in a
strain will be inherited by all of its descendants, it can be
used to trace back clonal complexes. On these bases, spoligotyping was developed. Spoligotyping is a simple and rapid
tool for molecular typing of M. bovis that does not require
purified DNA. Spoligotyping is a mixture of PCR and
hybridization techniques. In 1993, Clustered Regularly
Interspaced Palindromic Repeats (CRISPRs) were discovered in the MTBC. This direct repeat (DR) region consists
of multiple 36-bp direct repeats separated by spacers ranging in length from 25 to 41 bp. Meanwhile, a direct variant
repeat (DVR) consists of a single direct repeat (DR) and its
adjacent spacer. The typing of the strains depends on the
presence or absence of the different spacers. The use of
spoligotyping depends on the detection of 43 of 104 identified spacers. For spoligotyping, the DRs are targeted by
PCR oligonucleotides to be amplified and blotted to detect
the present spacers. The data can be digitalized and stored
as binary code, and every spoligotype pattern becomes a
unique identifier when submitted to the international bank
of spoligotype patterns at www.Mbovis.org. The code used
includes the prefix SB followed by one to four digits in
which 0 and 1 stand for the absence or presence of spacers,
respectively. This enables the comparison and tracing of the
evolutionary relationship among strains, as the spacers can
only be lost but cannot be regained. The spacers are frequently lost independently in different lineages (homoplasy) (Muller et al., 2008; Sahraoui et al., 2009; Berg et al.,
2011; Rodrıguez Campos, 2013).
Spoligotyping is considered to be the most common epidemiological molecular-typing tool for M. bovis. The application of spoligotyping for the spatial (geotyping) and
temporal (chronotyping) traceability of outbreaks is practical and helpful (Haddad et al., 2001). This depends on the
identification of the polymorphisms in the spacer units of
the direct repeat (DR) region of the chromosome. This
region consists of multiple, virtually identical direct variant
repeat (DVR) units of 36-bp sequences interspersed with
DNA spacers of a similar size. The DR region may contain
more than 60 DVR units. Nevertheless, 43 of these units
were chosen from the genome of the M. tuberculosis strain
(H37RV) and the M. bovis BCG strain (P3) to be used as
standard for spoligotyping of all members of M. tuberculosis complex. However, the amplification of some of the
spacers (14, 15, 18, 39 and 40) was found to be problematic
due to sequence mutations (Abadia et al., 2010; Rodrıguez
Campos, 2013).
Each spoligotype pattern has a given identifier and an
international name (e.g. ‘type 9’ in UK and SB0140, respectively) according to (Smith et al., 2006). The technique
delivers digital data which can be easily analysed and shared
between laboratories with a high distinguishing power
between the MTBC members. It can also define M. tuberculosis clades and families in phylogenetic studies (Brudey
et al., 2006). For the detection of the present spacers, Reverse Line Blotting step (RLB) is required. The blotting of
spoligotyping can be performed on a hybridization
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Restriction fragment length polymorphism
The typing of TB isolates using IS6110 restriction fragment
length polymorphism (RFLP) analysis was first described in
1991. It quickly became the standard tool for molecular
epidemiological investigations (van Soolingen et al., 1991)
and is even considered to be the current ‘gold standard’
and the most widely applied typing method of the M. tuberculosis complex (van Soolingen et al., 1991). The technique detects the variation in both the number and
genomic position of IS6110 and by so doing, it generates
reproducible strain-specific patterns (Hayward and Watson, 1998). However, the use of molecular typing of M.
bovis in England based on RFLP indicated that most strains
in the UK contain only one copy of IS6110, which limits
the usefulness of this method (Smith et al., 2006). Further,
it has a low discriminatory power, especially for strains
with a low number of IS6110 copies (Kremer et al., 1999;
Serraino et al., 1999).
Among all RFLP techniques for M. bovis typing, PGRSRFLP has the best discriminatory power. However, in contrast to all RFLP techniques, which require DNA extraction,
large amounts of DNA and high technicality, and which
have problems with their reproducibility and are time consuming, spoligotyping is not only rapid, simple and reproducible but can also be carried out on cell lysates or clinical
specimens (Dvorska et al., 2001; Haddad et al., 2001;
Jagielski et al., 2014).
Spoligotyping
A. El-Sayed et al.
membrane or a commercial spoligotyping membrane
(Luminex platform). The latter was found to be faster and
easier and to increase the reproducibility (Jeffries et al.,
2009).
A major disadvantage of spoligotyping is its inability to
deal with the mixed infection as the delivered data will be
from the present spacers of all existing strains. The VNTR,
in contrast, can be applied even if a mixed infection is suspected, as this will be visualized as a double band. In addition, and opposite to spoligotyping, variable number
tandem repeats typing (VNTR) also known as multilocus
variable number tandem repeat typing (MLVA) targets
polymorphic loci of tandem repeats distributed all over the
genome (Rodrıguez Campos, 2013).
Different Spoligotypes Reported from Different
Countries
The comparison of genetic profiles between animal and
human isolates of M. bovis enables us to detect the source(s)
of infection and the route(s) of transmission, and to predict
the future picture of disease progression. It is worth mentioning that the discriminatory power of the different typing
techniques available for M. bovis varies by country, and
M. bovis genotypes of human origin parallel those of livestock within the same country, which supports the presence
of an epidemiological link between human and animal
M. bovis TB cases (Lari et al., 2011). When comparing both
human and bovine isolates from Tuscany, Italy, the isolates
with the spoligotype profile ST482/SB0120 dominated in
both species. They were detected in 63.8% and 54.6% of
human isolates and infected herds, respectively (Boniotti
et al., 2009; Lari et al., 2011). The same spoligotype dominates among bovine isolates from France, Germany and
Spain (Aranaz et al., 1996; Haddad et al., 2001; Kubica
et al., 2003), but never in the United Kingdom or Ireland.
Other profiles were also reported in M. bovis isolates from
humans in Italy including the ST482/SB0120 spoligotype,
which was also detected in 20% of German patients and 1%
of British patients, the profile SIT665/SB0134, present in
5.8% of bovine isolates, the profile SB0934 in 1.3% of bovine
isolates and finally the less significant profiles SB0927,
SB0950 and SB0867, which were present in <1% of the herds
(Aranaz et al., 1996; Kubica et al., 2003; Lari et al., 2011).
The total number of spoligotypes in France is very high,
indicating more strain heterogeneity when compared to
other regions such as the UK, Australia, Cameroon and
Tanzania, and leading to the low prevalence of the dominant types among the M. bovis population. In France, the
most common spoligotype is the BCG-like group, which
resembles the vaccine strain M. bovis BCG isolated in
France in the 19th century. However, the prevalence of
the two main spoligotypes (BCG-like and GB54) in France
© 2015 Blackwell Verlag GmbH Zoonoses and Public Health, 2016, 63, 251–264
Molecular Epidemiology of Mycobacterium bovis
is low compared to the prevalence of the two major
spoligotypes present in the UK (70% of isolates), Ireland
(52% of isolates), Italy (66.6%), Spain (46%) and Australia,
Canada and Iran (88%) (Haddad et al., 2001).
It is remarkable that the spoligotypes in France differ
from those detected in the UK; the predominant spoligotypes in the UK were detected at a very low percentage in
France and vice versa. On the other hand, the strains that
dominate in the UK also dominate in places traditionally
linked to the UK, such as commonwealth countries and
South American countries. It is also unusual that the BCGlike type which dominates in France has never been
reported in the UK, but is frequently detected in other
European countries such as Belgium, Italy and Spain, and
also in North Africa (Haddad et al., 2001; Smith, 2012;
Lamine-Khemiri et al., 2014).
In France, the dominance of the BCG-like spoligotype
over decades indicates the high stability of the DR region
compared to other markers like IS6110. Meanwhile, the
detection of more variants of spoligotypes in the BCG-like
group along with the continuous decrease of the prevalence
of BCG isolates indicates the progressive evolution of the
spoligotypes (Haddad et al., 2001).
Mycobacterial Interspersed Repetitive UnitVariable Number of Tandem Repeats
Nowadays, most laboratories depend on PCR-based techniques such as multilocus variable analysis (MLVA) for
M. bovis typing as they are faster, easier, reproducible and
cost-effective (Romero et al., 2006). MLVA analysis, also
referred to as mycobacterial interspersed repetitive unit–
variable number of tandem repeats (MIRU-VNTR) (Allix
et al., 2006) depends on strain variation indicated by allelic
diversity at these multiple loci. The use of MLVA analysis
for M. bovis transmission research helps in delivering
highly informative data when combined with traditional
epidemiology (Allix et al., 2006). To increase the discriminatory power of spoligotyping, VNTR typing is used for
further subtyping of strain groups initially identified by
spoligotyping, as VNTR typing enables greater discrimination than spoligotyping (Muller et al., 2008; Sahraoui et al.,
2009). Moreover, spoligotyping allows for species differentiation based on the absence of certain spacers in certain
MTB members, for example the absence of spacer number
3, 9, 16 and 39–43 in M. bovis and spacers 1, 3–16, 28 and
39–43 in M. caprae (Dvorska et al., 2001; Rodrıguez Campos, 2013).
In general, the most commonly accepted molecular epidemiological tool is variable numbers of tandem repeats
(VNTR) typing. It is easy and cheap PCR-based typing that
delivers reproducible and accurate discrimination among
MTBC isolates. It includes typing of different genomic loci
257
Molecular Epidemiology of Mycobacterium bovis
A. El-Sayed et al.
such as exact tandem repeats (ETR) (Frothingham and
Meeker-O’Connell, 1998), mycobacterial interspersed
repetitive units (MIRUs) (Supply et al., 2000; Mazars et al.,
2001) and Queen’s University Belfast (QUB) (Skuce et al.,
2002). Variable nucleotide tandem repeat (VNTR) typing
detects the variation in the numbers of different repeat sets
dispersed throughout the genome and is similar to the concept of mini-satellite typing. The loci used are already well
characterized. However, no sharp demarcation exists
among these classifications, that is some genomic regions
are both ETR and MIRU at the same time (Smith et al.,
2006). Nonetheless, there are many differences between
MIRUs and other VNTRs, like the presence of some regulatory elements in the MIRUs as start and stop codons or
consensus sequences, and the fact that MIRUs usually overlap the start and stop codons of the flanking genes (Supply
et al., 2000; Romano et al., 2005).
Queen’s University in Belfast (QUB) and exact tandem
repeat (ETR) are other sets of VNTR sequence repeats.
They consist of many copies of 56- to 60-bp long units and
show high reproducibility. However, their major disadvantage is their low discrimination power for strains with a
high copy number of IS6110 in comparison with those with
a low copy number. In general, VNTR analysis allows for
the discrimination of unrelated strains of M. bovis better
than spoligotyping (Hilty et al., 2005; Skuce et al., 2005).
Six tandem repeats called exact tandem repeats (ETR)
were reported, namely A, B, C, D, E and F. They vary in size
from 53 to 79 bp. The first five (A to E) were found to offer
enough discrimination (Filliol et al., 2000). Also reported
were 40–50 mycobacterial interspersed repetitive units
(MIRUs) ranging in size from 46 to 101 bp. Although most
repeats are found in the intergenic regions, some of them
are located inside an ORF indicating variation in protein
sequence and eventually also physiological activity. While
some researchers suppose that the polymorphism in the
intergenic spaces might affect the expression of the downstream genes, for example ETR-A is located at the promoter
region of katG gene, others disagree with this theory
because the position of the loci is not always conserved
among the MTC members (Goyal et al., 1994; Rodrıguez
Campos, 2013). Nonetheless, an association between the
spoligotype-VNTR types and the virulence of M. bovis in
cattle was reported (Garbaccio et al., 2014).
Due to the increasing number of the discovered tandem
sequences (VNTRs, MIRUs, ETRs, and QUBs) and the
confusion in their nomenclature among researchers
(e.g. ETR-D and ETR-E are MIRUs at the same time), trials
to standardize a four-digit nomenclature system were
carried out to facilitate data comparison (Filliol et al.,
2000; Rodrıguez Campos, 2013).
In the last few years, VNTR typing has become the new
standard for typing of MTBC strains. However, it is not
yet standardized and allelic diversity of loci can vary in
different countries and among members of the
M. tuberculosis complex species. The reasons that VNTR is
the method of choice for typing of MTBC are its short
turn-around time, the fact that it offers a simple comparison of the digital results, and its applicability to small
volumes of DNA (Lindstedt, 2005). It also has a high discriminatory power for M. tuberculosis isolates (Andersen
et al., 1996; Le Fleche et al., 2001, 2002, 2006; Pourcel
et al., 2004). Only 12 of 41 well characterized MIRU loci
display polymorphism and have significant discriminatory
power (Supply et al., 2000). However, their combination
with other types of polymorphic loci like ETRs, which
contain large repeat units varying in size from 53 to
79 bp, would improve the discriminatory power (Frothingham and Meeker-O’Connell, 1998). In 2004, the Health
Protection Agency (HPA) standardized its typing protocols in different TB laboratories by combining both ETR
and MIRU loci into a set of 15 loci. Later on, in 2006, a
panel of 24 VNTRs was developed to optimize the discrimination and reproducibility of the test, which involved
the previously proposed 15 VNTRs plus an additional 9
VNTRs. In 2009, this protocol became the new global
standard panel for typing (Goyal et al., 1994; HPA, 2012;
Rodrıguez Campos, 2013) Moreover, this panel system
was also found to be useful for the typing of non-M. tuberculosis members of the MTBC (Stone et al., 2012). Different researchers reported that the application of
QUB3232, ETR-A, ETR-B, QUB11a, QUB11b and QUB
26 compared to MIRU4, 10, 16, 26 and 31 for M. caprae
resulted in the greatest discrimination among different
M. bovis isolates (Dvorska et al., 2001; Lari et al., 2011).
Thus far, none of the known typing techniques can be
applied on its own. Each tool has both advantages and disadvantages. However, the use of spoligotyping in combination with other MIRU-VNTR improves discriminatory
power among the strains (Ramos et al., 2014).
258
© 2015 Blackwell Verlag GmbH Zoonoses and Public Health, 2016, 63, 251–264
Novel DNA chip
In 2014, a new technique was developed by Srilohasin and
his team that was seen to be faster and simpler than other
techniques already in use. The multipurpose genotyping
tool can be efficiently applied for the performance of epidemiological and evolutionary studies of Mycobacterium
tuberculosis complex clinical isolates. It is very simple, flexible and quick, and allows for identification within 6 h
using a thermal cycler, a hybridization oven and a DNA
chip scanner. The technique, named Novel DNA Chip, is
based on a Modified DigiTag2 Assay. It depends on a multiplex PCR assay, followed by sizing of the amplicons using
capillary electrophoresis and automatic assignation of allelic values (Srilohasin et al., 2014).
A. El-Sayed et al.
Molecular Epidemiology of Mycobacterium bovis
Abadia, E., J. Zhang, T. D. Vultos, V. Ritacco, K. Kremer, E.
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© 2015 Blackwell Verlag GmbH Zoonoses and Public Health, 2016, 63, 251–264
259
Whole genome sequencing
The invention of new generations of sequencers enabled
the commercial use of Whole Genome Sequencing (WGS)
so that it became available for researchers. The technique
offers the potential for extraordinary insight into pathogens. The WGS-delivered data must be corroborated with
available epidemiological data in well-described systems. It
will likely become the typing method of choice in the future
as sequencing costs are reducing markedly (Biek et al.,
2012).
Conclusion
The development of molecular tools has added new aspects
to the conventional epidemiological approaches of M. bovis
infections. The data delivered significantly strengthened the
classical epidemiology, making the epidemiological data
more precise. Thus, understanding the advantages and disadvantages of these epidemiological tools is essential for a
more beneficial and accurate epidemiological evaluation of
the disease situation. This will be reflected in our understanding of the mycobacteria transmission dynamics and
pathogenesis within populations and between hosts, leading to the improvement of MTB control programs worldwide.
Conflict of Interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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