Gormley, 2014, M. Caprae

Research in Veterinary Science 97 (2014) S30–S43
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Research in Veterinary Science

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Bacteriological diagnosis and molecular strain typing of

Mycobacterium bovis and Mycobacterium caprae
E. Gormley a,*, L.A.L. Corner a, E. Costello b, S. Rodriguez-Campos c
a School of Veterinary Medicine, University College Dublin (UCD), Dublin 4, Ireland

b Central Veterinary Research Laboratory, Backweston, Celbridge, Co. Kildare, Ireland
c Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
A R T I C L E I N F O A B S T R A C T
Article history: The primary isolation of a Mycobacterium sp. of the Mycobacterium tuberculosis complex from an in-
Received 4 October 2013 fected animal provides a definitive diagnosis of tuberculosis. However, as Mycobacterium bovis and My-
Accepted 24 April 2014 cobacterium caprae are difficult to isolate, particularly for animals in the early stages of disease, success
is dependent on the optimal performance of all aspects of the bacteriological process, from the initial
Keywords: choice of tissue samples at post-mortem examination or clinical samples, to the type of media and con-
Mycobacterium bovis
ditions used to cultivate the microorganism. Each step has its own performance characteristics, which
M. caprae
can contribute to sensitivity and specificity of the procedure, and may need to be optimized in order to
Tuberculosis
Culture achieve the gold standard diagnosis. Having isolated the slow-growing mycobacteria, species identifica-
Diagnosis tion and fine resolution strain typing are keys to understanding the epidemiology of the disease and to
Strain typing devise strategies to limit transmission of infection. New technologies have emerged that can now even
Spoligotype discriminate different isolates from the same animal. In this review we highlight the key factors that con-
VNTR tribute to the accuracy of bacteriological diagnosis of M. bovis and M. caprae, and describe the develop-
ment of advanced genotyping techniques that are increasingly used in diagnostic laboratories for the purpose
of supporting detailed epidemiological investigations.
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction process has its own performance characteristic, determined by the

level of sensitivity and specificity of the particular test. For example,
In many countries, the principal reservoir host for Mycobacte- in animals at the very early stage of disease, diagnosis by clinical
rium bovis and Mycobacterium caprae infection are cattle and goats, examination is very insensitive and has low specificity.
respectively. Both cause tuberculosis in a wide range of domestic The maintenance and transmission of infection between cattle
and wild animals, also zoonotic tuberculosis in humans, and are re- and other domestic and wild animal species are poorly under-
garded as a cause of bovine tuberculosis (European Commission stood even though comprehensive surveillance can often reveal a
Health & Consumers Directorate-General Directorate G – Veterinary range of risk factors associated with disease transmission. The con-
and International Affairs Unit G2 – Animal Health, 2013; OIE, 2009). tribution of these factors can be difficult to quantify because of an
In well-established programmes to eradicate tuberculosis from cattle, inability to identify the source of infection and the lack of clear defi-
the majority of infected animals are detected when the disease is nitions of epidemiologically linked cases. The recent development
at an early stage of development, and it is rare to find animals of highly discriminating molecular genotyping technology is
showing clinical signs. From a public health perspective, the reduc- helping to address this problem by facilitating identification and fine
tion of disease levels in cattle, in combination with improved animal resolution of individual isolates that are associated with an out-
husbandry and routine pasteurization of milk, has served to lessen break. When applied alongside more traditional epidemiological
the risk of zoonotic transmission of infection to herd owners and methods, this can provide novel insights into the origin of break-
the general public. The disease can be diagnosed in cattle in many downs and the way in which the disease spreads both spatially and
ways, ranging from clinical examination and immunological assays temporally.
to post-mortem examination for gross lesions, collection of tissues The accurate diagnosis of M. bovis/M. caprae infection and fine
and other samples for histopathology and culture. Each diagnostic discrimination between isolates are therefore key to understand-
ing the origin and spread of disease and also for devising pro-
grammes for eradication of tuberculosis from cattle and other
* Corresponding author. Tel.: +353 1 716 6073; fax: +353 1 716 6091. domestic and wild animals. The availability of such information can
E-mail: [email protected] (E. Gormley). be of great value and can assist the risk manager to implement
http://dx.doi.org/10.1016/j.rvsc.2014.04.010
0034-5288/© 2014 Elsevier Ltd. All rights reserved.
E. Gormley et al./Research in Veterinary Science 97 (2014) S30–S43 S31
strategies to reduce the risk of transmission within and from popu- sensitivity of immunological diagnostic assays, or an underestima-
lations of different animal species. tion of test specificity. It is therefore essential to define the optimal
conditions for culture that take into account, for example, the con-
2. Bacteriological diagnosis of M. bovis and M. caprae centration of bacilli in the sample and the risk of contamination,
as these factors can influence the selection of decontamination pro-
Where bacteriological diagnosis is required, such as when seeking cedures, the duration of incubation, and the number and type of
confirmation of infection or when undertaking research on the epi- media slopes inoculated.
demiology and pathogenesis of infection in animal species, myco-
bacterial culture is used to identify and provide a definitive 2.2. Collection of post-mortem tissue samples for culture
confirmation of infection. The sensitivity of the bacteriological culture
is influenced by many factors and with little exception – the higher When using abattoir surveillance as the starting point for diag-
the sensitivity desired, the greater the resources required. For routine nosis, the range of samples collected for bacteriology is deter-
bacteriological diagnosis in a veterinary laboratory, the OIE (2009) mined during the post-mortem examination, and it is the detection
recommends for cattle, as a minimum, the culturing of suspect of gross lesions that largely dictates the choice of samples to be taken.
lesions, or the culturing of a pooled lymph node sample collected Specimens for bacteriological culture invariably include tissue with
from the head and thorax when no visible lesions are detected in visible lesions, such as caseous necrosis in lymph nodes (primarily
tuberculin skin test or interferon-gamma (IFN-γ) assay-positive the submandibular, retropharyngeal, tracheobronchial, mediasti-
animals. In some European countries, e.g., Spain, the isolation of nal, and mesenteric lymph nodes are the sites examined) and altered
M. bovis/M. caprae is required to confirm the presence of tubercu- parenchymatous organs, e. g. lung, liver or spleen. In addition to the
losis infection in a herd and for withdrawal of the Official TB Free bacteriological confirmation of suspect lesions, the sensitivity of di-
(OTF) status. This highlights the importance of incorporating diag- agnosis may be increased by the judicious selection of tissue without
nostic bacteriology into a tuberculosis control and eradication pro- visible pathology (NVL), as occurs in the early stages of infection
gramme. To date, the protocols for the bacteriological diagnosis of and in latently infected animals. As the anatomical distribution of
M. bovis and M. caprae infection have largely been based on allow- M. bovis-infected tissues is in general greater than that of visible
ing minimum time between specimen submission and the report- lesions (Corner et al., 2012a; Jackson et al., 1995; Lugton et al., 1997),
ing of a diagnosis, and the optimal conditions have not been increased sensitivity may be obtained by culturing lymph nodes from
rigorously defined for many of those factors that affect the success a wide range of body compartments, and organs. In infected tissues
of the primary isolation of M. bovis and M. caprae. that are NVL, the number of bacilli may be very low and therefore
sensitive bacteriology methods will be required to maximize re-
2.1. The gold standard covery of the pathogens.
A definitive diagnosis, the “gold standard”, is only achieved by 2.3. Factors influencing recovery of M. bovis and M. caprae
isolating the causative organism from clinical or post-mortem speci-
mens. An ideal gold standard diagnostic test procedure is one that Following selection of appropriate samples for culture, the key
identifies all cases of a disease without false positive diagnoses, i.e., determinants for optimal recovery of M. bovis and M. caprae will
100% sensitive and 100% specific (Dohoo et al., 2003). Direct mi- include conditions during storage and transport of samples, mac-
croscopy is the fastest, cheapest and simplest way for the detec- eration and decontamination of samples, and choice of growth media.
tion of acid-fast bacteria, including M. bovis and M. caprae, in tissue Where disease prevalence is high, culture can be routinely and con-
samples. For this purpose, a direct smear of a clinical sample or tissue veniently used to confirm a presumptive gross pathology or histo-
section can be stained following the Ziehl-Neelsen (ZN) technique pathology diagnosis. However, where a more sensitive bacteriological
(Cook, 1997) to provide a presumptive identification of M. bovis and diagnosis is required, such as when bacteriological procedures are
M. caprae. In several studies focusing on cattle, the sensitivity of ZN being compared, and also when diagnostic immuno-assays or vac-
staining was found poor (Costello et al., 1998; Gutiérrez Cancela and cines are being evaluated, then more comprehensive and strin-
García Marin, 1993; Varello et al., 2008; Watrelot-Virieux et al., 2006). gent diagnostic methods are required. The correct storage of samples
Alternatively, a fluorescent acid-fast stain, e. g., with auramine, can is important for maintenance of cell viability and to minimize the
be used that slightly increases sensitivity (Smithwick, 1976). growth of contaminating micro-organisms. Optimally, samples
For tuberculosis in animals, the isolation of M. bovis and M. caprae should be processed immediately after collection, but if this is not
is frequently taken as the gold standard for defining infection. available, then freezing and transport of samples immediately after
However, the number of infected animals identified on primary iso- collection are suitable, although often such facilities may not be avail-
lation, i.e. sensitivity, depends on the optimal performance of all el- able at sample collection. Chilling of samples at 4–6 °C after col-
ements of the bacteriological diagnostic process: from the manner lection and for transport has been shown to improve recovery rates
of collecting samples to the range of samples collected, also sample over maintaining them at ambient temperature (Corner, 1994).
storage and the bacteriological procedures employed. In most in- Samples stored chilled should be processed for culture within 24–
stances, there may be little doubt as to the accuracy of a positive 48 h post-collection and those at ambient temperature processed
isolation, except for the risk of cross-contamination between samples. immediately.
However, there can be no such confidence in a negative finding unless Appropriate pre-treatment and processing procedures (homog-
the quality of the methods employed can be assured. Any implicit enization, decontamination and concentration), and culture media
value placed on the term “gold standard” will be misplaced if sub- that inhibit contaminating organisms are employed to facilitate re-
optimal bacteriological procedures are employed. With tuberculo- covery of mycobacteria (Murray et al., 2007). A key factor that
sis there are a wide range of infection states, and suboptimal impacts on the success of primary culture is the decontamination
procedures may lead to severe bias in the cases detected. If the pro- procedures including the choice of decontaminant and the concen-
cedures favour only easily detected infection, then early infections tration of decontaminant used (Corner and Trajstman, 1988). De-
or latent infections may go undetected (Chambers et al., 2008). In contamination involves the use of toxic chemicals to which
situations where multiple tests are used concurrently, e.g., immu- mycobacteria are generally more refractory than the contaminat-
nological assays, an insensitive “gold standard”, may lead to an un- ing microorganisms. Although the toxicity to mycobacteria varies,
derestimation of prevalence of infection, an overestimation of the the chemicals generally decrease the number of viable bacilli in the
S32 E. Gormley et al./Research in Veterinary Science 97 (2014) S30–S43
specimens (Corner and Trajstman, 1988). Detergents can be used consistent with previous observations (Corner et al., 1995). The
for this purpose: cetylpyridinium chloride (CPC, also called decontaminants had adverse effects on the time to the first appear-
hexadecylpyridinium chloride, HPC) and benzylkonium chloride ance of colonies, the number of positive samples, and the number
(Corner and Trajstman, 1988; Corner et al., 1995), as well as acids, of colony recovered. The greatest number of colonies was isolated
oxalic acid (Claxton et al., 1979; Nassau, 1958) and sulphuric acid where there was no decontamination step, and each of the
(de Lisle and Havill, 1985), and alkali, sodium hydroxide (Corner and decontaminants decreased the number of colonies recovered. A
Trajstman, 1988; Krasnow and Wayne, 1969). In general, after the strength of this study was the use of a large number of samples and
addition of the decontaminant the mixture is shaken for 30 minutes using samples from animals that represented a wide range of disease
at room temperature, the suspension is centrifuged, the superna- states, from those that were NVL, representing early infection or
tant is discarded, and the sediment is used to inoculate the media. latent infection, to animals with single lesions or generalized disease.
Where the decontaminant is an acid or alkali, and if there is a concern To obtain the greatest sensitivity on primary isolation using solid
about the inhibitory effects of residual decontaminant, the pellet media, it was suggested that the use of multiple culture medium
can be resuspended in buffer and re-centrifuged, and the superna- types: agar based medium to exploit the faster growth rate and egg-
tant discarded. This latter procedure is used after CPC and oxalic acid based medium (Stonebrink’s or another egg based medium such as
decontamination. Although the usual recommendation is a minimum Löwenstein–Jensen with pyruvate) be used for greater sensitivity
of 8 weeks incubation, a significant increase in positive cultures is and the inhibition of contaminants (Corner and Trajstman, 1988);
obtained with an extended period of incubation, that is ≥12 weeks the use of two or more slopes of each medium; and an incubation
(Corner et al., 2012b). Due to the long incubation time and the en- time of ≥12 weeks for all media.
riched media employed, contaminating micro-organisms may still
overgrow the media. In those situations where there is wide- 2.5. Automated liquid broth-based culture systems
spread contamination on all culture media types, additional de-
contamination of the original stored specimens may be required and There are limitations in using solid media for high throughput
the culture repeated. screening for pathogenic mycobacteria, and improvements in the
time to detection and the recovery rate have been made by using
2.4. Impact of solid culture media broth-based culture systems such as the BACTEC 460, Mycobacte-
ria Growth Indicator Tube (BACTEC MGIT 960) and the VersaTREK
The M. bovis and M. caprae bacilli are small, non-motile rods, system (Thermo Fisher Scientific). In the BACTEC 460 system, the
1–4 µm long, characterized by a waxy mycolic acid cell wall that pro- mycobacteria are detected radiometrically after the processed sample
tects the cell from degradation within phagocytes, and is respon- is added to a modified Middlebrook 7H9 medium (BACTEC 12B) con-
sible for their acid-fastness. On primary isolation, M. bovis grow best taining 14C-labeled palmitic acid and an antibiotic complex, PANTA.
on enriched media but grow slowly, with colonies not appearing for Mycobacterial growth is measured by the liberation of 14CO2 and de-
>7 days. They are aerobic or microaerophilic, grow at 37 oC and show tected by BACTEC 460 instrument. The MGIT 960, a non-radiometric
cording when grown in liquid media. Growth of M. bovis is en- broth method, consists also of a modified Middlebrook 7H9 broth
hanced by the addition of pyruvate to the media but is inhibited by and a sensor embedded in silicone on the bottom of a tube. The ap-
glycerol. M. bovis is niacin negative, and sensitive to isoniazid, pearance of orange-coloured fluorescence in the sensor when excited
thiophene-2-carbxylic acid hydrazide, para-aminosalycylic acid and indicates the growth of acid fast mycobacteria. The VersaTREK system
neotetrazolium. is a fully-automated, continuous monitoring system for the growth
A variety of different media has been developed for the cultiva- and detection of mycobacteria. This technology is based on the de-
tion of mycobacteria and can be characterized by three basic types. tection of pressure changes (i.e. either the production or consump-
The first is egg-based media represented by Stonebrink’s medium tion of gas) inside the headspace of the broth culture medium.
and Löwenstein–Jensen with added pyruvate. The second type is With the discontinuation of the BACTEC 460 system, a small
agar-based media (sometimes enriched with serum and/or blood); number of studies have been carried out comparing the perfor-
the most commonly used are Middlebrook 7H10 and 7H11, and tu- mance of the two systems (BACTEC 460 and MGIT 960) to measure
berculosis blood-agar medium, B83. The third type is liquid media growth of M. bovis. In a study conducted on 506 bovine lymph node
such as Middlebrook 7H9. Several weeks of incubation may be re- samples collected at abattoirs in the USA and Mexico, the MGIT 960
quired for the earliest appearance of colonies of M. bovis on solid system had a higher recovery rate of M. bovis (122/129 specimens)
media. Growth rates are dependent on media and the concentra- than did the BACTEC 460 (102/129) and solid media (96/129) con-
tion of bacilli in the inoculum. Cultivation of the samples on a com- sisting of Middlebrook 7H10 and Middlebrook 7H11 both contain-
bination of solid and liquid media can increase the probability of ing sodium pyruvate and additional supplements (Hines et al., 2006).
maximal sensitivity, and has been recommended for human TB by The average time to detection was 15.8 days for the MGIT 960 system,
the international standards in mycobacteria laboratories (Tenover 28.2 days for the BACTEC 460 system, and 43.4 days for solid media.
et al., 1993). However, the contamination rates were 6.9% for the MGIT 960
The impact of media type and culture conditions on recovery rates system, 3.4% for the BACTEC 460 system, and 21.7% for solid media.
of M. bovis when using solid media has been investigated in a study The relatively poor growth success on the solid media was ex-
using specimens collected from cattle in Australia (Corner et al., plained by the fact that the bacteria were likely able to grow and
2012b). Two agar-based media, modified Middlebrook 7H11 and tu- spread more easily through liquid media than on solid media. As a
berculosis blood agar (B83), and an egg-based medium, Stonebrink’s, result, recovery rates on solid media may have been limited because
were compared. In addition, four decontamination procedures, 2% the bacteria are able to use the nutrients only in the vicinity of the
w/v sodium hydroxide (NaOH), 0.75% w/v and 0.075% w/v cetyl- colony. The difference in nutrients contained in the solid media tubes
pyridinium chloride, and 0.5% w/v benzalkonium chloride were and both liquid culture media could also be a factor affecting the
evaluated against treatment with sterile distilled water. Several recovery rates among culture systems because one mixture of nu-
factors were identified that significantly influenced the sensitivity trients may be more conducive to recovering M. bovis. The contam-
of the primary isolation of M. bovis from tissue samples. These in- ination rate of the MGIT 960 system may have been higher than the
cluded the duration of incubation, decontamination procedures, BACTEC 460 system because of the somewhat reduced concentra-
culture media, and the number of slopes of media inoculated. The tion of antibiotics supplementing the latter media. Contrasting results
toxicity of the decontaminants was clearly demonstrated and was were obtained in a more recent USA study where 3,168 specimens
were inoculated concurrently into both BACTEC 460 and MGIT 960 nology, there will be a requirement for ongoing performance val-
media (Robbe-Austerman et al., 2013). The samples included low- idation. In reality there are few true gold standard procedures due
risk, non-lesioned wildlife surveillance and high-risk samples. More to inherent imperfections in the procedures, but if perfect accura-
acid-fast bacteria were recovered with MGIT 960 than with BACTEC cy cannot be claimed, it is important to assess how far the new pro-
(269 vs. 252). However, of the 178 specimens where M. bovis was tocols and technologies produce results that deviate from the ideal
recovered, the BACTEC 460 significantly outperformed the MGIT for standard. In this way, the efforts and resources required to success-
M. bovis recovery (161 vs. 144). When 160 M. bovis positive samples fully cultivate M. bovis may be streamlined to strike the balance
were inoculated into both liquid systems, the sensitivity of the between costs involved and the optimal recovery rates required.
BACTEC (93.8% isolation) was significantly higher than the MGIT
(81.9%). However, the contamination rate in the MGIT 960 (14.4%) 3. Molecular methods for identification and typing of M. bovis
was also significantly higher than the BACTEC 460 (1.3%). It was sug- and M. caprae
gested that contamination was a major reason for the reduced sen-
sitivity of the MGIT 960 media and that the concentrations of Prior to the development of molecular methods, mycobacterial
antibiotics and stringency of decontamination conditions used was species were identified by a combination of phenotypic tests based
not equally optimal for both test formats. The contamination rates on culture, such as cellular and colony morphology, growth rates,
may have been influenced by the low concentration of sodium hy- preferred growth temperature, pigmentation, and also on a series
droxide (0.8%) and the short time of contact of the sample with the of biochemical tests. However, apart from the laborious nature of
detergent (7–10 minutes), which are not the standard conditions conducting these tests, they require good culture growth and can
used in veterinary diagnostic laboratories where up to 2% w/v NaOH occasionally give ambiguous results. This can lead to mis-
is used. identification because different species may have indistinguish-
Contamination of most specimens occurs during the collection able morphological and biochemical profiles. One useful phenotypic
procedure, and the employment of an aseptic collection proce- test is the presence or absence of serpentine cording in liquid culture
dure can help to decrease or avoid contamination. Norton et al. (1984) media (McCarter et al., 1998). It is relatively easy to perform and
used simple procedures (boiled instruments and wetting down the can differentiate MTBC isolates from MOTT. Although some iso-
carcase before incising the skin) to decrease contamination, but more lates can give intermediate results, it may be used in conjunction
complex procedures, where each specimen is collected using sep- with molecular identification tests.
arate sterile instruments have also been employed (Corner et al., Molecular typing makes use of genetic markers in order to search
2007). Collection of tissues using strict aseptic procedures may allow for outbreak sources, to track epidemic or pandemic spread of par-
the decontamination procedure to be dispensed with, altogether with ticular strains, or to reconstruct the evolution of a certain group of
a consequent increase in sensitivity (Corner and Trajstman, 1988). bacteria. The majority of the currently utilized methods of strain
The development of novel sensitive and specific screening tests identification have been developed since 1990. It was at this time
for M. bovis could help to circumvent some of the problems asso- that progress was made towards the standardization of typing pro-
ciated with decontamination, particularly where the bacilli numbers tocols in order to improve the quality of epidemiological investi-
are very low. The application of immuno-magnetic separation (IMS) gations (Cousins et al., 1998b; van Embden et al., 1993). The major
technology has shown some potential in this area. The rationale is genotyping techniques (Fig. 1) used for strain differentiation of
that IMS allows for selective capturing of M. bovis cells in the samples, members of the MTBC can be classified in two categories, based
but preserving the viability of M. bovis cells. In a study conducted either on whole or partial genome analysis (Durr et al., 2000). Whole
in Northern Ireland, the results showed evidence of higher M. bovis genome techniques were the first to be described and have the ad-
detection rates using the IMS-based methods (IMS-PCR and IMS- vantage of using all the potential genetic information of the organ-
MGIT culture) compared to conventional culture, notably with skin ism. However, these methods, which include restriction endonuclease
test reactor animals presenting with non-visible lesions (Stewart analysis (REA) and pulsed field gel electrophoresis (PFGE), are tech-
et al., 2013). Using this technology, viable M. bovis cells were re- nically demanding and difficult to automate. The partial genome
covered from NVL lymph node samples taken from skin test- techniques are more commonly used and can be subdivided into
positive animals, in situations where conventional culture methods (a) techniques that target specific repeated sequences comprising
failed to do so. The range of M. bovis spoligotypes isolated by the insertion sequences (IS), the direct repeat (DR) region, polymor-
IMS-MGIT culture method was very similar to the range isolated phic GC-rich sequences and tandem repeat loci; (b) targeting of
by culture, implying that IMS can select a very broad range of random sequences, e.g., random amplified polymorphic deoxyri-
spoligotypes. bonucleic acid (RAPD) analysis; (c) typing targeting house-keeping
With improved understanding of the factors that contribute to genes (MLST); (d) deletion typing targeting regions of deletion (RD);
optimal recovery of M. bovis, many other aspects of the bacterio- and (e) single nucleotide polymorphism (SNP) typing (Fig. 1).
logical diagnostic chain also need to be evaluated for their effect on A critical point that impacts on the success of the various typing
the sensitivity of a bacteriological diagnosis, for example, aspects techniques is knowledge of the mutation rate. The mutation rate de-
such as the temperature and duration of storage, the use of preser- scribes the frequency at which molecular fingerprint patterns change
vatives such as 1% CPC (Bobadilla-del-Valle et al., 2003; Jenkins et al., and is indispensable for the correct interpretation of molecular data
2008), sodium carbonate, or sodium tetraborate (Bobadilla-del-Valle for epidemiological or phylogenetic use (Grant et al., 2008; Wirth
et al., 2003). Continued developments in technology may improve et al., 2008). If the mutation rate is high, an overestimation of epi-
the specificity of detection of pathogenic mycobacteria, including demiologically unrelated tuberculosis is possible, and may happen,
M. bovis. For example, Capilia TB is a recently developed immune- for example, with hypervariable loci (Supply et al., 2006).
chromatographic assay for rapid discrimination between the My-
cobacterium tuberculosis complex (MTBC) and mycobacteria other 3.1. Whole genome techniques for strain typing
than tuberculosis (MOTT). It is based on detection of the MPT64/
MPB64 secreted antigen, specific for the MTBC. The kit can be easily 3.1.1. Restriction endonuclease analysis
used for rapid identification of the MTBC in combination with the Restriction endonuclease analysis (REA) was the first method de-
culture systems based on liquid media (Hillemann et al., 2005). veloped for typing of M. bovis isolates (Collins and de Lisle, 1985);
However, its performance has not yet been evaluated for M. bovis it uses three restriction endonuclease enzymes, BstEII, PvuII and BclI,
diagnosis in cattle. With each new improvement in culture tech- to cleave the DNA strands of whole genomic DNA at specific nucleo-
Fig. 1. Overview over the most important techniques used for typing of the Mycobacterium tuberculosis complex. REA: restriction endonuclease analysis; PFGE: pulsed field
gel electrophoresis; RAPD: random amplified polymorphic deoxyribonucleic acid; MLST: multilocus sequence typing; VNTR: variable number tandem repeat; RFLP: restric-
tion fragment length polymorphism; IS: insertion sequence; DR: direct repeat; PGRS: polymorphic GC-rich sequences; RD: region of difference; SNP: single nucleotide poly-
morphism. Adapted from Durr et al. (2000) with the kind authorization of the World Organisation for Animal Health (OIE) (http://www.oie.int).
tide sequences. This digest results in many small fragments, which in molecular tuberculosis research. Since then, whole genome se-
are then separated by conventional agarose gel electrophoresis. The quencing (WGS) has become more available and has revolution-
resolved fragment patterns are visualized and compared with other ized genotyping by providing the highest level of discrimination. WGS
isolates allowing for strain differentiation. When developed, REA is a promising tool for forensic epidemiology and phylogenetic
allowed for the first time to determine whether livestock had become studies enabling measurement of genetic variation over time and
infected on the farm or infection had entered the farm through pur- identification of fine scale transmission patterns at the individual
chase of an already infected animal (Collins et al., 1994a, 1994b). level (Bryant et al., 2013). In a study conducted in Northern Ireland
This technique has been applied for epidemiological studies in New on a small number of M. bovis isolates from cattle and badgers that
Zealand (Collins et al., 1993) and Ireland (Collins et al., 1994b), but were spatio-temporally linked, WGS revealed that most isolates and
has not become widely used due to technical issues and difficul- outbreaks were genetically unique, but that subsequent outbreaks
ties with interpretation of the patterns (Collins et al., 1993), but has on the same farm tended to involve the same genetic lineage as pre-
been used extensively as part of the bovine tuberculosis control viously detected in that location (Biek et al., 2012). Although WGS
scheme in New Zealand. can currently not be used in routine settings, mostly due to the costs
and the requirement of a bioinformatics pipeline, this rapidly de-
3.1.2. Pulsed field gel electrophoresis veloping technology should deepen our insight into the transmis-
Pulsed field gel electrophoresis (PFGE) has been used to over- sion of M. bovis infection and how it gives rise to local epidemics.
come the analysis problem of the excessive number of small DNA
fragments generated by REA. The PFGE technique uses restriction 3.1.4. Whole genome microarray
enzymes that generate a small number of fragments that are too The availability of whole genome sequence data for several strains
large to be separated by conventional agarose gel electrophoresis, of the MTBC has enabled the use of the microarray technique for
but can be resolved when subjected to a pulsed electrical field. The comparing a particular strain to sequenced reference strains (Butcher,
technique was first implemented for M. tuberculosis isolates (Zhang 2004). Bacterial microarrays have been mainly used for compara-
et al., 1992) and later set up for M. bovis BCG (Zhang et al., 1995) tive genomic hybridization (CGH), also referred to as comparative
and M. bovis (Feizabadi et al., 1996). However, PFGE has not been genomics (DNA microarray), and for comparison of gene expres-
established as a standard technique because it is technically diffi- sion, also named transcriptomics or expression profiling (RNA
cult and labour intensive. microarray). The basic principles are similar and independent of the
starting product (DNA or RNA): PCR products representing ORFs of
3.1.3. Whole genome sequencing the genes of the reference strain(s) are spotted onto a solid support,
The publications of the complete genome sequences of M. tu- e.g. glass slides, the samples are amplified (PCR amplification for DNA
berculosis H37Rv (Cole et al., 1998), M. bovis AF2122/97 (Garnier et al., and reverse transcription for RNA), labelled with fluorescent dyes
2003) and M. bovis BCG (Brosch et al., 2007) were breakthroughs and hybridized onto these slides (Holloway et al., 2002), subse-
quently, the spots are visualized in a fluorescence reader and quan-

tified by software. Comparative genomics elucidates differences
between members of the MTBC by surveying whole genomes and
has improved our understanding of the pathogenesis and host-
adaptation (Gordon et al., 2009). Although comparative genomics
has provided insights into virulence of different M. bovis strains
(Voskuil et al., 2004) and has identified chromosomal polymor-
phisms as markers for distinct clonal complexes (Berg et al., 2011;
Müller et al., 2009; Smith et al., 2011), the techniques are more suited
for research studies than for routine diagnostics.
3.2. Partial genome techniques for strain typing
3.2.1. Restriction fragment length polymorphism analysis

Restriction fragment length polymorphism (RFLP) analysis makes
use of the restriction enzyme PvuII or AluI followed by agarose gel
electrophoresis and Southern blotting (Southern, 1975) of the frag-
mented isolate DNA onto a nitro-cellulose or nylon filter. To iden-
tify the RFLP, labelled markers that are complements to specific
fragments of the DNA of the isolate, are hybridized to the immo-
bilized isolate DNA. Target regions that have been utilized for RFLP Fig. 2. Scheme of the direct repeat (DR) region and the spoligotyping technique.
include insertion sequences (IS) (van Embden et al., 1993), poly-
morphic GC-rich repeat sequences (PGRS) (Cousins et al., 1998b),
the direct repeat (DR) sequence (Cousins et al., 1998a), and the re- length ranging from 25 to 41 bp (van Embden et al., 2000); a DR and
petitive DNA element pUCD (O’Brien et al., 2000a). its adjacent spacer are termed a direct variant repeat (DVR). Since
Insertion sequences (IS) are mobile genetic elements that con- strains vary in the number of DVRs, the presence or absence of the
stitute a form of repetitive DNA in bacterial genomes. IS6110 is an spacers can be exploited for strain typing. Although 104 spacer se-
IS of 1,361 bp and is not present in mycobacteria other than members quences have been identified in the DR region, not all of them show
of the MTBC (McAdam et al., 1990). RFLP based on IS6110 is the most sufficient polymorphisms to be useful for strain typing (Caimi et al.,
widely used typing technique for M. tuberculosis strains, which 2001; van Embden et al., 2000). The current standard protocol for
harbour up to 20 copies of this IS (McAdam et al., 1990; Thierry et al., spoligotyping is based on 43 spacers (Kamerbeek et al., 1997). Two
1990), but the technique is difficult to standardize between labo- primers are used to target the individual DRs and amplify the whole
ratories (Braden et al., 2002). However, for low-copy-number strains DR region followed by hybridization of the fragments onto a blot
like M. bovis, this technique lacks discrimination and is therefore membrane, which is covalently linked to oligonucleotides repre-
not routinely used for typing purposes (Aranaz et al., 1998; Costello senting the spacer sequences, and subsequent visualization via che-
et al., 1999; Cousins et al., 1998a; Romano et al., 1996). Studies have miluminescence (Fig. 2); the membranes can be sourced either
also targeted IS1081 for use in the MTBC (Collins and Stephens, 1991; commercially (Ocimum Biosolutions Ltd., Hyderabad, India) or lab-
Poulet and Cole, 1995; van Soolingen et al., 1991). However, studies oratory produced. This reverse line blot hybridization method pro-
applying IS1081-RFLP to M. bovis isolates have resulted in limited duces data that are easy to store due to the translation of the patterns
or even no discrimination (Aranaz et al., 1998; Collins et al., 1993; into a binary code (0, absence of spacer; 1, presence of spacer), and
Skuce et al., 1994), except from M. bovis BCG (van Soolingen et al., internationally used authoritative names (prefix SB followed by four
1992). O’Brien and colleagues (2000a, 2000b) described the repet- digits or prefix SIT followed by one to four digits) for the spoligotype
itive element pUCD as a valid probe for RFLP typing of M. bovis patterns can be obtained from the major websites (http://
strains, offering an equivalent level of discrimination to that of RFLP www.Mbovis.org; http://www.pasteur-guadeloupe.fr/tb/bd_myco
typing with combined IS6110-PGRS-DR probes. .html) (Demay et al., 2012; Smith and Upton, 2012).
PGRS are short, repetitive sequences with a GC composition of Several spacers have been found to be potentially problematic
up to 80% that have been exploited for molecular typing (Cousins with respect to accuracy and reproducibility, which was con-
et al., 1998a; Poulet and Cole, 1995; Ross et al., 1992). This tech- firmed in an inter-laboratory comparison where errors at spacers
nique achieves high discriminatory levels, particularly when mul- 14, 15, 18, 39 and 40 were most frequent (Abadia et al., 2011). In
tiple copies of IS6110 are absent (Cousins et al., 1998a); however, the case of spacer 15, this might be due to a deletion of four nucle-
for M. bovis isolates, the banding patterns are quite complex. otides at the 5’ end of the adjacent DVR (DVR26), which hinders the
The DR region (Groenen et al., 1993; van Embden et al., 2000) proper amplification of this spacer (van Embden et al., 2000). In ad-
is one of the most important genomic targets used for molecular dition, characteristic spacer deletions have been recorded, often re-
typing of MTBC isolates and is exploited for spoligotyping as dis- ferred to as spoligotype signatures (Streicher et al., 2007) that can
cussed below. Although it is also suitable for the use with RFLP be exploited to provide additional information about lineage or clonal
(Cousins et al., 1998a; Zumárraga et al., 1999), it is difficult to in- complex membership within the different mycobacterial species
terpret and therefore not widely used. (Berg et al., 2011; Kato-Maeda et al., 2011; Kremer et al., 2004; Müller
et al., 2009; Rodriguez-Campos et al., 2012; Smith et al., 2011).
3.2.2. Spoligotyping Recently, the membrane-based assay has been transferred to the
Spacer oligonucleotide typing (spoligotyping: Kamerbeek et al., Luminex® platform (Luminex Corp.), a microbead-based multianalyte
1997) is a simple, rapid and robust technique that permits high- profiling system (Cowan et al., 2004). Spoligotyping on the Luminex
throughput typing of MTBC isolates without the need to purify the platform offers many advantages over traditional spoligotyping.
DNA. Groenen et al. (1993) identified a clustered regularly interspaced Firstly, turnaround time and labour involved are reduced, and
palindromic repeat (CRISPR) region unique to the MTBC desig- secondly, the reproducibility is increased. Although the spoligotyping
nated as the Direct Repeat (DR) region. It consists of multiple 36 bp assay is reproducible (>90%) (Kremer et al., 1999), it can be
DRs interspersed by unique sequences called spacers, with the spacer influenced by the subjective interpretation of the hybridization signal,
which changes with repeated use of the membrane, and by human the animal origin, geographical region, corresponding publica-
error arising from manual entry of data (Cowan et al., 2004). Both tions or any other information related to the pattern can be
membrane- and human-related quality assurance issues that can included. The implementation of a common nomenclature
affect the spoligotyping results can be improved, or even avoided, has provided an easy way to compare spoligotyping results
with the microbead-based method (Abadia et al., 2011; Zhang et al., between laboratories and hence is of great value for the scientific
2010). community.
Spoligotyping is the method of choice for IS6110-low-copy-
number strains, such as M. bovis or some M. tuberculosis strains 3.2.3. Variable number tandem repeat typing
(Cousins et al., 1998a; Thong-On et al., 2010). Several studies have Variable number tandem repeat (VNTR) typing is targeted at
observed improved discrimination by combining spoligotyping and genetic loci that are distributed throughout the genome; these loci
IS6110-RFLP (Aranaz et al., 1998; Costello et al., 1999; Roring et al., contain variable numbers of repeated sequences. The tandem repeats
1998); however, RFLP is technically more demanding, and high quan- have been successfully used for fingerprinting of bacterial genomes
tities of DNA are required. Since the implementation of spoligotyping because of the extensive polymorphism in the copy number of the
for typing of M. bovis (Aranaz et al., 1996; Roring et al., 1998), the repeats (Versalovic et al., 1991). VNTR typing is also known as multi-
technique has been considered useful as a fast method for the char- locus variable-number tandem repeat analysis (MLVA) and has many
acterization of isolates and has been applied on a large scale for first- advantages being relatively inexpensive, rapid and easy to perform,
line typing (Haddad et al., 2004; Milian-Suazo et al., 2008; Smith and yielding unambiguous results (Lindstedt, 2005; van Belkum,
et al., 2003). 2007). VNTR typing is based on PCR amplification of the targeted
It has been suggested that the use of the 43 selected spacers might loci with specific primer pairs followed by gel electrophoresis (Fig. 3).
not be appropriate for M. bovis isolates since these spacers were The use of automatic sequencers to assess the exact size of the am-
chosen in the context of discrimination of M. tuberculosis isolates plified fragments has also become established in many research and
(van der Zanden et al., 2002). To address this, a second-generation diagnostic laboratories and has enabled automated high-throughput
spoligotyping membrane was developed that compliments the stan- genotyping (Supply et al., 2001). For interpretation of the results,
dard membrane in order to assess all of the 104 spacers and this the use of an allele calling table that relates the different band
has resulted in improved strain discrimination (van der Zanden et al., sizes to the corresponding number of repeats for each locus is in-
2002). Another second-generation membrane with 25 additional dispensable (Fig. 3).
spacers has also been developed to specifically assess typing of The first VNTR locus identified in the M. tuberculosis genome con-
M. bovis and M. caprae isolates in Spain; the additional spacers did sists of a 75 bp tandem repeat located within a large open reading
improve the strain differentiation of the M. bovis but not of M. caprae frame (ORF) (Goyal et al., 1994). The implications of VNTR loci located
isolates (Javed et al., 2007), which commonly show less variation within ORFs are not yet understood, but the observation that the
in the DR locus than M. bovis isolates. A further limiting factor of DNA of the repeat unit is translated during expression of the ORF,
spoligotyping is its inability to detect mixed infections, because the leading to size variation in the expressed protein, hints at a possi-
pattern obtained may correspond to the cumulative presence of ble functional role. A study using VNTR to type MTBC isolates in-
spacers (Cohen et al., 2011; Romero et al., 2008). This is of impor- cluded the previously identified locus and additional five novel
tance when samples are pooled before culturing, especially when tandem repeats (exact tandem repeats, ETR); these ETRs (A to F)
different strains are present in different specimens which has been range in size from 53 to 79 bp (Frothingham and Meeker-O’Connell,
described in badgers (Furphy et al., 2012). 1998). In recent years, several new loci have been described and some
The discrimination of M. bovis isolates achieved with of them are also located within ORFs, for example QUB11a, QUB11b,
spoligotyping varies considerably between different countries and QUB18, QUB23, QUB26 (Skuce et al., 2002). All the loci that are
even geographical regions. While large population surveys using located within coding regions have repeat sizes that are multiples
spoligotyping in Australia (Cousins et al., 1998a), the Republic of of three. Most dispersed repeats are found in intergenic regions, and
Ireland (Costello et al., 1999), Northern Ireland (Skuce et al., 2005) are thought not to play any functional role in the bacterial genome.
and Great Britain (Hewinson et al., 2006) were hampered by low Supply et al. (1997) have designated these loci mycobacterial in-
discriminatory power, equivalent scale spoligotyping in France terspersed repetitive units (MIRUs), which range in size from 46 to
(Haddad et al., 2001), Portugal (Duarte et al., 2008), Italy (Boniotti 101 bp. Since the use of the MIRUs is commonly used, VNTR typing
et al., 2009), Spain (Rodriguez et al., 2011) and South Africa (Michel of mycobacteria is often referred to as MIRU-VNTR typing. The total
et al., 2008) revealed significantly better discrimination. number of MIRUs per genome is estimated to be about 40 to 50. It
In response to the need for an international nomenclature for is noteworthy that some of the intergenic loci are located up-
spoligotypes of animal origin, in 2003 the M. bovis Spoligotype Da- stream of ORFs in the same orientation, for example ETR-D (MIRU
tabase (http://www.Mbovis.org/) was created and hosted on the 4) which was found within the intergenic region of an operon en-
Animal Health Veterinary Laboratories Agency [(AHVLA) Weybridge, coding a mycobacterial two-component system (Supply et al., 1997).
UK] server and supported by the Department for Environment, Food Sequencing of one of the tandem repeats (ETR-D) has been shown
and Rural Affairs (DEFRA) (Smith and Upton, 2012). This database to differentiate members of the MTBC (Djelouadji et al., 2008). It
assigns authoritative names to the binary spoligotyping pattern has been speculated that variations in the number of tandem repeats
of MTBC members that lack the region of difference (RD) 9 might affect the expression of downstream genes (Magdalena et al.,
(M. africanum, oryx bacillus, dassie bacillus, Mycobacterium microti, 1998). Since the position of the loci is not always conserved among
Mycobacterium pinnipedii, Mycobacterium caprae, M. bovis and M. bovis different bacterial species, they might not influence the function of
BCG). The authoritative name consists of a prefix followed by four a given gene, but may be important for evolution of chromosome
digits (SBxxxx), the SB number. The nomenclature consisting of six structure and DNA rearrangement, such as tandem duplications,
blocks of two-digit hexadecimal numbers (HEX code), e.g. 1111111 chromosomal deletions and inversions (Petes and Hill, 1988).
1101110 1111110 1111111 11111000 1111101 is called 7F-6E-7E-7F- Many additional VNTR loci have been reported since the first de-
F8-7D (Dale et al., 2001), is not generally used and was removed from scription of tandem repeats, and it soon became evident that
the website in 2011. To date (28th August 2013), the website con- standardization of the nomenclature was necessary. Smittipat
tains 1660 different spoligotypes along with the corresponding in- et al. (2005) suggested the use of “VNTR” followed by four digits
formation about the country where it was first isolated, the date of corresponding to the locus position on M. tuberculosis H37Rv.
isolation and the person who submitted the pattern. Additionally, Nevertheless, the different loci aliases are still widely used
Fig. 3. Scheme of a variable number tandem repeat (VNTR) locus and VNTR typing.
(ETR = exact tandem repeat; MIRU = mycobacterial interspersed re- used, which can hamper inter-laboratory exchange. According to
petitive unit; QUB = Queen’s University Belfast). Many attempts have several reports, among the most discriminatory markers for M. bovis
been made to standardize the typing protocol and especially the use are QUB3232, ETR-A, ETR-B, QUB11a, QUB11b and QUB26 (Boniotti
of certain VNTR loci, in order to facilitate inter-laboratory compar- et al., 2009; Hilty et al., 2005; Lari et al., 2011; Rodriguez-Campos
ison of the results (Allix et al., 2006; Supply et al., 2006). The se- et al., 2013; Romero et al., 2008; Roring et al., 2002).
lection of particular sets of loci for VNTR typing is based on studies The use of VNTR typing for M. caprae isolates is less reported.
on their specificity, sensitivity and reproducibility (Kremer et al., 1999, Prodinger et al. (2005) analysed a large panel of M. caprae isolates
2004; Supply et al., 2001). Several loci, for example VNTR3232 and by spoligotyping and VNTR typing, focusing exclusively on 12 MIRU
VNTR2163a, have been described as hypervariable due to a higher markers (MIRU2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 and 40). In this
mutation rate and allelic diversity in M. tuberculosis (Supply et al., study, the best discrimination was found with MIRU 4, 10, 16, 26
2006). The mutation rate determines the discriminatory capacity and 31, which was not equivalent to the findings with M. bovis
of a locus, particularly where there are considerable variations (Prodinger et al., 2005). Generally, the same VNTR loci are as-
between the different loci. However, this issue remains controver- sessed in both M. caprae and M. bovis isolates, which might not
sial (Reyes and Tanaka, 2010; Supply et al., 2011) since the influ- provide optimal discrimination.
ence of geographic region (Hilty et al., 2005), the mycobacterial VNTR typing has been useful for epidemiological studies and, in
species, for example M. bovis (Lari et al., 2011; McLernon et al., 2010), contrast to spoligotyping, it is able to identify mixed infection (Furphy
or even the lineage (Velji et al., 2009) might generate conflicting et al., 2012; Romero et al., 2008). Double bands at a given locus hint
results for the best choice of VNTR loci. An overview of the differ- at mixed infection and a possible case of microevolution. The use
ent suggestions for the use of VNTR loci with M. bovis/M. caprae is of VNTRs in phylogenetic studies is not as straightforward as the
presented in Table 1. use of spoligotyping, because the evolution of VNTR loci is not uni-
Studies conducted to determine an ideal reference set of VNTR directional; repeats can be lost but also acquired so that the direc-
markers for M. bovis are fewer than studies on M. tuberculosis. tion of evolution is more difficult to estimate (Arnold et al., 2006).
Nevertheless VNTR typing has been used in many countries Yet the use of VNTR typing can form an integral part of the bovine
for epidemiological studies (Allix et al., 2006; Aranaz et al., 2004; tuberculosis control programmes (Boniotti et al., 2009; Duarte et al.,
Boniotti et al., 2009; Duarte et al., 2010; Etchechoury et al., 2010; 2010; Smith et al., 2003).
Figueiredo et al., 2012; Garbaccio et al., 2014; Martinez et al., 2008;
Richomme et al., 2010; Romero et al., 2008; Roring et al., 2002, 2004; 3.2.4. IS6110-ampliprinting
Skuce et al., 2002, 2005; Smith et al., 2006). In Europe, a network of Ampliprinting targets the major polymorphic tandem repeat
laboratories has agreed on a consensus of six VNTR loci for the use (MPTR) sequence (Hermans et al., 1992), which is similar to the DR
with M. bovis (Supply, 2006); however, these loci are still not widely region, consisting of 10 bp direct repeats separated by 5 bp unique
Table 1
Discriminatory variable number tandem repeat (VNTR) markers recommended for typing Mycobacterium bovis and/or Mycobacterium caprae isolates from different geo-
graphical regions.
VNTR Alias Locusa bpb ATc BEc EIc ESc ITc NAc NIc PTc TDc Vc
ETR-A 2165 75 X3 X X2 X X2 X X X3 X
ETR-B 2461 57 X2 X3 X1 X X X3 X1 X
ETR-C 0577 58 X X X2
ETR-D MIRU04 0580 77 X2 X X X X X
ETR-E MIRU31 3192 53 X3 X X X X
ETR-F 3239 79 X
MIRU10 0960 53 X
MIRU16 1644 53 X X
MIRU20 2059 77 X
MIRU24 2687 54 X X
MIRU26 2995 51 X1 X X3 X X X3 X3 X X
MIRU27 QUB5 3007 53 X
MIRU40 0802 54 X
QUB11a 2163a 69 X X X X X2 X2 X
QUB11b 2163b 69 X X X X X X X
QUB15 3155 54 X
QUB1895 1895 57 X1 X X
QUB23 1612 21 X
QUB26 4025 111 X2 X X X1 X
QUB3232 3232 56/57 X1 X1 X3 X1 X1 X X
QUB3336 3636 59 X2 X
QUB4156 4156 59 X
a VNTR locus corresponding to the first four digits of the locus position in M. tuberculosis H37Rv.
b
Repeat length.
c AT: Austria (Prodinger et al., 2005); BE: Belgium (Allix et al., 2006); EI: Republic of Ireland (McLernon et al., 2010); ES: Spain (Rodriguez-Campos et al., 2013); IT: Italy
(Boniotti et al., 2009); NA: North America (Martinez et al., 2008); NI: Northern Ireland (Roring et al., 2004); PT: Portugal (Duarte et al., 2010); TD: Chad (Hilty et al., 2005);
V: VENoMYC convention (Supply, 2006).
1–3
The three most discriminatory loci found in each study in descending manner.
spacers. The MPTR has been identified in atypical mycobacteria and determinants, or target the location of insertion sequences (Urwin
possesses limited polymorphism in the MTBC. Nevertheless, for and Maiden, 2003).
M. tuberculosis satisfactory results can be achieved in combination
with the IS6110, named IS6110-ampliprinting, which uses the vari- 3.2.7. RD typing
able distance between IS6110 and the copies of MPTR sequences Region of Deletion (RD) typing can be exploited for species dif-
(Plikaytis et al., 1993). Since the results obtained with ampliprinting ferentiation, e.g., RD9 differentiates all other MTBC members from
vary considerably in discrimination and reproducibility (Glennon M. tuberculosis and Mycobacterium canettii because it is only present
et al., 1997; Kremer et al., 1999), it is not applied as a routine in the latter species and RD4 which is absent from all isolates of
procedure. M. bovis. As a result, RDs have been used as targets for PCR as a rapid
species identification tool and additional RDs have been reported
3.2.5. Random amplified polymorphic deoxyribonucleic acid analysis more recently, including RD2seal (Bigi et al., 2005), RD1mic (Brodin
Random amplified polymorphic deoxyribonucleic acid (RAPD) et al., 2002) and RD1 das (Mostowy et al., 2004). Different ap-
analysis is a simple PCR-based fingerprinting technique and has been proaches have been described for the use of three primers, two flank-
widely used for typing bacteria (van Belkum, 1994). It was first de- ing and one internal (Mostowy et al., 2002; Talbot et al., 1997), or
scribed for typing of M. tuberculosis isolates by Palittapongarnpim four primers, two flanking and two internal primer pairs (Brosch
et al. (1993) but has rarely been applied to M. bovis, apart from a et al., 2002), to assess the presence or absence of the RDs. Several
study from Ireland, in which only poor discrimination was ob- combinations of PCRs targeting the different RDs have been rec-
tained (Glennon et al., 1997), and a study from Mexico (Milian-Suazo ommended for species identification (Huard et al., 2006; Parsons
et al., 2000). Due to problems with the reproducibility of the tech- et al., 2002; Warren et al., 2006). Moreover, due to the unidirec-
nique, it is not well established for typing MTBC members. tional evolution of the RDs (Gordon et al., 1999), these markers are
useful for the reconstruction of the evolution of the MTBC and to
3.2.6. Multilocus Sequence Typing (MLST) define clonal groups within the MTBC host-adapted members (Berg
MLST aims at the subdivision of microbes on the basis of neutral et al., 2011; Gagneux and Small, 2007; Müller et al., 2009; Smith
sequence diversity (Maiden et al., 1998) and is used for character- et al., 2011). Recently, evidence for variability within the RD4 was
izing isolates of bacterial species by sequence analysis of 450– provided by Domogalla et al. (2013) who described three RD4 vari-
500 bp of internal fragments of usually seven house-keeping genes. ants in German M. caprae isolates, and designed a panel of PCR
For each gene, the different sequences are assigned as distinct alleles, primers to differentiate between these variants, named Allgäu,
so that for every isolate an allelic profile or sequence type (ST) can Karwendel and Lechtal type. Differing results with the RD4 PCR have
be obtained by combining the alleles at each locus. House-keeping been described before in an Eastern European M. caprae isolate,
genes accumulate variation very slowly and this makes them likely hinting at a possible partial deletion of the locus (Rodriguez et al.,
to be selectively neutral. In contrast to most bacterial species that 2011). Possible variability in RDs should be taken into account for
have sufficient variation within house-keeping genes, the MTBC deletion typing. A multiplex real-time PCR assay targeting RD loci
members are genetically monomorphic and, hence, insufficient levels has recently been developed (Reddington et al., 2012). This may
of discrimination are achieved (Achtman, 2008). To increase dis- become available as a commercial kit (Seek TB). It consists of two
crimination, it might be necessary to characterize other housekeep- 5-plex real-time PCR assays which can be performed sequentially.
ing genes such as genes encoding antigens and antibiotic-resistance One of the assays enables identification of M. bovis, M. bovis BCG,
M. caprae, Mycobacterium africanum West African 2, M. microti and refined understanding of infectious disease epidemiology (Tompkins,
M. pinnipedii. The other assay identifies M. tuberculosis, M. canettii 1992; van Embden et al., 1993). In countries with well-developed
and M. africanum West African 1. diagnostic facilities, the resources committed to bacteriology for
animal TB and strain typing of isolates depend on the require-
3.2.8. Single nucleotide polymorphism (SNP) typing ments of the TB management programme in place. If the objective
SNP typing has proven useful for differentiation among the MTBC is to monitor and control disease incidence, then a less sensitive
members, e.g. for M. bovis (Espinosa de Los Monteros et al., 1998; culture methodology and presumptive identification of strains may
Scorpio and Zhang, 1996) and M. caprae (Aranaz et al., 2003). Re- be sufficient for maintenance of an efficient programme. Econom-
cently, Reddington et al. (2011) described a novel M. caprae- ic factors may dictate that liquid media culture systems are pre-
specific SNP in the M. tuberculosis H37Rv lepA (Rv 2404c) gene in ferred as they allow for high throughput and automation that
German and Dutch isolates, which has been confirmed in bovine and facilitates rapid detection of the growth of M. bovis. However, if a
cervine isolates from the Bavarian Alps (Domogalla et al., 2013). SNP more comprehensive diagnosis is required, for example, when trying
typing has also become a powerful tool for the identification of dis- to gain a deeper understanding of the pathogenesis of disease or
tinct lineages in M. tuberculosis (Abadia et al., 2010; Gagneux and assess how a vaccine might limit distribution of infection and trans-
Small, 2007; Hershberg et al., 2008; Schürch et al., 2011) and M. bovis mission, then there may be critical aspects of bacteriology and
(Allen et al., 2013; Garcia-Pelayo et al., 2009; Smith et al., 2006). A strain typing that require optimization to achieve success. This comes
disadvantage of SNP typing is that it relies on a two-step process, with a cost, however, as the higher the sensitivity desired the greater
either PCR and sequencing or PCR and restriction endonuclease anal- the resources required. Automated liquid culture systems may
ysis, which is cost- and time-intensive. Recently, Stucki et al. (2012) not be suitable when used alone, as there are as yet, few reports
described two new single-step approaches for the classification of on the comparative diagnostic sensitivity when used alongside the
the M. tuberculosis Beijing sublineage, M. bovis and M. caprae to over- conventional solid media. When maximum sensitivity is required,
come this disadvantage: multiplexed oligonucleotide ligation PCR all of the factors affecting sensitivity need to be addressed, includ-
and TaqMan® Real-time PCR. A commercially available DNA strip ing the manner of collecting samples to the range of samples col-
assay (Genotype MTBC, Hain Lifescience GmbH, Nehren, Germany) lected; also sample storage conditions and duration, and the
is largely based on SNPs in the gyrB gene. It also utilizes the RD1 bacteriological procedures employed, type of media, number of in-
deletion for the identification of M. bovis BCG. It can differentiate oculated media (slopes or bottle), decontamination, duration of in-
almost all members of the MTBC including M. bovis, M. bovis BCG cubation. The resource allocation issues will also apply to strain
and M. caprae (Richter et al., 2003). identification and typing. It may only be necessary to identify and
confirm an isolate as M. bovis or M. caprae, to establish that infec-
3.2.9. Molecular tests that distinguish MTBC from MOTT tion is present in an animal or in a herd. However, this will provide
There are a number of commercially available tests for differ- very limited epidemiological information on the nature of trans-
entiation between MTBC and MOTT isolates. The first developed was mission of infection. High-resolution typing systems can be very
the AccuProbe (Gen Probe Inc., San Diego, USA). This test is based useful in tracking of isolates as they move from herd to herd, and
on species-specific DNA probes that hybridize to rRNA. Another com- over long distances. The resources required to type each isolate are
mercially available test is the INNO-LiPA MYCOBACTERIA v2 (Inno- likely to be substantial but may be critical for identifying the source
genetics NV, Ghent, Belgium). This is a line probe assay based on of an outbreak and devising strategies to eradicate infection from
species-specific variation in the 16S-23S rRNA gene spacers. herds.
(Palomino, 2009). More recently, a number of tests based on the In developing countries where resources for diagnosis and
detection of MPT64/MPB64 secreted antigen specific to the MTBC molecular typing are scarce, development of control strategies
have become available. The tests consist of a simple and rapid is seriously constrained by the lack of facilities and knowledge
immunochromatic assay to detect the presence or absence of MPT64/ to accurately determine the prevalence of infection, and hence
MPB64 antigen. The commercially available kits include Capilia TB there is a weak understanding of the local epidemiology of
assay (Tauns Laboratories, Inc., Numazu, Japan), the Tibilia rapid test infection. This can lead to uncoordinated efforts and subsequent
(Hangzhou, China), the SD Bioline TB Ag MPT64 rapid test (Stan- failure of campaigns long before the basic information is available
dard Diagnostics, South Korea) and the MGIT TBc ID test (Yu et al., to inform the development of control strategies (Marcotty et al.,
2011). An immunochromatic assay based on detection of the MTBC- 2009).
specific secreted antigens ESAT-6 and CFP-10 has also been devel- Where resources are limited, comprehensive diagnostic bacte-
oped (Shen et al., 2011). A number of ‘in-house’ methods have also riology and molecular strain typing may also not be affordable.
been described. One such method is based on PCR amplification of However, progress towards eradication of tuberculosis in cattle
the gene encoding the 65-kDa heat shock protein and restriction frag- can be achieved in most circumstances, providing there is judi-
ment length polymorphism (RFLP) analysis of the amplified frag- cious use of the emerging test technologies. Ongoing commit-
ment (Plikaytis et al., 1992). However, this is a more difficult and ment from local veterinarians and national authorities and herd
time-consuming procedure than use of a commercial kit. owners can provide an environment that optimizes the chances of
It is likely that new tests will continue to be developed, driven success. Commitment is enhanced through education on im-
by the need to provide rapid cost-effective high-throughput diag- proved animal husbandry, awareness of and dealing with all likely
nostics. Most of the test evaluation is conducted on M. tuberculosis sources of infection, regular and efficient testing of cattle, quaran-
strains isolated from human TB cases and there are major chal- tine and removal of infected animals. Where commitment is sus-
lenges for veterinary research and diagnostic laboratories to fully tained in both the surveillance and disease control phases of a
evaluate new tests with M. bovis and M. caprae strains isolated from programme, an animal testing regime in itself has much to offer. It
bovine and wildlife cases of tuberculosis. may be difficult to immediately apply all facets of control simulta-
neously, particularly in developing countries, but by recognizing
4. Concluding remarks that different components of the diagnostics and strain typing pro-
grammes can be strategically used to effectively remove all poten-
From the early stages of the development of methodologies for tially infected animals, the eradication of tuberculosis in cattle can
cultivation and molecular typing of M. bovis strains, it has been rec- be accomplished.
ognized that these can make an important contribution to a more
Acknowledgements of the National Academy of Sciences of the United States of America 104,
5596–5601.
Bryant, J.M., Schürch, A.C., van Deutekom, H., Harris, S.R., de Beer, J.L., de Jager, V.,
We would like to acknowledge the support of all the partners et al., 2013. Inferring patient to patient transmission of Mycobacterium tuberculosis
funded through the EU project TB-STEP (KBBE-2007-1-3-04, no. from whole genome sequencing data. BMC Infectious Diseases 13, 110.
212414) – Strategies for the Eradication of bovine tuberculosis. SRC Butcher, P.D., 2004. Microarrays for Mycobacterium tuberculosis. Tuberculosis 84,
131–137.
is grateful for the support received from the Swiss Federal Veteri- Caimi, K., Romano, M.I., Alito, A., Zumarraga, M., Bigi, F., Cataldi, A., 2001. Sequence
nary Office and would like to acknowledge her former colleagues analysis of the direct repeat region in Mycobacterium bovis. Journal of Clinical
at the VISAVET Health Surveillance Centre and the Animal Health Microbiology 39, 1067–1072.
Chambers, M.A., Crawshaw, T., Waterhouse, S., Delahay, R., Hewinson, R.G.,
Department, Complutense University of Madrid (Spain), and the Lyashchenko, K.P., 2008. Validation of the BrockTB stat-pak assay for detection
Spanish Ministry of Agriculture, Food and Environment. of tuberculosis in Eurasian badgers (Meles meles) and influence of disease severity
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Research in Veterinary Science 97 (2014) S30–S43

Contents lists available at ScienceDirect

Research in Veterinary Science

Bacteriological diagnosis and molecular strain typing of

a School of Veterinary Medicine, University College Dublin (UCD), Dublin 4, Ireland

1. Introduction process has its own performance characteristic, determined by the

quently, the spots are visualized in a ﬂuorescence reader and quan-

3.2. Partial genome techniques for strain typing

3.2.1. Restriction fragment length polymorphism analysis

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