In addition toM. bovis and M. tuberculosis, the complex includes M. ...... Quigley F., Costello E. & Smith T. (1997). - PCR-based fingerprinting of Mycobacterium ...
Rev. sci. tech. Off. int. Epiz., 2000, 19 (3), 675-688
Molecular epidemiology of bovine tuberculosis I. Mycobacterium bovis genotyping P.A. Durr. R.G. Hewinson & R.S. Clifton-Hadley Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KJ15 3NB, United Kingdom Submitted for publication: 5 April 2000 Accepted for publication: 10 May 2000
Summary The lack of a typing system for Mycobacterium bovis has, un.til recently, been an impediment to undertaking sophisticated epidemiological studies to assist in the control and eradication of tuberculosis in domestic animals. Molecular biology techniques for mycobacteria have been in development since the mid-1980s, leading to the availability of a number of genetic typing systems forM. bovis. The authors summarise the available techniques, identify those which are most useful at present and those which might prove useful in the future. The present recommendation is to use spoligotyping analysis for rapid, large scale screening of M. bovis isolates, and to use restriction fragment length polymorphism analysis using the polymorphic guanine and cytosine-rich repeat sequences probe where greater differentiation of isolates is required. In the future, systematic analysis of the genome sequence of M. bovis will allow the development of improved techniques that combine good discrimination with ease of use. Keywords Genotyping- Molecular epidemiology- Mycobacterium bovis- Restriction endonuclease analysis- Restriction fragment length polymorphism- Spoligotyping -TuberculosisVariable number of tandem repeats.
Introduction Detailed epidemiological investigations of bovine tuberculosis have traditionally been hampered by the lack of an established typing system for Mycobacterium bovis. Strain differentiation has not proved possible using serotyping (33), and only limited typing proved possible using either phage typing (39) or biotyping (5). Consequently, with advances in molecular biology during the 1980s, typing systems based upon the bacterial genome ('genotyping') have been actively explored. Considerable advances have been made in developing techniques that discriminate between isolates of the Mycobacterium tuberculosis complex, of which M. bovis is a member. Nevertheless, problems remain to be overcome in the development of effective genotyping of M. bovis. Specifically,
no uniformly recognised system exists for sych typing. Although progress is now being made to develop standard protocols (16, 51), a lack of consensus on the 'best' technique remains. Even more problematic is the lack of a widely accepted method for the epidemiological application of the typing data, and few examples are available of the systematic use of genotyping techniques in regional or national bovine tuberculosis control (18). This review paper has been prepared to assist veterinary epidemiologists and disease control managers to select the most appropriate typing technique forM. bovis, according to particular circumstances. A further aim is to offer a succinct explanation of the molecular biological basis of each technique, thereby providing the background for understanding the potential of these techniques in practical control of tuberculosis.
676
The genome of
Mycobacterium bovis On account of a high genomic similarity, the mycobacterial 'species' pathogenic for mammals are now grouped together in theM. tuberculosis complex (23). In addition toM. bovis and M. tuberculosis, the complex includes M. microti and M. africanum, which cause tuberculous lesions in voles and humans, respectively. Recently, two further members of the complex have been recognised, namely M. caprae which causes tuberculosis of goats in Spain (4), and M. canetti which has been isolated from humans (55). The genomics of this complex have proved considerably more difficult to elucidate than those of many other bacteria, due to the difficulties presented by extremely slow growth, the unique cell wall composition of the organism, the need for protection of laboratory personnel and a paucity of cloning vehicles (44). Nevertheless, the resurgence of human tuberculosis has meant that the pathogen has become a research priority, and one result has been the considerable achievement of the sequencing of the entire genome of an important strain of M. tuberculosis, H37Rv (7). The sequencing of the genome of an M. bovis isolate from Great Britain is now well underway, and should be completed by the end of 2000 (l). The genome of the M. tuberculosis complex consists of a double strand of circular deoxyribonucleic acid (DNA), consisting of approximately 4. 4 million base pairs Cbp). This is comparatively large for an obligate intracellular bacterial pathogen, the majority of which have smaller genomes (6). The DNA of the genome has a high guanine and cytosine (G +C) content, of approximately 65% (7). A large fraction of the genome is devoted to enzymes for the metabolism oflipids such as the mycolic acids, which form an essential ingredient of the distinctive hydrophobic, waxy cell wall of the mycobacteria. The genome is also rich in gene regulatory circuits, consistent with the need to selectively activate genes in order to survive for prolonged periods within host macrophages, as well as in the outside environment during the transmission stage. Another peculiarity of the M. tuberculosis complex is the lack of interstrain genetic diversity. The reason for this genetic conservatism is uncertain, possibly reflecting recent evolution as a pathogen (47). An alternative explanation is the absence of mechanisms for genetic exchange between pathogenic mycobacteria, as neither conjugation nor transformation is known to occur, and limited contact occurs with bacteriophages needed for transduction. Much of the genomic polymorphism that has been detected is associated with mobile genetic elements, particularly insertion sequences (IS). These are small segments of DNA that can insert themselves at multiple sites within the genome, the purpose of which may be to provide genetic variability by deactivating genes (48). However, the recent sequencing of the genome of
Rev. sci. tech. Off. int. Epiz., 19 (31
M. tuberculosis H3 7Rv revealed that the IS are mainly
clustered in intergenic and non-coding regions (7), suggesting that in practice, functional gene deactivation is relatively uncommon.
Whole genomic techniques The numerous genotyping techniques that have been developed can be classified, based upon the genetic element used (Fig. 1). At the most basic level, this classification separates those techniques that use the whole genome for the typing from those that are based upon only part of the genome. The whole genomic techniques were the first to be developed and have the advantage that all the potential genetic information is used. However, the cell wall composition of the M. tuberculosis complex renders the organisms relatively refractive to DNA extraction, with the result that whole genomic based techniques are technically demanding. Combined with difficulties in automating the techniques, this has rendered these methods less popular than those that utilise only part of the genome.
Restriction endonuclease analysis The technique of restriction endonuclease analysis (REA) was the first to be developed for the intraspecific typing of M. bovis (9). After extraction from the cell, the whole genomic DNA is digested with the three restriction endonuclease enzymes, EstEll, Pvull and Bell, which cleave DNA strands at highly specific nucleotide sequences (Fig. 2). The resulting digest has many small fragments of DNA and these are then separated by agarose gel electrophoresis. The fragments migrate" in the gel in response to an electrical field, the smallest moving the furthest. Strains are then characterised based on the fragment patterns following staining with ethidium bromide. The fragment patterns are recorded photographically, following transillumination with ultraviolet light.
As the only typing technique available in the late 1980s and early 1990s, REA was used for molecular epidemiological studies in New Zealand (10) and Ireland (13). However, the method is technically demanding, and interpretation of the complex REA patterns is difficult (12). Furthermore, no method exists for numerically cataloguing types, which makes comparison between laboratories difficult. As a consequence, this technique is now generally rarely used (16). The exception is in New Zealand, where familiarity with the method and superior discrimination as compared to the newer techniques, means that it remains the technique of choice (8).
Pulsed field gel electrophoresis A solution to the problem of the excessive number of small DNA fragments encountered with REA is the use of pulsed field gel electrophoresis (PFGE). Essentially, all the steps are the same as for REA, the only difference being the use of restriction endonuclease enzymes to generate a relatively
677
Rev. sc1: tech. Off. Int. Epiz., 19 (31
I
Whole genome techniques
I
I
r
I
I
I
I
I
I Many small fragments: REA
Partial genome techniques
Specific repeated sequences
r
I
I
Few large fragments: PFGE
I
Non tandem repeated sequences
I
I
r
ITandem repeated sequences
J
I
I
Extra-tandem repeat loci VNTR*
I
Random sequences
RAPD analysis*
I
I Major polymorphic tandem repeat loci IS6111J.ampliprinting*
I
Insertion sequences IS6110-RFLP IS 1081-RFLP DR-RFLP GC IS PFGE PGRS-RFLP RAPD REA RFLP VNTR
: : : : : : : : :
Direct repeat region DR-RFLP Spoligotyping*
Polymorphic GCrich sequence PGRS-RFLP
direct repeat-restriction fragment length polymorphism guanine and cytosine insertion sequence pulsed field gel electrophoresis polymorphic GC-rich sequences-restriction fragment length polymorphism random amplified polymorphic deoxyribonucleic acid restriction endonuclease analysis restriction fragment length polymorphism variable number of tandem repeats
Fig.1 A classification of techniques (shown in bold) for typing Mycobacterium bovisand other members of the M. tuberculosis complex, based upon the genomic element used Those marked with an asterisk use the polymerase chain reaction
small number oflarge DNA fragments. Such fragments are too large to be separated by conventional electrophoresis, but can be discriminated when subjected to a constantly changing (pulsed) electrical field. Although PFGE is widely used for subtyping pathogenic bacteria, the technique has been comparatively rarely used for the M. tuberculosis complex. One of the earliest reports of the use of PFGE was by Zhang et al. for the typing of M. tuberculosis (56); Zhang et al. later reported the use of PFGE for differentiation of M. bovis bacillus Calmette-Guerin (BCG) substrains (57). To date, the only published report of the use of PFGE for differenLiation of isolates of M. bovis is by Feizabadi et al., where good discrimination between types for epidemiological purposes was reported (19). Unfortunately, application of PFGE to theM. tuberculosis complex is difficult and labour-imensive, which has restricted the utilisation of the technique.
Partial genomic techniques not based on the polymerase chain reaction A number of techniques are based upon identifying polymorphisms v.dthin specific regions of the M. bovis genome. The earliest, \Vb.ich essentially evolved as a refinement of REA, is restnctwn fragment length polymorphism (RFLP) analysis. The same procedure of DNA restriction enzyme digestion (using either Pvuii or Alui) and separation by agarose electrophoresis is followed (Fig. 3). An additional step is then added by Southem blotting of the DNA onto a nitro-cellulose or nylon filter and then adding probes bearing labels or markers. These probes are selected to be complementary to only a fraction of the DNA of the isolate,
678
Rev. sci. tech. Off. int. Epiz., 1913)
2
2
c
::::>
(c
::::>
)
3
c
::::>
.... c
::::>
c
::::>
--
3
.. ---------__________..,
....~
-:~------_-_:-..
.. ..------------- .... +
Step 1 Culture of Mycobacterium bovis
Step 2 Extraction of chromosomal DNA
Step 3 Digestion by restriction enzymes (Bst Ell, Pvull and Bell)
Step4 Agarose gel electrophoresis and staining with ethidium bromide
DNA: deoxyribonucleic acid
Fig.2 Basic steps for the detection of genetic polymorphism of Mycobacterium bovis using restriction enzyme analysis
and therefore after hybridisation, only some of the fragments will be visualised, resulting in a considerable simplification in comparison with REA. In the past, radioactive labelled probes, such as 32P were utilised, allowing visualisation by exppsure to X-ray film. Due to safety concerns, non-radioactive methods, particularly chemiluminescent systems, are currently recommended (16). The crucial first step for the technique is the selection of a DNA element, which when used as a probe, will reveal polymorphisms or DNA differences between isolates. Probes based upon repetitive DNA elements are preferred because these bind to a greater number of fragments and this can increase the potential to reveal polymorphisms. Those repetitive elements that have been utilised for RFLP include IS, polymorphic GC-rich repeat sequences (PGRS) and the direct repeat (DR) sequence (Fig. 4).
The potential of IS6110 for typing M. bovis has been investigated in some detail. Generally, fewer copies of IS611 0 are present in M. bovis than in M. tuberculosis, and the majority of bovine isolates have been found to have only a single copy located in the DR region (12, 17, 35, 40, 54). However, this may depend upon the infected species, as multiple copies have been reponed from various zoo and wild animals (54). Furthermore, isolates from some cattle populations may have high IS6110 numbers, as Liebana et al. demonstrated that almost 50% of bovine isolates from Spain had multiple copies (30). Consequently, the usefulness of this technique will. depend upon the characteristics of the local strains. The current recommendation is that if strains contain more than three copies of the sequence, then IS6110-RFLP should be considered the method of choice due to the good discrimination offered by this technique (16).
Restriction fragment length polymorphism using insertion sequence 6110
Restriction fragment length polymorphism using insertion sequence 1081
The most important insertion sequence identified in the M. tuberculosis complex is 1S6110, a 1,361 bp fragment. This IS was also isolated and referred to as IS986 and 1S987 before these elements were recognised as being essentially the same as IS6110 (38). In M. tuberculosis, up to twenty copies are present in the genome (31, 49), and the resulting degree of polymorphism has enabled IS6110-RFLP to become the 'gold standard' for molecular epidemiological studies of human tuberculosis (51).
Investigations have also focused on IS1 081 for use in the M. tuberculosis complex (ll, 38, 52). In studies where 1S1081-RFLP was applied to M. bovis isolates, limited polymorphism was observed and differentiation between strains was not possible (3, 12, 45). However, the capacity of 1Sl081-RFLP to distinguish M. bovis BCG from other strains of M. bovis is useful (53), and IS1081 has been used in combination with other probes to increase the discrimination ofRFLP (45).
679
Rev. sci. tech. Off. int. Epiz., 19 (3)
Acridinium, digoxigenin or Jzp labelled oligonucleotide probes
+
DNA extraction and endonuclease digestion (Pvull or Alui)
....
+
Step 1 Agarose gel electrophoresis
Step 2 Southern blot of DNA onto nylon membrane
Step 3 Hybridisation with labelled probes
Step4 Washing and visualisation of hybridised fragments by luminometer or autoradiography
DNA: deoxyribonucleic acid
Fig. 3 Basic steps for the detection of genetic polymorphism of Mycobacterium bovis using restriction fragment length polymorphism analysis
Restriction fragment length polymorphism using polymorphic guanine and cytosine-rich repeat sequences Mycobacteria have a very high G + C content, with G + C constituting 66% of the total M. tuberculosis genome (7). Within the genome, short, repeated sequences are present which have a GC composition in excess of 80% (37, 43). These polymorphic GC-rich sequences (PGRS) are present in multiple clusters scattered throughout the genome (Fig. 4). The PGRS are also present in several plasmids, and when PGRS-RFLP was originally developed, the plasmid pTBN12 was used to prepare a transcript probe (43). Currently, the recommended probe consists of a set of oligonucleotide primers comprising several PGRS sequences (16). Investigations of the potential of the PGRS-RFLP forM. bovis, have revealed that when used to probe M. bovis DNA digested by Alu1, a good degree of strain differentiation can be achieved (15, 54). Cousins et al. demonstrated that PGRS-RFLP offered a much higher strain differentiation than spohgotyping, 1S6110-RFLP or DR-RFLP for a large sample of isolates from Europe, Canad~, Australia and Iran (17). A similar conclusion was reached by a comparative assessment in the Republic of Ireland, using isolates from cattle, badgers and deer (14) . Consequently, PGRS-RFLP is recommended as the method of choice when maximum strain differentiation is required and multiple copies of 1S6110 are absent (16). However, the
technique is technically demanding and consequently, variable results can be obtained, even by experienced technicians.
Direct repeat restriction fragment length polymorphism The DR cluster is unique to isolates of the M. tuberculosis complex, consisting of multiple 36 bp 'DR' sequences interspersed with non-repetitive spacer sequences ('spacers'), from 35 bp to 41 bp in length (24, 26). Strains vary in the number of DRs and in the presence and absence of particular spacers, which are variable in length and sequence (24). The function of the DR region is uncertain, but may play a role in protein binding (24). Cousins et al. included DR-RFLP in a detailed comparison of typing techniques from a broad geographical area (17). Discrimination of M. bovis isolates was similar to that obtained by spoligotyping, which was consistent with the fact that both techniques target the same chromosomal locus. However, in a study of 224 isolates from South America, Zumarraga et al. reported that DR-RFLP was able to distinguish more types than spoligotyping (58). Nevertheless, where the discrimination is similar to spohgotyping, the latter would be recommended, as the method is technically simpler to perform and the results are easier to interpret.
Rev. sci. tech. Off. int. Epiz., 19 (3)
680
a) DR region
b)lnsertion sequences
IS6110
IS/081 IS/081 IS1081 IS6110 IS6110 IS1081 IS6110 IS6170 IS6110
c) PGRSs
IS61 10 IS6110
IS6110
d) ETRs and MPTRs
Mbp: megabase pairs a) the direct repeat (DR) region b) the insertion sequences IS611D and IS 1081. The IS611D shown in the square box is at the location of the single copy found in many Mycobacterium bovis isolates c) the polymorphic GC-rich sequences (PGRS) d) the extra-tandem repeat (ETRI and major polymorphic tandem repeat (MPTRI sequences
Fig. 4 Approximate locations within the genome of Mycobacterium tuberculosis H37Rv of the main repetitive sequences used for typing the M. tuberculosis complex genomes Data source: lnstitut Pasteur, Tuberculist website (http://genolist.pasteur.fr(Tuberculist)
Mixed probe restriction fragment length polymorphism Where maximum discrimination of types is required, combining the results of several RFLP probes is often useful, provided that the probes are not genetically linked. By using three probes (IS6110, IS1081 and PGRS), Skuce et al. were able to identify twenty-eight distinct RFLP types, whereas the maximum obtained by PGRS-RFLP was twelve (45). Similarly, Gutierrez et al. used a combination ofiS6110, PGRS and DR, to differentiate between caprine and bovine strains of M. bovis in Spain (25). Several other examples of improved strain discrimination by combining probes have been described (35, 40, 41, 46).
Partial genomic techniques based on the polymerase chain reaction While the various RFLP techniques successfully enable a reliable sub-typing of M. bovis, these methods do require a high concentration of DNA, which consequently requires culture of the organism and obviates the direct use of the technique on clinical specimens. Additionally, the techniques required for DNA extraction and manipulation of M. bovis are technically demanding and time-consuming. Consequently,
681
Rev. sci. tech. Off. int. Epiz., 19(3)
the development of more rapid, automated techniques, that will enable a high throughput of samples, has been of great interest. These techniques depend upon the polymerase chain reaction (PCR), an essential method in molecular biology for amplification of sequences of DNA.
isolates in Ireland, albeit with only eight isolates (22). However, in a more recent assessment using a larger sample size, the technique performed poorly, with reproducibility between duplicate samples of only 39% (29).
Spoligotyping Random amplified polymorphic deoxyribonucleic acid analysis The simplest PCR-based fingerprinting technique uses short oligonucleotide primers that target sequences randomly within the genome. Random primer-based PCR fingerprinting has also been referred to as 'arbitrary primer PCR' and 'DNA amplification fingerprinting'. Random amplified polymorphic DNA (RAPD) analysis has been applied widely to type bacteria (50). However, fragments are not all amplified with equal efficiency and amplification is extremely sensitive to reaction conditions. For these reasons, concerns have been raised regarding the reproducibility, and thereby the validity, of the methodology (21). The use of RAPD analysis for typing M. tuberculosis was first reported by Palittapongarnpim et al. (34), but to date, the technique has been rarely used to type M. bovis. Glennon et al. used the technique for a small number of samples in Ireland (22), but found that differentiation between isolates was poor compared to ampliprinting (see below). Nevertheless, in a more recent assessment in Mexico, using a large number of isolates from cattle, the technique performed reasonably well (32).
Ampliprinting using insertion sequence 6110 The major polymorphic tandem repeat (MPTR) sequence was first described in 1992 (27). The sequence is structurally similar to the DR cluster, consisting of 10 bp DRs separated by a 5 bp unique DNA spacer. The MPTR copy number has been estimated at approximately eighty per genome. The MPTR is not confined to the M. tuberculosis complex, and has been identified in M. hansasii and M. gordonae. In the M. tuberculosis complex, the MPTR possesses very limited restriction polymorphism and consequently the sequence has not been used alone for typing. However, when combined ·with the 156110, the MPTR has enabled the development of a novel typing technique, named ampliprinting, which was first described forM. tuberculosis by Plikaytis et al. (36). The basis of ampliprinting is the variable distance between IS6110 elements and copies of MPTR sequences which are amplified byPCR. Two reports are available on the use of this technique for M. bovis isolates. Gutierrez et al. used the technique to differentiate between strains of M. bovis infecting goats, and found the method to be as sensitive as RFLP (25). This · discrimination was only possible as the caprine strains contained multiple copies of the 15611 0; the technique would be less effective in single copy strains. Notwithstanding, Glennon et al. achieved good discrimination between bovine
Spoligotyping ('spacer oligonucleotide typing') is based on the detection of DNA polymorphism within the DR locus (see above), and represents the first PCR technique to be widely accepted (28). The individual DRs, by warrant of being identical, are all potential targets for in vitro PCR amplification, and the variation in spacers can be exploited to obtain different hybridisation patterns of the amplified DNA (24). The amplified products are then hybridised to a set of immobilised oligonucleotides, each corresponding to one of the forty-three potential unique spacer DNA sequences within the DR that have been sequenced from a number of M. tuberculosis complex strains (Fig. 5). This technique of 'reverse line blot hybridisation' is relatively simple and robust to perform and accelerates considerably the throughput of samples. Detection of hybridised DNA then follows, using a chemiluminescent system. The output of the spoligotyping is the result of the hybridisation of each of the forty-three consequent sequences as positive or negative. As the data is in a binary format, a word processor can be used to compare the forty-three res~lts. The value of spoligotyping for strain differentiation of M. bovis has been assessed critically in several studies. Aranaz et al. demonstrated that while differentiation of isolates was poorer than with 156110-RFLP, the technique did enable the grouping of the affected mammalian host species, which presumably reflected an underlying difference in epidemiology of the disease (2). Similar results showing limited type differentiation have been shown in subsequent studies (3, 14, 17, 41). Consequently, spoligotyping is now recommended to be used for rapid screening of isolates, followed by a more discriminatory technique such as PGRS-RFLP (16). As spoligotyping is PCR-based, the technique can potentially allow for simultaneous diagnosis and typing, an advantage over the RFLP techniques (28). However, this potential has not yet been realised using spoligotyping on M. bovis clinical specimens. Recently, Raring et al. investigated the potential for reducing the culture time by using radiometric culture followed by spoligotyping (4 2). Definitive spoligotype patterns were obtained for 85% of culture-positive samples within ten days of culture, suggesting that the possibility of early diagnosis and typing might soon be a reality.
Variable number of tandem repeats typing Genetic loci containing variable numbers of tandem repeats (VNTR loci) form the basis for human gene mapping, forensic analysis and paternity testing, In this technique, DNA containing variable numbers of tandem repeat sequences is
682
Rev. sci. tech. Off. int. Epiz., 19 (31
DR region
Amplified oligo(nucleotides)
~ 1 j:'t:''==!::!=t~
~
2:
-
~
O
.,,,,
:::
II II
Ill
Visualisation via chemiluminescence
1 "'2 Q)
•••••• •••••
~ DR : direct repeat PCR : polymerase chain reaction
Fig. 5 Basic steps for the detection of strain differences of Mycobacterium bovis using spoligotyping
amplified by PCR and the size of the product determined by gel electrophoresis (Fig. 6). Recently, Frothingham and Meeker-O'Connell identified six VNTR loci (ETR-A to F) for typing theM. tuberculosis complex (20). When compared to RFLP-15611 0 fingerprinting, VNTR was demonstrated to be less discriminatory for strains with a high copy number, but ETR!Al
I ~~ --~c }s=;) Sln1P1
'-· A
__,__1
1
--illii}- -{IIJ--
allowed improved discrimination for strains with only one or two copies ofiS611 0 (20). In a recent assessment of a range of typing techniques, VNTR performed well, with a reproducibility between duplicates of 97%, and discrimination into types only slightly less sensitive than spoligotyping (29).
~~~ -~ Strain2
Cell content extraction --+ PCR
..miD-
1
-ill)-
..miD-
- -= = -----
1
500bpor Strain 1
Electrophoresis (-6 h) -ethidium bromide staining
l
~Obp[Jj Strain 2
bp : base pairs ETA : extra-tandem repeat PCR : polymerase chain reaction
Fig. 6 Basic steps for the detection of strain differences of Mycobacterium bovis using variable number of tandem repeats (VNTR) to determine the number of tandem repeats in each of the six (A-F) extra-tandem repeat loci Analysis of VNTR loci A. Band Cfor two isolates of M. bovis which differ in the number of tandem repeats within the VNTR A locus
Rev. sci. tech. Off. int Epiz., 19 (3)
683
The usefulness of the technique has not yet been fully assessed for M. bovis, although evaluation of the technique for isolates from Great Britain is currently underway at the Veterinary Laboratories Agency (Weybridge). Preliminary results suggest that although VNTR gives a higher degree of discrimination than spoligotyping, best results are obtained by combining the two techniques.
problems can arise when the differences are minor. Another important advantage of spoligotyping and VNTR is that electronic data transfer is easy and therefore comparison of results between laboratories in different countries is simple. Currently, data libraries of M. bovis genotypes are being constructed, which should enable new strains to be compared for similarity to reference strains.
Choice of technique
The criterion of epidemiological usefulness is possibly the most critical, as the purpose of typing should ultimately be to assist in epidemiological investigations. A common epidemiological requirement is typing of the isolates to assist in inferring the source: of infection. An example of this is a multi-reactor herd breakdown, where the isolation of a single strain supports the hypothesis that the infection arose from a point source and spread within the herd. For these types of investigations, maximum discrimination is the main criterion for assessment, and either single or combined probe RFLP techniques are recommended. However, for those investigations concerned with the more global questions of geographical distribution and virulence of strains, high throughput and ease of interpretation are the critical characteristics to enabk the adequate sample number necessary for any rigorous quantitative analyses. Consequently, spoligotyping and VNTR would be the recommended techniques.
The technical achievement of molecular biologists in developing practical M. bovis typing methods has presented animal disease control managers with the problem of which method should be utilised for molecular epidemiological studies. This can be a difficult decision, as few laboratories can afford to invest in developing multiple techniques and each technique has considerable start-up costs in terms of equipment, staff training and site-specific problem solving. In the case of M. tuberculosis, this problem was largely resolved, as one of the early techniques (IS6110-RFLP) was quickly established as the gold standard. However, the inappropriateness of this technique for the majority of strains of M. bovis has meant that the choice of technique is less clear cut. This choice will largely depend upon the importance given to the different criteria that can be used to assess the methods, such as discrimination, ease of use, ease of interpretation and epidemiological usefulness. The original problem that motivated the development of genotyping techniques of M. bovis was the need for discrimination between isolates, and this has remained an important criterion. The REA, by using the entire genome, gives the greatest discrimination, but this in turns presents problems of interpretation. Of the RFLP techniques, PGRS-RFLP gives greatest discrimination, but several probes may be required for maximum separation of types. As regards the PCR techniques, spoligotyping . has only moderate discrimination, but the discriminatory capacity of VNTR for M. bovis holds promise. In terms of technical difficulty, PCR-based methods are clearly advantageous, with spoligotyping being one of the simplest methods. The whole genomic techniques (REA and PFGE) are at the opposite end of the spectrum, requiring considerable skill and experience to produce quality gels. The various RFLP techniques fall between these two extremes, with some, but not all laboratories reporting difficulty in producing quality gels. A third factor to consider is ease of interpretation of results. For this, the better techniques are those which produce results in an intrinsically quantitative form, such as spoligotyping and VNTR. The gel techniques are more problematic, in the case of REA, requiring a considerable degree of skill in interpreting differences. The ease of interprelalion of the RFLP techniques depends upon the quality of the gels, and
A final consideration is cost, for which discrimination is necessary between the cost of start-up and the cost per sample, as these can vary considerably between techniques. The REA has a low start-up cost, requiring electrophoresis equipment only, but each run is demanding in time of highly skilled staff. The various RFLP techniques require more equipment (a vacuum blotter and an oven for hybridisation) as well as incurring ongoing costs for oligonucleotides, chemiluminescence and autoradiography. For spoligotyping, the additional capital cost of PCR equipment and a specific perspex block to allow the reverse blot hybridisation must be considered; however, per unit staff costs are significantly lower, due to the relative ease of the technique. Agarose gel VNTR is a less expensive technique than spoligotyping to set up and run, requiring electrophoresis and PCR equipment only. Therefore at present, no clear recommendation can be given as to which is the best technique, as this will depend upon· local circumstances and the purpose of typing isolates. None of the established methods possess all the desired attributes, and this has motivated the intensive work on developing and adapting novel typing techniques. The anticipated availability of the entire genome of M. bovis will catalyse further developments, and a technique which combin5s high throughput with good discrimination should be available within the next five to ten years. Until that time, the recommendation by Cousins et al., to use spoligotyping for
684
Rev. sci. tech. Off. int. Epiz., 19131
rapid screening, followed by a RFLP technique for maximum discrimination of M. bovis isolates, remains valid (16).
Acknowledgements Thanks are extended to L. Gallagher and S. Hughes for practical information on VNTR and spoligotyping; S. Gordon for assistance in producing Figure 4, and R. Sainsbury, ]. Gallagher and D. Reynolds for helpful editorial comments.
This paper was funded by the Ministry of Agriculture, Fisheries and Food as part of investigations into the application of geographical information systems (GIS) and spoligotyping for the control of bovine tuberculosis in Great Britain (Project SE300l).
•
Epidemiologie moh~culaire de Ia tuberculose bovine I. Typage genomique de Mycobacterium bovis PA Durr, R.G. Hewinson & R.S. Clifton-Hadley Resume Jusqu'a une epoque recente, I' absence de systeme de typage de Mycobacterium bovis avait empeche de realiser des etudes epidemiologiques complexes permettant de contr61er et d'eradiquer Ia tuberculose au sein des populations d'animaux domestiques. Les techniques de Ia biologie moleculaire appliquees aux mycobacteries, mises au point depuis une quinzaine d'annees, ant permis d'elaborer plusieurs systemes de typage genetique de M. bovis. Les auteurs font une synthese des techniques existantes, et decrivent celles qui sont le plus utiles a l'heure actuelle et celles qui pourraient le devenir a l'avenir. Pour un reperage rapide et a grande echelle des isolats de M. bovis, il est actuellement recommande d'effectuer I' analyse par spoligotypage; lorsqu'une differentiation plus precise des isolats est souhaitable, il est recommande de recourir a I' analyse par polymorphisme de taille des fragments de restriction, en utilisant Ia sonde a sequences polymorphes repetees riches en guanine et en cytosine. Al'avenir, !'analyse systematique de Ia sequence genomique de M. bovis permettra de mettre au point des techniques plus elaborees, a Ia fois tres specifiques et plus faciles a realiser. Mots-cles Analyse par enzymes de restriction (endonucleases) - Epidemiologie moleculaire Mycobacterium bovis - Nombre variable de sequences repetees en tandem Polymorphisme de taille des fragments de restriction- Spoligotypage- TuberculoseTypage genomique .
•
685
Rev. sci. tech. Off int. Epiz., 19(3)
Epidemiologia molecular de Ia tuberculosis bovina I. Tipificaci6n genetica de Mycobacterium bovis P.A. Durr, R.G. Hewinson & R.S. Clifton-Hadley Resumen Hasta hace poco tiempo, Ia falta de un sistema de tipificaci6n de Mycobacterium bovis impedfa llevar a cabo estudios epidemiol6gicos sofisticados que sirvieran de apoyo a las campaiias de control y erradicaci6n de Ia tuberculosis en animales domesticos. Pero Ia aplicaci6n de tecnicas de biologfa molecular a las micobacterias empez6 a progresar a mediados de los anos ochenta,y hoy existen diversos sistemas de tipificaci6n genetica de M. bovis. Los autores pasan revista a las tecnicas existentes y evaluan su utilidad presente y sus posibilidades de cara al futuro. En las circunstancias actuales, recomiendan el analisis de spoligotipos para realizar pruebas de criba a gran escala capaces de detectar con rapidez cepas de M. bovis. Para obtener un mayor nivel de discriminaci6n entre cepas, recomiendan el analisis del polimorfismo de longitud de fragmentos de restricci6n, utilizando Ia sonda de secuencias polimorfas repetidas ricas en guanina y citosina. En aiios venideros, el analisis sistematico de Ia secuencia gen6mica de M. bovis hara posible elaborar tecnicas mas adecuadas, que combinen capacidad de discriminaci6n y facilidad de uso. Palabras clave Analisis por endonucleasas de restricci6n - Epidemiologfa molecular - Mycobacterium bovis - Numero variabl~ de repeticiones consecutivas - Polimorfismo de longitud de fragmentos de restricci6n- Spoligotipificaci6n- Tipificaci6n genetica- Tuberculosis .
•
References l.
Anon. (1999). - Bovine tuberculosis: plans to sequence
5.
Barrow P.A. (1981).- Aspects of the epidemiology of bovine tuberculosis in badgers and cattle. II. The development and use of a typing system for MycobacteJium bovis. ]. Hyg. (Camb.), 86, 247-257.
6.
Bishai W. (1998). -The MycobacteJium tuberculosis genomic sequence: anatomy of a master adapter. Trends Microbial., 6, 464-465.
7.
Cole S.T., Brosch R., Parkhill j., Gamier T., Churcher C., Harris D., Gordon S.V., Eiglmeier K., Gas S., Barry C.E., Tekaia F., Badcock K., Basham D., Brown D., Chillingworth T., Conner R., Davies R., Devlin K., Feltwell T., Gentles S., Hamlin N., Holroyd S., Hornsby T., Jagels K., Krogh A., McLean ]., Maule S., Murphy L, Oliver K., Osborne ]., Quail M.A., Rajandream M.A., Rogers ]., Rutter S., Seeger K., Skelton ]., Squares R., Squares S., Sulston j.E., Taylor K., Whitehead S. &: Barrell B.G. (1998). - Deciphering the biology of MycobacteJium tuberculosis from the complete genome sequence. Nature, 393, 537-544.
M. bovis. Vet. Rec., 144, 302. 2.
Aranaz A., Liebana E., Mateos A., Dominguez L, Vidal D., Domingo M., Gonzalez 0., Rodriguez-Fern E.F., Bunschoten A.E., van Embdenj.D.A. &: Cousins D. (1996). - Spacer oligonucleotide typing of Mycobactelium bovis strains from cattle and other animals: a tool for studying epidemiology of tuberculosis. ]. clin. Microbial., 34 (11), 2734-2740.
3.
Aranaz A., Liebana E., Mateos A., Dominguez L &: Cousins D. (1998). :- Restriction fragment length polymorphism and spacer oligonucleotide typing: a comparative analysis of fingerprinting strategies for Mycobactelium bovis. Vet. Microbial., 61, 311-324.
4.
Aranaz A., Liebana E., G6mez-Mampaso E., Galan j.C., Cousins D., Ortega A., Blazquez ]., Baquero F., Mateos A., Suarez G. &: Dominguez L (1999). - MycobacteJium tuberculosis subsp. caprae subsp. nov.: a taxonomic study of a new member of the Mycobactelium tuberculosis complex isolated from goats in Spain. Int. ]. syst. Bacte1iol., 49, 1263-1273.
686
Rev. sci. tech. Off. int. Epiz., 19 (3)
8.
Collins D.M. (1999).- DNA typing of Mycobacterium bovis strains from the Castlepoint area of the Wairarapa. N.Z. vet. ]., 47, 207-209.
9.
Collins D.M. & de Lisle G.W. (1985). - DNA restriction endonuclease analysis of Mycobacterium bovis and other members of the tuberculosis complex.]. clin. Microbial., 21, 562-564.
10.
Collins D.M., De L.G.W. & Gabric D.M. (1986). Geographic distribution of restriction types of Mycobacterium bovis isolates from brush-tailed possums (Trichosurus vulpecula) in New Zealand.]. Hyg. (Camb.), 96, 431-438.
11.
Collins D.M. &: Stephens D.M. (1991). -Identification of an insertion sequence IS1081, in Mycobacterium bovis. FEMS Microbial. Lett., 83, 11-16.
12.
Collins D.M., Erasmuson S.K., Stephens D.M., Yates G.F. & de Lisle G.W. (1993). - DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110.]. clin. Microbial., 31, 1143-114 7.
13. Collins D.M., de Lisle G.W., Collins ].D. & Costello E. (1994).- DNA restriction fragment typing of Mycobacterium bovis isolates from cattle and badgers in Ireland. Vet. Rec., 134, 681-682. 14.
15.
Costello E., O'Grady D., Flynn 0., O'Brien R., Rogers M., Quigley F., Egan]. & Griffin]. (1999).- Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection.]. clin. Microbial., 37,3217-3222. Cousins D.V., Williams S.N., Ross B.C. & Ellis T.M. (1993). - Use of a repetitive element isolated from Mycobacterium tuberculosis in hybridization studies with Mycobacterium bovis - a new tool for epidemiological studies of bovine tuberculosis. Vet. Microbial., 37, 1-17.
21.
Glennon M. & Smith T. (1995). - Can random amplified polymorphic DNA analysis of the 16S-23S spacer region of Mycobacterium tuberculosis differentiate between isolates? Letter to the editor.]. clin. Microbial., 33,3359.
22.
Glennon M., Jager B., Dowdall D., Maher M., Dawson M., Quigley F., Costello E. & Smith T. (1997). - PCR-based fingerprinting of Mycobacterium bovis isolates. Vet. Microbial., 54, 235-245.
23.
Grange j.M. (1996). - The biology of the genus Mycobacterium.]. appl. Bacterial., 81, 1S-9S.
24.
Groenen P.M.A., Bunschoten A.E., van Sooligen D. & van Embden j.D.A. (1993). -Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Malec. Microbial., 10, 1057-1065.
25.
Gutierrez M., Samper S., Gaviganj.-A., Garcia-Marinj.F. & Martin C. (1995). -Differentiation by molecular typing of Mycobacterium bovis strains causing tuberculosis in cattle and goats.]. clin. Microbial., 33, 2953-2956.
. 26.
Hermans P.W.M., van Soolingen D., Bik E.M., de Haas P.E.W., Dale].W. & van Embden].D.A. (1991).- The insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis strains. Infect. Immun., 59, 2695-2705.
27. Hermans P.W.M., van Soolingen D. & van Embden ].D.A. (1992). -Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae.]. Bacterial., 174, 4157-4165. 28.
Kamerbeek ]., Schouls L., KolkA., van Agterveld M., van Soolingen D., Kuijper S., Bunschoten A., Molhuizen H., Shaw R., Goyal M. & van Embdenj. (1997).- Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.]. clin. Microbial., 35,907-914.
29.
Kremer K., van Soolingen D., Frothingham R., Haas W.H., Hermans P.W.M., Martin C., Palittapongampim P., Plikaytis B.B., Riley L.W., Yakrus M.A., Musser ].M. & van Embden].D.A. (1999).- Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. ]. clin. Microbial., 37, 2607-2618.
16. Cousins D.V., Skuce R.A., Kazwala R.R. &: van Embden j.D.A. (1998). -Towards a standardized approach to DNA fingerprinting of Mycobacterium bovis. Int.]. Tuberc. Lung Dis., 2, 471-478. 17. Cousins D.V., Williams S., Liebana E., Aranaz A., Bunschoten A., van Embden ]. & Ellis T. (1998). Evaluation of four DNA typing techniques in epidemiological investigations of bovine tuberculosis.]. clin. Microbial., 36, 168-178. 18. Durr P.A., Clifton-Hadley R.S. & Hewinson R.G. (2000).Molecular epidemiology of bovine tuberculosis. II. Applications of genotyping. Rev. sci. tech. Off. int. Epiz., 19 (3), 689-701. 19. Feizabadi M.M., Robertson I.D., Cousins D.V. & Hampson D.J. (1996).- Genomic analysis of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex by isoenzyme analysis and pulsed-field gel electrophoresis.]. clin. Microbial., 34, 1136-1142. 20. Frothingham R. & Meeker-O'Connell W.A. (1998). Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology, 1"1;4, 1189-1196.
30. Liebana E., Aranaz A., Dominguez L., Mateos A., Gonzalez-Llamazares P., Rodriguez-Ferri E.F., Domingo M., Vidal D. & Cousins D. (1997). - The insertion element IS6110 is a useful tool for DNA fingerprinting of Mycobacterium bovis isolates from cattle and goats in Spain. Vet. Microbial., 54, 223-233. 31.
McAdam R.A., Hermans P.W.M., van Soolingen D., Zainuddin F., Catty D., van Emden ].D.A. & Dale ].W. (1990). - Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the 153 family. Malec. Microbial., 4, 1607-1613.
687
Rev. sci. tech. Off. int. Epiz.. 19 (3)
46.
Skuce R.A., Brittain D., Hughes M.S. & Neill S.D. (1996).Differentiation of Mycobacterium bovis isolates from animals by DNA typing.]. din. Microbial., 34,2469-2474.
47.
Palittapongampim P., Chomyc S., Fanning A. & Kunimoto D. (1993).- DNA fragment length polymorphism analysis of Mycobacterium tuberculosis isolates by arbitrarily primed polymerase chain reaction. ]. infect. Dis., 167, 975-978.
Sreevatsan S., Pan X., Stockbauer K.E., Connell N.D., Kreiswirth B.N., Whittam T.S. & Musser j.M. (1997). Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc. nat! Acad. Sci. USA, 94, 9869-9874.
48.
Perumaalla V.S., Adams L.G., Payeur J.B., Jarnagin J.L., Baca D.R., Guemes F.S. & Ficht T.A. (1996). -Molecular epidemiology of Mycobacterium bovis in Texas and Mexico. ]. din. Microbial., 34, 2066-2071.
Syvanen M. (1998). - Insertion sequences and their evolutionary role. In Bacterial genomes: physical structure and analysis (F. de Bruijn, JR. Lupski & G.M. Weinstock, eds). Chapman & Hall, New York, 213-220.
49.
Thierry D., Cave M.D., Eisenach K.D., Crawford J.T., Bates J.H., Gicquel B. & Guesdon J.L. (1990). - IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res., 18, 188.
32.
Milian-Suazo F., Salman M.D., Black W.C., Triantis J.M., Ramirez C., Payeur J.B. & Torres M.C. (2000). -Molecular epidemiologic analysis of Mycobacterium bovis isolates from Mexico. Am.]. vet. Res., 61,90-95.
33.
Morris J.A. & Ivanyi ]. (1985). - Immunoassays of field 'isolates of Mycobacterium bovis and other mycobacteria by use of monoclonal antibodies. ]. med. Microbial., 19, 367-373.
34.
35.
36.
37.
Plikaytis B.B., Crawford J.T., Woodley C.L., Butler W.R., Eisenach K.D., Cave M.D. & Shinnick T.M. (1993).- Rapid, amplification-based fingerprinting of Mycobacterium tuberculosis.]. gen. Microbial., 139, 1537-1542. Poulet S. & Cole S.T. (1995). - Characterization of th~ highly abundant polymorphic GC-rich-repetitive sequence (PGRS) present in Mycobacterium tuberculosis. Arch. Microbial., 163,87-95.
38.
Poulet S. & Cole S.T. (1995).- Repeated DNA sequences in mycobacteria. Arch. Microbial., 163, 79-86.
39.
Richards W.D. (1974). - Phage susceptibilities Mycobacterium bovis isolates. Ann. Sdavo, 17, 618-622.
40.
Romano M.l., Alita A., Fisanotti ].C., Bigi F., Kantor I., Cicuta M.E. & Cataldi A. (1996).- Comparison of different genetic markers for molecular epidemiology of bovine tuberculosis. Vet. Microbial., 50, 59-71.
51. Van Embden J.D.A., Cave M.D., Crawford J.T., Dale J.W., Eisenach K.D., Gicquel B., Hermans P., Martin C., McAdam R.> Shinnick T.M. & Small P.M. (1993). - Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology.]. din. Microbial., 31,406-409.
of
41. Raring S., Brittain D., Bunschoten A.E., Hughes M.S., Skuce R.A., Neill S., van Embdenj.D.A. & Neill S.D. (1998). - Spacer oligotyping of Mycobacterium bovis isolates compared to typing by restriction fragment length polymorphism using PGRS, DR and IS6110 probes .. Vet. Microbial., 61, 111-120. 42.
50. Van Belkum A. (1994). - DNA fingerprinting of medically important microorganisms by use of PCR. Clin. Microbial. Rev., 7, 174-184.
Raring S., Hughes M.S., Beck L.-A., Skuce R.A. & Neill S.D. (1998). - Rapid diagnosis and strain differentiation of Mycobacterium bovis in radiometric culture by spoligotyping. Vet. Microbial., 61, 71-80.
43. Ross B.C., Raios K., Jackson K. & Dwyer B. (1992). Molecular cloning of a highly repeated DNA element from Mycobacterium tuberculosis and its use as an epidemiological tool.]. din. Microbial., 30, 942-946. 44.
Shinnick T.M., King C. H. & Quinn F.D. (1995).- Molecular biology, virulence, and pathogenicity of mycobacteria. Am.]. med. Sci., 309, 92-98.
45.
Skuce R.A., Brittain D., Hughes M.S., Beck L.-A. & Neill S.D. (1994). - Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis.]. din. Microbial., 32, 2387-2392.
52. Van Soolingen D., Hermans P.W.M., de Haas P.E.W., Soll D.R. & van Embden J.D.A. (1991). -Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. ]. din. Microbial., 29, 2578-2586. 53. Van Soolingen D., Hermans P.W.M., de Haas P.E.W. & van Embden J.D.A. (1992). - Insertion element IS1081associated restriction fragment length polymorphism in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.]. din. Microbial., 30, 1772-1777. 54. Van Soolingen D., de Haas P.E.W., Haagsma ]., Eger T., Hermans P.W.M., Ritacco V., Alitb A. & Van Embdenj.D.A. (1994).- Use of various genetic markers in differentiation of Mycobacterium bovis strains from animals and humans and for studying epidemiology of bovine tuberculosis. ]. din. Microbial., 32, 2425-2433. 55. Van Soolingen D., Hoogenboezem T., de Haas P.E.W., Hermans P.W.M., Koedam M.A., Teppema K.S., Brennan P.]., Besra G.S., Portaels F., Top]., Schouls L.M. & van Embden J.D.A. (1997). -A novel pathogenic taxon of Mycobacterium tuberculosis complex, Canetti: the characterization of an exceptional isolate from Africa. Int. ]. syst. Bacterial., 4 7, 1236-1245.
688
Rev. sci. tech. Off. int. Epiz., 19 (3)
56.
ZhangY., Mazurek G.H., Cave M.D., Eisenach K.D., PangY., Murphy D.T. & Wallace R.]. (1992).- DNA polymorphisms in strains of Mycobacterium tuberculosis analyzed by pulse-field gel electrophoresis: a tool for epidemiology. ]. din. Microbial., 30, 1551-1556.
57.
Zhang Y., Wallace R.j. Jr & Mazurek G.H. (1995).- Genetic difference between BCG substrains. Tuberc. Lung Dis., 76, 43-50.
58.
Zumarraga M.]., Martin C., Samper 5., Alito A., Latini 0., Bigi F., Roxo E., Cicuta M.E., Errico F., Ramos M.C., Cataldi A., van Soolingen D. & Romano M.I. (1999). Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America. ]. din. Microbial., 37, 296-303.
-
