Veterinary Sciences Division, Department of Agriculture and Rural Development, Stormont, Belfast BT4 3SD, UK1
The Queens University of Belfast, Department of Veterinary Science, Stormont, Belfast BT4 3SD, UK2
Veterinary Laboratories Agency, Department for Environment, Food and Rural Affairs, Weybridge, Surrey KT15 3NB, UK3
Author for correspondence: Robin A. Skuce. Tel: +44 28 90 525771. Fax: +44 28 90 525745. e-mail: Robin.Skuce{at}dardni.gov.uk
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ABSTRACT |
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Keywords: bovine tuberculosis, Mycobacterium bovis, tandem repeat DNA, spoligotyping, molecular epidemiology
Abbreviations: DR, direct repeat; ETR, exact tandem repeat; HGDI, HunterGaston discrimination index; MIRU, mycobacterial interspersed repetitive units; PPE, novel glycine-asparagine-rich; QUB, Queens University Belfast; UPGMA, unweighted pair group method with arithmetic means; VNTR, variable-number tandem repeat
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INTRODUCTION |
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In recent years molecular epidemiology, i.e. the integration of robust strain-typing procedures with conventional epidemiological traceback approaches, has produced more specific data with which to inform, influence and monitor control and surveillance strategies for Mycobacterium tuberculosis (van Soolingen et al., 1999 ). Strain typing has often challenged accepted dogmas (van Helden, 1998
) and has been used to investigate important biological properties of strains (Kato-Maeda et al., 2001
). The recent integration of molecular epidemiology and mathematical modelling offers the potential to quantify the risks posed by different subpopulations of the community (Borgdorff et al., 2000
).
M. bovis belongs to the M. tuberculosis complex and has an extremely wide host range (OReilly & Daborn, 1995 ). Despite demonstrable phenotypic differences, members of the M. tuberculosis complex possess a remarkably high degree of genetic identity (Domenech et al., 2001
). They are rich in repetitive DNA (Cole et al., 1998
), a feature which has been exploited for molecular typing. Restriction fragment, or restriction enzyme, analysis has been successfully applied to the molecular epidemiology of M. bovis, most notably in New Zealand (Collins, 1998
). The restriction fragment length polymorphism (RFLP) analysis technique, which exploits repetitive DNA elements for use as probes in Southern blotting has proven to be a highly discriminatory tool (Skuce et al., 1996
). However, this technique is cumbersome and technically demanding, not least in the analysis, nomenclature and databasing of complex banding patterns (Heersma et al., 1998
). Most M. bovis isolates, particularly those of bovine origin, harbour one or more copies of IS6110. Therefore, the accepted IS6110-RFLP protocol agreed for M. tuberculosis (van Embden et al., 1993
) is not appropriate for M. bovis. To identify M. bovis strains, additional discrimination is required with further RFLP procedures, such as PGRS-RFLP analysis and direct repeat(DR)-RFLP analysis (Skuce et al., 1996
), or alternatives such as pUCD probing (OBrien et al., 2000a
). However, these are not ideally suited to inter-laboratory typing studies.
Spoligotyping is based on the detection of DNA polymorphisms within the DR cluster (Groenen et al., 1993 ; van Embden et al., 2000
), which is specific to the M. tuberculosis complex. The number of DR elements in the cluster can vary between strains of the M. tuberculosis complex. These 36 bp DRs are interspersed by non-repetitive DNA spacers of 3641 bp. Spacers have been sequenced, 37 from M. tuberculosis H37Rv and six from M. bovis BCG, synthesized as oligonucleotides and immobilized on a nylon membrane. Isolates are strain-typed on the basis of detecting the presence, or absence, of specific spacers using PCR and a reverse-line cross-blot technique (Kamerbeek et al., 1997
). However, spoligotyping was found to be less discriminatory than RFLP analysis (Roring et al., 1998
) for M. bovis isolates.
Tandem repeat loci, similar to eukaryotic minisatellites, have been identified in M. tuberculosis. These so-called variable-number tandem repeats (VNTRs) often differ in copy number between isolates (Frothingham & Meeker-OConnell, 1998 ). During the preparation of this manuscript several groups have described, classified and analysed tandem repeat loci within the available genome sequences of the M. tuberculosis complex (Supply et al., 2000
; Smittipat & Palittapongarnpim, 2000
). Structures consisting of 40100 bp repetitive sequences, called mycobacterial interspersed repetitive units (MIRUs; Magdalena et al., 1998a
, b
; Supply et al., 1997
, 2000
), were found scattered in 41 locations in the M. tuberculosis H37Rv chromosome; twelve were polymorphic in MIRU copy number between isolates. These novel targets offer the potential for the development of high-resolution, convenient and high-throughput typing methods. The key advantages of VNTR-typing are already evident for M. tuberculosis (Mazars et al., 2001
). VNTRMIRU-typing is PCR-based, and the typing data produced are numerical and easily managed; the data are also applicable to the global molecular epidemiology of the M. tuberculosis complex (Mazars et al., 2001
). VNTRMIRU-typing nomenclature is informative in as much that it is based on the repeat copy number at specific loci. This allows the calculation of relatedness for isolates. Identification of further VNTRMIRU loci should allow this approach to be extended to M. bovis, which has proven difficult to type by existing methods (Cousins et al., 1998
; Skuce & Neill, 2001
).
In this study, novel VNTR loci were identified by a bioinformatics approach using the available genome sequences for the M. tuberculosis complex. These were applied to a panel of M. bovis isolates from various animal sources, European locations and representative reference isolates of the M. tuberculosis complex. Results were assessed for their discriminatory power, and for the correlation of calculated genetic distances with an established panel of exact tandem repeats (ETRs; ETR-A to ETR-E; Frothingham & Meeker-OConnell, 1998 ) and spoligotyping.
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METHODS |
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Bioinformatics.
The lookup program of the SEQNET computing service, now merged with the Human Genome Mapping Project Resource Centre (http://www.hgmp.mrc.ac.uk), was used to interrogate the available annotation of the M. tuberculosis H37Rv sequence contigs (as of January 1998, http://www.sanger.ac.uk) for the keyword repeat. Cosmid sequences were downloaded and scanned manually for perfect, or near-perfect, tandem repeat loci in the range 20150 bp. VNTR loci were located and analysed on the M. tuberculosis H37Rv sequence, and displayed using the Artemis computer program (http://www.sanger.ac.uk). Genome sequences for M. tuberculosis CDC1551 and M. bovis AF2122/97 were searched at the Institute for Genome Research (http://www.tigr.org) and the Sanger Centre (http://www.sanger.ac.uk), respectively. Orthologous VNTR loci were identified in these sequences. PCR genotyping data were recorded in Microsoft Excel 97 and analysed using the programs GelCompar version 4.0 and BioNumerics 2.1 (Applied Maths).
VNTR-PCR.
VNTR-PCR primers (Table 2) were designed to anneal upstream and downstream of each tandem-repeat locus. PCR was performed in a total volume of 60 µl containing 10 ng template DNA and 45 µl PCR Supermix (Gibco-BRL Life Technologies) that contained 22 mM Tris/HCl pH 8·4, 55 mM KCl, 1·65 mM MgCl2, 220 µM of each of the four dNTPs and 1·1 U Taq DNA polymerase; 20 µM of each primer was used. PCRs were run in DNA thermal cyclers (model 480; Perkin Elmer) under the following conditions: 95 °C for 12 min, 40 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 2 min, followed by a final extension at 72 °C for 7 min. Isolates were amplified using primers for ETR-A through ETR-E (Frothingham & Meeker-OConnell, 1998
) according to the authors recommendations. PCR products were analysed by agarose gel electrophoresis, using 100 bp Stepladders (Promega) and 20 bp (FMC) DNA ladders, and visualized by ethidium bromide staining. Product sizes were estimated and the exact number of complete repeats present was calculated using a derived allele-naming table, based on the number of complete repeats which could theoretically be present in a PCR product of a given size, allowing for extra flanking nucleotides and primer size. Loci were named simply on the basis of the order in which they were found by the initial search. VNTR allele calls were entered and manipulated in BioNumerics as character data. Composite datasets were created for the six QUB(Queens University Belfast)-VNTRs (QUB-5, 11a, 11b, 18, 23 and 26) and the five ETRs (ETR-A, B, C, D and E) results. Distance trees were derived by clustering with the unweighted pair group method with arithmetic means (UPGMA), using categorical character table values.
Spoligotyping.
The M. bovis test panel (n=100) was spoligotyped as described by Kamerbeek et al. (1997) . DNA (
10 ng) was prepared from boiled cells and amplified by PCR using the primers DRa and DRb. The resultant spoligotype patterns were recorded and analysed using GelCompar. Spoligotype data were entered and manipulated in the BioNumerics package as character data. The Dice coefficient was used to plot dendrograms using UPGMA. Spoligotypes were determined for some of the reference isolates of the M. tuberculosis complex.
Allelic diversity and discrimination.
The HunterGaston equation (Hunter & Gaston, 1988 ), an application of Simpsons index of diversity (Simpson, 1949
), was used to calculate the allelic diversity, or the HunterGaston discrimination index (HGDI), at each locus. The equation reads:
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where D is the index of discriminatory power, aj is the number of strains in the population which are indistinguishable from the jth strain, and N is the number of strains in the population (Struelens et al., 1996 ).
Correlation and congruence.
Genetic relationships among isolates in the panel, with no known epidemiological associations (n=79), were estimated using UPGMA and plotted as dendrograms. Agreement between the genetic relationships inferred from the similarity matrices used to plot the dendrograms, based on the two sets of VNTR-PCR and spoligotyping datasets, was estimated by calculating the experiment congruence (using Pearsons correlation) and the BioNumerics software.
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RESULTS |
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A total of 33 different allele profiles were identified by the QUB-VNTRs, compared with 22 for the ETR set and 29 for spoligotyping (Table 4). When the allele profiles for the QUB and ETR sets were combined a total of 51 different profiles were identified. VNTR allele profiles and spoligotypes of M. bovis isolates and reference isolates of the M. tuberculosis complex are available as supplementary data at www.mic.sgmjournals.org
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Allelic diversity and discrimination
With our M. bovis test panel the HGDI for individual loci varied from 0·06 for QUB-23 to 0·62 for QUB-11a, and from 0·08 for ETR-D to 0·65 for ETR-A. QUB-5, QUB-11b, QUB-18 and QUB-23 showed striking differences in their HGDIs, despite the fact that three alleles were identified at each locus (Table 4). By combining the results at various loci the discrimination of VNTR-PCR was significantly improved (Table 4
). For example, 67·6% of the discrimination of the combined QUB and ETR sets was due to ETR-A alone. Similarly, 97·1% of the total discrimination of all 12 VNTRs was provided by just four VNTRs (ETR-A, QUB-11a, QUB-26 and ETR-B, in order of discrimination). Spoligotyping was also capable of resolving isolates which had the same VNTR profile (Table 3
).
Forty-five of the isolates were spoligotype ST140, the most common spoligotype among M. bovis strains from the UK and Ireland (Skuce et al., 1996 ; Costello et al., 1999
; Durr et al., 2000
). QUB-VNTRs resolved these 45 isolates into 14 VNTR profiles, with the largest subset being 15 isolates (HGDI=0·84). The ETRs resolved the ST140 isolates into seven profiles, with 27 isolates in the largest subset (HGDI=0·60). QUB-VNTRs and ETRs combined resolved these 45 ST140 isolates into 20 profiles, the largest subset comprising nine isolates (HGDI=0·92).
Correlation (experiment congruence) between VNTRs, ETRs and spoligotyping
Dendrograms were created using the UPGMA genetic distance matrix, calculated from each of the QUB-VNTR, ETR and spoligotyping datasets for 79 M. bovis isolates with no known epidemiological connections (i.e. the test panel, excluding the 21 outbreak isolates). The resulting dendrograms (not shown) were compared and the experiment congruence between the datasets was calculated using Pearsons correlation (). A weak to moderate, positive, linear correlation was found between the QUB-VNTR and ETR datasets (
=0·448), and between spoligotyping and QUB-VNTRs (
=0·416). A moderate to strong, positive, linear association was found between spoligotyping and the ETR data (
=0·678).
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DISCUSSION |
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Location and consequences
Of the VNTR loci described here, six are located in ORFs, where they potentially code protein repeat motifs of 737 aa, which differ in copy number between the three sequenced strains of the M. tuberculosis complex. Intriguingly, QUB-11a, QUB-11b and QUB-18 are located in PPE proteins, which have recently been cited as potential sources of antigenic variation in M. tuberculosis complex strains (Cole et al., 1998 ). The QUB-11a and QUB-11b VNTR loci map to Rv1917c and are largely responsible for the RFLPs attributed to the pUCD probe (OBrien et al., 2000b
). Apparent size differences have been detected amongst the novel glycine-alanine-rich (PE) proteins of the polymorphic gene sequence (PGRS) class of different isolates (Domenech et al., 2001
), and PE-PGRS genes of Mycobacterium marinum are upregulated in experimental granuloma models (Ramakrishnan et al., 2000
). The cellular location and function of PE and PPE proteins is currently unknown. From large amounts of sequence data, Musser and colleagues have extrapolated that
20 of the 167 PE/PPE proteins (
12%) in M. tuberculosis H37Rv would be polymorphic in other members of the M. tuberculosis complex (Musser et al., 2000
). In addition to the PPE protein Rv3135 (Musser et al., 2000
), we have confirmed that there is the potential for length variation in the protein products of ORFs Rv1917c (PPE) and Rv1753c (PPE) (Brosch et al., 2001
). Length variation in the protein products of ORFs Rv1435c and Rv3611 has not been previously reported.
Performance, discrimination and experiment congruence
With the test panel, the HGDI varied from 0·06 for QUB-23 to 0·65 for ETR-A. Using all 11 VNTRs and ETRs the HGDI was 0·96, which demonstrated significantly higher resolution than spoligotyping. The allelic diversity of the 12 VNTRMIRU loci (Mazars et al., 2001 ) was equivalent to the highly discriminatory IS6110-RFLP for M. tuberculosis isolates, although the authors cautioned against dispensing with certain VNTRs which had relatively low allelic diversity. Such loci resolved some strains which remained unresolved at other VNTR loci. ETR-PCR has been proposed as a novel method for genotyping within the M. tuberculosis complex (Frothingham & Meeker-OConnell, 1998
). However, although ETR-PCR was able to discriminate M. bovis isolates and M. bovis BCG substrains, it was subsequently shown to lack discrimination with isolates of the M. tuberculosis complex (Kremer et al., 1999
; Filliol et al., 2000
). We have shown that most of the discrimination attributed to the ETR-A to ETR-E set was due to ETR-A and ETR-B. Similarly, QUB-11a, QUB-11b and QUB-26 contributed most of the discrimination of the QUB-5 to QUB-26 set. We show that the discrimination of the VNTR technique can be greatly improved by combining VNTR loci (Table 4
).
Proposed strategy for use of VNTR-PCR
There is now an increasingly large panel of VNTR-type loci whose performance has not been systematically evaluated. The application of the HunterGaston equation to the genotyping of a comprehensive panel of isolates provides a mechanism for recording the discrimination provided by individual loci, or combinations of loci. The correlation between MIRU-typing and IS6110-RFLP for a test panel of M. tuberculosis isolates was highly significant (=0·512, Mazars et al., 2001
). The positive correlation determined between QUB-VNTR-, ETR-typing and spoligotyping suggests that the methods group isolates in a similar fashion. Polymorphisms found with different molecular markers show strong mutual association because the M. tuberculosis complex has a strongly clonal structure (Sreevatsan et al., 1997
).
VNTR-PCR proved to be a robust, convenient, highly discriminatory technique, which is reproducible and appropriate for typing isolates of the M. tuberculosis complex, including those with a low IS6110 copy number. If these loci are independent there would be 5100000 possible allelic variants using these markers with our test panel. The significance of VNTRMIRU-derived clusters remains to be determined empirically (Frothingham & Meeker-OConnell, 1998 ). VNTRMIRU loci appear to be sufficiently stable to allow meaningful epidemiological studies to be conceived and undertaken (Mazars et al., 2001
). A further attraction is that the performance of VNTR-PCR can be tailored to suit specific studies, where high throughput, convenience or discrimination may be issues. Nomenclature and databasing, which have proven so difficult for restriction-enzyme-analysis-, RFLP- and PFGE-based genotyping, are relatively simple and intuitive for VNTR-PCR. An added attraction of VNTR-typing would be the potential to detect and genotype bacteria of the M. tuberculosis complex directly in a range of clinical samples, as has been demonstrated for spoligotyping (Kamerbeek et al., 1997
; Roring et al., 2000
). The technique would be suitable for high-throughput automation using PCR workstations, and DNA sequencing platforms running allele-calling software.
Understanding the molecular basis of pathogen variation is not only important for discriminating and tracing clinically relevant strains, but also provides insights into pathogenesis, host adaptation and the origin of new pathogenic forms (Reid et al., 2001 ). For M. bovis, the integration of VNTR-typing with conventional epidemiological approaches, advanced animal movement recording and geographical information systems has the potential to be a powerful new technology, which should improve our understanding of bovine tuberculosis. The novel loci described here, when used in combination with other VNTR-type loci, should provide a robust and high-resolution tool for the molecular epidemiology of the M. tuberculosis complex.
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ACKNOWLEDGEMENTS |
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Received 13 August 2001;
revised 26 October 2001;
accepted 30 October 2001.