Genomic Analysis of Mycobacterium tuberculosis Complex Strains Used for Production of Purified Protein Derivative
TB Research Group, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB,1 Bacterial Microarray Group, Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, London SW17 0RE, United Kingdom2r\$t(, 百拇医药
Received 14 February 2003/ Returned for modification 12 May 2003/ Accepted 16 May 2003r\$t(, 百拇医药
ABSTRACTr\$t(, 百拇医药
The genomes of the tuberculin production strains Mycobacterium bovis AN5 and Mycobacterium tuberculosis DT were compared to genome-sequenced tubercle bacilli by using DNA microarrays. Neither the AN5 nor DT strain suffered extensive gene deletions during in vitro passage. This suggests that bovine tuberculin made from M. bovis AN5 is suitable to detect infection with presently prevalent M. bovis strains.r\$t(, 百拇医药
TEXTr\$t(, 百拇医药
Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease responsible for annual losses to global agriculture of $3 billion and with serious repercussions for public health and animal welfare. The program for the control of bovine tuberculosis in Great Britain (GB) involves regular testing of cattle with a crude preparation of mycobacterial antigens termed PPD (purified protein derivative [also called tuberculin]), followed by compulsory slaughter of positive reactors. However, in the last decade the number of herd breakdowns has been increasing across GB, especially in the Southwest, where prevalence has now risen to 3.5% of bovine herds (http://www.defra.gov.uk). This has worrying implications for the control strategy, which presently costs £25 million per year.
At the Veterinary Laboratories Agency Weybridge (VLA), the Mycobacterium tuberculosis strains DT, C, and PN were used for the production of bovine PPD prior to 1975 (11). These strains are the same as those employed for human tuberculin production, and they were used because the yield of M. tuberculosis bacilli from the glycerol-containing media employed was greater than that of M. bovis strains. However, since 1975, PPD production has switched to M. bovis AN5, a strain that was originally isolated in England circa 1948 (10) and is used worldwide for bovine PPD production. Its acceptance as a standard for tuberculin production was principally based on its high yield of cell mass on glycerinated media, a phenotype that was selected by repeated subculture of the bacillus on laboratory media (10). This selection for a desirable phenotype by passage through artificial media has parallels with the method used by Calmette and Guérin to attenuate a strain of M. bovis to generate M. bovis BCG (3). By using genomic technologies, it has been shown that during this in vitro culture the genome of BCG suffered a number of gene deletions and chromosomal rearrangements (1, 2). Hence, it is possible that the genome of M. bovis AN5 underwent similar events during in vitro passage that could have removed genes encoding potent antigens, as was the case with BCG.
Despite its widespread use as a diagnostic reagent, M. bovis AN5 is poorly defined at the genetic level. Hence, we sought to characterize its genome by using DNA microarrays and molecular typing technology. Furthermore, we analyzed the M. tuberculosis DT strain (ATCC 35810), originally used by Seibert and Glenn (14) to generate the international standard tuberculin (PPD-S), to determine whether there was any evidence of gene deletions from this strain.hl:@, 百拇医药
At VLA the present methods of choice for the molecular typing of M. bovis isolates are spacer-oligonucleotide typing (spoligotyping) and variable-number-of-tandem-repeat (VNTR) typing (4, 8, 17). Spoligotyping is based on a polymorphic region of the genome called the direct repeat (DR) locus that is composed of multiple 36-bp DR copies interspersed by unique sequences called spacers. Isolates of M. bovis differ in the presence or absence of spacers and adjacent DRs, allowing a barcode to be generated for each molecular type. Spoligotyping of M. bovis AN5 and M. tuberculosis DT was performed as described previously (8), with the resulting patterns compared to the VLA spoligotype database, which presently holds typing information on 20,000 M. bovis strains isolated from 1975 to 2003 (with 95% of data for strains isolated since 1997). From the results obtained with M. bovis AN5 (Fig. 1), it was clear that its profile was not shared by any strains in the database. This raised the possibility that the AN5 strain may not be optimal for the detection of infection by M. bovis strains presently prevalent in GB.
fig.ommitted)c!4c6, http://www.100md.com
FIG. 1. Spoligotype patterns of M. bovis AN5, M. tuberculosis DT, and the most prevalent GB M. bovis molecular types. Spacer numbers are shown at the top of the figure, with the spoligotype pattern depicted as a block to reflect the presence of a spacer. An empty lane signifies the absence of a spacer. The strain numbers in the far left column follow the VLA numbering convention, with the types shown in decreasing order of prevalence.)c!4c6, http://www.100md.com
The VNTR method uses six targets (A to F) originally described by Frothingham and Meeker-O'Connell (4). These loci vary in the length of internal repeat units, giving alleles that vary in size. Strain profiles can then be given on the basis of the number of repeats at each allele, i.e., 7-5-5-4-3-3 would have seven copies of allele A, five of B, etc. VNTR reactions were performed with fluorescently labeled primers directed against each allele as previously described (16). As with spoligotyping, the VNTR pattern for M. bovis AN5 showed no similarity to profiles from ~
3,000 typed M. bovis isolates from 1997 to date (Table 1).57juo, 百拇医药
fig.ommitted57juo, 百拇医药
TABLE 1. Ten most common VNTR profiles of GB M. bovis isolates based on the system of Frothingham and Meeker-O'Connellc57juo, 百拇医药
Spoligotyping records variation only at a single locus, while VNTR typing targeted six loci. However, DNA microarray technology allows the analysis of whole genomes. With this technology, we could therefore screen the genomes of tuberculin production strains for deletions by using an M. tuberculosis H37Rv microarray and exploiting the >99.9% sequence identity between the bacilli. The microarray that we used contained ~57juo, 百拇医药
4,000 genes, and its construction has been described elsewhere (15). The genome-sequenced M. bovis strain 2122/97,a 1997 GB isolate, was used in the hybridization experiments as a control to ensure that deletions determined by in silico comparisons and described by others could be identified by using the array (6, 12, 13). DNA labeling and slide hybridizations were performed as previously described (15). The microarrays were scanned by using an Affymetrix 428 scanner (Affymetrix, Santa Clara, Calif.), and then the resulting images were analyzed with ImaGene 4.1 (BioDiscovery, Marina Del Rey, Calif.) and GeneSpring (Silicon Genetics, Redwood City, Calif.) software tools. The validity of the array data was further checked by PCR amplification of the flanking arms of each deletion.
It should be noted that, as with all spotted microarrays, only part of the genome is actually represented on the array. The technique is therefore limited to detection of deletions that overlap the arrayed PCR products. Hence, analysis of the M. bovis 2122/97 control hybridizations revealed all the in silico predicted deletions except for N-RD17 (Fig. 2). This internal deletion of 713 bp from Rv3479 was not detected, as the arrayed PCR product was generated from a part of Rv3479 that is present in M. bovis; hence, the whole gene was scored as present. The deletion of this region was, however, confirmed by PCR (results not shown). The hybridization data for M. bovis AN5 disclosed a set of deletions almost identical to those of M. bovis 2122/97 (Fig. 1). The only difference was that AN5 had lost the RD3 region, a prophage locus that is deleted in approximately 84% of clinical isolates (9). Hence, it is probable that this locus was deleted prior to in vitro culture. The M. tuberculosis DT strain also showed no major differences with the H37Rv strain. The two RD loci missing from M. tuberculosis DT were the RD3 prophage and RD6, which corresponds to the insertion sequence element IS1532 (5). The RD6 locus has also been shown to be deleted from clinical strains of M. tuberculosis (7); hence, as with AN5, it is probable that RD3 and RD6 were deleted prior to the in vitro culturing of M. tuberculosis DT. Our analysis therefore shows that both M. bovis AN5 and M. tuberculosis DT strains have not suffered extensive gene deletion events.
fig.ommitted'#u$et, 百拇医药
FIG. 2. Graphical representation of regions of difference (RD) across the strains tested. Concentric circles denote the strain genomes, with the following abbreviations: H37Rv, M. tuberculosis H37Rv; DT, M. tuberculosis DT; 2122/97, M. bovis 2122/97; and AN5, M. bovis AN5. Black zones correspond to RD loci. The naming of RD loci is shown on the outer circle and follows that of Gordon et al. (6), except for N-RD17 and RD* (our notation), which are described by Salamon et al. (13) and Rauzier et al. (12), respectively.'#u$et, 百拇医药
Our analysis shows that, at least at the gross genomic level, there is no reason to suspect that M. bovis AN5 should be suboptimal for the detection of infection by M. bovis strains that are presently prevalent. However, there are some limitations to this microarray-based approach. The array used would not have detected single-nucleotide polymorphisms between the strains that may affect gene expression. Genome rearrangements such as translocations or inversions could also not be detected with this method. However, physical mapping studies have shown that the genomes of the M. tuberculosis complex are remarkably stable, with duplication events having been reported so far only in the genomes of BCG substrains (2). Furthermore, we would clearly not have detected alterations to cell wall components, such as surface-exposed sugars or lipids. However, our initial proteome analysis of M. bovis AN5 has shown that there are no major differences in protein profile between this strain and M. bovis 2122/97 (unpublished observations).
This study presents the first description at the genetic level of strains used for the production of PPD, the only reagent at present for the diagnosis of infection by M. tuberculosis complex strains in animals or humans. Our analyses have shown that the PPD strains do not possess any dramatic differences in their genomes compared to other strains of the same species. While the possibility exists that small deletions or single-nucleotide polymorphisms may have been missed by our approach, our findings underline the high degree of genetic identity shared by members across the M. tuberculosis complex.?(dvm3, 百拇医药
ACKNOWLEDGMENTS?(dvm3, 百拇医药
We thank Val Barnard of the Tuberculin Production Unit, VLA Weybridge, for the gift of the M. bovis AN5 strain and helpful advice.?(dvm3, 百拇医药
This work was funded by the Department for Environment, Food and Rural Affairs (Defra, London, United Kingdom) and by The Wellcome Trust, which funded the multicollaborative microbial pathogen microarray group at St. George's under its Functional Genomics Development Initiative.
REFERENCES&+, http://www.100md.com
Behr, M. A., M. A. Wilson, W. P. Gill, H. Salamon, G. K. Schoolnik, S. Rane, and P. M. Small. 1999. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284:1520-1523.&+, http://www.100md.com
Brosch, R., S. V. Gordon, C. Buchrieser, A. S. Pym, T. Garnier, and S. T. Cole. 2000. Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur. Yeast 17:111-123.&+, http://www.100md.com
Calmette, A., and C. Guérin. 1909. Sur quelques propriétés du bacille tuberculeux d'origine, cultivé sur la bile de boeuf glycérinée. C. R. Acad. Sci. 149:716-718.&+, http://www.100md.com
Frothingham, R., and W. A. Meeker-O'Connell. 1998. Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 144:1189-1196.&+, http://www.100md.com
Gordon, S. V., R. Brosch, A. Billault, T. Garnier, K. Eiglmeier, and S. T. Cole. 1999. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol. Microbiol. 32:643-655.
Gordon, S. V., K. Eiglmeier, T. Garnier, R. Brosch, J. Parkhill, B. Barrell, S. T. Cole, and R. G. Hewinson. 2001. Genomics of Mycobacterium bovis. Tuberculosis (Edinb.) 81:157-163.'%p{m, http://www.100md.com
Gordon, S. V., B. Heym, J. Parkhill, B. Barrell, and S. T. Cole. 1999. New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv. Microbiology 145:881-892.'%p{m, http://www.100md.com
Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden. 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35:907-914.'%p{m, http://www.100md.com
Mahairas, G. G., P. J. Sabo, M. J. Hickey, D. C. Singh, and C. K. Stover. 1996. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178:1274-1282.'%p{m, http://www.100md.com
Paterson, A. B. 1948. The production of bovine tuberculoprotein. J. Comp. Pathol. 58:302-313.
Pritchard, D. G. 1988. A century of bovine tuberculosis 1888-1988: conquest and controversy. J. Comp. Pathol. 99:357-399.:z%9, 百拇医药
Rauzier, J., E. Gormley, M. C. Gutierrez, E. Kassa-Kelembho, L. J. Sandall, C. Dupont, B. Gicquel, and A. Murray. 1999. A novel polymorphic genetic locus in members of the Mycobacterium tuberculosis complex. Microbiology 145:1695-1701.:z%9, 百拇医药
Salamon, H., M. Kato-Maeda, P. M. Small, J. Drenkow, and T. R. Gingeras. 2000. Detection of deleted genomic DNA using a semiautomated computational analysis of GeneChip data. Genome Res. 10:2044-2054.:z%9, 百拇医药
Seibert, F. B., and J. T. Glenn. 1941. Tuberculin purified protein derivative: preparation and analyses of a large quantity for standard. Am. Rev. Tuberc. 44:9-25.:z%9, 百拇医药
Stewart, G. R., L. Wernisch, R. Stabler, J. A. Mangan, J. Hinds, K. G. Laing, D. B. Young, and P. D. Butcher. 2002. Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148:3129-3138.:z%9, 百拇医药
Supply, P., S. Lesjean, E. Savine, K. Kremer, D. van Soolingen, and C. Locht. 2001. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39:3563-3571.:z%9, 百拇医药
Supply, P., E. Mazars, S. Lesjean, V. Vincent, B. Gicquel, and C. Locht. 2000. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol. Microbiol. 36:762-771.(Jacqueline Inwald Jason Hinds Si Palmer James Dale Philip D. Butcher R. Glyn Hewinson and Stephen V.)
Received 14 February 2003/ Returned for modification 12 May 2003/ Accepted 16 May 2003r\$t(, 百拇医药
ABSTRACTr\$t(, 百拇医药
The genomes of the tuberculin production strains Mycobacterium bovis AN5 and Mycobacterium tuberculosis DT were compared to genome-sequenced tubercle bacilli by using DNA microarrays. Neither the AN5 nor DT strain suffered extensive gene deletions during in vitro passage. This suggests that bovine tuberculin made from M. bovis AN5 is suitable to detect infection with presently prevalent M. bovis strains.r\$t(, 百拇医药
TEXTr\$t(, 百拇医药
Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease responsible for annual losses to global agriculture of $3 billion and with serious repercussions for public health and animal welfare. The program for the control of bovine tuberculosis in Great Britain (GB) involves regular testing of cattle with a crude preparation of mycobacterial antigens termed PPD (purified protein derivative [also called tuberculin]), followed by compulsory slaughter of positive reactors. However, in the last decade the number of herd breakdowns has been increasing across GB, especially in the Southwest, where prevalence has now risen to 3.5% of bovine herds (http://www.defra.gov.uk). This has worrying implications for the control strategy, which presently costs £25 million per year.
At the Veterinary Laboratories Agency Weybridge (VLA), the Mycobacterium tuberculosis strains DT, C, and PN were used for the production of bovine PPD prior to 1975 (11). These strains are the same as those employed for human tuberculin production, and they were used because the yield of M. tuberculosis bacilli from the glycerol-containing media employed was greater than that of M. bovis strains. However, since 1975, PPD production has switched to M. bovis AN5, a strain that was originally isolated in England circa 1948 (10) and is used worldwide for bovine PPD production. Its acceptance as a standard for tuberculin production was principally based on its high yield of cell mass on glycerinated media, a phenotype that was selected by repeated subculture of the bacillus on laboratory media (10). This selection for a desirable phenotype by passage through artificial media has parallels with the method used by Calmette and Guérin to attenuate a strain of M. bovis to generate M. bovis BCG (3). By using genomic technologies, it has been shown that during this in vitro culture the genome of BCG suffered a number of gene deletions and chromosomal rearrangements (1, 2). Hence, it is possible that the genome of M. bovis AN5 underwent similar events during in vitro passage that could have removed genes encoding potent antigens, as was the case with BCG.
Despite its widespread use as a diagnostic reagent, M. bovis AN5 is poorly defined at the genetic level. Hence, we sought to characterize its genome by using DNA microarrays and molecular typing technology. Furthermore, we analyzed the M. tuberculosis DT strain (ATCC 35810), originally used by Seibert and Glenn (14) to generate the international standard tuberculin (PPD-S), to determine whether there was any evidence of gene deletions from this strain.hl:@, 百拇医药
At VLA the present methods of choice for the molecular typing of M. bovis isolates are spacer-oligonucleotide typing (spoligotyping) and variable-number-of-tandem-repeat (VNTR) typing (4, 8, 17). Spoligotyping is based on a polymorphic region of the genome called the direct repeat (DR) locus that is composed of multiple 36-bp DR copies interspersed by unique sequences called spacers. Isolates of M. bovis differ in the presence or absence of spacers and adjacent DRs, allowing a barcode to be generated for each molecular type. Spoligotyping of M. bovis AN5 and M. tuberculosis DT was performed as described previously (8), with the resulting patterns compared to the VLA spoligotype database, which presently holds typing information on 20,000 M. bovis strains isolated from 1975 to 2003 (with 95% of data for strains isolated since 1997). From the results obtained with M. bovis AN5 (Fig. 1), it was clear that its profile was not shared by any strains in the database. This raised the possibility that the AN5 strain may not be optimal for the detection of infection by M. bovis strains presently prevalent in GB.
fig.ommitted)c!4c6, http://www.100md.com
FIG. 1. Spoligotype patterns of M. bovis AN5, M. tuberculosis DT, and the most prevalent GB M. bovis molecular types. Spacer numbers are shown at the top of the figure, with the spoligotype pattern depicted as a block to reflect the presence of a spacer. An empty lane signifies the absence of a spacer. The strain numbers in the far left column follow the VLA numbering convention, with the types shown in decreasing order of prevalence.)c!4c6, http://www.100md.com
The VNTR method uses six targets (A to F) originally described by Frothingham and Meeker-O'Connell (4). These loci vary in the length of internal repeat units, giving alleles that vary in size. Strain profiles can then be given on the basis of the number of repeats at each allele, i.e., 7-5-5-4-3-3 would have seven copies of allele A, five of B, etc. VNTR reactions were performed with fluorescently labeled primers directed against each allele as previously described (16). As with spoligotyping, the VNTR pattern for M. bovis AN5 showed no similarity to profiles from ~
3,000 typed M. bovis isolates from 1997 to date (Table 1).57juo, 百拇医药
fig.ommitted57juo, 百拇医药
TABLE 1. Ten most common VNTR profiles of GB M. bovis isolates based on the system of Frothingham and Meeker-O'Connellc57juo, 百拇医药
Spoligotyping records variation only at a single locus, while VNTR typing targeted six loci. However, DNA microarray technology allows the analysis of whole genomes. With this technology, we could therefore screen the genomes of tuberculin production strains for deletions by using an M. tuberculosis H37Rv microarray and exploiting the >99.9% sequence identity between the bacilli. The microarray that we used contained ~57juo, 百拇医药
4,000 genes, and its construction has been described elsewhere (15). The genome-sequenced M. bovis strain 2122/97,a 1997 GB isolate, was used in the hybridization experiments as a control to ensure that deletions determined by in silico comparisons and described by others could be identified by using the array (6, 12, 13). DNA labeling and slide hybridizations were performed as previously described (15). The microarrays were scanned by using an Affymetrix 428 scanner (Affymetrix, Santa Clara, Calif.), and then the resulting images were analyzed with ImaGene 4.1 (BioDiscovery, Marina Del Rey, Calif.) and GeneSpring (Silicon Genetics, Redwood City, Calif.) software tools. The validity of the array data was further checked by PCR amplification of the flanking arms of each deletion.
It should be noted that, as with all spotted microarrays, only part of the genome is actually represented on the array. The technique is therefore limited to detection of deletions that overlap the arrayed PCR products. Hence, analysis of the M. bovis 2122/97 control hybridizations revealed all the in silico predicted deletions except for N-RD17 (Fig. 2). This internal deletion of 713 bp from Rv3479 was not detected, as the arrayed PCR product was generated from a part of Rv3479 that is present in M. bovis; hence, the whole gene was scored as present. The deletion of this region was, however, confirmed by PCR (results not shown). The hybridization data for M. bovis AN5 disclosed a set of deletions almost identical to those of M. bovis 2122/97 (Fig. 1). The only difference was that AN5 had lost the RD3 region, a prophage locus that is deleted in approximately 84% of clinical isolates (9). Hence, it is probable that this locus was deleted prior to in vitro culture. The M. tuberculosis DT strain also showed no major differences with the H37Rv strain. The two RD loci missing from M. tuberculosis DT were the RD3 prophage and RD6, which corresponds to the insertion sequence element IS1532 (5). The RD6 locus has also been shown to be deleted from clinical strains of M. tuberculosis (7); hence, as with AN5, it is probable that RD3 and RD6 were deleted prior to the in vitro culturing of M. tuberculosis DT. Our analysis therefore shows that both M. bovis AN5 and M. tuberculosis DT strains have not suffered extensive gene deletion events.
fig.ommitted'#u$et, 百拇医药
FIG. 2. Graphical representation of regions of difference (RD) across the strains tested. Concentric circles denote the strain genomes, with the following abbreviations: H37Rv, M. tuberculosis H37Rv; DT, M. tuberculosis DT; 2122/97, M. bovis 2122/97; and AN5, M. bovis AN5. Black zones correspond to RD loci. The naming of RD loci is shown on the outer circle and follows that of Gordon et al. (6), except for N-RD17 and RD* (our notation), which are described by Salamon et al. (13) and Rauzier et al. (12), respectively.'#u$et, 百拇医药
Our analysis shows that, at least at the gross genomic level, there is no reason to suspect that M. bovis AN5 should be suboptimal for the detection of infection by M. bovis strains that are presently prevalent. However, there are some limitations to this microarray-based approach. The array used would not have detected single-nucleotide polymorphisms between the strains that may affect gene expression. Genome rearrangements such as translocations or inversions could also not be detected with this method. However, physical mapping studies have shown that the genomes of the M. tuberculosis complex are remarkably stable, with duplication events having been reported so far only in the genomes of BCG substrains (2). Furthermore, we would clearly not have detected alterations to cell wall components, such as surface-exposed sugars or lipids. However, our initial proteome analysis of M. bovis AN5 has shown that there are no major differences in protein profile between this strain and M. bovis 2122/97 (unpublished observations).
This study presents the first description at the genetic level of strains used for the production of PPD, the only reagent at present for the diagnosis of infection by M. tuberculosis complex strains in animals or humans. Our analyses have shown that the PPD strains do not possess any dramatic differences in their genomes compared to other strains of the same species. While the possibility exists that small deletions or single-nucleotide polymorphisms may have been missed by our approach, our findings underline the high degree of genetic identity shared by members across the M. tuberculosis complex.?(dvm3, 百拇医药
ACKNOWLEDGMENTS?(dvm3, 百拇医药
We thank Val Barnard of the Tuberculin Production Unit, VLA Weybridge, for the gift of the M. bovis AN5 strain and helpful advice.?(dvm3, 百拇医药
This work was funded by the Department for Environment, Food and Rural Affairs (Defra, London, United Kingdom) and by The Wellcome Trust, which funded the multicollaborative microbial pathogen microarray group at St. George's under its Functional Genomics Development Initiative.
REFERENCES&+, http://www.100md.com
Behr, M. A., M. A. Wilson, W. P. Gill, H. Salamon, G. K. Schoolnik, S. Rane, and P. M. Small. 1999. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284:1520-1523.&+, http://www.100md.com
Brosch, R., S. V. Gordon, C. Buchrieser, A. S. Pym, T. Garnier, and S. T. Cole. 2000. Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur. Yeast 17:111-123.&+, http://www.100md.com
Calmette, A., and C. Guérin. 1909. Sur quelques propriétés du bacille tuberculeux d'origine, cultivé sur la bile de boeuf glycérinée. C. R. Acad. Sci. 149:716-718.&+, http://www.100md.com
Frothingham, R., and W. A. Meeker-O'Connell. 1998. Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 144:1189-1196.&+, http://www.100md.com
Gordon, S. V., R. Brosch, A. Billault, T. Garnier, K. Eiglmeier, and S. T. Cole. 1999. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol. Microbiol. 32:643-655.
Gordon, S. V., K. Eiglmeier, T. Garnier, R. Brosch, J. Parkhill, B. Barrell, S. T. Cole, and R. G. Hewinson. 2001. Genomics of Mycobacterium bovis. Tuberculosis (Edinb.) 81:157-163.'%p{m, http://www.100md.com
Gordon, S. V., B. Heym, J. Parkhill, B. Barrell, and S. T. Cole. 1999. New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv. Microbiology 145:881-892.'%p{m, http://www.100md.com
Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden. 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35:907-914.'%p{m, http://www.100md.com
Mahairas, G. G., P. J. Sabo, M. J. Hickey, D. C. Singh, and C. K. Stover. 1996. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178:1274-1282.'%p{m, http://www.100md.com
Paterson, A. B. 1948. The production of bovine tuberculoprotein. J. Comp. Pathol. 58:302-313.
Pritchard, D. G. 1988. A century of bovine tuberculosis 1888-1988: conquest and controversy. J. Comp. Pathol. 99:357-399.:z%9, 百拇医药
Rauzier, J., E. Gormley, M. C. Gutierrez, E. Kassa-Kelembho, L. J. Sandall, C. Dupont, B. Gicquel, and A. Murray. 1999. A novel polymorphic genetic locus in members of the Mycobacterium tuberculosis complex. Microbiology 145:1695-1701.:z%9, 百拇医药
Salamon, H., M. Kato-Maeda, P. M. Small, J. Drenkow, and T. R. Gingeras. 2000. Detection of deleted genomic DNA using a semiautomated computational analysis of GeneChip data. Genome Res. 10:2044-2054.:z%9, 百拇医药
Seibert, F. B., and J. T. Glenn. 1941. Tuberculin purified protein derivative: preparation and analyses of a large quantity for standard. Am. Rev. Tuberc. 44:9-25.:z%9, 百拇医药
Stewart, G. R., L. Wernisch, R. Stabler, J. A. Mangan, J. Hinds, K. G. Laing, D. B. Young, and P. D. Butcher. 2002. Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148:3129-3138.:z%9, 百拇医药
Supply, P., S. Lesjean, E. Savine, K. Kremer, D. van Soolingen, and C. Locht. 2001. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39:3563-3571.:z%9, 百拇医药
Supply, P., E. Mazars, S. Lesjean, V. Vincent, B. Gicquel, and C. Locht. 2000. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol. Microbiol. 36:762-771.(Jacqueline Inwald Jason Hinds Si Palmer James Dale Philip D. Butcher R. Glyn Hewinson and Stephen V.)