Use of various genetic markers in differentiation of Mycobacterium bovis strains from animals and humans and for studying epidemiology of bovine tuberculosis.

One hundred fifty-three Mycobacterium bovis strains from cattle, various animal species from zoos and wild parks, and humans were analyzed for three different genetic markers for use in the epidemiology of bovine tuberculosis. M. bovis strains isolated from cattle were found to carry a single IS6110 element, whereas the majority of strains from other animals such as antelopes, monkeys, and seals harbored multiple IS6110 elements, suggesting that the reservoirs in cattle and wild animals are separated. Because the single IS6110 element in cattle strains is located at the same chromosomal position, strain differentiation by insertion sequence fingerprinting was hampered. Therefore, we investigated the usefulness of the direct repeat and polymorphic GC-rich repeat elements for strain differentiation. Both markers allowed sufficient strain discrimination for epidemiological purposes. Evidence is presented that in Argentina, most human M. bovis infections are due to transmission from cattle, whereas M. bovis infections among humans in the Netherlands are mainly contracted from animals other than cattle. Various outbreaks of M. bovis among animals and humans are described, including a small one which likely involved transmission from human to human.     

Differentiation by molecular typing of Mycobacterium bovis strains causing tuberculosis in cattle and goats.

Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis.     

Evaluation of spoligotyping in a study of the transmission of Mycobacterium tuberculosis.

Spoligotyping (for spacer oligotyping) is an easy, economical, and rapid way of typing Mycobacterium tuberculosis complex strains with the DR spacer markers (J. Kamerbeek et al., J. Clin. Microbiol. 35:907-914, 1997; D. van Soolingen et al., 33:3234-3248, 1995). The stability of the markers was demonstrated by showing that all the Mycobacterium bovis BCG strains tested gave the same spoligotyping pattern. None of the 42 atypical mycobacterial strains tested gave a spoligotyping signal, indicating the specificity of the technique for M. tuberculosis complex. The utility of the spoligotyping method was demonstrated by analyzing 106 isolates of M. tuberculosis obtained over 1 year in three Paris hospitals. The results obtained by this technique were compared to those obtained by Torrea et al. (G. Torrea et al., J. Clin. Microbiol. 34:1043-1049, 1996) by IS6110-based restriction fragment length polymorphism (RFLP) analysis. Strains from patients with epidemiological relationships that were in the same IS6110-RFLP cluster were also in the same spoligotyping group. Spoligotyping was more discriminative than RFLP analysis for strains with one or two copies of IS6110. RFLP analysis did not discriminate between the nine strains with one or two IS6110 bands with no known epidemiological relation, whereas spoligotyping distinguished between eight different types. IS6I10-RFLP analysis split some of the spoligotyping clusters, particularly when the IS6110 copy number was high. Therefore, we propose a strategy for typing M. tuberculosis strains in which both markers are used.     

Usefulness of Spoligotyping To Discriminate IS6110 Low-Copy-Number Mycobacterium tuberculosis Complex Strains Cultured in Denmark

Mycobacterium tuberculosis complex strains cultured in Denmark have been analyzed by IS6110 restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS6110 has increased steadily. Since the discriminatory power of IS6110 fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14 Mycobacterium bovis BCG, 48 M. bovis, and 249 M. tuberculosis strains. Spoligotyping correctly differentiated M. bovis and M. bovis BCG from M. tuberculosis strains, but it did not differentiate M. bovis from M. bovis BCG. All M. bovis BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number M. tuberculosis strains was higher than that of IS6110 fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number M. tuberculosis strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of the IS6110 element.     

DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110.

Strains of Mycobacterium bovis, the causative organism of bovine tuberculosis, can be clearly distinguished from each other by restriction fragment analysis. This method of DNA fingerprinting has been used for many epidemiological studies in New Zealand, but the technique presents practical difficulties that hinder its widespread use. The insertion element IS6110 is being widely used as a DNA probe for distinguishing restriction fragment polymorphisms among strains of Mycobacterium tuberculosis. Both this element and another recently sequenced element, IS1081, are also present in M. bovis. We assessed the usefulness of these two elements for distinguishing between 160 strains of M. bovis. These strains, most of which were isolated in New Zealand, were selected to be representative of the 95 different types that were identified among 530 strains that were previously typed by restriction fragment analysis. Fifteen IS6110 types were identified, but more than half of the strains representing 46 restriction types had the same IS6110 type. Virtually all M. bovis strains as well as strains of M. tuberculosis and Mycobacterium africanum had the same IS1081 type. The results indicate that for M. bovis, IS1081 cannot be used to type strains, IS6110 can be used to distinguish strains into broad groups, but only restriction fragment analysis is sufficiently sensitive for detailed epidemiological studies. An investigation of the host range of IS1081 revealed that, apart from its presence in species of the tuberculosis complex, it is also present in a strain of Mycobacterium xenopi.     

Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis.

Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis of M. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in different countries.     

Spacer oligonucleotide typing of Mycobacterium bovis strains from cattle and other animals: a tool for studying epidemiology of tuberculosis.

The spacer oligonucleotide typing (spoligotyping) method was evaluated for its ability to differentiate Mycobacterium bovis strains. This method detects the presence or absence of spacers of the direct repeat locus of the M. bovis genome. The spacers in the direct repeat locus are amplified by PCR and are detected by hybridization of the biotin-labelled PCR product with a membrane containing oligonucleotides derived from spacer sequences that have previously been bound to a membrane. One hundred eighty-two M. bovis isolates from domestic animals (cattle, goat, sheep, and cats) and wild animals (deer and wild boar) were spoligotyped, and the results were compared with those obtained by IS6110 restriction fragment length polymorphism analysis. Two rather homogeneous clusters of isolates containing 20 and 4 types, respectively, were identified by spoligotyping. The first cluster included isolates from cattle, cats, and feral animals. By spoligotyping, isolates from the Spanish wild boar and deer had the same pattern as some bovine isolates, suggesting transmission between these animals and cattle and highlighting the importance of the study of these reservoirs. The second cluster included all the caprine and ovine isolates. Within each cluster, the patterns of the different strains differed only slightly, suggesting that the spoligotypes may be characteristic of strains from particular animal species. Spoligotyping proved to be useful for studying the epidemiology of bovine M. bovis isolates, especially of those isolates containing only a single copy of IS6110. In view of our results, we suggest fingerprinting all M. bovis strains by the spoligotyping method initially and then by IS6110 restriction fragment length polymorphism typing of the strains belonging to the most common spoligotypes.     

Application of four molecular techniques for typing outbreak-associated Mycobacterium tuberculosis strains

We applied four molecular techniques for the typing of strains of Mycobacterium tuberculosis associated with outbreaks: RFLP of the IS6110 insertion sequence, spoligotyping, RAPD, and PCR-IS6110. All 4 techniques were applied to 18 strains which were shown by epidemiological data to be involved in 6 outbreaks. All the methods classified the strains into the same groups as the classical epidemiological data did, but RFLP of the IS6110 insertion sequence and spoligotyping are laborious techniques requiring more than a full day's work, whilst RAPD and PCR IS6110 are simple methods easily incorporated into the daily routine. Nevertheless, a large-scale process of standardization and evaluation is necessary in order to be able to establish the true value of the latter two methods as intraspecific characterization markers for M. tuberculosis isolates.     

Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of Mycobacterium bovis Infection

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.     

Use of variable-number tandem-repeat typing to differentiate Mycobacterium tuberculosis Beijing family isolates from Hong Kong and comparison with IS6110 restriction fragment length polymorphism typing and spoligotyping

The Mycobacterium tuberculosis Beijing family isolates may cause more than a quarter of all tuberculosis cases worldwide, are emerging in some areas, and are often associated with drug resistance. Early recognition of transmission of this genotype is therefore important. To evaluate the usefulness of variable-number tandem-repeat (VNTR) typing to discriminate and recognize strains of the Beijing family, M. tuberculosis isolates from Hong Kong were subjected to VNTR analysis, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. The allelic diversity of the 14 VNTR loci included in the analysis varied from 0 to 0.618 among Beijing strains. The discriminatory power of VNTR analysis was slightly lower than that of IS6110 RFLP. Our analysis shows that VNTR typing, which has many practical advantages over RFLP typing, can be used for epidemiological studies of Beijing strains. However, VNTR-defined clusters should be subtyped with IS6110 RFLP for maximal resolution.     

Identification of a novel DNA probe for strain typing Mycobacterium bovis by restriction fragment length polymorphism analysis

Bovine tuberculosis caused by Mycobacterium bovis remains a significant disease of farmed cattle in many countries despite ongoing tuberculosis eradication programs. Molecular typing methods such as restriction fragment length polymorphism (RFLP) analysis and spoligotyping have been used to identify related herd breakdowns in an attempt to identify more precisely the route of infection into cattle herds and to trace the transmission of bovine tuberculosis. A recent geographical survey of Irish M. bovis isolates demonstrated that a significant proportion of isolates ( approximately 20%) exhibit a common strain type, limiting the value of current strain typing methods as an epidemiological tool. We have identified and cloned a region of the M. bovis genome, pUCD, which generates a clear, highly polymorphic banding pattern when used as an RFLP probe on AluI restriction-digested M. bovis genomic DNA and which effectively subdivides this common strain type. When used to type 60 Irish M. bovis isolates, pUCD exhibited greater discriminatory power than the commonly used mycobacterial RFLP probes IS6110, PGRS, and DR and detected an equivalent number of strain types to a combination of these three probes. pUCD also detected significantly more strain types than the spoligotyping technique, while maintaining a high level of concordance between epidemiologically related and unrelated herd breakdowns. The polymorphic element within pUCD remains to be fully characterized, however the potential for this probe to greatly decrease the workload necessary to genotype M. bovis by RFLP analysis is compelling.     

Genetic characterization of multidrug-resistant Mycobacterium bovis strains from a hospital outbreak involving human immunodeficiency virus-positive patients.

Nineteen multidrug-resistant (MDR) Mycobacterium complex strains isolated in a nosocomial outbreak were characterized at the molecular level. The strains were microbiologically characterized as Mycobacterium bovis. The mpt40 sequence was not present in chromosomal DNA from these strains, supporting the fact that they were M. bovis. All of the isolates were resistant to isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, para-aminosalicylic acid, clarithromycin, cycloserine, ethionamide, ofloxacin, capreomycin, and amikacin. By performing the standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP) analysis, we were able to differentiate two groups (groups A and B) containing two (16 isolates) and three (3 isolates) IS6110 copies, respectively. These strains were typed by spoligotyping, developed to distinguish M. bovis strains and also to distinguish them from M. tuberculosis strains (J. Kamerbeek et al., J. Clin. Microbiol. 35:907-914, 1997). All the strains were confirmed to be M. bovis. In addition, spoligotyping showed a difference in only 1 of 43 spacers between RFLP groups A and B. The rpo beta region of several strains representative of each identified group was cloned and sequenced, and identical mutations (Ser-531 to Leu) responsible for the rifampin resistance phenotype were found. To our knowledge, this is the first characterization at the molecular level of an MDR M. bovis strain responsible for a nosocomial outbreak.     

Mycobacterium africanum subtype II is associated with two distinct genotypes and is a major cause of human tuberculosis in Kampala,Uganda

The population structure of 234 Mycobacterium tuberculosis complex strains obtained during 1995 and 1997 from tuberculosis patients living in Kampala, Uganda (East Africa), was analyzed by routine laboratory procedures, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. According to biochemical test results, 157 isolates (67%) were classified as M. africanum subtype II (resistant to thiophen-2-carboxylic acid hydrazide), 76 isolates (32%) were classified as M. tuberculosis, and 1 isolate was classified as classical M. bovis. Spoligotyping did not lead to clear differentiation of M. tuberculosis and M. africanum, but all M. africanum subtype II isolates lacked spacers 33 to 36, differentiating them from M. africanum subtype I. Moreover, spoligotyping was not sufficient for differentiation of isolates on the strain level, since 193 (82%) were grouped into clusters. In contrast, in the IS6110-based dendrogram, M. africanum strains were clustered into two closely related strain families (Uganda I and II) and clearly separated from the M. tuberculosis isolates. A further characteristic of both M. africanum subtype II families was the absence of spoligotype spacer 40. All strains of family I also lacked spacer 43. The clustering rate obtained by the combination of spoligotyping and RFLP IS6110 analysis was similar for M. africanum and M. tuberculosis, as 46% and 49% of the respective isolates were grouped into clusters. The results presented demonstrate that M. africanum subtype II isolates from Kampala, Uganda, belong to two closely related genotypes, which may represent unique phylogenetic branches within the M. tuberculosis complex. We conclude that M. africanum subtype II is the main cause of human tuberculosis in Kampala, Uganda.     

Epidemiologic usefulness of spoligotyping for secondary typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110

Restriction fragment length polymorphism (RFLP) analysis of IS6110 is commonly used to DNA fingerprint Mycobacterium tuberculosis. However, low-copy (< or =5) IS6110 M. tuberculosis strains are poorly differentiated, requiring secondary typing. When spoligotyping was used as the secondary method, only 13% of Maryland culture-positive tuberculosis (TB) patients with low-copy IS6110-spoligotyped clustered strains had epidemiologic linkages to another patient, compared to 48% of those with high-copy strains clustered by IS6110 alone (P < 0.01). Spoligotyping did not improve a population-based molecular epidemiologic study of recent TB transmission.     

Assessment of an optimized mycobacterial interspersed repetitive- unit-variable-number tandem-repeat typing system combined with spoligotyping for population-based molecular epidemiology studies of tuberculosis

An optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for the typing of Mycobacterium tuberculosis. Here, we evaluated the performances of this MIRU-VNTR typing system in combination with spoligotyping for the detection of transmission chains in a population-based study comprising 91% of culture-confirmed tuberculosis patients reported in 2003 in Hamburg, Germany. Of the 154 isolates investigated, more than 90% had high IS6110 copy numbers (>/=6). IS6110 restriction fragment length polymorphism (RFLP) typing resulted in 13 clusters, 5 of which had a confirmed epidemiological link. All five, as well as six of the eight IS6110 clusters with no identified epidemiological link, were perfectly matched by MIRU-VNTR typing with the 24 loci. Two IS6110 clusters were split by differences into 6 to 12 MIRU-VNTR loci, clearly supporting the absence of a link, as judged by contact tracing data. In contrast, only one MIRU-VNTR cluster, grouping what were probably epidemiologically unlinked isolates, was split by IS6110 RFLP. However, these isolates were also distinguished by spoligotyping. Both the optimized 24-locus and 15-locus sets thus showed a comparable to slightly better predictive value, especially when combined with spoligotyping, than the current gold standard IS6110 RFLP for the study of tuberculosis transmission in Hamburg. Because the epidemiological characteristics of this setting are similar to those of many developed countries, these results support the wide applicability of this real-time genotyping approach for population-based studies of M. tuberculosis transmission.     

Comparative Evaluation of Ligation-Mediated PCR and Spoligotyping as Screening Methods for Genotyping of Mycobacterium tuberculosis Strains

Spoligotyping has been suggested as a screening test in multistep genotyping of Mycobacterium tuberculosis strains. Relying on restriction fragment length polymorphism (RFLP) analysis with IS6110 (IS6110 RFLP analysis) as a "gold standard," we performed a comparative evaluation of spoligotyping and ligation-mediated PCR (LMPCR), a recently described PCR-based typing method, as rapid screening tests for fingerprinting of 158 M. tuberculosis strains collected in Verona, Italy. LMPCR seemed to be comparable to spoligotyping in terms both of feasibility with rapidly extracted DNA and of generation of software-analyzable images. Moreover, LMPCR grouped considerably fewer strains than spoligotyping (38 versus 67%) and was found to reduce the cluster overestimation rate (26.3 versus 58%) and to give a better discriminatory index (0.992 versus 0.970) compared to spoligotyping. In our geographical region, where there was no evidence of clustered strains carrying fewer than six IS6110 copies, LMPCR was found to be more discriminatory than spoligotyping. We also evaluated two models of three-step typing strategies, involving the use of spoligotyping and LMPCR as screening methods and IS6110 RFLP analysis as a further supporting test. LMPCR proved to be a more effective first-step test than spoligotyping, significantly reducing the need for subtyping. LMPCR should be considered an alternative to spoligotyping as a rapid screening method for M. tuberculosis fingerprinting, particularly in areas with a low prevalence of M. tuberculosis strains carrying few copies of IS6110.     

Analysis of the regions responsible for IS6110 RFLP in a single Mycobacterium tuberculosis strain.

A high degree of IS6110 restriction fragment length polymorphism (RFLP) is observed amongst the different strains of the Mycobacterium tuberculosis complex. The sequences of the IS6110 flanking regions from a M. tuberculosis strain harbouring four IS6110 copies were determined. Duplication of 3-4 nucleotides was found at the extremities of the four IS6110 copies, suggesting that IS6110 RFLP is due to transposition of the IS element. One of the copies of IS6110 analysed in the study was shown to be located at the same site in the genome of M. tuberculosis as the single copy present in an M. bovis BCG strain.     

Molecular fingerprinting of Mycobacterium bovis subsp. caprae isolates from central Europe

To study the dissemination of Mycobacterium bovis subsp. caprae, 79 European isolates from cattle, humans, and other hosts were examined by spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) analysis. Among a total of 11 different spoligotypes identified, type C1 proved to be predominant (n = 62). Five of the spoligotypes are described for the first time. A total of 43 different RFLP types were identified, thus allowing further differentiation for epidemiological tracking. Isolates from a series of outbreaks in one village proved to be of the same spoligotype and of identical or closely related RFLP types.     

Evaluation of the epidemiological relevance of variable-number tandem-repeat genotyping of Mycobacterium bovis and comparison of the method with IS6110 restriction fragment length polymorphism analysis and spoligotyping

Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, [corrected] taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen.     

Evolution and clonal traits of Mycobacterium tuberculosis complex in Guinea-Bissau

Two hundred twenty-nine consecutive isolates of Mycobacterium tuberculosis complex from patients with pulmonary tuberculosis in Guinea-Bissau, which is located in West Africa, were analyzed for clonal origin by biochemical typing and DNA fingerprinting. By using four biochemical tests (resistance to thiophene-2-carboxylic acid hydrazide, niacin production, nitrate reductase test, and pyrazinamidase test), the isolates could be assigned to five different biovars. The characteristics of four strains conformed fully with the biochemical criteria for M. bovis, while those of 85 isolates agreed with the biochemical criteria for M. tuberculosis. The remaining 140 isolates could be allocated into one of three biovars (biovars 2 to 4) representing a spectrum between the classical bovine (biovar 1) and human (biovar 5) tubercle bacilli. By using two genotyping methods, restriction fragment length polymorphism analysis with IS6110 (IS6110 RFLP analysis) and spoligotyping, the isolates could be separated into three groups (groups A to C) of the M. tuberculosis complex. Group A (n = 95), which contained the majority of classical human M. tuberculosis isolates, had large numbers of copies of IS6110 elements (mean number of copies, 9) and a distinctive spoligotyping pattern that lacked spacers 33 to 36. Isolates of the major group, group B (n = 119), had fewer IS6110 copies (mean copy number, 5) and a spoligotyping pattern that lacked spacers 7 to 9 and 39 and mainly comprised isolates of biovars 1 to 4. Group C isolates (n = 15) had one to three IS6110 copies, had a spoligotyping pattern that lacked spacers 29 to 34, and represented biovar 3 to 5 isolates. Four isolates whose biochemical characteristics conformed with those of M. bovis clustered with the group B isolates and had spoligotype patterns that differed from those previously reported for M. bovis, in that they possessed spacers 40 to 43. Interestingly, isolates of group B and, to a certain extent, also isolates of group C showed a high degree of variability in biochemical traits, despite genotypic identity in terms of IS6110 RFLP and spoligotype patterns. We hypothesize that isolates of groups B and C have their evolutionary origin in West Africa, while group A isolates are of European descent.