Characterization of the Mycobacterium bovis restriction fragment length polymorphism DNA probe pUCD and performance comparison with standard methods

In this study, the newly described Mycobacterium bovis restriction fragment length polymorphism (RFLP) typing probe pUCD was characterized by sequence analysis and the previously observed polymorphic banding pattern was reproduced with a combination of three oligonucleotide probes in a single, mixed hybridization. In addition, the ability of pUCD to distinguish between 299 M. bovis isolates from the Republic of Ireland was assessed in relation to established methods and a statistical function for objective comparison of RFLP probes was derived. It was found that typing with pUCD alone produced greater discrimination between M. bovis isolates than typing with the commonly used mycobacterial DNA probes IS6110, PGRS, and DR and also by the spoligotyping technique. pUCD and DR in combination produced the highest level of discrimination while maintaining a high level of concordance with known epidemiological data relating to the samples. The reduction of pUCD to the level of oligonucleotides should in future allow pUCD and DR to be included together in a mixed hybridization, thus producing a high level of M. bovis strain type discrimination from a single round of RFLP analysis.     

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.     

Molecular typing of Mycobacterium bovis isolates from Cameroon

In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.     

Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovis strains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.     

Evaluation of the Mycobacterium bovis Restriction Fragment Length Polymorphism Probe pUCD, in Combination with the Direct Repeat Probe, for Molecular Typing of Mycobacterium tuberculosis Strains in Ireland

A mycobacterial restriction fragment length polymorphism probe, pUCD, has recently been described which represents an effective tool for the strain typing of Mycobacterium bovis. The present study evaluated this probe, in combination with the direct repeat probe (DR), for the molecular typing of 90 strains of Mycobacterium tuberculosis from 87 patients, looking at a group (62 isolates) of nonselected samples to assess pUCD combined with DR as a general tool and a subset of 32 isolates with a common specific IS6110 strain type in Ireland. Within the group of 62 isolates, pUCD-DR identified 42 strains and was comparable to both IS6110 (41 strains) and polymorphic guanine-cytosine-rich sequence (PGRS) (37 strains) analysis. pUCD-DR was found to be comparable to IS6110 and PGRS in identifying four separate clusters of isolates which were confirmed to be clinically related. pUCD-DR divided the common IS6110 isolates into six distinct types and was comparable to PGRS (seven strain types). The usefulness of this probe as an epidemiological tool is discussed.     

Usefulness of Spoligotyping in Molecular Epidemiology of Mycobacterium bovis-Related Infections in South America

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.     

Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of Mycobacterium tuberculosis Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility

In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.     

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.     

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.     

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 Four DNA Typing Techniques in Epidemiological Investigations of Bovine Tuberculosis

DNA fingerprinting techniques were used to type 273 isolates of Mycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.     

Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping

Various genetic markers have been exploited for fingerprinting the Mycobacterium tuberculosis complex (MTBC) in molecular epidemiological studies, mainly through identifying restriction fragment length polymorphisms (RFLP). In large-scale studies, RFLP typing has practical processing and analysis limitations; therefore, attempts have been made to move towards PCR-based typing techniques. Spoligotyping (spacer oligotyping) and, more recently, variable-number tandem repeat (VNTR) typing have provided PCR-derived typing techniques. This study describes the identification and characterization of novel VNTR loci, consisting of tandem repeats in the size range of 53 to 59 bp in the MTBC, and their assessment as typing tools in 47 Mycobacterium bovis field isolates and nine MTBC strains. Spoligotyping and the previously described set of exact tandem repeats (ETRs) (R. Frothingham and W. A. Meeker-O'Connell, Microbiology 144:1189-1196, 1998) were also applied to the same panel of isolates. The allelic diversity of the individual VNTR loci was calculated, and a comparison of the novel VNTRs was made against the results obtained by spoligotyping and the existing set of ETRs. Eleven unique spoligotypes were discriminated in the panel of 47 M. bovis isolates. Greater resolution was obtained through the combination of the most-discriminating VNTRs from both sets. Considerable discrimination was achieved, with the 47 M. bovis isolates resolved into 14 unique profiles, while all nine MTBC isolates were uniquely differentiated. The novel VNTR markers described increased the discrimination possible in strain typing of M. bovis, with the added benefit of an intuitive digital nomenclature, with the allele copy number of the individual VNTRs providing a profile. VNTR typing was shown to be a valuable technique with great potential for further development and application to epidemiological tracing of tuberculosis transmissions.     

Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides

The direct repeat (DR) region in Mycobacterium tuberculosis complex strains is composed of multiple well-conserved 36-bp DRs interspersed with nonrepetitive DNA spacer sequences of similar size. Clinical isolates show extensive polymorphism in this DR region, and this has led to the development of a 43-spacer reversed line blot methodology: spoligotyping. Although this method has contributed significantly to the molecular epidemiology of tuberculosis in the last decade, the discriminatory power and the readability of this method were not found to be optimal. In order to improve the discriminatory power, the usefulness of 43 redesigned oligonucleotides and the usefulness of 51 new spacer oligonucleotides were evaluated. For 314 M. tuberculosis complex strains isolated in the central part of The Netherlands over a 5-year period, 264 different IS6110 RFLP types could be distinguished, and 160 different spoligotype patterns were identified by traditional spoligotyping. After the introduction of 51 new spacer oligonucleotides, 14 additional spoligotypes were recognized. This enabled us to split 11 clusters of isolates identified by the traditional spoligotyping. Furthermore, on the basis of the new spacer oligonucleotides a dichotomy was found among the Beijing genotype isolates. Among 76 Mycobacterium bovis strains, 20 patterns were found by traditional spoligotyping and 30 patterns were found by novel probe spoligotyping, respectively. Nine M. bovis subsp. caprae isolates yielded six patterns by traditional spoligotyping and eight patterns by novel probe spoligotyping. A part of the redesigned oligonucleotides slightly improved the reading of spoligotype patterns. The reproducibility of spoligotyping, based on internal control probes, invariably yielded a high score; only 4 (1%) of the 314 patient isolates gave discrepant results. Analysis of a set of 31 duplicate M. tuberculosis complex strains demonstrated a 10% error rate for the identification of blinded duplicate samples. In a redundancy analysis, 40 essential spacer oligonucleotides of the 94-spacer sequences were selected, yielding the same number of spoligotype patterns. We propose to leave the traditional commercialized first-generation membrane for spoligotyping unchanged for current applications and to introduce a second-generation spoligotyping membrane whenever extended discrimination is required, e.g., for low-copy-number IS6110 strains or for phylogenetic studies of Beijing genotype strains.     

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.     

Differentiation of Mycobacterium bovis isolates from animals by DNA typing.

The insertion sequence IS6110 and the direct repeat (DR) specific to tuberculosis complex mycobacteria and the highly repeated DNA sequence, the polymorphic GC-rich repeat sequence (PGRS), were systematically used to identify restriction fragment length polymorphisms (RFLPs) within 210 isolates of Mycobacterium bovis. The isolates were primarily of bovine origin, but isolates from badgers, feral deer, sheep, humans, and a pig were included. The RFLP probes IS6110, DR, and PGRS individually identified 17, 18, and 18 different RFLP types, respectively, but in combination these probes identified a total of 39 different M. bovis RFLP types. The recommendations (J. D. A. van Embden, M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small, J. Clin. Microbiol. 31:406-409, 1993) for a standardized RFLP analysis for M. tuberculosis were adapted to facilitate gel documentation, image analysis, and construction of a database of RFLP types. In the present study the same M. bovis RFLP types were evident in the various animal species included, indicating that the strains were not host restricted. Application of these techniques to defined field studies should help elucidate more accurately aspects of the epidemiology of bovine tuberculosis in different countries.     

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.     

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.     

Mixed tuberculosis infections in rural South Vietnam

Tuberculosis patients may be infected with or have disease caused by more than one Mycobacterium tuberculosis strain, usually referred to as "mixed infections." These have mainly been observed in settings with a very high tuberculosis incidence and/or high HIV prevalence. We assessed the rate of mixed infections in a population-based study in rural Vietnam, where the prevalences of both HIV and tuberculosis are substantially lower than those in previous studies looking at mixed infections. In total, 1,248 M. tuberculosis isolates from the same number of patients were subjected to IS6110 restriction fragment length polymorphism (RFLP) typing, spoligotyping, and variable-number-tandem-repeat (VNTR) typing. We compared mixed infections identified by the presence of (i) discrepant RFLP and spoligotype patterns in isolates from the same patient and (ii) double alleles at ≥ 2 loci by VNTR typing and assessed epidemiological characteristics of these infections. RFLP/spoligotyping and VNTR typing identified 39 (3.1%) and 60 (4.8%) mixed infections, respectively (Cohen's kappa statistic, 0.57). The number of loci with double alleles in the VNTR pattern was strongly associated with the proportion of isolates with mixed infections according to RFLP/spoligotyping (P < 0.001). Mixed infections occurred more frequently in newly treated than in previously treated patients, were significantly associated with minor X-ray abnormalities, and were almost significantly associated with lower sputum smear grades. Although the infection pressure in our study area is lower than that in previously studied populations, mixed M. tuberculosis infections do occur in rural South Vietnam in at least 3.1% of cases.     

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.

Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.     

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.