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.     

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.     

Molecular epidemiological studies of Mycobacterium bovis infections in humans and animals in Sweden.

Forty-nine isolates of Mycobacterium bovis from humans and animals in Sweden were analyzed by restriction fragment length polymorphism (RFLP) patterns probed by the insertion element IS6110. Most isolates had patterns indicating the presence of only one or two genomic copies of the IS6110 insertion element. This simple type of pattern was found in all human isolates. In contrast, isolates from M. bovis infections in five herds of farmed deer in Sweden showed a specific RFLP pattern with seven bands, indicating seven copies of the IS6110 sequence. In 1958, Sweden was declared free from M. bovis in cattle. However, in 1987, M. bovis was reintroduced with imported farmed deer, and since 1991, 11 outbreaks in deer herds, but not in other livestock or wildlife, have been diagnosed. Continued RFLP studies of the new Swedish M. bovis isolates can reveal possible transmission of this deer strain to other animals or humans.     

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.     

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.     

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.     

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.     

Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex.

It is important to correctly identify species within the Mycobacterium tuberculosis complex because of the zoonotic implications of bovine tuberculosis, especially in developing countries. We assessed the use of various genetic markers for species-specific identification of mycobacteria from the M. tuberculosis complex. A multiplex PCR designed for detection of the mtp40 and IS1081 elements was optimized and evaluated in 339 mycobacterial strains from different animal and geographic origins. The host range of the IS6110, MPB70, and 16S rRNA genes was also studied by PCR in all the strains. Finally, the usefulness of the genetic markers was compared by an immunoperoxidase test for specific identification of Mycobacterium bovis strains. The mtp40 sequence was detected in 87 of the 91 strains of M. tuberculosis and in 9 of the 11 Mycobacterium africanum strains but not in any of the M. bovis or Mycobacterium microti strains, indicating that the mtp40 element was also found in all of the M. tuberculosis complex strains isolated from seals. This organism is considered to be a true seal pathogen, but its origin is essentially unknown. The finding of the mtp40 element in the strains from seals suggests a closer relationship of these strains with a human origin than to an animal origin. The mtp40 element was not found in any other mycobacterial species included in the study. As a result of this study, we suggest that biochemical tests or alternate genetic markers are still needed to differentiate M. tuberculosis from M. africanum when these species coexist as causative agents of tuberculosis. The immunoperoxidase test worked well for the identification of M. bovis strains. We also report, for the first time, PCR amplification of the repetitive element IS6110 in an isolate of Mycobacterium ulcerans and an isolate of Mycobacterium gilvum, which emphasizes the need for further investigation of the host range of this sequence.     

Molecular markers demonstrate that the first described multidrug-resistant Mycobacterium bovis outbreak was due to Mycobacterium tuberculosis

We genetically characterized multidrug-resistant Mycobacterium tuberculosis complex strains which caused a nosocomial outbreak of tuberculosis affecting six human immunodeficiency virus (HIV)-positive patients and one HIV-negative staff member (E. Bouvet, E. Casalino, G. Mendoza-Sassi, S. Lariven, E. Vallée, M. Pernet, S. Gottot, and F. Vachon, AIDS 7:1453-1460, 1993). The strains showed all the phenotypic characteristics of Mycobacterium bovis. They presented a high copy number of IS6110, the spacers 40 to 43 in the direct repeat locus, and the mtp40 fragment. They lacked the G-A mutation at position 285 in the oxyR gene and the C-G mutation at position 169 in the pncA gene. These genetic characteristics revealed that these were dysgonic, slow-growing M. tuberculosis strains mimicking the M. bovis phenotype, probably as a consequence of cellular alterations associated with the multidrug resistance. Spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) analysis confirmed that the outbreak was due to a single strain. However, the IS6110 RFLP pattern of the strain isolated from the last patient, diagnosed three years after the index case, differed slightly from the patterns of the other six strains. A model of a possible genetic event is presented to explain this divergence. This study stresses the value of using several independent molecular markers to identify multidrug-resistant tubercle bacilli.     

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.     

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample.     

The mtp40 gene is not present in all strains of Mycobacterium tuberculosis.

A multiple PCR-based assay that targets IS6110 and the mtp40 gene was evaluated for the rapid differentiation of Mycobacterium bovis and M. tuberculosis, two of the causative agents of tuberculosis. The IS6110 target is present in both species, whereas the mtp40 gene was thought to be specific for M.tuberculosis (P.Del Portillo, L.A. Murillo, and M.E. Patarroyo, J. Clin. Microbiol. 29:2163-2168, 1991). However, the mtp-40 gene is not present in all M. tuberculosis strains and, hence, is not useful for differentiating M.tuberculosis and M.bovis.     

Identification of one insertion site of IS6110 in Mycobacterium tuberculosis H37Ra and analysis of the RvD2 deletion in M. tuberculosis clinical isolates

Mycobacterium tuberculosis H37Rv and the attenuated strain H37Ra were used as a model to investigate the virulence properties of M. tuberculosis at the genetic level. To test whether transposition of the insertion element IS6110 might be involved in the loss of virulence of strain H37Ra, the nucleotide sequence of a differential IS6110-positive restriction fragment detected in strain H37Ra, but not in strain H37Rv, was determined. The region flanking the 3' end of the IS6110 element showed partial sequence homology with internal sequences of M. tuberculosis H37Rv genes plcA, plcB and plcC, each one coding for phospholipase C, a well-known bacterial virulence factor. A 100% homology was found between the IS6110-flanking region and an internal sequence of M. bovis plcD, a further phospholipase C gene that is truncated and partly lost in strain H37Rv in the so-called RvD2 deletion. This result indicates that the differential restriction fragment of strain H37Ra originally stems from the plcD gene interrupted by the insertion of the IS6110 element. The occurrence of the RvD2 deletion was then investigated in 45 clinical isolates of M. tuberculosis by Southern blot. The deletion was demonstrated in 15 isolates; the entire RvD2 region (including the undisrupted plcD gene) was detected in 29 isolates, whereas only one isolate showed the RvD2 region in which the plcD gene was interrupted by an IS6110 insertion. It is concluded that disruption of the plcD gene and deletion of the RvD2 region by IS6110 insertion have no consequence for the virulence of M. tuberculosis, although the role of phospholipase C as a virulence factor of M. tuberculosis remains debatable.     

A combination of two genetic markers is sufficient for restriction fragment length polymorphism typing of Mycobacterium tuberculosis complex in areas with a high incidence of tuberculosis

The incidence of tuberculosis (TB) in Madagascar is 150 cases per 100,000 people. Because of this endemicity, we studied the genetic diversity of Mycobacterium tuberculosis strains isolated in four big cities in 1994 to 1995 with the aim of monitoring TB transmission. Isolates from 316 cases of pulmonary TB (PTM(+)) were typed by Southern hybridization with genetic markers IS6110 and DR. Of the 316 PTM(+) strains, 66 (20.8%) had a single IS6110 band and were differentiated by the DR marker into 33 profiles. Using both markers, 37.7% (119) of the patients were clustered, a proportion similar to that in countries with a high prevalence of TB. There was no significant difference between clustered and nonclustered patients in age, sex, Mycobacterium bovis BCG status, and drug susceptibility of strains. Clustering was significantly greater in the capital, Antananarivo, than in the other cities, suggesting a higher rate of transmission. However, most of the patients in clusters were living in different areas, and, within a distance of 0.7 km, we did not find epidemiologically unrelated strains with the same restriction fragment length polymorphism profile. Despite an apparently low polymorphism, genetic markers such as IS6110 are potentially valuable for monitoring TB transmission. However, the high proportion of Malagasy isolates with a single IS6110 copy makes this marker alone unsuitable for typing. Additional markers such as DR are necessary for the differentiation of the isolates and for epidemiological surveys.     

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 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.     

Molecular characteristics of strains of the cameroon family, the major group of Mycobacterium tuberculosis in a country with a high prevalence of tuberculosis

A preliminary investigation of the genetic biodiversity of Mycobacterium tuberculosis complex strains in Cameroon, a country with a high prevalence of tuberculosis, described a group of closely related M. tuberculosis strains (the Cameroon family) currently responsible for more than 40% of smear-positive pulmonary tuberculosis cases. Here, we used various molecular methods to study the genetic characteristics of this family of strains. Cameroon family M. tuberculosis strains (i) are part of the major genetic group 2 and lack the TbD1 region like other families of epidemic strains, (ii) lack spacers 23, 24, and 25 in their direct repeat (DR) region, (iii) have an identical number of repeats in 8 of 12 variable-number tandem repeats of mycobacterial interspersed repetitive unit (MIRU-VNTR) loci, (iv) have similar IS6110-restriction fragment length polymorphism (RFLP) multiband patterns (10 to 15 copies) with seven common IS6110 bands, (v) do not have an IS6110 element in their DR locus, and (vi) have four IS6110 elements in open reading frames (adenylate cyclase, phospholipase C, moeY, and ATP binding genes). Analysis by spoligotyping, MIRU-VNTR, and IS6110-RFLP typing methods revealed differences not observed in previous studies; polymorphism as assessed by MIRU-VNTR typing was lower than suggested by spoligotyping, and in rare cases, strains with identical IS6110-RFLP patterns had spoligotypes differing by as much as 15 spacers. Our findings confirm the recent expansion of this family in Cameroon and indicate that the interpretation of molecular typing results has to be adapted to the characteristics of the strain population within each setting. The knowledge of this particular genotype, with its large involvement in tuberculosis in Cameroon, allows greater refinement of tuberculosis transmission studies by interpreting data in the context of this geographic area.     

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.