Differentiation of Mycobacterium bovis Isolates from Animals by DNA Typing

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1996, p Vol. 34, No /96/$ Copyright 1996, American Society for Microbiology Differentiation of Mycobacterium bovis Isolates from Animals by DNA Typing R. A. SKUCE,* D. BRITTAIN, M. S. HUGHES, AND S. D. NEILL Veterinary Sciences Division, Department of Agriculture for Northern Ireland, Belfast BT4 3SD, United Kingdom Received 25 March 1996/Returned for modification 13 May 1996/Accepted 10 July 1996 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: , 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. Tuberculosis (TB) caused by the bacterium Mycobacterium bovis is an infectious disease affecting a large number of animal species (16). Infections in cattle and other farmed animals in developed countries can cause considerable economic loss to agricultural communities (15). In developing countries bovine TB also potentially represents a significant zoonosis, the extent of which is currently unknown (27). Concerted attempts to eradicate the disease from cattle in some countries have not been successful. In New Zealand a wildlife reservoir of M. bovis infection in the possum has clearly hampered tuberculosis eradication (4). In Ireland and the United Kingdom the significance of a reservoir of M. bovis infection identified in the badger and to some extent deer remains unclear (7, 9). Interbovine transmission is also considered to be of primary importance in maintaining disease in the cattle population, for example, in Northern Ireland (15). A more precise understanding of the distribution and transmission of disease between and within cattle and wildlife populations is therefore required to effect a better disease control strategy. The integration of novel molecular techniques for the accurate identification specific strains of M. bovis by conventional epidemiological tracing approaches could provide much useful information. M. bovis belongs to the homogeneous M. tuberculosis complex group of mycobacteria, which also includes M. tuberculosis, M. africanum, and M. microti (26). It has been suggested that M. bovis should be considered a biovar of M. tuberculosis rather than a distinct species (8). Restriction enzyme analysis (REA) of mycobacterial genomic DNA for studying the epidemiology of bovine TB has not gained widespread acceptance because of the technical difficulty in comparing complex fragment patterns. However, a large number of apparently stable M. bovis types can be differentiated by this technique (3). Recognition of various repetitive DNA elements within TB complex mycobacteria has led to the recent exploitation of * Corresponding author. Phone: Fax: Electronic mail address: RSKUCE@QUB.AC.UK. DNA restriction fragment length polymorphism (RFLP) analysis for mycobacteria, and there is an increasing consensus that RFLP patterns are a reliable indicator of strain identity. By RFLP analysis only the polymorphic restriction fragments are visualized, thus simplifying their analysis, documentation, and comparison. Five different genetic elements have been shown to contribute to DNA polymorphism within the TB complex mycobacteria (17). Two of these elements are the TB complex-specific insertion sequences IS6110 and IS1081. RFLP analysis with IS6110 to probe restriction enzyme-digested DNA from M. tuberculosis isolates has now become accepted in the molecular epidemiology of human tuberculosis (22). IS6110 is often present in multiple copies at various sites in the genome, and because of the ability of IS6110 to transpose, the DNA restriction fragments carrying IS6110 are polymorphic (13, 24). This has not been the case for M. bovis, isolates of which, particularly from cattle, harbor only one or a few copies of IS6110, making IS6110 of relatively limited use in the strain typing of M. bovis (3, 6, 11, 20, 23). Strains carrying one copy of IS6110 do so at one chromosomal location, a hot spot within the cluster of direct repeats (DRs) (12). The assertion of clonality is weaker for these isolates with low IS6110 copy numbers and particularly so for isolates with only a single copy of IS6110. For example, Collins and colleagues (3) used IS6110 to analyze RFLPs in 160 M. bovis isolates representing 95 types by REA. These were differentiated into 15 IS6110 types, and all but five isolates contained one copy of IS6110 (3). In contrast to the majority of M. bovis isolates from cattle, M. bovis isolates from other animals such as antelopes, monkeys, and seals were shown to contain multiple copies of IS6110, and their fingerprints were highly polymorphic, suggesting that the M. bovis reservoirs in cattle and these wild animals were separate and distinct (23). The insertion sequence IS1081 was identified in species of the TB complex, but it reveals very few RFLPs and does not distinguish between species of the TB complex (5). Therefore, IS1081 is not considered a suitable genetic marker for the epidemiology of bovine tuberculosis, although it can clearly distinguish M. bovis BCG from other M. bovis strains 2469

2 2470 SKUCE ET AL. J. CLIN. MICROBIOL. (25). The remaining three short repetitive genetic elements which contribute to DNA polymorphism in mycobacterial DNA include the polymorphic GC-rich repeat sequence (PGRS) (19), the DR (12), and the major polymorphic tandem repeat (MPTR) (14). PGRS consists of short, repeated polymorphic sequences of 24 bp separated by spacer sequences of variable lengths scattered in at least 26 clusters throughout the genome (18). Although PGRS is not specific to the TB complex, when it is used to probe AluI-digested M. bovis DNA a very acceptable level of strain differentiation can be achieved (6, 20, 23). The DR region, unique to TB complex isolates, was identified in M. bovis BCG DNA flanking the single IS6110 element and comprises directly repeated sequences of 36 bp separated by unique spacer sequences varying from 36 to 41 bp in length. DNA polymorphism in TB complex isolates relates to the number of DRs and associated spacers in the DR region and is believed to be a function of DNA recombination. The DR cluster has been the target for the development of novel fingerprinting techniques based on DNA amplification and digital typing (10). The MPTR sequence has not been extensively analyzed in TB complex isolates (14). Previously, a systematic comparison of the abilities of IS6110, IS1081, and PGRS to identify epidemiologically significant RFLP types among M. bovis isolates was reported (20). A refinement to the analytical procedure by replacing IS1081 with DR in a systematic RFLP analysis is presented here. DNAs from M. bovis isolates from cattle, badgers, deer, pigs, sheep, and humans were probed with PGRS, DR, and IS6110. We confirm that DR-associated RFLP is superior to IS1081 for typing M. bovis strains. The RFLP patterns assigned to each isolate for probes PGRS, DR, and IS6110 can be used in combination to increase the discriminatory power of the original technique. MATERIALS AND METHODS Mycobacterial isolates. M. bovis isolates (n 210) from different geographic and temporal origins (n 130) and from various animal species were included in the study. The majority originated from Northern Irish cattle (n 177) and were cultured from lymph gland or lung tissues. Single Northern Irish isolates from a feral deer and a badger were also included. M. bovis isolates from the Republic of Ireland (n 31) and originating from cattle (n 13), badgers (n 9), feral deer (n 3), sheep (n 2), pigs (n 1), and humans (n 3) were obtained from the Veterinary Research Laboratories, Abbotstown, Dublin, Ireland. Of the latter group of isolates, three cattle isolates and 3 badger isolates originated from the same farm premises. The origin of each bovine isolate and the history and movement of individual cattle in Northern Ireland were recorded on a Unix-based computer workstation. Primary isolation in BACTEC 7H12 medium, subculturing onto Lowenstein- Jensen (LJ) medium, and heat inactivation were performed as described previously (20). Presumptive identification was based on Ziehl-Neelsen staining, colony morphology, growth on LJ medium, and cording in BACTEC 7H12 medium. DNA techniques. M. bovis genomic DNA was extracted as described previously (24). DNA was digested with PvuII for the IS6110 probe and AluI for the PGRS and DR probes. Electrophoresis was performed as specified previously (20). DNA fragments were transferred onto nylon membranes (Hybond N ; Amersham International plc) exactly as recommended by the manufacturer. Molecular weight markers were incorporated into each sample as recommended for IS6110 fingerprinting of M. tuberculosis, except that AluI digests of M. bovis DNA were heat inactivated prior to loading. The first and last gel lanes were marked with a lane marker, as recommended previously (22). The IS6110 probe used in the present study was the entire 1,358-bp IS6110 sequence generated by PCR as described previously (20) and confirmed by restriction analysis. The PGRS probe was the 3.8-kb EcoRI-HindIII restriction enzyme product of plasmid ptbni2 (19). The DR probe was the 36-mer oligonucleotide described previously (12). DNA fragment probes for RFLP analysis were labelled with the Enhanced Chemiluminescence Gene Detection System (Amersham International plc) by using ECL Direct for IS6110 and PGRS and ECL 3 oligonucleotide labelling for the DR probe exactly as specified by the manufacturer. Hybridization conditions were as described previously (23). Fluorograms were analyzed by computer with the BioImage Whole Band Analyzer (Millipore). Molecular weights were assigned to mycobacterial DNA fragments by superimposing the image of the internal molecular weight markers as described previously (20). RFLP patterns were assigned for each probe. For FIG. 1. RFLP lane maps of all patterns obtained from 210 among M. bovis isolates digested with AluI and probed with PGRS (A) and DR (B) and digested with PvuII and probed with IS6110 (C). The images were generated by using BioImage Whole Band Analysis, version 3.0, running on a Sun IPX workstation. the PGRS probe only DNA fragments larger than 2 kb were considered since it proved difficult to accurately resolve and compare fragments of lower molecular sizes. Adobe Photoshop, version 3.0 was used to produce the images in Fig. 2; however, no such manipulation was used during image analysis. RESULTS In an attempt to improve the discriminatory potential of the RFLP analysis and to further subdivide the majority of M. bovis isolates carrying one IS6110 element, Southern blots of AluIdigested M. bovis DNA were probed sequentially with labelled PGRS and DR probes. The PGRS probe identified a total of 18 different RFLP types (Fig. 1A); PGRS type A accounted for 130 of 210 isolates. PGRS was able to resolve the IS6110 RFLP type A (n 135) isolates into eight different groups. The

3 VOL. 34, 1996 DNA TYPING OF M. BOVIS 2471 TABLE 1. RFLP types assigned to M. bovis isolates by combination of individual probe patterns, numbers of isolates of each type, sources of origin, and identified host species No. of isolates (no. of sources) a RFLP pattern with the following probe: PGRS DR IS6110 Identified host(s) (no. of isolates) 1 (1) A A C Cattle 89 (49) A C A Cattle (83), badger (2), deer (2), sheep (1), human (1) 2 (2) A C C Cattle 4 (3) A C G Cattle 1 (1) A C N Cattle 1 (1) A C Q Cattle 1 (1) A C R Cattle 25 (6) A D A Cattle (24), sheep (1) 1 (1) A J B Badger 1 (1) A N P Cattle 2 (2) A O B Cattle, human 1 (1) A R A Cattle 15 (15) B A C Cattle (12), badger (1), deer (1), pig (1) 9 (7) B A F Cattle 1 (1) B A H Cattle 2 (2) B Q F Cattle 10 (2) C A C Cattle 12 (11) D C A Cattle 1 (1) D G A Cattle 1 (1) D L C Cattle 2 (1) E I J Cattle, badger 1 (1) F B E Cattle 1 (1) G C A Cattle 1 (1) H E C Cattle 3 (2) H E N Cattle 1 (1) H F L Cattle 2 (2) J O B Cattle 1 (1) L C A Cattle 4 (1) L C M Cattle (3), badger (1) 1 (1) M G A Badger 2 (1) N H D Badger 2 (2) N K E Badger, human 1 (1) R P E Cattle 1 (1) S C A Cattle 1 (1) S M K Deer 3 (3) T F O Cattle 1 (1) U C A Cattle 1 (1) V C A Cattle 1 (1) W D A Cattle a A total of 210 isolates were obtained from 130 sources. PGRS fingerprint of M. bovis DNA consists of a large number of fragment lines, but most of the DNA polymorphism between isolates was associated with DNA fragments larger than 2 kb (see Fig. 2A). A natural gap occurs in the PGRS fingerprint at about 2.2 kb. The DR probe generated RFLP patterns containing fragment lines ranging in number from three to seven and up to 2 kb in size. The DR probe identified a total of 18 different DR RFLP types (Fig. 1B). The most common RFLP pattern was pattern C, accounting for 119 of 210 isolates. IS6110 RFLP type A (n 135) was further subdivided into four groups of isolates by DR. The three probes, PGRS, DR, and IS6110, identified different sets of isolates, so the RFLP types for each probe can be combined to give an overall RFLP type represented by a three-letter code. For example, IS6110 RFLP type A(n 135) was further subdivided into 12 different types by using this combination of three probes. Individual strains of M. bovis were subsequently identified by this three-letter code with individual RFLP types written in the order PGRS, DR, and IS6110 (Table 1). By using the entire IS6110 sequence to probe blots of PvuIIdigested M. bovis DNA, a total of 17 different IS6110 RFLP types were identified in the 210 M. bovis isolates (Fig. 1C). The majority (n 194) of isolates contained only one copy of IS6110. Eleven isolates contained two copies and five isolates contained three copies, as determined by the number of IS6110-hybridizing fragments in the RFLP profile (Fig. 1C). No more than three copies of IS6110 have been demonstrated in isolates from Ireland to date. Among the 194 isolates with one copy of IS6110 were 10 different types, clearly differentiated on the basis of slight but reproducible differences in the molecular weights of the hybridizing fragments, 3 of which are shown in Fig. 2C. Isolates from cattle. The 190 cattle isolates were subdivided into 34 different strains identified by using the combined RFLP types. The majority of M. bovis isolates from cattle in Northern Ireland and the small number of isolates analyzed from the Republic of Ireland contained one IS6110 copy. Most of these (n 83) were of RFLP type ACA (Table 1). These isolates were from 49 different sources and included 30 isolates from one large disease outbreak affecting 30 animals in one herd. The remaining RFLP type ACA strains were from other

4 2472 SKUCE ET AL. J. CLIN. MICROBIOL. FIG. 2. Restriction enzyme digests of 20 M. bovis field isolates digested with AluI and probed with ECL (Amersham International plc)-labelled PGRS (A) and DR (B) and digested with PvuII and probed with ECL-labelled IS6110 (C). The images were scanned with a Kodak megaplus camera into a Sun IPX workstation running Whole Band Analysis, version 3.0 (BioImage). The images were then cropped and labelled with Adobe Photoshop, version 3.0 on a Viglen DX2. sources geographically distributed throughout both Northern Ireland and the Republic of Ireland. Isolates from Northern Ireland were recovered over a number of years (1989 to 1994). Type ACA appears to be widely distributed throughout the country. Some RFLP types appear to be more geographically clustered than type ACA. For example, type BAF was isolated from seven separate bovine sources in an area of South Down and South Armagh, Northern Ireland. Six of these strains were isolated in 1993 and one was isolated in Similarly, 12 isolates of type DCA originating from 11 different sources were shown to cluster in an area around Ballymoney, County FIG. 3. Restriction enzyme digests of five M. bovis isolates digested with PvuII and probed with ECL (Amersham International plc)-labelled IS6110. Lanes 1 and 2, isolates from badgers; lanes 3, 4, and 5, isolates from cattle. All five isolates were from a single farm in Kinsale, Republic of Ireland. The images were scanned with a Kodak megaplus camera into a Sun IPX workstation running BioImage Whole Band Analysis, version 3.0. The image was then cropped and labelled with Adobe Photoshop, version 3.0, on a Viglen DX2. Antrim, Northern Ireland, in 1992 and M. bovis RFLP type CAC was isolated from 10 tuberculin skin test-reactive cattle on one farm (farm A) and also in 1 skin test-reactive animal on another farm (farm B) 48 km from farm A. It is now known from tracing the movement of these animals on the computerized database for animal movement in Northern Ireland that the single reactive animal on farm B had spent a short time on farm A and that this was a likely source of infection for that animal. M. bovis isolates (n 13) from cattle in the Republic of Ireland were resolved into 10 different types by this procedure. These 13 isolates originated from 10 different sources. Two isolates, from independent sources, were of the ubiquitous ACA type. Only 4 of these 10 RFLP types have been seen in Northern Ireland M. bovis isolates from cattle to date. Isolates from other animals. Eight different RFLP types were seen among the nine M. bovis isolates from badgers, most of which contained one IS6110 copy. Three RFLP types recognized in these badger isolates have not been found in cattle isolates analyzed in the study or the previously reported study (20). Interestingly, M. bovis isolates from three cattle in the same herd on a farm in the Republic of Ireland had RFLP types (type LCM) identical to those of isolates from two badgers trapped on this farm. RFLP type LCM is unusual in that it possesses three copies of IS6110 (Fig. 3, lanes 1, 3, 4, and 5). Another isolate from a third badger on this farm had a totally different type, type MGA (Fig. 3, lane 2). This distinction was clear when all three probes were used separately and in combination. M. bovis RFLP type NHD was isolated from two badgers on another farm. Unfortunately, no bovine isolates were available from this farm. One bovine isolate identified as M. bovis RFLP type EIJ was the same RFLP type as the M. bovis isolate recovered from a badger trapped 6 km away. M. bovis was isolated from four feral deer, one Sika stag in Northern Ireland and three deer from the Republic of Ireland. M. bovis RFLP types ACA, ADA, and BAC were identified. These have commonly been isolated from cattle in Ireland to date. M. bovis RFLP type ACA was also isolated from a sheep in Northern Ireland in This animal was known to be grazing adjacent to a farm which suffered a large outbreak of disease involving 30 cattle, all of which yielded M. bovis RFLP type ACA. In the absence of other disease in the area, it is quite possible that the sheep became infected from those cattle. Two further M. bovis isolates were obtained from sheep in the Republic of Ireland. Again, the RFLP types of both of

5 VOL. 34, 1996 DNA TYPING OF M. BOVIS 2473 these isolates, types ACA and ADA, are commonly seen in cattle. M. bovis RFLP type BAC was isolated from a farmed pig in the Republic of Ireland, and again, isolates of type BAC are commonly isolated from cattle. Three isolates of M. bovis were obtained from humans originating in the Republic of Ireland. One was RFLP type ACA, very commonly seen in cattle, and one was type AOB, which in the present study was only isolated from cattle. The third RFLP type was NKE, the same RFLP type as that of one of the badger isolates. DISCUSSION The TB complex-specific insertion sequence IS6110 together with the repetitive sequences PGRS and DR were systematically used to identify RFLPs within a relatively large number of field isolates of M. bovis from Northern Ireland and the Republic of Ireland. The isolates were primarily from cattle, but they also came from badgers, deer, sheep, humans, and a pig. As several other researchers have recognized (11, 20, 23), the majority of M. bovis isolates from cattle contained one copy of IS6110, integrated presumably into the DR cluster hot spot for IS6110. Multiple copies of IS6110 are present in strains of other species of the TB complex and, indeed, in M. bovis isolates from zoo and wild-park animals (23). The reason why most M. bovis isolates from cattle in Ireland, New Zealand, Australia, and several other countries possess only one IS6110 copy, however, remains unclear. The IS6110 element retains the ability to transpose to other areas of the genome without showing much sequence preference. IS6110 RFLP patterns have been shown to be relatively stable for M. tuberculosis isolates in culture, in small animal models, and in humans with clinical cases of infection (21). The IS6110 RFLP is considered a reliable indicator of strain identity and is sufficiently stable to be used in epidemiological studies. The time scale required for IS6110 transposition is sufficiently long to make these epidemiological studies meaningful. We have shown previously that if the entire IS6110 element is used to probe Southern blots of PvuII-digested M. bovis DNA, a considerable amount of polymorphism can be detected, significantly more than if the 3 PvuII fragment alone is used (20). Gel comparisons and the compilation of a database of RFLP types were greatly facilitated by adoption of the RFLP analysis protocol advocated (22) for the typing of M. tuberculosis isolates with IS6110. Several IS6110-hybridizing fragments were of a similar size, but careful superimposition of in-lane and origin molecular weight markers allows these different fragments to be distinguished. The number of polymorphisms detected by the PGRS probe (n 18) was similar to the number detected by IS6110 when PGRS was used to probe Southern blots of AluI-digested M. bovis DNA, but it did not group the same isolates. The PGRS fingerprint is relatively complex. The element is present in at least 26 different loci in M. tuberculosis and comprises multiple tandem repeats of the consensus sequence CGGCAGGAA. Polymorphism associated with PGRS is believed to have arisen by means of triplet expansion (18). PGRS used alone to probe AluI-digested M. bovis DNA has been advocated as a sufficient tool with which to study the epidemiology of bovine tuberculosis (6). Since the element is not entirely TB complex specific, the identities of the isolates would need to be confirmed in advance. However, by using multiple RFLP probes in combination, including IS6110, we have demonstrated that it is possible to significantly improve upon the discrimination offered by PGRS alone. Similarly for the DR probe, different groups of strains were identified within this set of isolates. Although polymorphism within the DR cluster, caused by homologous recombination between DR elements and resulting in different numbers of DRs, must contribute to RFLP when using IS6110 as a probe, the connection between DR and IS6110 types is not immediately apparent. Both PGRS and DR were able to resolve further IS6110 RFLP type A (Table 1). This is likely to be of considerable importance when tracing the point source of infection caused by M. bovis isolates with similar IS6110 fingerprints. The DR fingerprint can readily be generated from the AluI-digested DNA blotted for analysis by PGRS, provided that the electrophoresis run times are sufficiently short to retain the DR-hybridizing fragments on the gel, the smallest of which is approximately 250 bp. By using PGRS, DR, and IS6110, it proved possible to identify a total of 39 different M. bovis RFLP types among the 210 isolates analyzed (Table 1). DR is a superior RFLP probe compared with IS1081. For example, in a set of random M. bovis isolates (n 69) common to both the present study and a previous study (20), IS1081 identified two types, whereas DR identified seven types. In combination with IS6110, IS1081 identified seven types, whereas DR identified 12 types. Overall, PGRS, IS1081, and IS6110 identified 14 types, whereas PGRS, DR, and IS6110 identified 15 types (data not shown). It appears that some RFLP types, notably, types ACA and ADA, are predominant within the cattle population. This may be a somewhat artificial perception since outbreaks with a large number of affected cattle were included and the frequency of laboratory submissions may actually reflect multiple isolations from a few geographic areas currently considered to be hot spots for disease in cattle. Type ACA was also widespread in its distribution and has consistently been isolated from cattle over a number of years. Several isolations of some RFLP types were found to be clustered in relatively small geographic areas. In the present study, using isolates from a country with an ongoing disease problem, we have shown that the M. bovis RFLP types in cattle and other farmed animals, wild and feral animal species, and humans are shared. This is in contrast to the situation in which M. bovis strains from cattle were compared with those isolated from other animals such as antelopes, monkeys, and seals from zoos and wild parks. Strains from cattle were clearly different from strains isolated from these exotic animals, principally on the basis of IS6110 fingerprinting (23), suggesting that these reservoirs were separated. Similarly, RFLP analysis with IS6110 demonstrated that different M. bovis strains were implicated in bovine and caprine TB (11). TB is endemic in the badger population in Ireland and the United Kingdom (1). The badger is considered to be the most important wildlife reservoir for M. bovis infection and has been cited as a significant obstacle to the effective control and eradication of tuberculosis from cattle. In the small number of M. bovis isolates from badgers typed in the present study, the majority were of RFLP types commonly seen in cattle. In one set of isolates from the Republic of Ireland, three cattle and two of three badgers trapped on the same farm premises were shown to be infected with the same strain. However, one of the three badgers was infected with an entirely different strain, a strain apparently not responsible for the outbreak of disease in cattle. RFLP patterns were sufficiently different to speculate that strain of types LCM and MGA were not even closely related. Although two of the badgers harbored the same strain which caused the disease outbreak in cattle, without more detailed epidemiological investigations it is impossible to speculate on the route, extent, and more importantly, actual direc-

6 2474 SKUCE ET AL. J. CLIN. MICROBIOL. tion of transmission in this case. This result is in broad agreement with REA typing data for 20 isolates of M. bovis isolated from cattle (n 12) and badgers (n 8) in nine separate herd breakdowns in four counties of the Republic of Ireland (2). Six of the eight badger isolates were of the same REA type as at least one of the cattle in the nine herd breakdowns. Further defined epidemiological studies are required to investigate the potential transmission of disease from wildlife to cattle, but it appears that the M. bovis strains circulating in cattle and badgers are largely the same and that the reservoir of M. bovis strains in the wild badger population is not distinct from that in cattle. Epidemiological data have identified several risk factors in the spread of TB within the cattle population in Ireland. The majority of disease outbreaks for which sources are identified are thought to be due to cattle-to-cattle transmission. Cattle trading practices, lateral spread from a neighboring farm, and/or poor farming practices which facilitate the spread of disease are considered risk factors (15). The spread of infection by these routes must be addressed if eradication is to be achieved. Molecular techniques have been described here. These techniques can and should be integrated into standard control and conventional epidemiological approaches to assist in detecting and tracing point source outbreaks and as a tool in disease control and surveillance. More extensive sampling will be required to confirm the true frequency of the strains identified in the present study in the human, farmed, and wild animal populations. ACKNOWLEDGMENTS We acknowledge and thank the mycobacteriology laboratory staff for primary isolation, maintenance, and subculture of M. bovis isolates. Gratitude is expressed to L. O Reilly for contributing M. bovis isolates from the Republic of Ireland and to Bruce Ross for providing plasmid ptbn12. REFERENCES 1. Cheeseman, C. L., J. W. Wilesmith, and F. A. Stuart Tuberculosis: the disease and it s epidemiology in the badger, a review. Epidemiol. Infect. 103: Collins, D. M., G. W. de Lisle, J. D. Collins, and E. Costello DNA restriction fragment typing of Mycobacterium bovis isolates from cattle and badgers in Ireland. Vet. Rec. 134: Collins, D. M., S. K. Erasmuson, D. M. Stephens, A. F. Yates, and G. W. de Lisle DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110. J. Clin. Microbiol. 31: Collins, D. M., D. M. Gabric, and G. W. de Lisle Typing of Mycobacterium bovis isolates from cattle and other animals in the same locality. N. Z. Vet. J. 36: Collins, D. M., and D. M. Stephens Identification of an insertion sequence IS1081 in Mycobacterium bovis. FEMS Microbiol. Lett. 83: Cousins, D. V., S. N. Williams, B. C. Ross, and T. M. Ellis Use of a repetitive element isolated from Mycobacterium tuberculosis in hybridisation studies with Mycobacterium bovis: a new tool for epidemiological studies on bovine tuberculosis. Vet. Microbiol. 37: Dunnet, G. M., D. M. Jones, and J. P. McInerney Badgers and bovine tuberculosis review of policy. MAFF report. Her Majesty s Stationary Office, London. 8. Grange, J. M Koch s tubercle bacillus a centenary reappraisal. Zentrabl. Bakteriol. Parsitenk. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 252: Grange, J. M., J. D. Collins, L. M. O Reilly, E. Costello, and M. D. Yates Identification and characterisation of Mycobacterium bovis isolated from cattle, badgers and deer in the Republic of Ireland. Ir. Vet. J. 43: Groenen, P. M. A., A. E. Bunschoten, D. van Soolingen, and J. D. A. van Embden Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; applications for strain differentiation by a novel typing method. Mol. Microbiol. 10: Gutierrez, M., S. Samper, J.-A. Gavignan, J. F. Garcia Marin, and C. Martin Differentiation by molecular typing of Mycobacterium bovis strains causing tuberculosis in cattle and goats. J. Clin. Microbiol. 33: Hermans, P. W. M., D. van Soolingen, E. M. Bik, P. E. W. de Haas, J. W. Dale, and J. D. A. van Embden Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot region for insertion elements in Mycobacterium tuberculosis complex strains. Infect. Immun. 59: Hermans, P. W. M., D. van Soolingen, J. W. Dale, A. R. J. Schutsma, R. McAdam, D. Catty, and J. D. A. van Embden Insertion element IS986 from Mycobacterium tuberculosis, a useful tool for diagnosis and epidemiology of tuberculosis. J. Clin. Microbiol. 28: Hermans, P. W. M., D. van Soolingen, and J. D. A. van Embden Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae. J. Bacteriol. 174: Neill, S. D., J. Pollock, T. D. G. Bryson, and J. Hanna Pathogenesis of Mycobacterium bovis in cattle. Vet. Microbiol. 40: O Reilly, L. M., and C. J. Daborn The epidemiology of Mycobacterium bovis infections in animals and man: a review. Tubercle Lung Dis. 76(Suppl. 1): Poulet, S., and S. T. Cole Repeated DNA sequences in mycobacteria. Arch. Microbiol. 163: Poulet, S., and S. T. Cole Characterisation of the polymorphic GC rich repetitive sequence (PGRS) present in Mycobacterium tuberculosis. Arch. Microbiol. 163: Ross, B. C., K. Raios, K. Jackson, and B. Dwyer Molecular cloning of a highly repeated DNA element from Mycobacterium tuberculosis and its use as an epidemiological tool. J. Clin. Microbiol. 30: Skuce, R. A., D. Brittain, M. S. Hughes, L.-A. Beck, and S. D. Neill Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis. J. Clin. Microbiol. 32: Small, P., and J. D. A. van Embden Molecular epidemiology of tuberculosis, p In B. R. Bloom (ed.), Tuberculosis: pathogenesis, protection, and control. ASM Press, Washington, D.C. 22. van Embden, J. D. A., 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 Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J. Clin. Microbiol. 31: van Soolingen, D., P. E. W. de Haas, J. Haagsma, T. Eger, P. W. M. Hermans, and J. D. A. van Embden Strain differentiation of Mycobacterium bovis from animals and man: use of various genetic markers in the epidemiology of bovine tuberculosis. J. Clin. Microbiol. 32: van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, D. R. Soll, and J. D. A. van Embden Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains, evaluation of an insertion sequence dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29: van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, and J. D. A. van Embden Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG. J. Clin. Microbiol. 30: Wayne, L. G Mycobacterial speciation, p In G. P. Kubrica and L. G. Wayne (ed.), The mycobacteria: a source book, part A. Marcel Dekker, Inc., New York. 27. World Health Organization Report of the WHO meeting on zoonotic tuberculosis (Mycobacterium bovis) with the participation of FAO. World Health Organization, Geneva.