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Bovine tuberculosis (bovine TB) caused by Mycobacterium bovis remains a significant disease of farmed cattle in the Republic of Ireland, incurring substantial annual economic costs through surveillance and eradication programs (21, 29). Despite the implementation in this country of a comprehensive testing and eradication scheme operated by the Department of Agriculture and Food since 1954, which has dramatically reduced the incidence of the disease, bovine TB persists within the Irish cattle population (11). Various reasons have been proposed to explain this residual level of infection, including a relatively high level of cattle movement within the country (20) and reservoirs of infection maintained in other domestic, feral, and wild species (15, 21). Although cattle are the definitive host of this pathogen, M. bovis has an exceptionally broad host range and can be maintained in a variety of mammalian species to a greater or lesser extent (35). In Ireland and the United Kingdom, the badger (Meles meles) is known to represent a significant source of infection outside of the cattle population and is thought to be a contributory factor in the repeated herd breakdowns observed in certain areas, although the precise dynamics of TB transmission between badgers and cattle have never been firmly established (17, 20). Epidemiological analyses of M. bovis infection have been hampered in the past by the difficulty associated with distinguishing between isolates originating from different sources and species. In recent years, molecular biological techniques have been employed to examine M. bovis bacilli at the DNA level and to identify genotypically distinct strain types that may be presumed to be epidemiologically unrelated. Such data may provide information pertinent to routine field investigations undertaken following herd breakdowns and provide a greater understanding of disease transmission within the national cattle herd and between cattle and nonbovine transmission vectors. Several strategies for typing M. bovis isolates on the basis of DNA polymorphisms have arisen in recent years. Techniques commonly used internationally include restriction fragment length polymorphism (RFLP) analysis (4, 7, 9, 25, 30, 31, 37), spoligotyping (4, 7, 16, 27), pulsed-field gel electrophoresis (12), and PCR-based techniques such as “ampliprinting” (13, 22). RFLP analysis has been demonstrated to be a robust and highly discriminatory typing procedure due to the availability of multiple DNA probes for the detection of polymorphic loci within the M. bovis genome and has been the method of choice, alongside spoligotyping, for typing M. bovis isolates from cattle and other hosts in Ireland (4, 5, 30, 31). The most commonly used genetic markers for this type of analysis include the mycobacterial insertion sequence IS6110 (33, 34), the highly repetitive polymorphic GC-rich repeat sequence (PGRS) (27), and the direct repeat (DR) region of the mycobacterial genome (14). IS6110 has been widely used as a tool for genotyping Mycobacterium tuberculosis (19, 36), in which it is usually present in up to 20 copies (24). This DNA element has since been adopted for use as an RFLP probe for typing M. bovis isolates also (8, 18). In M. bovis however, IS6110 is generally found in one to five copies, greatly limiting the ability of this element to discriminate between different strains of this organism (12, 30). The majority of Irish bovine-derived M. bovis isolates typed to date contain a single, monomorphic copy of IS6110 which is insufficiently sensitive for epidemiological purposes, necessitating supplementary typing with additional probes (4, 30, 31). The PGRS-based RFLP probe appears to be the single most discriminatory of the probes currently available for M. bovis strain typing (4, 7, 9, 25) and can be present in up to 30 copies in members of the M. tuberculosis complex (23, 28). PGRS is present in multiple copies interspersed throughout the genome and exhibits a high level of polymorphism between unrelated isolates. However, the result of a PGRS DNA fingerprint is relatively complex, containing a great many bands that can be difficult to interpret and which can hinder computer-assisted band analysis, particularly when the final image is less than ideal. The third most commonly used RFLP probe for this kind of analysis is the DR region. Unique to members of the TB complex, the DR region consists of a series of identical 36-bp DR sequences interspersed with variable spacer sequences from 35 to 41 bp in length (14). This region has found application both as a target for RFLP typing and for the PCR amplification-based spoligotyping technique, both of which give a satisfactory level of strain discrimination (1, 7). The DR RFLP probe is targeted at the 36-bp DR sequences found in multiple copies within the DR region, whereas spoligotyping targets the spacer sequences in between. As both DR RFLP analysis and spoligotyping target the same genomic locus, albeit different areas within that same locus, the results of these two methods tend to be comparable in terms of strain discrimination and sensitivity (8). While having the advantages of being a considerably faster and less labor-intensive method to perform than RFLP analysis, spoligotyping alone does not usually provide sufficient discrimination between strains of M. bovis to be used as a sole typing method and is often combined with supplementary techniques such as RFLP typing with IS6110 and PGRS (4, 8, 26). A recent geographical survey of Irish M. bovis isolates in which 452 M. bovis isolates were examined by RFLP analysis with IS6110, PGRS, and DR and also by spoligotyping (4) showed that 20% of isolates analyzed exhibited a common RFLP strain type, designated strain type A1 A1 A (IS6110, PGRS, and DR, respectively). This strain type, which cannot be further subdivided by existing RFLP methods, occurred in a variety of host species and was widely distributed across the country, appearing in geographically distant regions of the country where genetically isolated genotypes might be expected to arise. This strain type is similar to the most frequently occurring strain type identified from isolates in Northern Ireland (30). This may be due to an overall genetic homogeneity within the Irish M. bovis population and may represent a significant limitation on the usefulness of strain typing as an aid to the epidemiological investigation of herd breakdowns. As the collection and cultivation of isolates for typing is time-consuming and labor-intensive, there has been a need for supplementary methods of dividing this common type in order to maximize the amount of data that can be obtained from cultured material. In this study, a new RFLP probe has been identified from an M. bovis genomic DNA library which is capable of splitting the most common M. bovis strain type observed in Irish isolates. Preliminary analysis indicates that this probe yields a clear, highly polymorphic banding pattern that has a discriminatory ability comparable to the current combination of IS6110, PGRS, and DR probes.

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS 6110 , 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 IS 6110 or DR probes. The majority of isolates from all species carried a single IS 6110 copy. In four RFLP types IS 6110 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.

Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. MIRU-VNTR typing and spoligotyping were evaluated in this study, and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers, and deer in the Republic of Ireland that had previously been typed by IS 6110 , polymorphic GC-rich sequence (PGRS), and direct-repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26, and 1895) were performed on the samples. RFLP analysis was the method that gave the greatest differentiation of strains, with a Hunter-Gaston discriminatory index (HGDI) of 0.927; the HGDI recorded for MIRU-VNTR typing was marginally lower at 0.918, and spoligotyping was the least discriminatory method, with an HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140, there was a much lower level of concordance between RFLP and MIRU-VNTR typing than for groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol, the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A, and 4052) while maintaining a high level of strain differentiation.

In this study, the newly described Mycobacterium bovisrestriction 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. bovisisolates 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.