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This study surveys 2,593,348 cattle slaughtered between 1996 and 2000, and further investigates 571 (0.02%) animals found to have tuberculous lesions. Culture of 346 randomly selected tissue samples from animals younger (n = 215) and older (n = 131) than 2 years, isolated mycobacteria from 91 animals (26.3%). These included 74 Mycobacterium avium subsp. avium isolates of IS901+ and IS1245+ genotype and serotype 2, 13M. avium subsp. hominissuis isolates of IS901- and IS1245+ genotype and serotypes 8 (n = 7) and 4 (n = 6), two M. chelonae, one M. avium subsp. paratuberculosis (RFLP type B-C1), and one M. terrae. Culture of mesenteric lymph node samples obtained 66 isolates of M. avium complex (MAC) and four isolates of other mycobacterial species. M. bovis was significantly absent from all samples. Mycobacteria were more frequently (P = 0.01) isolated from tissues of animals under 2 years (34.4%) than animals over 2 years (13.0%). IS901 and IS1245 RFLP methods were used to type 17 randomly selected MAC isolates, virulent after intramuscular inoculation of pullets, from 17 different cattle herds. These revealed 11 distinct IS901 RFLP types and three IS1245 RFLP profiles. Polyclonal infection of individual animals was detected by IS901/IS1245 typing in 2 of the 17 selected isolates.

Mycobacterial interspersed repetitive units (MIRU) comprise short tandem repeat structures found at multiple loci throughout the Mycobacterium tuberculosis genome and have been used for typing these pathogens. We have identified MIRU at 18 conserved loci throughout the common portions of the Mycobacterium avium subspecies paratuberculosis (MAP) and M. avium subspecies avium (MAA) genomes. Six of these loci were found to differ between MAA and MAP in the number of tandem repeat motifs occurring at each MIRU locus. Locus specific PCR at 4 of these loci segregated MAP into two major groups, which could be differentiated from ovine-pigmented strains of MAP and the MAP vaccine strain 316F. The same PCR differentiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other M. avium complex (MAC) tested. PCR at both loci 1 and 4 also distinguished MAP from Mycobacterium intracellulare. MIRU typing may provide an additional simple and rapid procedure for the differentiation between MAP and other MAC.

Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.

A new insertion sequence, IS1512, from Mycobacterium gordonae was cloned and sequenced. This element is present in up to 10 copies which provides a high diversity for restriction fragment length polymorphism analysis. We have also identified truncated IS1512-like elements, including a truncated IS1512 and another truncated insertion sequence which displays homology with IS1512 and was designated IS1511. Sequences homologous to the previously described transposon Tn554 are inserted into these truncated insertion sequences. Insertion loci of IS1511/IS1512 are shown to be highly related to those described for IS256-like elements in other species. Alignment of the putative transposases from Rhodococcus and Mycobacterium suggests these insertion sequences may form a distinct closely homologous subclass within the IS256 family. Analysis comparing phylogenetic divergence of these elements with that of 16S rRNA and superoxide dismutase genes suggests horizontal transfer of a IS1511/IS1512 precursor into M. gordonae, and the occurrence of horizontal transfer between Mycobacteriaceae and Rhodococcaceae.

Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M intracellulare that is absent in M. avium. This antigen cross reacts immunologically with a major 38 kDa antigen of M. tuberculosis, and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli. Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.

Optimal therapy of mycobacterial infections in acquired immunodeficiency syndrome (AIDS) is difficult to achieve because of the time needed for a conventional culture to differentiate between Mycobacterium avium-intracellulare complex (MAIC) and Mycobacterium tuberculosis complex (MTBC). The recent commercial availability of gene probing techniques has introduced the potential for more rapid differentiation. We have evaluated the suitability of this technique. The specificity of the chemiluminescent gene probe system 'AccuProbe' was determined for differentiating mycobacterial isolates from 114 AIDS patients. 'AccuProbe' was 100% specific for MTBC isolates (21 of 21 isolates). Using two separate probes to MAIC and 'M. avium-intracellulare complex subtype X' (MAIC-X), 'AccuProbe' was 96% specific (87 of 91 isolates) with 5% of isolates belonging to the MAIC-X group. There were no cross-reactions between any of the probes. Using a modification of the manufacturer's protocol, 'AccuProbe' was used in a 6-month trial for the rapid differentiation of mycobacteria grown from 'Bactec' blood culture. Fifty-seven isolates (31 patients) from 805 Bactec 13A blood cultures (510 patients) were investigated. Ninety-nine per cent of isolates were able to be identified after a mean incubation period of 2.1 weeks (SD 1.2 weeks). Ninety-three per cent of isolates were reported with a presumptive identification the same day and 99% by the day after the culture flagged positive.