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BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission.Results164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900 - restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property.ConclusionThe results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe 3؉ and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island.Paratuberculosis, also called Johne's disease, is a severe and incurable enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis and has a considerable economic impact on the livestock industry (21, 28). In cattle, infection most commonly occurs in newborn calves by the fecal-oral route. However, clinical symptoms, predominantly including persistent diarrhea and weight loss, are usually delayed in animals until 3 to 5 years of age (13). Paratuberculosis is prevalent in domestic animals and many species of wildlife worldwide, including primates (31). In addition, M. avium subsp. paratuberculosis has been isolated from intestinal tissue of Crohn's disease patients, and its potential role as a zoonotic pathogen is currently being investigated (12,22).Previous attempts to determine the molecular cause for pathogenicity of M. avium subsp. paratuberculosis have focused on the identification of antigenic proteins (16, 26) and the characterization of secreted components by using monoclonal antibodies (32). Further studies have identified an M. avium subsp. paratuberculosis-specific low-GC cassette carrying genes with putative functions related to lipopolysaccharide or extracellular polysaccharide biosynthesis (47) and have identified cross-reactive, species-and subspecies-specific epitopes (32). In addition, an M. avium subsp. paratuberculosis-specific ferric reductase which is secreted in vivo and in vitro has been described (25). Other potential virulence factors reported include a serine protease (10) and a fibronectin attachment protein (41). However, closely related homologues of these factors can be found in M. avium subsp. avium and Mycobacterium tuberculosis, and they are unlikely to be M. avium subsp. paratuberculosis specific.In ...