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In domestic animal husbandry the maintenance of tuberculosis free status is of primary importance. As immunological cross-reactions due to NTM hamper the diagnosis of bovine tuberculosis, the precise identification of NTM strains would be essential in the veterinary diagnostics, especially for potentially zoonotic strains. This is the first study investigating the strain diversity of non-avium NTM in Hungary.

bAccurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.T he species Mycobacterium avium is divided into three subspecies according to the currently valid taxonomical classification: Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease; Mycobacterium avium subsp. avium, the pathogen of avian tuberculosis; and Mycobacterium avium subsp. silvaticum (1). In addition, the designation "Mycobacterium avium subsp. hominissuis" has been proposed for human/porcine-type M. avium isolates (2). M. avium subsp. silvaticum, previously called "wood pigeon Mycobacterium," was assigned to the Mycobacterium avium complex (MAC) in 1990 (3). Besides wood pigeons, it was isolated from crane (3), penguin (4), roe deer (5), and hazel hen (5). The zoonotic potential of MAC members is renowned (6), but less is known about M. avium subsp. silvaticum; hence, the detection and study of this subspecies are hindered by its unreliable and ambiguous identification.Accurate identification of mycobacterial species and subspecies is essential for evaluation of their significance, pathogenicity, and epidemiology. The four subspecies of M. avium differ greatly in their host range, growth potential, and environmental occurrence (4). Three subspecies can be easily differentiated by molecular biological methods thanks to distinct molecular differences. IS900 is the specific insertion sequence (IS) of M. avium subsp. paratuberculosis. "M. avium subsp. hominissuis" and M. avium subsp. avium both harbo...

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Our results demonstrate the circulation and transmission of different MAP strain types among individuals, herds and even wildlife reservoirs in Hungary and other European countries; correlation between production type or breed and MAP genotype is hypothesized.