Severe
emaciation and mortalities suggestive of mycobacterial
infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis
infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp.
paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp.
paratuberculosis in mycobacterial
infections of deer.
DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp.
paratuberculosis and M. avium subsp. avium
infections, whereas the restriction step differentiated single M. avium subsp.
paratuberculosis or M. avium subsp. avium
infections from mixed M. avium subsp.
paratuberculosis/M. avium subsp. avium
infections. The
endonuclease Hpy188I cleaves
DNA between
nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34
initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp.
paratuberculosis strains tested, independent of their origin,
Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis
infection and to identify single (M. avium subsp.
paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp.
paratuberculosis/M. avium subsp. avium)
infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp.
paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.