In situ-PCR for the detection of Mycobacterium paratuberculosis DNA in paraffin-embedded tissues

Molecular biological techniques have permitted the rapid and sensitive dete ction of the Mycobacterium paratuberculosis genome in infected tissues, mos t commonly by polymerase chain reaction amplification of sequences in the I S900 DNA insertion sequence. The aim of this work was the detection of M. p aratuberculosis DNA in ovine tissues by in situ-polymerase chain reaction, which is sensitive and localises the signal within the tissue sample. Paraf fin embedded tissues from three acid-fast positive ovine guts with classica l lesions of paratuberculosis, and from negative control samples were teste d. A 413-bp fragment of the IS900 sequence was amplified in-situ and hybrid ised to an internal PCR-synthesised digoxygenin-labelled probe. The samples from sheep affected by paratuberculosis clearly showed cell-specific cytop lasmic signals in mucosal and submucosal macrophages. This technique could be useful both in the diagnosis and study of the pathogenesis of infections in which involvement of M. paratuberculosis is suspected.