Identification of cell wall deficient forms of M-avium subsp paratuberculosis in paraffin embedded tissues from animals with Johne's disease by in situ hybridization

M. avium subsp. paratuberculosis (M. paratuberculosis) is the causative age nt of Johne's disease (JD) in ruminants leading to enormous economical loss es in dairy and meat industries worldwide. During the subclinical stage of the disease, the infected animals are difficult if not impossible to detect by the available diagnostic tests including the PCR based ones. Although o nly considered an animal pathogen, cell wall deficient (CWD) forms of M. pa ratuberculosis have been isolated from patients with sarcoidosis and Crohn' s disease (idiopathic diseases) in humans. Hence, the CWD form of this orga nism has been suspected to play a role in the pathogenesis of these disease s by persisting in the affected tissues and triggering a localized immune r esponse and pathology. Differentiating between the CWD and acid-fast forms of this organism may lead to the determination of whether the CWD form is t he pathogenic form in the subclinical cases of JD in animals and/or the eti ologic agent for the above human diseases. To localize such organisms in ti ssue sections, CWD forms of mycobacteria were prepared in vitro and injecte d into beef cubes which were then formalin fixed and paraffin embedded. An in situ hybridization (ISH) technique, combined with the IS900 M. paratuber culosis-specific probe labeled with digoxigenin, was developed for the dete ction of nucleic acids specifically from the CWD forms but not their acid-f ast forms in tissue sections. Specificity was confirmed by the negative fin ding with an irrelevant probe and with control tissue preparations containi ng CWD cells of related mycobacteria and unrelated organisms. This ISH proc edure provides a way to distinguish between the acid-fast and CWD forms of M. paratuberculosis and to localize them in tissue sections. ISH may prove useful to evaluate the significance of CWD forms of M. paratuberculosis in the pathogenesis of JD, Crohn's disease and sarcoidosis. (C) 2000 Elsevier Science BN. All rights reserved.