In situ hybridization method for studies of cell wall deficient M-paratuberculosis in tissue samples

Cell wall deficient forms of mycobacteria may be important in the pathogene sis of Crohn's disease and sarcoidosis. However, no method has been availab le to localize this type of organisms in tissue sections. We developed an i n situ hybridization method for the demonstration of Mycobacterium paratube rculosis spheroplasts (cell wall deficient forms) in paraffin embedded tiss ue sections. M. paratuberculosis spheroplasts were prepared by treatment with glycine an d lysozyme. Pieces of beef were injected with the prepared spheroplasts. Th e samples were fixed in buffered formalin and paraffin embedded. A M. parat uberculosis-specific probe derived from the IS900 gene was used. Specificit y was controlled by using an irrelevant probe and by hybridizing sections w ith spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only s amples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis Bs well as the acid- fast forms of M. paratuberculosis did not hybridize with the probe. Unrelat ed bacterial controls, i.e. Helicobacter pylori and Escherichia coli were a lso negative in the assay. In situ hybridization with the IS900 probe provi des a specific way to localize M. paratuberculosis spheroplasts in tissue s ections and may be useful for studies of the connection between M. paratube rculosis and Crohn's disease and sarcoidosis. The assay may also be valuabl e for studies on Johne's diseased animals. (C) 2000 Elsevier Science B.V. A ll rights reserved.