Title

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

Authors

Authors

K. Hulten; T. J. Karttunen; H. M. T. El-Zimaity; S. A. Naser; A. Almashhrawi; D. Y. Graham;F. A. K. El-Zaatari

Abbreviated Journal Title

Vet. Microbiol.

Keywords

In situ hybridization; M. paratuberculosis; Crohn's disease; spheroplast; POLYMERASE CHAIN-REACTION; CROHNS-DISEASE; MYCOBACTERIUM-PARATUBERCULOSIS; SPHEROPLASTS; ACID; DNA; Microbiology; Veterinary Sciences

Abstract

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections. M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only samples 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. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals. (C) 2000 Elsevier Science B.V. All rights reserved.

Journal Title

Veterinary Microbiology

Volume

77

Issue/Number

3-4

Publication Date

1-1-2000

Document Type

Article; Proceedings Paper

Language

English

First Page

513

Last Page

518

WOS Identifier

WOS:000166140400029

ISSN

0378-1135

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