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United States Department of Agriculture

Agricultural Research Service

Title: Identification of a Gene Unique to Mycobacterium Avium Subspecies Paratuberculosis and Application to Diagnosis of Paratuberculosis

Authors
item Ellingson, Jay
item Bolin, Carole
item Stabel, Judith

Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 31, 1998
Publication Date: N/A

Interpretive Summary: Johne's disease or ruminant paratuberculosis is considered to be one of the most serious diseases in the cattle industry. Johne's disease is characterized by a chronic granulomatous enteritis caused by Mycobacterium paratuberculosis. Clinical disease is characterized by profuse, nontreatable diarrhea, emaciation, and eventual death. Johne's disease causes economic losses to the cattle industry worldwide primarily as a result of reduced milk production, decreased reproductive efficiency, and death. It is estimated that annual losses in the United States exceed $1.5 billion. Diagnosis of subclinical paratuberculosis presents a major problem in control of this disease. Currently, cultivation of M. paratuberculosis from fecal or tissue specimens is the most definitive method for detecting animals with Johne's disease. The procedure requires 8 to 16 weeks of incubation and specialized medium for propagation of the organism. Because of this, researchers have strived to improve the efficiency of detection by developing diagnostic nucleic acid methodologies. Nucleic acid diagnostic methodology has been used as a rapid and sensitive way to identify specific species of mycobacteria. In this study, we describe the cloning and sequencing of a gene unique to M. paratuberculosis and the use of this gene in the development of an efficient nucleic acid diagnostic test for Johne's disease. This nucleic acid diagnostic test distinguishes M. paratuberculosis from all closely related mycobacterial species. Improved diagnostics are critical for accurate detection of infected animals by diagnostic laboratories and overall reduction in disease incidence.

Technical Abstract: Mycobacterium paratuberculosis is the etiologic agent of paratuberculosis (Johne's disease), a chronic granulomatous enteritis in ruminants. Currently, there is a need for improved diagnostic tests because of the lack of methods for accurate, rapid and reliable detection of M. paratuberculosis infection. A M. paratuberculosis gene (hspX) was cloned, sequenced, and a 30 bp species-specific oligonucleotide was synthesized. As an internal control to identify all mycobacterial species, a 33 bp oligonucleotide was synthesized based on the conserved 5' terminus of the mycobacterial recA gene. Dioligonucleotide hybridization (dOH) analysis identified mycobacterial species and specifically identified reference (ATCC 19698), bovine, and human isolates of M. paratuberculosis. The M. paratuberculosis-specific oligonucleotide distinguished M. paratuberculosis isolates from related mycobacteria, including all closely related Mycobacterium avium and Mycobacterium intracellulare strains tested in thi study. Hybridization was not observed with DNA extracted from a selected group of other bacterial pathogens. The experiments indicate that the dOH analysis is a useful diagnostic tool to detect mycobacterial infection, specifically M. paratuberculosis, and will be adapted for analysis of bacteriologic cultures, feces and tissue specimens.

Last Modified: 9/2/2014
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