title: Evaluation of an automated system for non-radiometric detection of Mycobacterium avium paratuberculosis in bovine feces.
abstract: Cultivation of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from feces remains the most reliable method to detect infected animals. The purpose of this study was to evaluate a broth-based automated system used for cultivation of mycobacteria such as M. tuberculosis from human hosts, for the detection of M. paratuberculosis in bovine feces. Bovine feces was spiked with tenfold serial dilutions of M. paratuberculosis (5x10(5) to 5x10(-1) organisms), then processed with a double-centrifugation technique that included disinfection prior to inoculation into broth tubes. The same pathogen dilution series was also inoculated directly into broth and broth with uninfected processed feces. All of the system signal-positive bottles were identified within 30 days, with the highest concentration of M. paratuberculosis detected by the system in as few as 8 days. The presence of the pathogen was confirmed with acid-fast staining and an IS900-based PCR assay when growth of M. paratuberculosis was indicated by the system. However, some of the signal-negative cultures inoculated with the equivalent of 0.5 organisms tested PCR-positive 56 days post-inoculation, indicating that longer culture periods may lead to detection of small quantities of the organisms. Additionally, it was indicated that the processing step had a detrimental effect on detection of the organism. Comparison of the broth- and Herrold's egg yolk medium (HEYM) solid media-based culture methods with defined check test specimens corroborated the experimental evaluation of this system, indicating that broth-based detection could provide a more rapid assay for M. paratuberculosis. These results suggest that this automated system could be used to detect this organism in bovine feces, but that new approaches to processing the feces for culture should be explored.
authors.affiliation: Department of Veterinary Preventive Medicine, The Ohio State University, 1900 Coffey Rd., Columbus, OH 43210-1092, USA. stich.2@osu.edu
authors.forename: Roger W
authors.initials: RW
authors.lastname: Stich
authors.forename: Beverly
authors.initials: B
authors.lastname: Byrum
authors.forename: Brenda
authors.initials: B
authors.lastname: Love
authors.forename: Natalie
authors.initials: N
authors.lastname: Theus
authors.forename: Linda
authors.initials: L
authors.lastname: Barber
authors.forename: William P
authors.initials: WP
authors.lastname: Shulaw
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D015169:Colony Count, Microbial / Q000662:veterinary; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000254:growth & development*; D010283:Paratuberculosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000662:veterinary
chemical_list: D004269:DNA, Bacterial