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New Latex Bead Agglutination Assay for Differential Diagnosis of Cattle Infected with Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis

Department of Microbiology, College of Veterinary Medicine, and School of Agricultural Biotechnology, Seoul National University, Seoul, South Korea,¹ Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa,² Department of Veterinary Microbiology and Pathology,³ Department of Veterinary Clinical Sciences, Washington State University, Pullman, Washington,⁴ Department of Bacteriology, Immunology and Mycology, College of Veterinary Medicine, Minufiya University, Minufiya, Egypt,⁵ Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, New York⁶

Received 30 March 2004/ Returned for modification 6 July 2004/ Accepted 20 August 2004

Extensive studies have shown that the current assays used to identify cattle infected with Mycobacterium bovis or Mycobacterium avium subsp. paratuberculosis are not sufficiently sensitive and specific to detect all infected animals, especially animals recently infected with the pathogens. In the present report we show that these limitations might be overcome with a latex bead agglutination assay (LBAA). With the specific immunodominant epitope (ESAT6-p) of M. bovis, we developed an LBAA and enzyme immunoassay (EIA) for that purpose and compared them with the "gold standard" culture method and skin test for their efficacy in detecting bovine tuberculosis. When sera from control healthy cows (n = 10), M. avium subsp. paratuberculosis-positive cattle (naturally infected, n = 16; experimentally infected, n = 8), and M. bovis-positive cattle (naturally infected, n = 49;experimentally infected, n = 20) were applied to an EIA and an LBAA developed with ESAT6-p, the two tests showed similar sensitivity (97.1% by EIA, 95.7% by LBAA), high specificity (94.2% by EIA, 100% by LBAA), and a positive correlation (kappa value, 0.85; correlation rate, 93.2%; correlation coefficient, 0.64). Receiver operating characteristic analysis of EIA results and comparison with the culture method determined a suitable cutoff value at 0.469, with an area under the curve of 0.991 (95% confidence interval, 0.977 to 1.0). As LBAA didn't show any positive reactions with sera from uninfected control cows or M. avium subsp. paratuberculosis-infected cattle, which were confirmed to be free of M. bovis by culture or PCR, LBAA using the ESAT6-p can be a rapid and useful M. bovis diagnostic assay. The data suggest that rapid, sensitive, and specific assays can be developed with peptides containing immunodominant epitopes present in proteins uniquely expressed in M. bovis or M. avium subsp. paratuberculosis for differential diagnosis of cattle infected with M. bovis or M. avium subsp. paratuberculosis.

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• Koo, H. C., Park, Y. H., Ahn, J., Waters, W. R., Palmer, M. V., Hamilton, M. J., Barrington, G., Mosaad, A. A., Park, K. T., Jung, W. K., Hwang, I. Y., Cho, S.-N., Shin, S. J., Davis, W. C. (2005). Use of rMPB70 Protein and ESAT-6 Peptide as Antigens for Comparison of the Enzyme-Linked Immunosorbent, Immunochromatographic, and Latex Bead Agglutination Assays for Serodiagnosis of Bovine Tuberculosis. J. Clin. Microbiol. 43: 4498-4506 [Abstract] [Full Text]