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Clinical and Vaccine Immunology, August 2006, p. 837-844, Vol. 13, No. 8
1071-412X/06/$08.00+0     doi:10.1128/CVI.00148-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Highly Sensitive and Subspecies-Specific Surface Antigen Enzyme- Linked Immunosorbent Assay for Diagnosis of Johne's Disease

Shigetoshi Eda,1 John P. Bannantine,2 W. R. Waters,2 Yasuyuki Mori,3 Robert H. Whitlock,4 M. Cathy Scott,1 and C. A. Speer1*

Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, the University of Tennessee, Knoxville, Tennessee 37996,1 Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa,2 Paratuberculosis Research Team, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki, Japan 305-0856,3 New Bolton Center, University of Pennsylvania, Kennett Square, Pennsylvania 19348-16924

Received 18 April 2006/ Returned for modification 22 May 2006/ Accepted 5 June 2006

Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 µg/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.

* Corresponding author. Mailing address: Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, the University of Tennessee, P.O. Box 1071, Knoxville, TN 37901-1071. Phone: (865) 974-0467. Fax: (865) 974-4714. E-mail: caspeer{at}utk.edu.

Clinical and Vaccine Immunology, August 2006, p. 837-844, Vol. 13, No. 8
1071-412X/06/$08.00+0     doi:10.1128/CVI.00148-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hughes, V., Bannantine, J. P., Denham, S., Smith, S., Garcia-Sanchez, A., Sales, J., Paustian, M. L., Mclean, K., Stevenson, K. (2008). Immunogenicity of Proteome-Determined Mycobacterium avium subsp. paratuberculosis-Specific Proteins in Sheep with Paratuberculosis. CVI 15: 1824-1833 [Abstract] [Full Text]  
  • Bannantine, J. P., Paustian, M. L., Waters, W. R., Stabel, J. R., Palmer, M. V., Li, L., Kapur, V. (2008). Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays. Infect. Immun. 76: 739-749 [Abstract] [Full Text]  
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