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Clinical and Vaccine Immunology, December 2008, p. 1824-1833, Vol. 15, No. 12
1071-412X/08/$08.00+0     doi:10.1128/CVI.00099-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Immunogenicity of Proteome-Determined Mycobacterium avium subsp. paratuberculosis-Specific Proteins in Sheep with Paratuberculosis{triangledown}

Valerie Hughes,1* John P. Bannantine,2 Susan Denham,1 Stuart Smith,1 Alfredo Garcia-Sanchez,1,{dagger} Jill Sales,3 Michael L. Paustian,2 Kevin Mclean,1 and Karen Stevenson1

Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, United Kingdom,1 National Animal Disease Center, U.S. Department of Agriculture-Agricultural Research Service, Ames, Iowa 50010,2 Biomathematics and Statistics Scotland, James Clerk Maxwell Building, The King's Buildings, Edinburgh EH9 3JZ, United Kingdom3

Received 19 March 2008/ Returned for modification 16 July 2008/ Accepted 21 September 2008

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionization-time of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis.

* Corresponding author. Mailing address: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, United Kingdom. Phone: 44 (0) 131 445 5111. Fax: 44 (0) 131 445 6111. E-mail: Val.Hughes{at}moredun.ac.uk

{triangledown} Published ahead of print on 8 October 2008.

{dagger} Present address: Patología Infecciosa, Facultad de Veterinaria, Universidad de Extremadura, Avda. de la Universidad s/n, 10071 Cáceres, Spain.

Clinical and Vaccine Immunology, December 2008, p. 1824-1833, Vol. 15, No. 12
1071-412X/08/$08.00+0     doi:10.1128/CVI.00099-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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