Proceedings of the 10th International Colloquium on Paratuberculosis

#35 Evaluation <strong>of</strong> subspecies-specific proteins for <strong>the</strong> diagnosis <strong>of</strong> Mycobacterium avium
subspecies paratuberculosis infections
Valerie Hughes 1 , John P Bannantine 2 , Susan Denham, Jim McNair, Samuel Strain, Mike L Paustian 2 ,
Karen Stevenson 1
1 Moredun Research Institute, United Kingdom; 2 National Animal Disease Center, USDA-ARS, Ames, Iowa, USA;
Agri-Food and Biosciences Institute, United Kingdom
Mycobacterium avium subspecies paratuberculosis (Map)-specific proteins (35) were identified by comparing
<strong>the</strong> proteomes <strong>of</strong> Map isolates with those <strong>of</strong> <strong>the</strong> genetically similar subspecies IS901 + Mycobacterium avium
subspecies avium or silvaticum. This approach identified subspecies-specific proteins including <strong>the</strong> products
<strong>of</strong> differential expression that would not have been detected by a comparative genomics approach. The genes
encoding <strong>the</strong> proteins <strong>of</strong> interest were cloned into pMAL-c2X and expressed in Escherichia coli and <strong>the</strong> immunogenicity
<strong>of</strong> <strong>the</strong> recombinant proteins determined to assess <strong>the</strong>ir potential as specific immunological reagents
for <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis and epidemiological studies. Immunogenicity was evaluated using <strong>the</strong>
interferon-gamma enzyme-linked immunosorbent assay (IFN-γ ELISA), serum ELISA and immunoblotting.
Responses to <strong>the</strong> recombinant Map-specific proteins were compared to those <strong>of</strong> PPD from M.avium subspecies
avium (PPD-A) and Map (PPD-J). Immune responses were evaluated in naturally Map-infected sheep with
subclinical and clinical disease and in calves experimentally infected with ei<strong>the</strong>r Map or IS901 + M.avium. Three
proteins were found to have diagnostic potential when incorporated in <strong>the</strong> IFN-γ ELISA. Seventeen proteins
were detected in at least one <strong>of</strong> <strong>the</strong> immunoassays and eleven proteins were detected by serum ELISA with
an optical density in excess <strong>of</strong> <strong>the</strong> cut<strong>of</strong>f <strong>of</strong> 0.1 in four <strong>of</strong> six sheep sera tested. The immunoreactivity <strong>of</strong> <strong>the</strong>se
proteins indicates <strong>the</strong>ir potential as unique diagnostic antigens for <strong>the</strong> specific immunodiagnosis <strong>of</strong> paratuberculosis.
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