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United States Department of Agriculture

Agricultural Research Service

Title: Identification of Differences in the Protein Content of Mycobacterium Avium Subspecies Paratuberculosis Purified Protein Derivative Preparations with Different Skin Test Specificities

Authors
item Paustian, Michael
item Robbe Austerman, Suelee
item Lippolis, John
item Stabel, Judith
item Bannantine, John

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 20, 2006
Publication Date: May 20, 2006
Citation: Paustian, M., Robbe Austerman, S., Lippolis, J.D., Stabel, J.R., Bannantine, J.P. 2006. Identification of Differences in the Protein Content of Mycobacterium avium subspecies paratuberculosis Purified Protein Derivative Preparations with Different Skin Test Specificities [abstract]. American Society for Microbiology. Paper No. U-016.

Technical Abstract: Mycobacterium avium subspecies paratuberculosis (Map) strain ATCC 19698 has been used to produce a purified protein derivative (PPD) that can be utilized as a diagnostic reagent. Two Map PPD lots (Johnin 9801 and Johnin 0202) were used to skin test sheep and cattle for exposure to Map. Johnin 9801 was 97.6% ± 2.1% and 98.9% ± 1.0% specific in cattle and sheep, respectively, while Johnin 0202 was 84.2% ± 3.8% and 84.9% ± 5.4% specific in cattle and sheep. We hypothesize that identifying differences between the PPD preparations that are correlated with skin test performance will lead to the identification of proteins that can be utilized as an alternative diagnostic reagent. Strong cation exchange liquid chromatography followed by tandem mass spectrometry was utilized to examine the relative abundance of proteins from the two PPD preparations that had been differentially labeled with isotope tags. A total of 193 proteins were identified in both PPD preparations based on peptide matches. Notably, the protein with the greatest change in abundance between the two PPD lots was the surface layer protein SbsA from Geobacillus stearothermophilus, which was present at nearly 8-fold higher concentration in Johnin 9801. The effect of SbsA on PPD specificity is not clear and it may represent a trace contaminant from the autoclave used for PPD production, as G. stearothermophilus is commonly used as a test organism for autoclave performance. Twenty three other proteins were present at 2-fold or higher concentrations in Johnin 9801, most of which have no known function. Sixty two proteins Johnin 0202 were identified at greater than 2-fold higher concentrations and approximately half of which have no known function. A significant number of the remaining proteins that were enriched in Johnin 0202 are predicted to be involved in lipid biosynthesis or degradation. The results of this study indicate that an overabundance of specific proteins within different preparations of PPD is responsible for the observed differences in skin test specificity in cattle and sheep.

Last Modified: 9/21/2014