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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGICAL CHARACTERISTICS OF JOHNE'S DISEASE Title: Production and Evaluation of an Improved Mycobacterium avium subsp. paratuberculosis Purified Protein Derivative for Use in In-Vivo and In-Vitro Diagnostic Testing

Authors
item Capsel, Randy -
item Bannantine, John
item Hoffman, Lorraine -
item Thoen, Charles -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 9, 2009
Publication Date: August 9, 2009
Citation: Capsel, R., Bannantine, J.P., Hoffman, L., Thoen, C. 2009. Production and Evaluation of an Improved Mycobacterium avium subsp. paratuberculosis Purified Protein Derivative for Use in In-Vivo and In-Vitro Diagnostic Testing [abstract].

Technical Abstract: Purified protein derivatives (PPD’s) were prepared from the cultured filtrate of Mycobacterium avium subsp. paratuberculosis (MAP) ATCC strain 19698. Production of PPD has historically been problematic for maintaining optimal floating cultures yielding defined immunogenic components. To obtain more consistent and potent PPD preparations, production methods must be re-evaluated for process improvements and culture selection. PPD production was conducted using Mycobacterium avium subsp. paratuberculosis (MAP) ATCC strain 19698 and two recent bovine field isolates utilizing both Povitsky bottles and Erlenmeyer flasks. Traditional production consisted of floating culture incubation at 37oC, live organism inactivation by autoclaving 30 minutes at 121oC, and coarse filtration to remove cellular debris. Proteins were subsequently precipitated by adding trichloroacetic acid to a final 4% concentration in the suspension. Floating cultures were readily maintained from the ATCC 19698 culture, but similar suspension cultures were difficult to obtain from the field isolates due to difficulties to grow on the liquid media. Culture production in Erlenmeyer flasks was superior to that of Povitsky bottles as determined by floating culture mat density, incubation duration, and consistent media suspension characteristics. SDS-PAGE evaluation of four production lots did identify specific protein bands, but were difficult to discern due to protein smearing. Rabbit antiserum raised against NVSL Johnin Lot 1 was utilized to evaluate four ATCC 19698 production lots by immunoblot. Immunoblot results from the four new production lots indicated reactivity equal to or greater than Johnin Lot 1. Results indicate that the current production procedures are capable of producing Johnin PPD equivalent to the current NVSL Johnin Lot 1, but a need exists to further evaluate processing procedures, with a goal of producing a PPD which is better defined. The antisera raised against Johnin Lot 1 will be used to screen a MAP DNA expression library to identify proteins present within the PPD.

Last Modified: 4/23/2014
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