Proceedings of the 10th International Colloquium on Paratuberculosis

#210 Production and evaluation <strong>of</strong> an Mycobacterium avium subsp. paratuberculosis Purified
Protein Derivative for use in in-vivo and in-vitro diagnostic testing
Randy Capsel 1 , John P. Bannantine 2 , Lorraine H<strong>of</strong>fman 3 , Charles O. Thoen 4
1 National Veterinary Services Laboratories, U.S. Department <strong>of</strong> Agriculture, Ames, Iowa, USA; 2 National Animal Disease
Center, USDA-ARS, Ames, Iowa, USA; 3 Veterinary Diagnostic and Production Animal Medicine, Iowa State University,
College <strong>of</strong> Veterinary Medicine, Ames, Iowa, USA; 4 Department <strong>of</strong> Veterinary Microbiology and Preventive Medicine,
Iowa State University, College <strong>of</strong> Veterinary Medicine, Ames, Iowa, USA
Purified protein derivatives (PPD’s) were prepared from <strong>the</strong> cultured filtrate <strong>of</strong> Mycobacterium avium subsp. paratuberculosis
(MAP) ATCC strain 19698. The production <strong>of</strong> PPD has historically been problematic because <strong>of</strong> <strong>the</strong>
difficulty with 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 <strong>of</strong> floating culture incubation at 37 o C, live organism inactivation by autoclaving 30 minutes at
121 o C, and coarse filtration to remove cellular debris. Proteins were subsequently precipitated by adding trichloroacetic
acid to a final 4% concentration in <strong>the</strong> suspension. Floating cultures were readily maintained from <strong>the</strong>
ATCC 19698 culture, but similar suspension cultures were difficult to grow using <strong>the</strong> field isolates. Culture production
in Erlenmeyer flasks was superior to that <strong>of</strong> Povitsky bottles as determined by floating culture mat density, time
required for growth, and consistent media suspension characteristics. SDS-PAGE evaluation <strong>of</strong> four production
lots did identify specific protein bands, but was 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 <strong>the</strong> four new production lots indicated reactivity equal to or greater than Johnin Lot 1. Results indicate
that <strong>the</strong> current production procedures are capable <strong>of</strong> producing Johnin PPD equivalent to <strong>the</strong> current NVSL
Johnin Lot 1, but a need exists to fur<strong>the</strong>r evaluate processing procedures, with a goal <strong>of</strong> 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 clones expressing specific immunogenic proteins in PPD.
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