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Title: Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives

item CAPSEL, RANDAL - Animal And Plant Health Inspection Service (APHIS)
item THOEN, CHARLES - Iowa State University
item Reinhardt, Timothy
item Lippolis, John
item OLSEN, RENEE - Animal And Plant Health Inspection Service (APHIS)
item Stabel, Judith
item Bannantine, John

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2015
Publication Date: 5/2/2016
Publication URL:
Citation: Capsel, R.T., Thoen, C.O., Reinhardt, T.A., Lippolis, J.D., Olsen, R., Stabel, J.R., Bannantine, J.P. 2016. Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives. Veterinary Microbiology. 11(5):e0154685. doi: 10.1371/journal.pone.0154685.

Interpretive Summary: Johne’s disease in livestock such as dairy cattle and sheep is caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). A major obstacle to controlling this disease is early detection after infection and before the animal begins shedding the bacterium on farm. In this manuscript, we examined a long-standing tradition of antigen production for skin testing of cows. This antigen, termed PPD for “purified protein derivative”, when injected into the skin of cows will produce a swelling at the injection site only if the cow was exposed to the disease causing bacterium. However, some long-held traditions that are not scientifically based, but rather are based on empirical results have been incorporated into production methods for PPD. We examined a reference PPD produced at NVSL that is currently used in the field by a variety of approaches, including mass spectrometry. We determined that the heat treatment used in traditional PPD production could be dispensed with and that gsome recombinant proteins could supplant the need for PPD production entirely. This research is of primary interest to state veterinarians, stakeholders and other researchers in the field.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain ATCC 19698. Traditional production consists of floating culture incubation at 37oC, organism inactivation by autoclaving, coarse filtration, and protein precipitation. PPDs produced by the traditional method used in this study included lot 9801, which served as a reference and is currently used in the field, along with two additional lots (0802 and 0803A). Two alternative production lots, which removed the autoclaving step, were prepared in this study and compared with these traditional PPDs. SDS-PAGE analysis revealed protein smearing in traditional PPD lots, but distinct bands were observed in the alternative PPD preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, whereas an immunoreactive smear was observed in the traditional PPDs. PPDs were further evaluated by mass spectrometry and tested for hypersensitivity utilizing the guinea pig potency test. Mass spectrometry identified 130 MAP proteins among three PPD lots representing the two different production methods, ten of which were present in all PPDs. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon-gamma production in blood from Johne’s positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents.