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Open AccessArticle

Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

National Animal Disease Center, USDA-Agricultural Research Service, Ames, IA 50010, USA
Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, TN 37996, USA
Author to whom correspondence should be addressed.
Vet. Sci. 2019, 6(4), 88;
Received: 30 September 2019 / Revised: 24 October 2019 / Accepted: 28 October 2019 / Published: 31 October 2019
(This article belongs to the Special Issue Mycobacterial Diseases in Animals)
Antigens extracted using ethanol (EtOH) and incorporated in the EtOH vortex ELISA (EVELISA) test have previously shown high specificity and sensitivity for detecting Mycobacterium avium subspecies paratuberculosis (Map) and M. bovis infections in cattle. The objective of this study is to define the components present in the EtOH extract. We show that this extract is composed of lipid, carbohydrate, and proteins on the surface of the bacilli, and that EtOH removes the outer layer structure of Map which comprise these elements. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a Map genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits, but this protein is not strongly immunogenic in cattle with Johne’s disease. A panel of monoclonal antibodies was used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that a well-known antigen, termed MPB83, is present in M. bovis EtOH extracts and a fatty acid desaturase (MAP_2698c) is present in Map EtOH extracts, while lipoarabinomannan was common to both. The lipid and carbohydrate components of the extract were analyzed using thin layer chromatography and lectin binding, respectively. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle. View Full-Text
Keywords: Johne’s disease; paratuberculosis; ELISA; lipid; antigens Johne’s disease; paratuberculosis; ELISA; lipid; antigens
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MDPI and ACS Style

Bannantine, J.P.; Wadhwa, A.; Stabel, J.R.; Eda, S. Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis. Vet. Sci. 2019, 6, 88.

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