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

Area of Science:

• Veterinary Immunology
• Microbial Diagnostics
• Bacteriology

Background:

• Ethanol (EtOH) extracts and the EtOH vortex ELISA (EVELISA) test show high specificity and sensitivity for detecting Mycobacterium avium subspecies paratuberculosis (Map) and M. bovis infections in cattle.
• Understanding the precise composition of these EtOH antigen extracts is crucial for improving diagnostic accuracy.

Purpose of the Study:

• To define the specific molecular components present in the EtOH antigen extract used for cattle mycobacterial diagnostics.
• To identify the key antigenic molecules responsible for the diagnostic efficacy of the EtOH extract.

Main Methods:

• Screening of a Map genomic expression library using polyclonal antibodies against the EtOH prep.
• Recombinant production and characterization of identified protein candidates (MAP_0585).
• Analysis of lipid and carbohydrate components using thin layer chromatography and lectin binding; assessment of antigenicity via protease treatment and monoclonal antibody panels.

Main Results:

• The EtOH extract contains lipids, carbohydrates, and proteins from the mycobacterial outer layer.
• MAP_0585, a hypothetical protein, showed reactivity in rabbit sera but not strongly in cattle with Johne's disease.
• MPB83 (in M. bovis) and a fatty acid desaturase (MAP_2698c) (in Map) were identified, along with common lipoarabinomannan.
• Lectin binding and protease treatment indicated that the primary antigenic component is carbohydrate, not protein.

Conclusions:

• The EtOH extract's antigenic activity is primarily attributed to its carbohydrate components.
• This detailed characterization provides valuable insights into the diagnostic antigen preparation for bovine mycobacterial diseases.
• Further research may focus on optimizing carbohydrate-based antigens for enhanced cattle diagnostics.