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Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine
Raul G Barletta1, John P Bannantine2, Judith R Stabel2
1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA.
Abstract:
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 106 CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.
Insights
This study shows that Mycobacterium avium subsp. paratuberculosis (MAP) deletion mutants induce apoptosis in macrophages. This pro-apoptotic property in MAP vaccine candidates correlates with their reduced virulence and immunogenicity.
Area of Science:
- Veterinary Microbiology
- Cellular Immunology
- Bacteriology
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a significant gastrointestinal illness in ruminants.
- Developing effective vaccines against MAP is crucial for animal health.
- Previous research indicated that certain MAP deletion mutants are attenuated and immunogenic in bovine cells.
Purpose of the Study:
- To establish a cell culture model for rapidly screening MAP mutants for vaccine potential.
- To investigate the induction of apoptosis and necrosis by different MAP strains in murine macrophages.
- To correlate the pro-apoptotic activity of MAP mutants with their vaccine potential.
Main Methods:
- Utilized RAW 264.7 murine macrophages infected with wild-type and mutant MAP strains (MOI of 10).
- Assessed cell death kinetics using real-time luminescence (apoptosis) and fluorescence (necrosis) assays.
- Quantified apoptosis through DAPI staining of nuclear morphology and validated with flow cytometry.
Main Results:
- All MAP strains exhibited similar growth rates, but deletion mutants showed altered cell morphology (elongated, bulging cell walls).
- A 6-hour infection period was optimal for detecting apoptosis, which preceded secondary necrosis.
- Both deletion mutants were confirmed to be pro-apoptotic in RAW 264.7 macrophages.
Conclusions:
- The developed cell culture system effectively identifies pro-apoptotic MAP mutants.
- Increased apoptosis induced by MAP deletion mutants in macrophages correlates with their attenuated phenotype and immunogenicity.
- Pro-apoptotic activity is a promising indicator for identifying MAP vaccine candidates.
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