Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine Macrophages

subsp. (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 (...

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Bibliographic Details
Published in:Vaccines (Basel) 2023-06, Vol.11 (6), p.1085
Main Authors: Barletta, Raul G, Bannantine, John P, Stabel, Judith R, Muthukrishnan, Ezhumalai, Anderson, Dirk K, Dutta, Enakshy, Manthena, Vamsi, Hanafy, Mostafa, Zinniel, Denise K
Format: Article
Language:English
Subjects:
Apoptosis
Bacteria
Cattle
Cell culture
Cell death
Cell morphology
Cell walls
Combined vaccines
Cytology
Deletion
Deletion mutant
Flow cytometry
Fluorescence
Gastroenteritis
Immunogenicity
Infections
Macrophages
Milk production
Morphology
Mutants
Mycobacterium avium
Mycobacterium avium subsp. paratuberculosis
Necrosis
Paratuberculosis
Pasteurization
Pathogens
Phenotypes
vaccine
Vaccines
virulence
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Summary:subsp. (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 × 10 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.
ISSN:2076-393X
2076-393X
DOI:10.3390/vaccines11061085