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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Publications at this Location » Publication #358710

Research Project: Characterization of Antigens, Virulence Markers, and Host Immunity in the Pathogenesis of Johne’s Disease

Location: Infectious Bacterial Diseases Research

Title: In-vitro characterization of Mycobacterium avium subsp. paratuberculosis attenuated mutants with DIVA potential

item BARLETTA, R - University Of Nebraska
item LAMONT, E - University Of Minnesota
item Stabel, Judith
item FENTON, R - University Of Minnesota
item RATHNAIAH, G - University Of Nebraska
item ZINNIEL, D - University Of Nebraska
item POSEY, J - University Of Georgia
item MCGARVEY, J - US Department Of Agriculture (USDA)
item Bannantine, John
item SREEVATSAN, S - Michigan State University

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/5/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Objective The immunodominant Mycobacterium avium subsp. paratuberculosis (MAP) antigens MAP_1152 (PPE protein) and MAP_1156 (diacylglycerol acyltransferase) may be used to distinguish infected from non-infected animals by serology. In the current study, we constructed and characterized the corresponding MAP deletion mutants DMAP52 and DMAP56 in-vitro. Methods About 2.0 × 107 monocyte-derived macrophages were infected with 1.0 × 108 wild type K-10 and DMAP52 and DMAP56 for 2 h (invasion incubation), washed to remove non-adherent bacteria and incubated at 37oC in RPMI 1640 supplemented with 2% serum for post-infection times: 0, 2, 6, 12, 24 and 48 h after the completion of the invasion incubation period. Intracellular bacteria were recovered and enumerated. Mutants were also tested for their immunogenicity in peripheral blood mononuclear cells by cell proliferation assays to determine elicitation of activation markers (flow cytometry) relevant to cell-mediated immune responses. Results At the invasion point (2 h), strain DMAP52 was about 30-fold less invasive than K-10 and DMAP56. For the intracellular killing phase (6 h post-invasion), the decrease in the log percent survival per h were K-10 (-0.14), DMAP52 (-0.16) and DMAP56 (-0.19) indicating that DMAP56 was killed the fastest. In the post-killing period (12 to 48 h), DMAP52 was unable to replicate and DMAP56 replicated somewhat but less than K-10. Considering the total bacillary burden vs K-10, both DMAP56 and DMAP52 displayed attenuation. For the immune responses after 72 h, the percent of activated cells for CD4, CD8 and 'dTCR were similar for all strains and greater than the non-stimulated response, suggesting that these mutants are immunogenic as required for a good vaccine strain. Macrophage proliferation (CD14/CD86 and CD14/MHCII) remained unaltered upon the different treatments. Conclusions Attenuation was observed for DMAP52 (invasion and late replication) and DMAP56 (killing and replication). We expect these deletion mutants will offer the possibility of differentiating infected from vaccinated animals (DIVA formulation) by serological tests.