Real-Time Automated Fluorescence Imaging Assay for Quantifying GFP-Expressing Mycobacterium avium subsp. paratuberculosis

Measurement of Mycobacterium avium subsp. paratuberculosis (MAP) load within macrophages using a mycobacterial growth inhibition assay (MGIA) is an accurate indicator of host immune control of MAP infection. Real-time fluorescent microscopy allows the continuous analysis of infected cells and the quantification of fluorescence intensity, which directly correlates with concentration of fluorescent bacteria. The objective of this study was to develop an automated and real-time live-cell imaging assay using the IncuCyte SX1® platform for quantifying green fluorescence protein (GFP)-expressing MAP (MAP-GFP) within infected macrophages. The fluorescence of serially diluted free MAP-GFP was quantified using the IncuCyte SX1® demonstrating that the fluorescence signal of 3 × 107 CFU was significantly different from lowest MAP concentrations. Significant differences between uninfected and infected BOMAC cells (MOI; 1:10) in DMEM-F12, DMEM-F12 supplemented with fetal bovine serum (FBS), and RPMI-1640 supplemented with FBS were observed, indicating the suitability of these culture media for measuring MAP-GFP-host cell interaction in the IncuCyte SX1®. For the optimization of the MOI and cell density, bovine monocyte-derived macrophages (MDMs) and the BOMAC cells were seeded at different cell densities and infected with MAP-GFP at different MOIs. After 2 h of infection, the supernatant was removed, the cells were washed three times with HBSS, and fresh RPMI-1640 supplemented with FBS was added to the cells. Then, the fluorescence was quantified in the IncuCyte SX1® at different times p.i. Our results demonstrated that MAP phagocytosis is pathogen number- and cell density-dependent. Overall, the results showed that infection of 1 × 106 cells at an MOI of 1:10 maintained a stable fluorescence signal of approximately 400 to 500 GCU ⋅ μm2/image across 2 h to 4 days p.i. In addition, good correlations between MAP-GFP (logCFUs) estimated in the BACTEC MGIT™ 960 system and fluorescence intensity measured in the IncuCyte SX1® were observed for free MAP-GFP bacilli and from MAP-GFP within infected BOMAC cells, r = 0.79 and r = 0.78, respectively. In conclusion, real-time fluorescent microscopy enables continuous monitoring of MAP-GFP within infected macrophages.