Detalle de: A High-Throughput and Real-Time Imaging Assay to Quantify <em>Mycobacterium avium</em> Subsp. <em>Paratuberculosis</em>

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. However, this method has not yet been evaluated for real-time quantification of free MAP bacilli or MAP-infected macrophages. The objective of this study was to develop an assay based on automated and real-time live-cell imaging (IncuCyte®) for quantifying green fluorescence protein (GFP)-expressing MAP and to monitor MAP growth within infected macrophages. The fluorescence of serially diluted free MAP–GFP bacilli was quantified using the IncuCyte® 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®. For the optimization of the MOI and cell density; bovine monocyte-derived macrophages (MDMs) and the BOMAC cells were seeded at different cell densities, infected with MAP-GFP at different MOIs, and after 2 h p.i. the fluorescence was quantified in the IncuCyte® 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® were observed for free MAP-GFP bacilli and from MAP-GFP within infected BOMAC cells; r2 = 0.79 and r2 = 0.78, respectively. In conclusion, real-time fluorescent microscopy is a reliable method for quantifying fluorescent MAP and enables continuous monitoring of bacterial load within infected macrophages.