Introduction: The intracellular bacterium Coxiella burnetii causes the zoonotic disease Q fever and, in Australia, human cases are most commonly reported as associated with cattle. However there remains limited understanding of mammary gland C. burnetii infection dynamics and subsequent milk shedding. Three-dimensional (3D) cell models potentially bridge the physiological gap between in vitro and in vivo experiments. To address the increased morphological complexity of the 3D model, an automated, high-content, quantitative C. burnetii bovine mammary disease model was developed to investigate bacterial uptake in bovine mammary epithelial cells (bMEC).
Methods: The bMEC line, MAC-T, was cultured on extracellular matrix for formation of 3D spheroids replicating the ultrastructure of mammary alveoli. Fluorescently labelled, fixed C. burnetii phase II Clone 4 was inoculated at a range of multiplicity of infections (MOI: 0, 100, 250, 500, 750, 1000). Fluorescent confocal (PerkinElmer Opera Phenix Plus) images were captured using the automated PreciScan routine of an initial 5x scan to locate spheroids in the X, Y and Z planes followed by a high resolution 20x scan. Image analysis measured internal C. burnetii fluorescence per MOI treatment, standardised per µm3 and spheroid morphology (diameter, height, volume, surface area, sphericity). Replicated experiments were completed in triplicate with results analysed using a linear mixed model.
Results: The 3D cell culture conditions and image analysis protocols were optimised for compatibility with an automated, high-content quantitative assay. The model was further characterised, including validating the quantitation method, determining the bacterial uptake saturation point and the spheroid morphology variability. Preliminary results show average bacterial fluorescence intensity significantly increased between MOIs 0, 250, 500 and 750 but there was no significant difference between MOI 750 to 1000.
Discussion: The 3D architecture and objective assay that define this disease model may expand the scope of investigation of C. burnetii mammary epithelial cell pathogenesis and improve in vitro predictive power. Future cell uptake work can use an optimal MOI to maximise measuring range of any effect. Further, the high-content capabilities combined with the PreciScan routine balances the trade-off between Z plane detail and unusable data to increase analysis speed, making this approach feasible.