Establishment of placental 3-dimensional-(3D)-spheroid cultures to study the tropism of Coxiella burnetii for ovine trophoblasts

The tropism of the zoonotic, obligate intracellular bacterium Coxiella burnetii (C. burnetii) [Q-fever agent] for ovine trophoblasts in placen-tal tissues is difficult to study. Thus, we established 3D-spheroid models deploying ovine AH-1 trophoblasts to investigate the spread of C. burnetii in tissue-like culture models. Spheroids were formed via hanging drops (named: AH-1HD) or poly-HEMA (AH-1PH) and characterized by immunohistochemistry (IHC: cytoskeleton, cell turnover) and transmission electron microscopy [TEM]. AH-1HD (diameter: 200 μm) developed within 3 days and AH-1PH (diameter: 1000 μm) within 10 days. AH-1HD consisted of intact cells, while AH- 1PH had an inner degenerative core surrounded by intact AH-1 cells. All cells of AH-1HD and the intact AH-1 cells of AH-1PH expressed cytokeratin 18, ezrin, SHMT2 and Ki-67. Active caspase-3 was mainly detected in the core of AH-1PH. AH1-HD spheroids were infected with C. burnetii (axenically [cell free] grown,100 genome equiva-lents as quantified by icd- qPCR) for 5–17 days. C. burnetii infection was detected by IHC. Following infection, C. burnetii spread rapidly through AH-1HD until 14 days post-infection (dpi) at which all cells contained Coxiella-containing parasitophoric vacuoles (PV). Upon transfer of supernatants collected from infected AH-1HD at 14 dpi to AH-1PH spheroids, newly infected cells were detected in AH-1PH within 3 dpi. Both spheroidal models developed typically in vivo-like lesions like the formation of intracellular C. burnetii- containing para-sitophoric vacuoles after infection. Therefore, the AH-1 trophoblast spheroids are promising ex vivo tools to study the tropism of C. bur-netii for ovine trophoblasts under controlled conditions.