The South African clawed frog epithelial cells (XTC-2) were grown in Leibovitz L-15 (Gibco) medium supplemented with 10% NBC (Gibco), 0.4% tryptose phosphate broth (Oxoid, UK) and 1% L-glutamine (Gibco) and incubated at 28 °C in 5% CO2. Six 24-well trays (IWAKI, Japan) each containing the four cell culture types were grown to confluency. Each well contained

2 mL of medium. Dilutions 10−6–10−11 (Arandale isolate) or 10−5–10−10 (Henzerling PI3K inhibitor strain) were used to infect the cell cultures. Six wells of each cell culture type were inoculated with 100 μL of each dilution of C. burnetii. Cultures were incubated for 6 weeks before the monolayer from each well was harvested by scraping. Cells were pelleted by centrifugation for 5 min at 4500  g and resuspended in 300 μL of phosphate-buffered saline (PBS; Belnacasan nmr Oxoid) and analysed by DNA extraction and Com1 PCR. The DNA

was extracted from 200 μL by Qiagen Extraction Kit (Qiagen, Germany), following the manufacturers’ instructions, eluted into 50 μL and analysed by specific PCR targeting a 76-bp sequence of the com1 gene (Lockhart et al., 2011). Extracted DNA (5 μL) was analysed for each reaction. The cycling threshold resulting form the PCR was used to calculate the approximate C. burnetii DNA concentration (μg μL−1) in each reaction. The C. burnetii dose that would infect 50% of cultures (ID50) was calculated using the Spearman–Kärber method (Anellis & Werkowski,

1968). The dilutions of the inoculum were analysed by PCR, and a standard curve was made (data not shown) and used to convert the ID50 calculation from a dilution into a number of bacterial copies required for 50% infection. By determining which wells contained C. burnetii DNA in amounts to suggest growth of the bacteria, the ID50 could be determined for each cell line and C. burnetii isolate. The cell line most susceptible (sensitive) to infection was different for the two C. burnetii isolates (Table 1). Fludarabine For the Arandale isolate the Vero cell line was the most sensitive with an ID50 of 0.1 copies of C. burnetii, followed by the L929 cell line with an ID50 of 3.2 copies. For the Henzerling strain, the DH82 cell line was the most sensitive with an ID50 of 14.6 copies of C. burnetii followed by the L929 cell line with an ID50 of 22.0 copies. Number of C. burnetii (copy numbers per 100 μL) required for 50% infection of cell line Number of C. burnetii (copy numbers per 100 μL) required for 50% infection of cell line During the growth of C. burnetii the monolayers were routinely observed under light microscopy. Only in Veros could infection with C. burnetii be seen as large vacuoles in the cell cytoplasm.