When 80% confluent, the cells were infected with a predetermined dilution of O. tsutsugamushi (isolate UT76) inoculum and incubated at 35 °C with 5% CO2 using maintenance media (5% FBS + RPMI 1640, (Gibco, Carlsbad, CA, USA)) for 8 hours. Following incubation, the infected cells were fixed and permeabilized in acetone for 10 min at −20 °C and allowed to air dry. Indirect immunofluorescence (IFA) was performed to visualize the intracellular O.

tsutsugamushi organisms. The coverslips were incubated with pooled human serum (diluted 1:320 in PBS) from O. tsutsugamushi confirmed-patients at 37 °C for 30 min, washed twice with PBS, then further incubated with FITC-conjugated goat antihuman IgG (Gibco) diluted ERK inhibitor mouse 1:40 in PBS for 30 min at 37 °C. The monolayer was then washed twice with PBS and the cells were counterstained with 0.00125% (w/v) Evans blue. The infected cells were visualized by epifluorescence microscopy (Nikon Eclipse 80i, Nikon Corp., Chiyoda-ku, Tokyo, Japan). Images of O. tsutsugamushi infected in cell culture were captured by digital camera (Nikon Digital Sight DS-5M-L1, Japan) at a 400× magnification. The method for enumeration of O. tsutsugamushi using ImageJ required the

image file to be converted from RGB color to 8-bit grayscale. The manual counting of the O. tsutsugamushi particles was performed using the built-in cell-counter plugin of the ImageJ program. After opening the image to be counted, the cell-counter plugin was opened (commands used: Plugins > Analyze > Cell Counter), ‘internalize’ and

‘Type 1’ selected. The Orientia particles Ruxolitinib mw were manually counted by the operator by moving the crosshairs over the particle and confirming the identity of Casein kinase 1 the particle by clicking the mouse button. The number of Orientia particles selected was then displayed within the plugin. Automated counting of the O. tsutsugamushi particles uses threshold algorithms to discriminate the features of interest from background. The threshold level is dependent on the algorithm selected and in this study Minimum, MaxEntropy, RenyiEntropy and Yen threshold algorithms 4, 5 and 6 were used however another twelve algorithms were assessed and found to be unsuitable for this application. To set the counting threshold following opening the selected image, the following commands Image > Adjust > Threshold > select algorithm to be applied > Apply were used and the image converted to a binary image by selecting Process > Binary > Make binary. O. tsutsugamushi particles were counted using the commands Analyze > Analyze Particles, with the the upper and lower limits for the particle size set at 0–infinity, selected to ‘Show outlines’ and checked box to ‘Summarize’. Each counted particle was outlined and numbered in a new window. Twenty-five IFA image fields were digitally photographed and the images processed as described above. O.