Quantifying Bacterial Loads in Spleen and Lung

To determine bacterial loads, portions of spleens and lungs were collected and homogenized by using a FastPrep-24 homogenizer (MP Biomedical). DNA was then extracted using a DNeasy Blood & Tissue Kit (Qiagen) and used for qPCR assays, as previously described (5 (link)). The primers were OtsuF630 (5’-AACTGATTTTATTCAAACTAATGCTGCT-3’) and OtsuR747 (5’-TATGCCTGAGTAAGATACGTGAATGGAATT-3’) (Integrated DNA Technologies). Bacterial loads were normalized to total nanogram (ng) of DNA per µL for the same sample, and data are expressed as the gene copy number of 47-kDa protein per ng of DNA. The copy number for the 47-kDa gene was determined by known concentrations of a control plasmid containing single-copy insert of the gene. Gene copy numbers were determined via serial dilution (10-fold) of the O. tsutsugamushi Karp 47-kDa plasmid.