The ticks were screened for spotted fever group rickettsia using a Power SYBR Green real-time PCR Mastermix kit (Life Technologies, Carlsbad, CA, USA) and primers for the romp A gene (Eremeeva et al., 2003). Reactions were carried out in a total volume of 20 μL composed of 10 µL Power SYBR Green Mastermix, 0.125 μL each of primers RR190.547F (20 μM) and RR190.701R (20 μM), 7.75 μL of nuclease-free water, and 2 μL of template DNA (Eremeeva et al., 2003; Wikswo et al., 2008). Real-time PCR cycling conditions were: 3 min at 95 °C; 40 cycles of: 20 s at 95 °C, 30 s at 57 °C, 30 s at 65 °C; a holding cycle of 5 min at 72 °C; and a continuous cycle of: 15 s at 95 °C, 1 min at 55 °C, 30 s at 95 °C, 10 s at 55 °C; and a final holding temperature of 4 °C.