Dna extraction

DNA extraction and molecular analysis

Individual ticks were bisected longitudinally using sterile scalpels and washed with distilled water to remove residual ethanol. DNA was extracted using the commercial kit GeneJET Genomic DNA Purification Kit (Thermo Scientific, Lithuania) following manufacturer instructions. To confirm nymph tick species, ticks were analyzed through PCR amplification of a ~460 base pair (bp) fragment of the tick mitochondrial 16S rRNA gene ( Mangold et al., 1998).

For the detection of microorganisms, extracted DNA was tested by a battery of PCR protocols targeting Rickettsia , Anaplasmataceae family organisms, Borrelia , Babesia and Hepatozoon spp. , using specific primers and published protocols for each agent ( Table 2). Molecular grade water was used as negative control in all PCR reactions. Positive controls used included Rickettsia parkeri strain Toledo, Ehrlichia canis isolate P1091, Borrelia anserina PL , Babesia bovis Paysandú, Hepatozoon sp. (Z78S, P1244), and Ixodes fuscipes S18IpH1. Five microliters of PCR products were separated by electrophoresis in a 1.5% agarose gel, stained with GoodView TM Nucleic Acid Stain (Beijing SBS Genetech Co., Ltd), and examined under UV transillumination. Amplicons of expected size were purified using GeneJET PCR purification kit and Sanger sequenced (Macrogen, Korea).