Dnaeasy tissue kit

DNA was extracted from all isolates using a DNAeasy Tissue Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. The 16S rRNA gene was amplified and sequenced using 27F and 1492R primers, according to the conditions described by Lane (1991) . PCR products were sequenced and used to identify isolates. Once a strain was identified as Bacillus, Exiguobacterium or Pseudomonas, we amplified and sequenced a set of housekeeping genes commonly used for MLST (Cerritos et al., 2011 (link); Yamamoto et al., 2000 (link); Sarkar & Guttman, 2004 (link); Rodrigues et al., 2006 (link)). Genus-specific PCR primers (Table S1) were used. All PCR products were sequenced at the University of Washington’s High Throughput Genomics Center. Sequences were deposited in the GenBank database with the following accession numbers: Bacillus citC (KC900996–KC901178); Bacillus gltx (JQ241465–JQ241624); Bacillus hsp70 (KC901179–KC901361); Bacillus recA (JQ247793–JQ247952); Bacillus spo0A (KC901362–KC901540); Exiguobacterium citC (JF916988–JF917080, JF952020–JF952109); Exiguobacterium hsp70 (JF952111–JF952292); Exiguobacterium recA (JF952293–JF952475); Exiguobacterium rpoB (JF952476–JF952658); Pseudomonas acnB (KC953704–KC953753); Pseudomonas gyrB (KC920532–KC920576); Pseudomonas recA (KC961435–KC961462); Pseudomonas rpoD (KC920481–KC920531).

Genomic DNA was extracted from liver tissue of five individuals of Cnemaspis (Table 1) using the Qiagen DNAeasy tissue kit (Valencia, CA, USA). A 1,251 bp fragment of mitochondrial (mt) DNA consisting of the NADH dehydrogenase subunit 2 (ND2) gene and the flanking tRNAs Trp, Ala, Asn, and Cys was amplified using polymerase chain reaction (PCR) under the following conditions: initial denaturation at 95 °C for 2 min, followed by a second denaturation at 95 °C for 35 sec, annealing at 52 °C for 35 sec, followed by a cycle extension at 72 °C for 35 sec, for 33 cycles using the light strand primer L4437b (5’-AAGCAGTTGGGCCCATACC-3’; Macey et al. 1997 (link)) and heavy strand primer H5934 (5’ AGRGTGCCAATGTCTTTGTGRTT-3’; Macey et al. 1997 (link)). PCR products were purified using the AccuPrep PCR Purification Kit (Bioneer, Daejeon, Korea), and were sequenced using the amplifying primers on an ABI 3730 automatic sequencer (Applied Biosystems, CA, USA). Sequences were edited and aligned using Geneious R11 (Biomatters, Ltd, Auckland, New Zealand). All new sequences were deposited in GenBank under accession numbers MT112890–MT112894 (Table 1).

Total RNAs from stored skin biopsies were extracted using the RNAeasy fibrous tissue mini kit (Qiagen S.A., Courtaboeuf, France) and treated with RNase-free DNase to remove contaminant genomic DNA according to the manufacturer's instructions. Genomic DNA was also isolated using the DNAeasy tissue kit (Qiagen S.A., Courtaboeuf, France) according to the manufacturer's instruction. The quality and quantity of isolated RNA and DNA were measured using a GENESYS 10 UV spectrophotometer (Thermo, USA) and by calculating the ratio of optical density at A260/A280. RNA and DNA integrity were checked using 1.5% formamide-agarose gel electrophoresis and 0.8% agarose gel electrophoresis, respectively. RNA and DNA samples with good quantity and quality were stored at −80°C for further analysis. The first strand cDNA was synthesized using 2 μg of total RNA with 10 pmol OdTm primer (Table 1), 0.5 mM dNTPs, 1 × RT buffer, 20 U RNase inhibitor, and 200 U PrimeScript Reverse Transcriptase (Takara Biotech, Japan) in a 20 μL total reaction volume according to the manufacturer's instructions. The reaction mixture was incubated for 45 min at 50°C and then at 70°C for 15 min; the resulting cDNA was used in coding sequence and 3′UTR amplification. All PCR reactions were carried out using a Perkin-Elmer Thermal Cycler (Perkin-Elmer Corporation, Norwalk, CT, USA).

Genomic DNA was extracted from the foot tissue using a DNAeasy Tissue Kit (QIAGEN Inc.). The mitochondrial 16S ribosomal RNA (16S rRNA) region and cytochrome c oxidase subunit I (COI) gene region were PCR amplified using the 16sar and 16sbr²⁸ and the LCO1490 and HCO2198^{29 (link)} primer pairs, respectively. The nuclear 28S ribosomal RNA (28S rRNA) region was PCR amplified using primers 28SF4 and 28SR5^{30 (link)}. The thermal cycling conditions for amplification of the 16S rRNA and 28S rRNA were 2 min at 94 °C, followed by 36 cycles of 30 s at 94 °C, 30 s at 50 °C and 90 s at 72 °C, and then a final 5 min at 72 °C. That for the amplification of COI was performed in the same condition except that the annealing stage was changed to 2 min at 42 °C and the extension time to 2 min. Products were resolved and visually checked by 1% (w/v) agarose gel electrophoresis with SYBR Safe staining and blue light transillumination. A QIAquick purification Kit (QIAGEN Inc.) was used to purify the PCR products before being sent for commercial automatic cycle-sequencing at Macrogen, Inc. (Seoul, South Korea).

DNA extraction was done following the chelex protocol described by Estoup et al. [33 ]. Briefly, a small sample of the tissue was cut and placed in a 1.5 mL tube; then, 500 μL of chelex and 7.5 μL of proteinase k were added. The samples were then placed in an oven at 55 °C for 1 h ½ and shaken every 15 min. To inactivate the proteinase k, the samples were heated at 100 °C for 20 min. The samples that did not give a successful DNA yield were re-extracted using the DNAeasy tissue kit (Qiagen, Hileden Germany) following the manufacturers’ protocol.

A total of 543 questing ticks and 295 bird-fed I. ricinus ticks were tested for the prevalence of tick-borne bacteria. DNA was isolated from ticks individually. For questing ticks, alcaline-hydrolysis method [65 (link)] was used. DNA from bird-fed ticks was isolated with the commercial extraction kit (DNAeasy tissue kit, Qiagen, Hilden, Germany). DNA samples were stored at −20 °C. A 620 bp fragment of tick mitochondrial gene cytochrome b was amplified in each extracted tick DNA to confirm the presence of tick DNA [66 (link)]. Only positive samples were further analyzed for the presence of tick borne agents.