PCRs were performed to characterize notable bacterial species detected in tick samples through the NGS screening. The bacteria Anaplasma, Ehrlichia, Bartonella, Coxiella, Francisella and Rickettsia were targeted in the PCR amplification and sequencing. The details of all primers used for the bacteria species identification are described in Table S1.
PCRs targeting the citrate synthase gene (gltA), which amplified a 694 bp fragment, and cell division protein gene (ftsZ), which amplified a 900 bp fragment of Bartonella, were conducted in semi-nested and single PCR, respectively. For Francisella species characterization, a fraction of the T-cell epitope gene (tul4) and 16S rDNA of Francisella were targeted in a single PCR to amplify 248 bp and 1 kb fragments, respectively. Single PCRs were conducted for Rickettsia species characterization by targeting six genes: gltA gene for 580 bp, outer membrane A gene (ompA) for 542 bp, outer membrane protein B gene (ompB) for 816 bp, 17 kDa common antigen gene (htrA) for 550 bp, 16S rDNA for 1.3 kb and surface cell antigen-4 gene (Sca4) for 928 bp fragment. All PCRs for Bartonella, Francisella and Rickettsia were conducted using Ex Taq Hot Start Version (Takara Bio) in a reaction mixture of 20 µl. The conditions used in the PCR assays were as follows: 35 or 40 cycles of denaturation at 94 °C for 30 s, annealing temperature according to each respective primer set for 30 s, and extension at 72 °C for 30 s, 60 s or 90 s depending on the targeted amplicon size.
Next, to characterize the species of Anaplasma, Ehrlichia and Coxiella, we used Tks Gflex DNA Polymerase (Takara Bio) with a 25 µl reaction mixture preparation. Nested PCR was conducted for Anaplasma and Ehrlichia by amplifying a 1.3 kb fragment of 16S rDNA of Anaplasmatacea with the following conditions: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation step at 95 °C for 30 s, 48 or 54 °C of annealing for 30 s, and extension at 68 °C for 90 s, with a final extension at 68 °C for 5 min. A total of five genes were used for Coxiella species characterization, which included chaperone protein DnaK gene (dnaK) for 512 bp, chaperone protein GROEL gene (groEL) for 619 bp, β subunit of bacterial RNA polymerase gene (rpoB) for an estimate of 550 bp, 16S rDNA for an estimate of 1 kb and large ribosomal subunit (23S rDNA) for a 583–867 bp fragment. DNA of Coxiella was amplified with nested or semi-nested PCRs, with the following conditions: initial denaturation at 94 °C for 1 min, followed by 40 cycles of denaturation at 98 °C for 10 s, 54 or 56 °C of annealing for 15 s, and extension at 68 °C for 1 min, with a final extension at 68 °C for 5 min.
Finally, the amplicon size was verified with electrophoresis and visualized as described above. Sanger sequencing was performed on the successfully amplified samples using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems). The obtained sequencing products were analysed on an ABI Prism 3130X genetic analyzer (Applied Biosystems), as per the manufacturer’s instructions. The resulting sequences were assembled and trimmed using the ATGC software version 9.0.0 (GENETYX) and compared with the sequences available in the public databases using the Nucleotide Basic Local Alignment Search Tool (BLASTn) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
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