Nucleospin plasmid quickpure

For sequencing, 3–5 positive samples per detected pathogen were randomly selected. Amplicons were purified using a QIAquick Gel Extraction Kit (Qiagen, Germany) per the manufacturer’s protocol. The concentration of the extracts was checked using the Nano Drop 2000 spectrophotometer. The template (6 µL) was ligated into a pGEM-T Easy vector (2 µL) (Promega, US), with T4 DNA ligase and restriction buffer (each 1 µL) added and incubated at 16 °C for 3 hours and then at 4 °C overnight. Transformation of the plasmid into Escherichia coli DH5α competent cells (prepared in-house) was performed. Lysogeny broth (LB) was added and incubated at 37 °C in a shaker incubator for 1 h and then inoculated on LB agar plates and incubated at 37 °C overnight. Colonies were picked and put in LB broth with an antibiotic (Ampicillin) 50 µg/mL (Wako, Saitama, Japan) incubated at 37 °C overnight in a shaker incubator. Plasmid was extracted using the NucleoSpin^® Plasmid QuickPure (Macherey-Nagel-Germany) kit. The samples were sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, US) and a 3100 Genetic Analyzer (Applied Biosystems, Foster city, Calafornia, US). The alignment of the sequences was performed using Lasergene v14.1 (DNASTAR, Madison, WI, US). The nucleotide sequence identities and similarities were determined by using a GenBank BLASTn analysis.

Following PCR assays, B. ovis, T. ovis, A. ovis, and A. phagocytophilum amplicons were selected for sequencing. Agarose gel elution was employed to purify amplified PCR products using QIAquick Gel Extraction Kit (Qiagen, Germany), and eluted DNA concentrations were measured on a NanoDrop 2000 spectrophotometer. The extracts were then cloned into a pGEM vector according to the commercial protocol of pGEM^®-T Easy Vector System (Promega, USA). The template (6 µL) was ligated into pGEM-T easy vector (2 µL) using T4 DNA ligase and restriction buffer. Thereafter, the mixture was incubated at 16 °C for 2.5–3 h and kept at 4 °C overnight. Plasmid was transformed into Escherichia coli DH5α competent cells. LB broth was added to every tube and incubated at 37 °C in a shaker incubator for at least 1 h. Meanwhile, LB agar plates were warmed up at 37 °C. After centrifugation (2500 rpm for 3 min) and removal of the supernatant, the remaining mixture was spread on LB agar plates using a spreader, followed by incubation at 37 °C overnight. Recombinant clones from this transformation were then selected to be sequenced, transferred in LB broth with ampicillin (50 µg/mL) (Wako, Saitama, Japan), and incubated at 37 °C overnight in a shaker incubator. The plasmid was extracted from this culture using the Nucleospin^® Plasmid QuickPure (Macherey-Nagel-German) Kit.