Genomic DNA was extracted from whole blood samples using the DNeasy Blood and Tissue Kit (Qiagen, Melbourne, Australia) according to the manufacturer’s protocol, and its quantity and quality were measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) before storage at − 20 °C until analysis.
Screening was performed by nested PCR using the AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Korea) and designated primer sets. Anaplasma spp. were detected based on amplification of the 16S rRNA gene using the primer sets EE1/EE2 and EE3/EE4 [19 (link)]. Borrelia spp. were identified based on the presence of the 5S (rrf)–23S (rrl) intergenic spacer using primer sets Bb23S3/Bb23Sa and Bb23SnF/Bb23SanR, and B. burgdorferi was detected by amplification of the outer surface protein A gene fragment using primer sets N1/C1c and N2/C2c [20 (link)]. Hemoplasmas were first identified based on the amplification of 16S rRNA with universal primers fHf1/rHf2 and M. suis-specific primers f2/r2 [16 (link), 21 (link)]; positive results were then confirmed at the species level by PCR using cmsf2/cmsr2 and msf2/msf2 primer sets to amplify the 16S rRNA gene of M. suis, M. parvum, and the novel hemotropic M. haemosuis [12 (link)].
Screening was performed by nested PCR using the AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Korea) and designated primer sets. Anaplasma spp. were detected based on amplification of the 16S rRNA gene using the primer sets EE1/EE2 and EE3/EE4 [19 (link)]. Borrelia spp. were identified based on the presence of the 5S (rrf)–23S (rrl) intergenic spacer using primer sets Bb23S3/Bb23Sa and Bb23SnF/Bb23SanR, and B. burgdorferi was detected by amplification of the outer surface protein A gene fragment using primer sets N1/C1c and N2/C2c [20 (link)]. Hemoplasmas were first identified based on the amplification of 16S rRNA with universal primers fHf1/rHf2 and M. suis-specific primers f2/r2 [16 (link), 21 (link)]; positive results were then confirmed at the species level by PCR using cmsf2/cmsr2 and msf2/msf2 primer sets to amplify the 16S rRNA gene of M. suis, M. parvum, and the novel hemotropic M. haemosuis [12 (link)].
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