B. miyamotoi Culture Isolate

The B. miyamotoi strain Fr74B was obtained by the Centers for Disease Control and Prevention (Fort Collins, CO, USA), as a culture isolate. This strain was originally isolated from an infected Apodemus argenteus field mouse from Japan. The DNA from this strain was isolated by diluting the culture 1:10 with phosphate-buffered saline and heating to 95°C for 10 min. The raw lysate was then used in the Borrelia PCR/ESI-MS genotyping assay (Abbott Laboratories, Des Plaines, IL, USA) at 1 μL per PCR well (20).

Ixodes Tick Collection and Extractions

Ticks were obtained from most locations by flagging during 2008–2012. In Germany, a subset of ticks were also obtained after they were removed from persons. The species of Ixodes tick was determined by an entomologist and confirmed by the detection of the species-specific endosymbionts (19). The numbers and locations of the collection sites are described in Table 1.

Nucleic acids were extracted from ticks according to a published protocol by using bead-beating homogenization followed by isolation of RNA and DNA with DNeasy Blood and Tissue Kit columns (QIAGEN, Valencia, CA, USA) instead of the published QiaAmp Virus Elute Kits (21). A negative control consisting of a lysis buffer without a tick was with each set of extractions. Ticks from the United States were processed at Ibis Biosciences (Carlsbad, CA, USA). Ticks collected from the European countries were isolated at their respective sources. Nucleic acid samples from Germany and the Czech Republic were shipped to Ibis at ambient temperatures; those from Czech Republic were shipped after being stabilized by RNAstable (Biomatrica, San Diego, CA, USA) per the manufacturer’s instructions.

Molecular Detection and Genotyping of B. miyamotoi from Nucleic Acid Extracts

B. miyamotoi was detected and identified by using a previously described broad-range PCR/ESI-MS assay designed to detect tickborne pathogens (16). For each set of samples analyzed with the assay, an extraction negative control sample as well as a PCR plate negative-control sample of water was included. A PCR-positive control was already built into the plate for each well in the form of a calibrant (20). Amplicons were analyzed by using a research use only PLEX-ID system (Abbott Laboratories). Samples positive for B. miyamotoi were further characterized by using a Borrelia PCR/ESI-MS genotyping assay as described that is designed to differentiate between Borrelia species and genotypes (20). PCR/ESI-MS assay provides genetic information about the PCR amplicon in the form of A, G, C, and T basecounts, and B. miyamotoi detection was defined as positive when one or more primer pairs produced an amplicon basecount signature that was unique to B. miyamotoi. Although most researchers agree that the nymphal stage of Ixodes ticks is the most epidemiologically essential life stage for transmission of B. burgdorferi sensu lato, because little is known about the transmission of B. miyamotoi from Ixodes ticks to humans, the data for both nymphs and adults were combined.

Sequence Confirmation of B. miyamotoi Detections

Representative samples positive for B. miyamotoi were selected for 16S Sanger sequencing. Primers were designed to amplify a 676-bp region of the 16S rRNA gene for Borrelia. A M13 tag was added to each primer for sequencing. The M13 forward sequence tag was 5′-CCC AGT CAC GAC GTT GTA AAA CG-3′, and the reverse tag was 5′-AGC GGA TAA CAA TTT CAC ACA GG-3′. The forward primer used was 5′-M13-CGC TGG CAG TGC GTC TTA AG-3′, and the reverse primer was 5′-M13-GCG TCA GTC TTG ACC CAG AAG TTC-3′. The amplification of the 16S rRNA genes was performed in a 50 μL reaction containing 1 μL nucleic acid extract, 1 unit of Platinum Taq High Fidelity polymerase (Invitrogen, Carlsbad, CA, USA) or Immolase Taq (Bioline, Randolph, MA, USA), the manufacturer’s PCR buffer, 2.0 mmol/L MgSO₄, 200 μmol/L dATP, 200 μmol/L dCTP, 200 μmol/L dTTP, 200 μmol/L dGTP (Bioline), and 250 nmol/L of each primer. The following PCR cycling conditions were used on an MJ Dyad 96-well thermocycler (Bio-Rad Inc., Hercules, CA, USA): 95°C for 2 min, followed by 8 cycles of 95°C for 15 s, 50°C for 45 s, and 68°C for 90 s, with the 50°C annealing temperature increasing 0.6°C for each cycle. PCR was continued for 37 additional cycles of 95°C for 15 s, 60°C for 15 s, and 68°C for 60 s. The PCR cycle ended with a final extension of 4 min at 72°C. Reactions were visualized by electrophoresis on 1% agarose gels to ensure the presence of appropriately-sized products before being sent to SeqWright (Houston, TX, USA) for purification and sequencing with M13 primers. Resulting sequences were trimmed of primer sequences and a consensus created. The consensus sequence was analyzed with NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the nucleotide database to determine the species.

Multilocus PCR/ESI-MS Genotyping of B. miyamotoi

The multilocus Borrelia PCR/ESI-MS genotyping assay differentiates strains and species of Borrelia by their unique combination of basecount signatures. To characterize the prevalence of B. miyamotoi in Ixodes ticks we examined the basecount signatures from ticks that were positive for B. miyamotoi. Positive specimens from each of the 3 regions (United States, Europe, and Japan) typically produced basecount signatures at 5 of the 8 loci evaluated in the Borrelia genotyping assay. Based upon these 5 signatures, B. miyamotoi from the United States, Europe, and Japan are distinct genotypes (Table 2). All the specimens from North America had the same basecount signatures for the 5 detecting primer pairs. A separate signature combination was found for all of the European isolates detected in ticks from Germany and the Czech Republic. A third signature was observed from the CDC culture isolate from the Japanese strain. Although all 3 genotypes shared the same basecount for the locus BCT3515, the European genotype did not have any other basecount signatures in common with the other 2 genotypes. The North American and Japanese genotypes had the same signatures for 2 of the 4 remaining loci, BCT 3519 and BCT3511. We detected B. miyamotoi with 3 or more primers in the Borrelia genotyping assay in all but 4 of the 68 positive specimens. Several factors may explain why all 5 primers did not detect the bacteria, including nucleic acid quality and quantity or differences in primer sensitivities.

Prevalence of B. miyamotoi in Europe and the United States

I. ricinus ticks from the Czech Republic and Germany in Europe and I. scapularis and I. pacificus ticks from 5 states in the United States were screened for B. miyamotoi by PCR/ESI-MS. B. miyamotoi was found in all regions examined in varying degrees (Table 1) and in all 3 Ixodes species examined. Germany had a low incidence rate; only 4 of the 226 ticks tested were infected (1.8%). Incidence of B. miyamotoi infection of ticks from the Czech Republic varied by region and ranged from 0% to 3.2% with an average infection rate of 2%. In North America, the infection rates of ticks varied from 0% to 15.4%. All negative controls were negative and all positive controls were positive.

Sequence Confirmation of B. miyamotoi detections

Representative samples were selected for 16S rRNA sequencing: 1 sample from Pennsylvania in the United States, 1 from Germany, and 1 from the Czech Republic. The samples from Germany and the Czech Republic were identical (KF740842 and KF740841, respectively) and matched 99.11% (669 bp out of 675 bp) of the B. miyamotoi LB-2001 sequence, a North American isolate from the East Coast (GenBank accession no. NC_022079). The sample from Pennsylvania (KF740843) was identical (675 bp of 675 bp) to the B. miyamotoi LB-2001 sequence.