Prevalence of Borrelia miyamotoi in Ixodes Ticks in Europe and the United States

Infection rates of Ixodes ticks with Borrelia miyamotoi in Europe and the United States vary greatly based upon location.

I xodes ticks can transmit a variety of pathogens, including viruses, bacteria, and protozoa (1). Borrelia spirochetes are one of the genera of bacteria transmitted by Ixodes ticks. Most Borrelia that infect ticks belong to the Borrelia burgdorferi senso lato group and include B. burgdorferi senso stricto, B. garinii, and B. afzelii, all of which cause Lyme disease in humans (1). Borrelia miyamotoi has been found in a variety of Ixodes ticks and is more closely related to the relapsing fever spirochetes that infect soft ticks than to the bacteria that cause Lyme disease (2).
B. miyamotoi found in Europe and the United States also cause disease in humans (3)(4)(5). A study in Russia has shown that the spirochete B. miyamotoi has the ability to infect humans; infections with B. miyamotoi cause symptoms similar to those seen with relapsing fever, as well as erythema migrans-like skin lesions on rare occasions (6). B. miyamotoi has been found in ticks of the following species: Ixodes scapularis and I. pacifi cus in the United States, I. persulcatus in Japan, and I. ricinus and I. persulcatus in Europe and Asia (2,(7)(8)(9)(10)(11). In North America, B. miyamotoi has been found as far north as the Canadian provinces of Ontario and Nova Scotia (12). In the United States, the geographic range of B. miyamotoi is from the Northeast to California and has been reported as far south as Tennessee (7,8,(13)(14)(15). Previous studies have shown that B. miyamotoi can be placed into different genetic groups based upon its geographic location and has some variation within the genographic groups (6,9).
To examine the prevalence distribution and diversity of B. miyamotoi in Ixodes ticks, we screened individual ticks by PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to detect tickborne pathogens, including B. miyamotoi (16). This approach has been used to characterize tickborne microorganisms, including Ehrlichia and Borrelia, from clinical specimens, heartworms in canine blood, and naturally occurring tick endosymbionts (16)(17)(18)(19). Ticks that tested positive for B. miyamotoi were further characterized by using a Borrelia genotyping assay to assess genetic diversity (20).  (20).

Ixodes Tick Collection and Extractions
Ticks were obtained from most locations by fl agging 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 confi rmed by the detection of the species-specifi c 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 (Carls-bad, 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 identifi ed 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 defi ned 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. 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 fi nal 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 purifi cation 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. pacifi cus 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.

Discussion
In this study, we identifi ed 3 distinct B. miyamotoi genotypes in the United States, Europe, and Japan. Results show that B. miyamotoi is widely distributed across North America and Europe.We observed no genotypic differences using this PCR/ESI-MS assay between the B. miyamotoi detected in I. scapularis from the eastern US states and the midwest or between these bacteria and the B. miyamotoi detected in I. pacifi cus from California. In a study by Mun et al., a 766-bp region of the fl agellin gene sequence were shown to have and a 0.9% difference between B. miyamotoi found in I. pacifi cus and those found in I. scapularis in the United States (8). However, our fl agellin primers targeted a region of the fl agellin gene that does not contain the differences identifi ed by Mun et al., thus explaining why we found a single North American genotype. Previous studies that examined the sequence of the 16S rRNA gene from multiple B. miyamotoi strains indicated that strains from the United States and Europe were located in their own clusters (6). The Japanese strain FR64b grouped with isolates found in infected humans and I. persulcatus ticks in Russia, whereas the B. miyamotoi found in I. ricinus ticks from Russia grouped with those found in Europe (6). In our genetic analysis, the Japanese strain also differed from that found in I. ricinus in Europe.
Our study demonstrates that the presence of B. miyamotoi in Ixodes ticks is widespread across the regions examined and was observed in all 3 species of fi eld-collected Ixodes ticks. In Europe we observed B. miyamotoi in 2.0% of I. ricinus ticks tested, consistent with the detection rates in other studies examining I. ricinus prevalence at other locations in Europe (9,10). Our detection rates were also similar to those seen in an earlier study on ticks from Mendocino County, California (8). I. scapularis ticks from the East Coast region (New York, Connecticut, and Pennsylvania) were found to have infection rates ranging from 0% to 6.8% for ticks. In Indiana, however, a much higher percentage, 12%, of I. scapularis ticks examined were infected with B. miyamotoi. Other stu dies have also shown that local site-to-site prevalence of B. miyamotoi can vary greatly from the overall regional mean (13).
Our study indicates that B. miyamotoi is likely present in any region where Ixodes ticks reside but that infection rates can vary greatly by region. Since the original description of B. miyamotoi as a human pathogen, studies have shown clinical infection in both healthy and immunocompromised patients in both Europe and the United States (3)(4)(5)(6)22). If physicians know the regional infection rate in ticks, they will be alert for possible exposure risks for their patients. Standard Lyme borreliosis serologic tests offered by commercial laboratories cannot be relied on to detect B. miyamotoi infection in patients. B. miyamotoi has been shown to have transovarial transmission, suggesting that larval ticks may also pose a risk (7). Little is yet known about the transmission rates to humans, and further studies are required to better gauge the risk to humans in these B. miyamotoi-endemic regions.