Paired real-time PCR assays for detection of Borrelia miyamotoi in North American Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae)

Abstract

Borrelia miyamotoi is an emerging, tick-borne human pathogen. In North America, it is primarily associated with Ixodes scapularis and Ixodes pacificus, two species known to bite humans. Here we describe the development and evaluation of a pair of real-time TaqMan PCR assays designed to detect B. miyamotoi in North American ticks. We sought to achieve sensitivity to B. miyamotoi strains associated with ticks throughout North America, the full genetic diversity of which is unknown, by targeting sequences that are largely conserved between B. miyamotoi strains from the eastern United States and genetically distinct B. miyamotoi strains from Japan. The two assays target different loci on the B. miyamotoi chromosome and can be run side by side under identical cycling conditions. One of the assays also includes a tick DNA target that can be used to verify the integrity of tick-derived samples. Using both recombinant plasmid controls and genomic DNA from North American and Japanese strains, we determined that both assays reliably detect as few as 5 copies of the B. miyamotoi genome. We verified that neither detects B. burgdorferi, B. lonestari or B. turicatae. This sensitive and specific pair of assays successfully detected B. miyamotoi in naturally-infected, colony-reared nymphs and in field-collected I. scapularis and I. pacificus from the Northeast and the Pacific Northwest respectively. These assays will be useful in screening field-collected Ixodes spp. from varied regions of North America to assess the risk of human exposure to this emerging pathogen.

Prevalence and Geographic Distribution of Borrelia miyamotoi in Host-Seeking Ixodes pacificus (Acari: Ixodidae) Nymphs in Mendocino County, California

“…DNA had been previously extracted using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for animal tissue (Girard et al 2009), eluted in 50 μl deionized sterile water, and stored at −20°C. We used a pair of real-time TaqMan PCR assays (Graham et al 2016) to detect B. miyamotoi : a singleplex assay targeting the glycerophosphodiester phosphodiesterase ( glpQ ) gene of B. miyamotoi , and a duplex assay targeting the adenylosuccinate lyase ( purB ) gene of B. miyamotoi and the actin gene in I. scapularis and I. pacificus . The actin target allowed us to verify the presence of amplifiable DNA in each sample.…”

“…The actin target allowed us to verify the presence of amplifiable DNA in each sample. Each reaction included 4.8 μl DNA extract and 5.2 μl master mix as described in Graham et al (2016).…”

“…Controls on each plate included a no-template control (elution buffer), DNA extracted from uninfected, colony-reared I. scapularis nymphs (Centers for Disease Control and Prevention, Fort Collins, CO), and recombinant plasmid DNA containing the purB and glpQ target sequences as previously described (Graham et al 2016). Samples were considered positive if Cq values for both B. miyamotoi targets were ≤40.…”

Transmission of the relapsing fever spirochete, Borrelia miyamotoi, by single transovarially-infected larval Ixodes scapularis ticks

“…Fed larval ticks were homogenized in 350 μl of lysis buffer using a Mini-Beadbeater-96 (BioSpec Products, Inc., Bartlesville, OK, USA) as previously described (Graham et al, 2016, 2018). Spent female ticks were homogenized by hand in 500 μl of lysis buffer using a 1.5 ml microcentrifuge tube and a pestle.…”

“…The subsequent real-time TaqMan PCR reactions included primers and probes for the following DNA targets: a pan Borrelia 16S rDNA target (Kingry et al, 2018) and the adenylosuccinate lyase ( purB ) target for B. miyamotoi (Graham et al, 2016). PCR and DNA purification controls included the I. scapularis actin target (Hojgaard et al, 2014) for tick samples and the rodent GAPDH target (Applied Biosystems ® TaqMan ® Rodent GAPDH ControlReagents kit; ThermoFisher) for mouse blood samples.…”