Lyme borreliosis (LB) is a multisystem disease caused by spirochetes of the Borrelia burgdorferi sensu lato (Bbsl) complex (Steere, 2001; Stanek et al., 2012). It is the most common vector-borne disease in temperate regions of the northern hemisphere, causing an estimated 300 000 annual cases in the United States and 85 000 annual cases in Europe (Lindgren & Jaenson, 2006; Hinckley et al., 2014). To date, 20 Bbsl genospecies have been described, with a subset of these considered pathogenic for humans. Whereas Borrelia garinii, Borrelia afzelii and Borrelia bavariensis cause the majority of LB cases in Eurasia, B. burgdorferi sensu stricto is the predominant cause of LB in the USA (Margos et al., 2013; Schotthoefer & Frost, 2015; Stanek et al., 2012). Among humans, LB is characterized by tissue localization and low levels of spirochetemia (van Dam et al., 1993; Aguero-Rosenfeld et al., 2005; Liveris et al., 2012; Babady et al., 2008). This is in contrast to infection by relapsing fever borreliae in which spirochete loads range from 10⁵ to >10⁶ ml⁻¹ of blood (Dworkin et al., 2008).

We recently reported the identification of a novel Bbsl genospecies in six patient specimens (five blood and one synovial fluid) submitted to the Mayo Clinic laboratory (Rochester, MN, USA) for LB testing using a real-time PCR assay that uses hybridization probes and targets the oligopeptide permease A2 gene (oppA2) (Pritt et al., 2016). This PCR assay detects and differentiates Bbsl genospecies by melting temperature (T_m) analysis (Babady et al., 2008; Pritt et al., 2016). The patients were all residents of the upper midwestern USA (Minnesota, Wisconsin and North Dakota) and reported tick exposure in Minnesota and Wisconsin during 2012–2014. Patients presented with somewhat differing clinical presentations when compared with patients infected with B. burgdorferi sensu stricto including diffuse macular rash not typical of erythema migrans (EM), nausea or vomiting and symptoms potentially consistent with neurological involvement. By oppA2 quantitative PCR and dark-field microscopy, the genome and spirochete load, in blood of acutely ill patients, was estimated to be approximately 10⁵ ml⁻¹, which is higher than previously reported among patients infected with other Bbsl genospecies (Bil-Lula et al., 2015; Liveris et al., 2012). Motile spirochetes were cultured from blood of two patients under microaerophilic conditions at 34 °C in modified Barbour–Stoenner–Kelly (BSK) medium. After freezing at −80 °C, the isolates, MN14-1420 and MN14-1539, were transferred to BSK-II (without gelatin) (Zückert, 2007) and grown to densities of over 10⁷ spirochetes ml⁻¹.

Nucleic acid of the novel Bbsl genospecies was also detected by oppA2 PCR in Ixodes scapularis ticks collected near the approximate sites of possible patient exposures in Barron County, Wisconsin and Clearwater County, Minnesota during 2013–2015 and in I. scapularis ticks collected in three other counties (Eau Claire County, Wisconsin and Morrison and Pine Counties, Minnesota) during 2010–2015 (Table S1, available in the online Supplementary Material) (Pritt et al., 2016). Overall, 29 (2.1 %, range 0%–5.2 %) out of 1381 adult and 6 (1.5 %, range 0%–3.7 %) out of 399 nymphal I. scapularis ticks tested were PCR-positive for the atypical Bbsl genospecies (Supplemental Methods, available in the online Supplementary Material). Of the 35 total ticks testing positive for DNA of the novel Bbsl genospecies, 13 (37.1 %) were also positive for B. burgdorferi sensu stricto (Table S1).

Sequencing performed on the oppA2 (149 bp) PCR products directly amplified from both patients and an I. scapularis tick collected in Wisconsin showed 89–95% similarity to genospecies of the Bbsl complex (Pritt et al., 2016). The closest sequence identity for the 16S rRNA gene (1327 bp), ospC (561 bp), flaB (435 bp) and rrf–rrl (253 bp) sequences from the two patient isolates was to genospecies of the Bbsl complex at 99, 85, 97 and 95 %, respectively (Pritt et al., 2016). The taxonomic relationship of the atypical Bbsl isolates, MN14-1420 and MN14-1539, was determined using an eight-housekeeping-gene multi-locus sequence analysis (MLSA) (clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA) developed by Margos and colleagues for delineation of Bbsl genospecies (Margos et al., 2009, 2010). A genetic-distance analysis using concatenated sequences of the eight MLSA loci (4785 nucleotides) demonstrated that the distance to B. burgdorferi sensu stricto (~0.051–0.053) and other species of the genus Borrelia (>0.053) exceeded the previously determined threshold for species determination (0.017) (Table S2). The MLST sequence types (STs) are ST674 for MN14-1420 and ST675 for MN14-1539. By seven-gene MLSA, the genospecies of the Bbsl complex detected in I. scapularis ticks was confirmed to be the same as that identified in patients (Pritt et al., 2016). Taken together, these analyses reveal a distinct genospecies of the Bbsl complex among patients with LB and I. scapularis ticks from the upper midwestern USA. Further work is needed to determine the geographic distribution of infected humans and ticks. The novel species is named Borrelia mayonii sp. nov. in honor of William James Mayo and Charles Horace Mayo, founders of the Mayo Clinic, where this novel organism was first discovered.