Third Borrelia Species in White-footed Mice

To the Editor: The white-footed mouse, Peromyscus leucopus, is a natural reservoir host of several pathogens, including Borrelia burgdorferi, an agent of Lyme borre-liosis (LB) (1). B. burgdorferi spiro-chetes are transmitted in the mouse population by Ixodes scapularis ticks. This tick vector also bears B. miyamo-toi, a sister species to the relapsing fever group of spirochetes (2,3). B. miyamotoi infects P. leucopus in the laboratory (2), but the role of this mouse as a reservoir was not known. Here we report that P. leucopus is a reservoir for B. miyamotoi in nature and, in addition, that this mouse is host for a third, hitherto unknown, species of Borrelia. In a recent study of a 9-hectare site in a mixed hardwood forest in eastern Connecticut, we found that ≈35% of I. scapularis nymphs were infected with B. burgdorferi and ≈6% were infected with B. miyamotoi (4). For that study of a field vaccine we also collected blood from P. leucopus mice captured from June to early September of 2001. DNA was extracted from the blood and then subjected to quantitative polymerase chain reaction (PCR) assay for the presence of B. burgdorferi as described (4). In the present study, we analyzed the extracts of 556 blood samples from 298 mice from the non-vaccine control grids by a multiplex, quantitative real-time PCR for 16S rDNA that discriminated between B. burgdorferi and B. miyamotoi at the site (4). Sixty-nine (12%) of the samples were positive for B. burgdorferi and 36 (6%) were positive for B. miyamotoi; 5 (0.9%) of the samples were positive for both species. In infected mice, the mean number of B. miyamotoi cells per milliliter of blood was 251 (95% confidence limits of 126–631), 5-fold greater than that of B. burgdorferi at 50 cells/mL (40–63). A standard PCR assay of the blood samples with primers for the 16S–23S rDNA intergenic spacer (IGS) was performed as described by Bunikis et al. (5); results suggested the presence of a third species of Borrelia among the blood samples of the mice. A uniquely sized amplicon of ≈350 bp was observed in the reactions of 6 of 100 samples that were positive for B. burgdorferi and or B. miyamotoi by 16S PCR, and of 2 of 31 randomly selected samples that were negative for both B. burgdorferi and B. miyamotoi (p = 0.3 by 2-sided exact chi-square test). Samples with the 350-bp amplicon were further …