Borrelia miyamotoi is a relapsing fever spirochete in Ixodes ticks that has been recently identified as a human pathogen causing hard tick-borne relapsing fever (HTBRF) across the Northern hemisphere. No validated serologic test exists and current serologic assays have low sensitivity in early HTBRF. To examine the humoral immune response against B. miyamotoi, we infected C3H/HeN mice with B. miyamotoi strain LB-2001 expressing variable small protein 1 (Vsp1) and demonstrated that spirochetemia was cleared after 3 days - coinciding with anti-Vsp1 IgM production. Clearance was also observed after passive transfer of immune sera to infected SCID mice. Next, we showed that anti-Vsp1 IgG eliminates Vsp1-expressing B. miyamotoi, selecting for spirochetes expressing a variable large protein (VlpC2) resistant to anti-Vsp1. The viability of Asian isolate B. miyamotoi HT31, expressing Vlp15/16 and Vlp18, was also unaffected by anti-Vsp1. Finally, in nine HTBRF patients, we demonstrated IgM reactivity to Vsp1 in two and against Vlp15/16 in four around one week after these patients tested positive for B. miyamotoi by PCR. Our data show that B. miyamotoi is able to express various variable major proteins (VMPs) to evade humoral immunity and that VMPs are antigenic in humans. We propose that serologic tests based on VMPs are of additional value in diagnosing HTBRF.

Ixodes ticks transmit Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis (LB). These tick species also transmit Borrelia miyamotoi, which was recently found to cause infections in humans. We were interested in the prevalence of B. miyamotoi infection in ticks and natural hosts in The Netherlands, and to what extent ticks are co-infected with B. burgdorferi. In addition, erythema migrans has been sporadically described in B. miyamotoi-infected patients, but these skin lesions might as well represent co-infections with B. burgdorferi s.l. We therefore investigated whether B. miyamotoi was present in LB-suspected skin lesions of patients referred to our tertiary Lyme disease clinic. 3360 questing Ixodes ricinus nymphs as well as spleen tissue of 74 rodents, 26 birds and 10 deer were tested by PCR for the presence of B. miyamotoi. Tick lysates were also tested for the presence of B. burgdorferi s.l. Next, we performed a PCR for B. miyamotoi in 31 biopsies from LB-suspected skin lesions in patients visiting our tertiary Lyme center. These biopsies had been initially tested for B. burgdorferi s.l. by PCR, and the skin lesions had been investigated by specialized dermatologists. Out of 3360 unfed (or questing) nymphs, 313 (9.3%) were infected with B. burgdorferi s.l., 70 (2.1%) were infected with B. miyamotoi, and 14 (0.4%) were co-infected with B. burgdorferi s.l. and B. miyamotoi. Co-infection of B. burgdorferi s.l. with B. miyamotoi occurred more often than expected from single infection prevalences (p=0.03). Both rodents (9%) and birds (8%) were found positive for B. miyamotoi by PCR, whereas the roe deer samples were negative. Out of 31 LB-suspected skin biopsies, 10 (32%) were positive for B. burgdorferi s.l. while none were positive for B. miyamotoi. The significant association of B. burgdorferi s.l. with B. miyamotoi in nymphs implies the existence of mutual reservoir hosts. Indeed, the presence of B. miyamotoi DNA indicates systemic infections in birds as well as rodents. However, their relative contributions to the enzootic cycle of B. miyamotoi requires further investigation. We could not retrospectively diagnose B. miyamotoi infection using biopsies of LB-suspected skin lesions, supporting the hypothesis that B. miyamotoi is not associated with LB-associated skin manifestations. However, this warrants further studies in larger sets of skin biopsies. A prospective study focused on acute febrile illness after a tick bite could provide insight into the incidence and clinical manifestations of B. miyamotoi infection in The Netherlands.

Ticks are one of the principal arthropod vectors of human and animal infectious diseases. Whereas the prevalence of tick-borne encephalitis virus in ticks in Europe is well studied, there is less information available on the prevalence of the other tick-borne viruses (TBVs) existing worldwide. The aim of this study was to improve the epidemiological survey tools of TBVs by the development of an efficient high-throughput test to screen a wide range of viruses in ticks.In this study, we developed a new high-throughput virus-detection assay based on parallel real-time PCRs on a microfluidic system, and used it to perform a large scale epidemiological survey screening for the presence of 21 TBVs in 18,135 nymphs of I. ricinus collected from five European countries. This extensive investigation has (i) evaluated the prevalence of four viruses present in the collected ticks, (ii) allowed the identification of viruses in regions where they were previously undetected.In conclusion, we have demonstrated the capabilities of this new screening method that allows the detection of numerous TBVs in a large number of ticks. This tool represents a powerful and rapid system for TBVs surveillance in Europe and could be easily customized to assess viral emergence.

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