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BackgroundLyme borreliosis is the most common tick-borne human disease and is caused by Borrelia burgdorferi sensu lato (s.l.). Borrelia miyamotoi, a relapsing fever spirochaete, is transmitted transovarially, whereas this has not been shown for B. burgdorferi (s.l). Therefore, B. burgdorferi (s.l) is considered to cycle from nymphs to larvae through vertebrates. Larvae of Ixodes ricinus are occasionally B. burgdorferi (s.l) infected, but their vector competence has never been studied.MethodsWe challenged 20 laboratory mice with field-collected larvae of I. ricinus. A subset of these larvae was analysed for infections with B. burgdorferi (s.l) and B. miyamotoi. After three to four challenges, mice were sacrificed and skin and spleen samples were analysed for infection by PCR and culture.ResultsField-collected larvae were naturally infected with B. burgdorferi (s.l) (0.62 %) and B. miyamotoi (2.0 %). Two mice acquired a B. afzelii infection and four mice acquired a B. miyamotoi infection during the larval challenges.ConclusionWe showed that larvae of I. ricinus transmit B. afzelii and B. miyamotoi to rodents and calculated that rodents have a considerable chance of acquiring infections from larvae compared to nymphs. As a result, B. afzelii can cycle between larvae through rodents. Our findings further imply that larval bites on humans, which easily go unnoticed, can cause Lyme borreliosis and Borrelia miyamotoi disease.

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.

BackgroundBorrelia miyamotoi is a relapsing fever spirochete found in Ixodes ticks in North America, Europe, and Asia, and has recently been found to be invasive in humans. Cultivation of this spirochete has not yet been described, but is important for patient diagnostics and scientific purposes. Host specificity of Borrelia species is dependent on resistance to host complement (serum resistance), and since B. miyamotoi has been identified as a human pathogen we were interested whether B. miyamotoi is resistant to human complement.MethodsWe inoculated B. miyamotoi strains LB-2001 and HT31 in modified-Kelly-Pettenkofer medium with 10% fetal calf serum (MKP-F), and used standard non-laborious Borrelia culture methods to culture the spirochetes. Next, we assessed serum sensitivity by a direct killing assay and a growth inhibition assay.ResultsWe were able to passage B. miyamotoi over 10 times using a standard culture method in MKP-F medium, and found B. miyamotoi to be resistant to human complement. In contrast to B. miyamotoi, Borrelia anserina - a relapsing fever spirochete unrelated to human infection- was serum sensitive.ConclusionsUsing a variation on MKP medium we were able to culture B. miyamotoi, opening the door to in vitro research into this spirochete. In addition, we describe that B. miyamotoi is resistant to human complement, which might play an important role in pathogenesis. We have also found B. anserina to be sensitive to human complement, which might explain why it is not related to human infection. Summarizing, we describe a novel culture method for B. miyamotoi and show it is resistant to human complement.

We clearly demonstrate here the diagnostic potential of these seromarkers. Our findings will facilitate future epidemiological and clinical studies on BMD and lead to the development of a serologic test to be used in clinical practice.

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.

Borrelia (B.) miyamotoi, an emerging tick-borne relapsing fever spirochete, resists complement-mediated killing. To decipher the molecular principles of immune evasion, we sought to identify determinants contributing to complement resistance. Employing bioinformatics, we identified a gene encoding for a putative Factor H-binding protein, termed CbiA (complement binding and inhibitory protein A). Functional analyses revealed that CbiA interacted with complement regulator Factor H (FH), C3, C3b, C4b, C5, and C9. Upon binding to CbiA, FH retained its cofactor activity for Factor I-mediated inactivation of C3b. The Factor H-binding site within CbiA was mapped to domain 20 whereby the C-terminus of CbiA was involved in FH binding. Additionally, CbiA directly inhibited the activation of the classical pathway and the assembly of the terminal complement complex. Of importance, CbiA displayed inhibitory activity when ectopically produced in serum-sensitive B. garinii G1, rendering this surrogate strain resistant to human serum. In addition, long-term in vitro cultivation lead to an incremental loss of the cbiA gene accompanied by an increase in serum susceptibility. In conclusion, our data revealed a dual strategy of B. miyamotoi to efficiently evade complement via CbiA, which possesses complement binding and inhibitory activities.