Hard Tick-borne Relapsing Fever Research Articles

Borrelia miyamotoi in wild rodents from four different regions of Turkey

Borrelia miyamotoi is a tick-borne zoonotic agent that causes hard tick-borne relapsing fever, an emerging disease in humans. Some small mammalian and bird species are reported to be reservoirs of B. miyamotoi. This study aims to examine Borrelia species present in rodents captured from rural areas of Turkey. Blood samples of rodents were initially screened with Borrelia 16S rRNA qPCR. The Borrelia flaB gene was subsequently amplified by conventional PCR, after which all positive samples were sequenced. Borrelia miyamotoi was observed in nine out of 536 blood samples (1.7%) collected from wild rodents. Phylogenetic analysis showed that all positive samples belonged to the European genotype clade of B. miyamotoi. PCR positivity was 5.3%, 3.7%, and 1.8% in Apodemus uralensis, Apodemus flavicollis, and Myodes glareolus, respectively. Borrelia burgdorferi sensu lato that causes Lyme borreliosis in humans could not be detected in the rodents. In this study, presence of B. miyamotoi DNA is reported for the first time in rodents in Turkey.

Borrelia miyamotoi: A Comprehensive Review.

Borrelia miyamotoi is an emerging tick-borne pathogen in the Northern Hemisphere and is the causative agent of Borrelia miyamotoi disease (BMD). Borrelia miyamotoi is vectored by the same hard-bodied ticks as Lyme disease Borrelia, yet phylogenetically groups with relapsing fever Borrelia, and thus, has been uniquely labeled a hard tick-borne relapsing fever Borrelia. Burgeoning research has uncovered new aspects of B. miyamotoi in human patients, nature, and the lab. Of particular interest are novel findings on disease pathology, prevalence, diagnostic methods, ecological maintenance, transmission, and genetic characteristics. Herein, we review recent literature on B. miyamotoi, discuss how findings adapt to current Borrelia doctrines, and briefly consider what remains unknown about B. miyamotoi.

Metatranscriptomic profiling reveals diverse tick-borne bacteria, protozoans and viruses in ticks and wildlife from Australia.

Tick-borne zoonoses are emerging globally due to changes in climate and land use. While the zoonotic threats associated with ticks are well studied elsewhere, in Australia, the diversity of potentially zoonotic agents carried by ticks and their significance to human and animal health is not sufficiently understood. To this end, we used untargeted metatranscriptomics to audit the prokaryotic, eukaryotic and viral biomes of questing ticks and wildlife blood samples from two urban and rural sites in New South Wales, Australia. Ixodes holocyclus and Haemaphysalis bancrofti were the main tick species collected, and blood samples from Rattus rattus, Rattus fuscipes, Perameles nasuta and Trichosurus vulpecula were also collected and screened for tick-borne microorganisms using metatranscriptomics followed by conventional targeted PCR to identify important microbial taxa to the species level. Our analyses identified 32 unique tick-borne taxa, including 10 novel putative species. Overall, a wide range of tick-borne microorganisms were found in questing ticks including haemoprotozoa such as Babesia, Theileria, Hepatozoon and Trypanosoma spp., bacteria such as Borrelia, Rickettsia, Ehrlichia, Neoehrlichia and Anaplasma spp., and numerous viral taxa including Reoviridiae (including two coltiviruses) and a novel Flaviviridae-like jingmenvirus. Of note, a novel hard tick-borne relapsing fever Borrelia sp. was identified in questing H. bancrofti ticks which is closely related to, but distinct from, cervid-associated Borrelia spp. found throughout Asia. Notably, all tick-borne microorganisms were phylogenetically unique compared to their relatives found outside Australia, and no foreign tick-borne human pathogens such as Borrelia burgdorferi s.l. or Babesia microti were found. This work adds to the growing literature demonstrating that Australian ticks harbour a unique and endemic microbial fauna, including potentially zoonotic agents which should be further studied to determine their relative risk to human and animal health.

Suppressive effects of Ixodes persulcatus sialostatin L2 against Borrelia miyamotoi-stimulated immunity

Borrelia miyamotoi infection is an emerging tick-borne disease that causes hard tick-borne relapsing fever. B. miyamotoi is transmitted through the bite of ticks, including Ixodes persulcatus. Although accumulating evidence suggests that tick salivary proteins enhance the infectivity of other tick-borne pathogens, the association of B. miyamotoi with tick-derived proteins remains unknown. In this study, the effect of I. persulcatus sialostatin L2 (Ip-sL2), a tick-derived cystatin, on specific immunity to B. miyamotoi was preliminarily investigated in vitro. Mice were immunized with heat-killed B. miyamotoi and in vitro analyses of the splenocytes of the immunized mice indicated that the expression levels of the activation markers of CD11c+ and CD3+ cells were significantly upregulated by B. miyamotoi stimulation. Spleen cells from B. miyamotoi-immunized mice were used to determine whether Ip-sL2 regulates murine immune responses against B. miyamotoi. Treatment with Ip-sL2 in vitro inhibited the activation of CD11c+ and CD3+ cells as well as inflammatory cytokine production by cultured splenocytes. These findings show that Ip-sL2 has modulatory effects on murine immune responses to B. miyamotoi. Therefore, it is necessary to clarify in the future whether Ip-sL2 is involved in the enhanced infectivity of B. miyamotoi.

Borreliae Part 2: Borrelia Relapsing Fever Group and Unclassified Borrelia.

Simple SummaryFollowing Borreliae Part 1, this second review describes Borreliae of the relapsing fever group (RFG) and unclassified Borreliae. The RFG is further divided according to vector transmission and geographical distribution in other subgroups, namely the soft-tick-borne relapsing fever (STBRF) group, the hard-tick-borne relapsing fever (HTBRF) one, the louse-borne relapsing fever (LBRF) group, and the Avian relapsing fever group. Where possible, according to the literature description, each sub-group of the RFG Borreliae is organized here in sections explaining the geographical distribution, the vectors, the hosts, the epidemiology, and the microbiology. In case of human infectiveness, clinical aspects are also discussed. Isolation and sequencing of Borrelia species is ongoing, and in addition to the groups mentioned in these reviews, there are Borreliae that at present cannot be cultivated, but according to sequencing data they share some characteristics with one specific group. Nevertheless, they still cannot be classified in one of them. This is the case of Borreliae with not-yet-identified pathogenicity for humans or animals, which are here named “unclassified Borreliae” and described separately, recalling the similarities with Borreliae already classified. In the future, we expect that those Borreliae are going to be characterized, including them in one of the previous groups.Borreliae of the relapsing fever group (RFG) are heterogenous and can be divided mainly into three groups according to vectors, namely the soft-tick-borne relapsing fever (STBRF) Borreliae, the hard-tick-borne relapsing fever (HTBRF) Borreliae, the louse-borne relapsing fever (LBRF) Borreliae, and the avian relapsing fever ones. With respect to the geographical distribution, the STBRF Borreliae are further subdivided into Old World and New World strains. Except for the Avian relapsing fever group Borreliae, which cause avian spirochetosis, all the others share infectivity in humans. They are indeed the etiological agent of both endemic and epidemic forms of relapsing fever, causing high spirochaetemia and fever. Vectors are primarily soft ticks of Ornithodoros spp. in the STBRF group; hard ticks, notably Ixodes sp., Amblyomma sp., Dermacentor sp., and Rhipicephalus sp., in the HTBRF group; and the louse pediculus humanus humanus in the TBRF one. A recent hypothesis was supported for a common ancestor of RFG Borreliae, transmitted at the beginning by hard-body ticks. Accordingly, STBRF Borreliae switched to use soft-bodied ticks as a vector, which was followed by the use of lice by Borrelia recurrentis. There are also new candidate species of Borreliae, at present unclassified, which are also described in this review.

The evolution of hard tick-borne relapsing fever borreliae is correlated with vector species rather than geographical distance

BackgroundRelapsing fever (RF) borreliae are arthropod-borne spirochetes and some of them cause human diseases, which are characterized by relapsing or recurring episodes of fever. Recently, it has been classified into two groups: soft tick-borne RF (STRF) borreliae and hard tick-borne RF (HTRF) borreliae. STRF borreliae include classical RF agents and HTRF borreliae, the latter of which include B. miyamotoi, a human pathogen recently identified in Eurasia and North America.ResultsIn this study, we determined the genome sequences of 16 HTRF borreliae strains: 15 B. miyamotoi strains (9 from Hokkaido Island, Japan, 3 from Honshu Island, Japan, and 3 from Mongolia) and a Borrelia sp. tHM16w. Chromosomal gene synteny was highly conserved among the HTRF strains sequenced in this study, even though they were isolated from different geographic regions and different tick species. Phylogenetic analysis based on core gene sequences revealed that HTRF and STRF borreliae are clearly distinguishable, with each forming a monophyletic group in the RF borreliae lineage. Moreover, the evolutionary relationships of RF borreliae are consistent with the biological and ecological features of each RF borreliae sublineage and can explain the unique characteristics of Borrelia anserina. In addition, the pairwise genetic distances between HTRF borreliae strains were well correlated with those of vector species rather than with the geographical distances between strain isolation sites. This result suggests that the genetic diversification of HTRF borreliae is attributed to the speciation of vector ticks and that this relationship might be required for efficient transmission of HTRF borreliae within vector ticks.ConclusionsThe results of the present study, together with those from previous investigations, support the hypothesis that the common ancestor of borreliae was transmitted by hard-bodied ticks and that only STRF borreliae switched to using soft-bodied ticks as a vector, which was followed by the emergence of Borrelia recurrentis, lice-borne RF borreliae. Our study clarifies the phylogenetic relationships between RF borreliae, and the data obtained will contribute to a better understanding of the evolutionary history of RF borreliae.

Detection of Asian-Type Borrelia miyamotoi from Ixodes ricinus Inhabiting Tver Province (Russia): A Sympatric Region for I. ricinus and Ixodes persulcatus

Borrelia miyamotoi, a hard tick-borne relapsing fever agent, was sampled in Ixodes ricinus and Ixodes persulcatus ticks from the Tver province in Russia (a sympatric region of both tick species) and examined by TaqMan-PCR targeting the 16S rRNA gene. Borrelia was detected in 4 out of 168 ticks: 2 out of 58 I. ricinus ticks (infection rate 2.9%) and 2 out of 110 I. persulcatus ticks (1.8%). The agent was identified as B. miyamotoi on the basis of the 16S and 23S rDNA intergenic spacer and glycerophosphodiester phosphodiesterase gene sequencing analyses. Interestingly, the genes sequences detected from one I. ricinus tick were identical to those of Asian-type B. miyamotoi from I. persulcatus. This tick was identified as I. ricinus by sequencing analysis of internal transcribed spacer 2 and mitochondrial cytochrome oxidase subunit 1. The results suggest that the I. ricinus ticks were infected with Asian-type B. miyamotoi in a sympatric region for I. ricinus and I. persulcatus.

An immunocompromised mouse model to infect Ixodes scapularis ticks with the relapsing fever spirochete, Borrelia miyamotoi

The hard tick-borne relapsing fever spirochete, Borrelia miyamotoi, has recently gained attention as a cause of human illness, but fundamental aspects of its enzootic maintenance are still poorly understood. Challenges to experimental studies with B. miyamotoi-infected vector ticks include low prevalence of infection in field-collected ticks and seemingly inefficient horizontal transmission from infected immunocompetent rodents to feeding ticks. To reliably produce large numbers of B. miyamotoi-infected ticks in support of experimental studies, we developed an animal model where immunocompromised Mus musculus SCID mice were used as a source of B. miyamotoi-infection for larval and nymphal Ixodes scapularis ticks. Following needle inoculation with 1 × 105 spirochetes, the SCID mice developed a high spirochetemia (greater than 1 × 107 copies of B. miyamotoi purB per mL of blood) that persisted for at least 30 d after inoculation. In comparison, immunocompetent M. musculus CD-1 mice developed transient infections, detectable for only 2–8 d within the first 16 d after needle inoculation, with a brief, lower peak spirochetemia (8.5 × 104 – 5.6 × 105purB copies per mL of blood). All larval or nymphal ticks fed on infected SCID mice acquired B. miyamotoi, but frequent loss of infection during the molt led to the proportion infected ticks of the resulting nymphal or adult stages declining to 22–29%. The ticks that remained infected after the molt had well-disseminated infections which then persisted through successive life stages, including transmission to larval offspring.

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

The relapsing fever spirochete, Borrelia miyamotoi, is increasingly recognized as a cause of human illness (hard tick-borne relapsing fever) in the United States. We previously demonstrated that single nymphs of the blacklegged tick, Ixodes scapularis, can transmit B. miyamotoi to experimental hosts. However, two recent epidemiological studies from the Northeastern United States indicate that human cases of hard tick-borne relapsing fever peak during late summer, after the spring peak for nymphal tick activity but coincident with the peak seasonal activity period of larval ticks in the Northeast. These epidemiological findings, together with evidence that B. miyamotoi can be passed from infected I. scapularis females to their offspring, suggest that bites by transovarially-infected larval ticks can be an important source of human infection. To demonstrate experimentally that transovarially-infected larval I. scapularis ticks can transmit B. miyamotoi, outbred Mus musculus CD1 mice were exposed to 1 or 2 potentially infected larvae. Individual fed larvae and mouse blood taken 10 d after larvae attached were tested for presence of B. miyamotoi DNA, and mice also were examined for seroreactivity to B. miyamotoi 8 wk after tick feeding. We documented B. miyamotoi DNA in blood from 13 (57%) of 23 mice exposed to a single transovarially-infected larva and in 5 (83%) of 6 mice exposed to two infected larvae feeding simultaneously. All 18 positive mice also demonstrated seroreactivity to B. miyamotoi. Of the 11 remaining mice without detectable B. miyamotoi DNA in their blood 10 d after infected larvae attached, 7 (64%) had evidence of spirochete exposure by serology 8 wk later. Because public health messaging for risk of exposure to Lyme disease spirochetes focuses on nymphal and female I. scapularis ticks, our finding that transovarially-infected larvae effectively transmit B. miyamotoi should lead to refined tick-bite prevention messages.

The Complement Binding and Inhibitory Protein CbiA of Borrelia miyamotoi Degrades Extracellular Matrix Components by Interacting with Plasmin(ogen).

The emerging relapsing fever spirochete Borrelia (B.) miyamotoi is transmitted by ixodid ticks and causes the so-called hard tick-borne relapsing fever or B. miyamotoi disease (BMD). More recently, we identified a surface-exposed molecule, CbiA exhibiting complement binding and inhibitory capacity and rendering spirochetes resistant to complement-mediated lysis. To gain deeper insight into the molecular principles of B. miyamotoi-host interaction, we examined CbiA as a plasmin(ogen) receptor that enables B. miyamotoi to interact with the serine protease plasmin(ogen). Recombinant CbiA was able to bind plasminogen in a dose-dependent fashion. Moreover, lysine residues appear to play a crucial role in the protein-protein interaction as binding of plasminogen was inhibited by the lysine analog tranexamic acid as well as increasing ionic strength. Of relevance, plasminogen bound to CbiA can be converted by urokinase-type plasminogen activator (uPa) to active plasmin which cleaved both, the chromogenic substrate S-2251 and its physiologic substrate fibrinogen. Concerning the involvement of specific amino acids in the interaction with plasminogen, lysine residues located at the C-terminus are frequently involved in the binding as reported for various other plasminogen-interacting proteins of Lyme disease spirochetes. Lysine residues located within the C-terminal domain were substituted with alanine to generate single, double, triple, and quadruple point mutants. However, binding of plasminogen to the mutated CbiA proteins was not affected, suggesting that lysine residues distant from the C-terminus might be involved in the interaction.

A relapsing fever group Borrelia sp. is widely distributed among wild deer in Japan

A relapsing fever group Borrelia sp. was detected from the blood of wild deer (Cervus nippon) in Japan. The Borrelia sp. was distributed nationwide among deer with an overall prevalence of 26% in blood samples. The prevalence of infection was significantly higher in fawns (48.4%) compared to adult deer (23.6%). Sequencing analysis reveals that this Borrelia sp. belongs to the hard tick-borne relapsing fever borreliae, and that it forms a single lineage based on sequences of the flagellin and glycerophosphodiester phosphodiesterase genes. Borrelial genome copy number was estimated at 8.8 × 103 genome copies/μl of blood. Other hard tick-borne relapsing fever borrelia (e.g. Borrelia miyamotoi) were not detected in deer blood in this study. These findings suggest that wild deer may act as reservoirs for this Borrelia sp. in Japan.

Human seroprevalence of Borrelia miyamotoi in Manitoba, Canada, in 2011-2014: a cross-sectional study.

Hard tick-borne relapsing fever caused by Borrelia miyamotoi has been reported in Russia, the Netherlands, Germany, Japan and the northeastern and upper midwestern United States. We sought to investigate the presence of B. miyamotoi infection in humans in Manitoba, Canada. Two hundred fifty sera collected from residents of Manitoba with suspected Lyme disease between 2011 and 2014 were tested for Borrelia burgdorferi antibody using a C6 peptide enzyme-linked immunosorbent assay (ELISA) followed by Western blot. Residual sera were then anonymized, stored at -80°C and subsequently thawed and tested for B. miyamotoi antibody using a 2-step glycerosphosphodiester phosphodiesterase-based ELISA and Western blot assay. Twenty-four of the 250 (9.6%) sera tested positive for B. miyamotoi immunoglobulin G. Participants who were B. miyamotoi seropositive were predominantly male (54%) and younger on average than those who were seronegative (32 and 44 yr of age, respectively). Participants who were seropositive for B. burgdorferi were significantly more likely to be B. miyamotoi seropositive than those who were B. burgdorferi seronegative (20.3% v. 6.6%, respectively, odds ratio 3.6, 95% confidence interval 1.5-8.5). This initial report of human B. miyamotoi infection in Canada should raise awareness of hard tick-borne relapsing fever among clinicians and residents of areas in Canada and western North America where Lyme disease is endemic.

Development and optimization of an in vitro cultivation protocol allows for isolation of Borrelia miyamotoi from patients with hard tick-borne relapsing fever

ObjectivesBorrelia miyamotoi has been shown to infect humans in Eurasia and North America causing hard tick-borne relapsing fever (HTBRF). In vitro cultivation of B. miyamotoi was described recently; but clinical isolation of relapsing fever Borrelia is cumbersome. Our aim was to develop a straightforward protocol enabling B. miyamotoi isolation directly from the blood of patients. MethodsModified Kelly-Pettenkorfer (MKP-F) medium, with or without anticoagulants, or blood from healthy human volunteers, was spiked with B. miyamotoi spirochaetes in vitro. Subsequently, either media or plasma was used for cultivation directly, or after an additional centrifugation step. This isolation protocol was tested in a clinical setting on patients suspected of HTBRF. ResultsDipotassium-EDTA, trisodium citrate and lithium heparin inhibited growth of B. miyamotoi at concentrations ≥250 μg/mL, 2.5 mM and 1 IU/mL, respectively. However, when plasma originating from human blood containing B. miyamotoi spirochaetes was subjected to an additional centrifugation step at 8000 g, suspended and inoculated into fresh MKP-F media, positive cultures were observed within 2 weeks. Of importance, this straightforward protocol allowed for isolation of B. miyamotoi from six out of nine patients with confirmed HTBRF. ConclusionsDirect culture from K2-EDTA, trisodium citrate and lithium heparin plasma containing B. miyamotoi is hampered due to anticoagulants. Using a simple centrifugation protocol we were able to circumvent this detrimental effect, allowing for the first clinical isolation of B. miyamotoi. This will be of value for future research on the pathogenesis, genetics, diagnosis, therapy and epidemiology of HTBRF and other tick-borne relapsing fevers.

Detection and Characterization of the Emerging Relapsing Fever Pathogen, Borrelia miyamotoi, from the Ixodes ricinus Tick in the Rural Trakya (Thrace) Region of Northwestern Turkey.

The hard tick-borne relapsing fever agent, Borrelia miyamotoi infection in Ixodes ricinus ticks sampled from Istanbul and the countryside of Kirklareli in northwestern Turkey, was examined by TaqMan-PCR targeting 16S rDNA, nested PCR targeting 16S rDNA, the flagellin gene (flaB), and the 16S and 23S rDNA intergenic spacer (IGS), and sequencing analyses of these amplicons. B. miyamotoi was detected in 1 out of 248 I. ricinus ticks (infection rate 0.4%). The tick infected with B. miyamotoi was collected in Longos, Kirklareli province on the European side of Turkey near the Bulgarian border. The 16S rDNA, flaB, and IGS sequences from the infected tick showed high similarities to those of B. miyamotoi detected in I. ricinus in Europe.

Variable Major Proteins as Targets for Specific Antibodies against Borrelia miyamotoi.

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 d, 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 ∼1 wk 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.