Agent Of Tick-borne Relapsing Fever Research Articles

Isolation and Genetic Characterization of the Human Relapsing Fever Spirochete Borrelia persica from a Dog with Improved Cultivation Techniques

Borrelia persica is the causative agent of tick-borne relapsing fever in Israel and is prevalent in the Eastern Mediterranean basin and parts of Asia. Infection with B. persica causes severe illness and potentially life-threatening complications in humans and companion animals. Isolation and in vitro cultivation of B. persica in culture medium is difficult and there are only two previous isolates of this spirochete. Here, we describe the first cultivation of B. persica from an ill dog. Isolation from the dog’s blood was performed with the Pettenkofer-LMU Bp medium and spirochetes with vibrant motility and cell density of 2 × 106/ml were observed in culture by dark-field microscopy 3 days after sampling. The isolate was passaged every 3–4 days with cell densities reaching up to 3 × 107/ml achieved over 30 passages. Cryopreservation was made at −80°C without any cryoprotectant additive, and successful growth from thawed culture samples was observed up to 3 months after freezing with repeated freeze and thaw cycles. Generation time during the exponential growth phase was 14.6 hr. Genetic characterization by polymerase chain reaction amplification and DNA sequencing of the flagellin, glycerophosphodiester phosphodiesterase, and 16S rRNA genes indicated that the isolate is a B. persica genotype I strain associated with human relapsing fever. The new canine isolate would be helpful for research on the pathogenesis of relapsing fever and the new modifications in cultivation and preservation methods may assist in future studies of relapsing fever by simplifying previously reported in vitro methods.

Pathogen Detection in Ornithodoros sonrai Ticks and Invasive House Mice Mus musculus domesticus in Senegal.

Ornithodoros sonrai (O. sonrai) ticks are the only known vectors of Borrelia crocidurae, an agent of tick-borne relapsing fever (TBRF) borreliosis. Rodents serve as principal natural reservoirs for Borrelia. Our research objective was to detect TBRF Borrelia and other zoonotic bacterial infections in ticks and in house mice Mus musculus domesticus, an invasive species currently expanding in rural northern Senegal. Real-time and conventional PCR were utilized for detecting Borrelia and other bacterial taxa. The analyses were performed on 253 specimens of O. sonrai and 150 samples of brain and spleen tissue from rodents. Borrelia crocidurae was found in one O. sonrai tick and 18 Mus musculus domesticus samples, with prevalences of 0.39 percent and 12 percent, respectively, as well as Ehrlichia sp. in one Mus musculus domesticus. Further, we were able to detect the presence of a potentially infectious novel species belonging to the Anaplasmataceae family for the first time in O. sonrai ticks. More attention should be paid to the house mouse and O. sonrai ticks, as they can be potential hosts for novel species of pathogenic bacteria in humans.

Diversity and distribution of the tick-borne relapsing fever spirochete Borrelia turicatae.

Borrelia turicatae is a causative agent of tick-borne relapsing fever (TBRF) in the subtropics and tropics of the United States and Latin America. Historically, B. turicatae was thought to be maintained in enzootic cycles in rural areas. However, there is growing evidence that suggests the pathogen has established endemic foci in densely populated regions of Texas. With the growth of homelessness in the state and human activity in city parks, it was important to implement field collection efforts to identify areas where B. turicatae and its vector circulate. Between 2017 and 2020 we collected Ornithodoros turicata ticks in suburban and urban areas including public and private parks and recreational spaces. Ticks were fed on naïve mice and spirochetes were isolated from the blood. Multilocus sequence typing (MLST) was performed on eight newly obtained isolates and included previously reported sequences. The four chromosomal loci targeted for MLST were 16S ribosomal RNA (rrs), flagellin B (flaB), DNA gyrase B (gyrB), and the intergenic spacer (IGS). Given the complexity of Borrelia genomes, plasmid diversity was also evaluated. These studies indicate that the IGS locus segregates B. turicatae into four genomic types and plasmid diversity is extensive between isolates. Furthermore, B. turicatae and its vector have established endemic foci in parks and recreational areas in densely populated settings of Texas.

Seroprevalence for the tick-borne relapsing fever spirochete Borrelia turicatae among small and medium sized mammals of Texas.

BackgroundIn low elevation arid regions throughout the southern United States, Borrelia turicatae is the principal agent of tick-borne relapsing fever. However, endemic foci and the vertebrate hosts involved in the ecology of B. turicatae remain undefined. Experimental infection studies suggest that small and medium sized mammals likely maintain B. turicatae in nature, while the tick vector is a long-lived reservoir.Methodology/principal findingsSerum samples from wild caught rodents, raccoons, and wild and domestic canids from 23 counties in Texas were screened for prior exposure to B. turicatae. Serological assays were performed using B. turicatae protein lysates and recombinant Borrelia immunogenic protein A (rBipA), a diagnostic protein that is unique to RF spirochetes and may be a species-specific antigen.Conclusions/significanceSerological responses to B. turicatae were detected from 24 coyotes, one gray fox, two raccoons, and one rodent from six counties in Texas. These studies indicate that wild canids and raccoons were exposed to B. turicatae and are likely involved in the pathogen’s ecology. Additionally, more work should focus on evaluating rodent exposure to B. turicatae and the role of these small mammals in the pathogen’s maintenance in nature.

Isolation and molecular characterization of a relapsing fever Borrelia recovered from Ornithodoros rudis in Brazil

In South America, early reports from more than 50 years ago incriminated Ornithodoros rudis as vector of Borrelia venezuelensis, an agent of tick-borne relapsing fever (TBRF). Herein we report the rediscovery of O. rudis by means of morphological, biological and molecular analyses, which also comprise the first report of this tick species in Brazil. Phylogenetic analysis using partial fragments of mitochondrial 16S rRNA gene suggested that O. rudis forms a monophyletic group with Ornithodoros erraticus. By using laboratory rodents as hosts, we isolated a relapsing fever Borrelia from an infected O. rudis female. Phylogenetic analysis inferred from the rrs, flaB, and glpQ genes of Borrelia spp. placed the spirochete harbored by O. rudis closely related to Borrelia turicatae. Until further genetic evidence is not obtained we are referring to this O. rudis spirochete as B. venezuelensis. This is the first in vitro isolation of a TBRF Borrelia from South America. The presence of O. rudis in Brazil should not be overlooked, since this tick has been historically implicated in human cases of TBRF in Colombia, Panama, and Venezuela. This study provides new reports of O. rudis and B. venezuelensis after decades of scientific silence on these agents.

Cyclic-di-GMP binding induces structural rearrangements in the PlzA and PlzC proteins of the Lyme disease and relapsing fever spirochetes: a possible switch mechanism for c-di-GMP-mediated effector functions.

The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R145 of the PilZ domain motif, R145xxxR. Comparative sequence analyses led to the identification of Borrelia consensus sequences for the PilZ domain signature motifs. This study provides insight into c-di-GMP:Plz receptor interaction and identifies a possible switch mechanism that may regulate Plz protein effector functions.

Relapsing fever group Borrelia in Southern California rodents.

Wild rodent reservoir host species were surveyed prospectively for infection with Borrelia hermsii, the causative agent of tick-borne relapsing fever in the western United States. Trapping occurred during the summer of 2009-2012 at field sites surrounding Big Bear Lake, CA, a region where human infection has been reported for many years. Using quantitative polymerase chain reaction (qPCR), we tested 207 rodents from 11 species and found chipmunks (Tamias spp.) and a woodrat (Neotoma macrotis) infected. Chipmunks represented the majority of captures at these sites. Sixteen of the 207 (7.7%; CI = 4.6-12.4) animals were qPCR-positive for Borrelia spp. associated with relapsing fever, and of those, we obtained bacterial DNA sequences from eight. The phylogram made from these sequences depict a clear association with B. hermsii genomic group I. In addition, we identified an infection with Borrelia coriaceae in a Tamias merriami, a potentially nonpathogenic member of the tick-borne relapsing fever group. Our findings support the hypothesis that chipmunk species play an important role in the maintenance of Borrelia species that cause tick-borne relapsing fever in the western United States, and therefore the risk of infection to people.

BorreliaBase: a phylogeny-centered browser of Borrelia genomes.

BackgroundThe bacterial genus Borrelia (phylum Spirochaetes) consists of two groups of pathogens represented respectively by B. burgdorferi, the agent of Lyme borreliosis, and B. hermsii, the agent of tick-borne relapsing fever. The number of publicly available Borrelia genomic sequences is growing rapidly with the discovery and sequencing of Borrelia strains worldwide. There is however a lack of dedicated online databases to facilitate comparative analyses of Borrelia genomes.DescriptionWe have developed BorreliaBase, an online database for comparative browsing of Borrelia genomes. The database is currently populated with sequences from 35 genomes of eight Lyme-borreliosis (LB) group Borrelia species and 7 Relapsing-fever (RF) group Borrelia species. Distinct from genome repositories and aggregator databases, BorreliaBase serves manually curated comparative-genomic data including genome-based phylogeny, genome synteny, and sequence alignments of orthologous genes and intergenic spacers.ConclusionsWith a genome phylogeny at its center, BorreliaBase allows online identification of hypervariable lipoprotein genes, potential regulatory elements, and recombination footprints by providing evolution-based expectations of sequence variability at each genomic locus. The phylo-centric design of BorreliaBase (http://borreliabase.org) is a novel model for interactive browsing and comparative analysis of bacterial genomes online.

The Borrelia hermsii factor H binding protein FhbA is not required for infectivity in mice or for resistance to human complement in vitro.

The primary causative agent of tick-borne relapsing fever in North America is Borrelia hermsii. It has been hypothesized that B. hermsii evades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement. In vitro, B. hermsii produces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen for B. hermsii infection in humans. The ability to test the hypothesis that FhbA is a critical virulence factor in vivo has been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated a B. hermsii fhbA deletion mutant (the B. hermsii YORΔfhbA strain) through allelic exchange mutagenesis. Deletion of fhbA abolished FH binding by the YORΔfhbA strain and eliminated cleavage of C3b on the cell surface. However, the YORΔfhbA strain remained infectious in mice and retained resistance to killing in vitro by human complement. Collectively, these results indicate that B. hermsii employs an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice.

Development of Genetic System to Inactivate a Borrelia turicatae Surface Protein Selectively Produced within the Salivary Glands of the Arthropod Vector

Background Borrelia turicatae, an agent of tick-borne relapsing fever, is an example of a pathogen that can adapt to disparate conditions found when colonizing the mammalian host and arthropod vector. However, little is known about the genetic factors necessary during the tick-mammalian infectious cycle, therefore we developed a genetic system to transform this species of spirochete. We also identified a plasmid gene that was up-regulated in vitro when B. turicatae was grown in conditions mimicking the tick environment. This 40 kilodalton protein was predicted to be surface localized and designated the Borrelia repeat protein A (brpA) due to the redundancy of the amino acid motif Gln-Gly-Asn-Val-Glu.Methodology/Principal FindingsQuantitative reverse-transcriptase polymerase chain reaction using RNA from B. turicatae infected ticks and mice indicated differential regulation of brpA during the tick-mammalian infectious cycle. The surface localization was determined, and production of the protein within the salivary glands of the tick was demonstrated. We then applied a novel genetic system for B. turicatae to inactivate brpA and examined the role of the gene product for vector colonization and the ability to establish murine infection.Conclusions/SignificanceThese results demonstrate the complexity of protein production in a population of spirochetes within the tick. Additionally, the development of a genetic system is important for future studies to evaluate the requirement of specific B. turicatae genes for vector colonization and transmission.

Interleukin 10 Protects the Brain Microcirculation From Spirochetal Injury

Spirochetal infections are an important cause of neurological disease. In previous studies of the pathogenesis of spirochetal brain infection, mice inoculated with Borrelia turicatae, an agent of tick-borne relapsing fever in North America, developed mild meningitis and parenchymal activation/infiltration by interleukin 10 (IL-10)-producing microglia/macrophages. Here, we investigated the neuroprotective effects of IL-10 during spirochetal infection by comparing the outcomes of B. turicatae infection in wild-type and IL-10-deficient RAG2-deficient mice. Mice were infected with either serotype 1 (Bt1), which causes more brain infection but lower bacteremia, or Bt2, which causes less brain infection but higher bacteremia. Interleukin 10 deficiency resulted in early death from subarachnoid/intraparenchymal brain hemorrhage in Bt2-infected mice. These mice had marked apoptosis of brain microvascular endothelial cells as assessed by terminal transferase-mediated DNA nick end-labeling staining. In contrast, Bt1 infection caused milder subarachnoid hemorrhage. Neuronal apoptosis was observed in mice infected with both serotypes and was prominent in the cerebellum. Neutralization of tumor necrosis factor prevented death and reduced morbidity and brain injury in mice infected by both serotypes. We conclude that IL-10 plays a critical role protecting the cerebral microcirculation from spirochetal injury possibly by inhibition effects of tumor necrosis factor.

Identification of an Antiparallel Coiled-Coil/Loop Domain Required for Ligand Binding by theBorrelia hermsiiFhbA Protein: Additional Evidence for the Role of FhbA in the Host-Pathogen Interaction

Borrelia hermsii, an etiological agent of tick-borne relapsing fever in North America, binds host-derived serum proteins including factor H (FH), plasminogen, and an unidentified 60-kDa protein via its FhbA protein. Two distinct phylogenetic types of FhbA have been delineated (FhbA1 and FhbA2). These orthologs share a conserved C-terminal domain that contains two alpha helices with a high predictive probability of coiled-coil formation that are separated by a 14-amino-acid loop domain. Through site-directed mutagenesis, we have identified residues within these domains that influence the binding of both mouse and human FH, plasminogen, and/or the 60-kDa protein. To further investigate the involvement of FhbA in the host-pathogen interaction, strains that are either FhbA(+) (isolate YOR) or FhbA(-) (isolate REN) were tested for serum sensitivity. Significant differences were observed, with YOR and REN being serum resistant and serum sensitive (intermediate), respectively. To test the abilities of these strains to infect and persist in mice, mice were needle inoculated, and infectivity and persistence were then assessed. While both strains REN and YOR infected mice, only the FhbA(+) YOR strain persisted beyond day 4. Survival of the YOR isolate in blood correlated with the upregulation of the fhbA gene, as demonstrated by real-time reverse transcriptase PCR. These data advance our understanding of the unique interactions of FhbA with individual serum proteins and provide support for the hypothesis that FhbA is an important contributor to the pathogenesis of the relapsing fever spirochete B. hermsii.

Molecular Analyses of the Interaction ofBorrelia hermsiiFhbA with the Complement Regulatory Proteins Factor H and Factor H-Like Protein 1

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative complement pathway. The ability of FH-binding protein A (FhbA) to bind FH-like protein 1 (FHL-1) has not been assessed previously. In this study, using a whole-cell absorption assay, we demonstrated that B. hermsii absorbs both FH and FHL-1 from human serum. Consistent with this, affinity ligand binding immunoblot analyses revealed that FH constructs spanning short consensus repeats 1 to 7 and 16 to 20 bind to FhbA. To investigate the molecular basis of the interaction of FhbA with FH/FHL-1, recombinant FhbA truncated proteins were generated and tested for FH/FHL-1 binding. Binding required determinants located in both the N- and C-terminal domains of FhbA, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHL-1-binding pocket. To identify specific FhbA residues involved in binding, random mutagenesis was performed. These analyses identified a loop region of FhbA that may serve as a contact point for FH/FHL-1. The data presented here expand our understanding of the pathogenic mechanisms of the relapsing fever spirochetes and of the molecular nature of the interaction between FH/FHL-1 and FhbA.

New World relapsing fever Borrelia found in Ornithodoros porcinus ticks in central Tanzania.

Ticks were collected from 8 houses in Mvumi Mission village, near Dodoma, Tanzania. All ticks were examined for Borrelia infestation by flagellin gene-based nested polymerase chain reaction. All houses were highly infested with ticks, and all ticks collected were of the Ornithodoros porcinus species. Fifty-one out of 120 ticks were infected with spirochetes, and a flagellin gene sequence comparison showed that most of the spirochetes belonged to Borrelia duttonii, which is the causative agent of tick-borne relapsing fever in East Africa. The rest of the spirochetes were quite different from B. duttonii and instead resembled the New World tick-borne relapsing fever borreliae. Phylogenetic analysis using 16S ribosomal RNA gene sequences also supported the interpretation that the spirochete was a Borrelia species distinct from previously described members of the genus.

A newly identified tick-borne Borrelia species and relapsing fever in Tanzania

Tick-borne relapsing fever caused by the spirochaete Borrelia duttonii is a common cause of serious illness in central Tanzania. Screening of Ornithodoros sp ticks from infested houses for the presence of B duttonii had detected a previously unidentified species of Borrelia. We investigated whether this species infected the human population in a central Tanzanian village, by use of blood slide examination and PCR. PCR was twice as sensitive in detection of infections, showing Borrelia sp in six (11%) of 54 children with fever, and in 13 (4%) of 307 otherwise healthy children. Genotyping Borrelia from 17 infections identified Borrelia duttonii and an unnamed species. Our findings show that the newly discovered species is a causal agent of tick-borne relapsing fever.

The Relapsing Fever Spirochete Borrelia hermsii Contains Multiple, Antigen-Encoding Circular Plasmids That Are Homologous to the cp32 Plasmids of Lyme Disease Spirochetes

Borrelia hermsii, an agent of tick-borne relapsing fever, was found to contain multiple circular plasmids approximately 30 kb in size. Sequencing of a DNA library constructed from circular plasmid fragments enabled assembly of a composite DNA sequence that is homologous to the cp32 plasmid family of the Lyme disease spirochete, B. burgdorferi. Analysis of another relapsing fever bacterium, B. parkeri, indicated that it contains linear homologs of the B. hermsii and B. burgdorferi cp32 plasmids. The B. hermsii cp32 plasmids encode homologs of the B. burgdorferi Mlp and Bdr antigenic proteins and BlyA/BlyB putative hemolysins, but homologs of B. burgdorferi erp genes were absent. Immunoblot analyses demonstrated that relapsing fever patients produced antibodies to Mlp proteins, indicating that those proteins are synthesized by the spirochetes during human infection. Conservation of cp32-encoded genes in different Borrelia species suggests that their protein products serve functions essential to both relapsing fever and Lyme disease spirochetes. Relapsing fever borreliae replicate to high levels in the blood of infected animals, permitting direct detection and possible functional studies of Mlp, Bdr, BlyA/BlyB, and other cp32-encoded proteins in vivo.

Homology between Borrelia burgdorferi OspC and members of the family of Borrelia hermsii variable major proteins

Synthesis of the Borrelia burgdorferi outer surface protein C (OspC) is quite variable. We have cloned and sequenced the ospC gene from B. burgdorferi isolate CA-11.2A, a clone in which ospC expression varies. The 5' flanking region of the gene contains at least two consensus promoter regions, as well as two large overlapping inverted repeats. Sequence comparison to other OspC proteins indicated that the CA-11.2 A OspC is as closely related to OspC from two different genospecies of Lyme disease spirochetes as it is to OspC from the prototype B. burgdorferi strain, B31. Comparisons of the OspC amino acid (aa) sequence with those in aa sequence databases revealed partial identity with the variable major proteins Vmp3 and Vmp24 of B. hermsii, a causative agent of tick-borne relapsing fever. An ospC probe hybridized to B. hermsii restriction fragments and linear plasmids that also were recognized by the vmp3 and vmp24 probes. OspC and these Vmp appear to be related, but their synthesis is regulated differently in the two species of spirochetes. This represents a fascinating example of the evolution of the number, position, regulation and perhaps function of homologous genes in two related pathogens. These parameters may relate to characteristic properties of the pathogens and their separate tick vectors.

Pathophysiology of the Lyme disease spirochete, Borrelia burgdorferi, in ixodid ticks.

The pathophysiology of Borrelia burgdorferi, the Lyme disease spirochete, is unique in tick/vector relationships, differing substantially from that of other spirochetes, e.g., Borrelia duttonii, the agent of tick-borne relapsing fever, and Borrelia recurrentis, the agent of louse-borne relapsing fever, in their respective vectors. Following ingestion by a tick, B. burgdorferi lodges in the midgut diverticula, in some instances penetrating the gut wall and invading various tissues. Certain investigators suggest that transmission of the spirochete occurs via infectious saliva, although, in light of the fact that only 5% of adult ticks are systemically infected, this mechanism is open to question. Alternatively, transmission may occur via periodic regurgitation of gut fluids during the feeding process. While ticks of the genus Ixodes were once thought to be the only vectors, it now appears that other genera, and possibly other hematophagous arthropods, may also be involved.

Antigenic variation of surface proteins of Borrelia species.

During relapsing fever, the etiologic spirochetes employ antigenic variation to avoid early immune clearance. In Borrelia hermsii, an agent of tick-borne relapsing fever, a switch in serotype is associated with a change in the predominant protein on the surface of the cells. The variable major proteins differ from one another in primary structure along their lengths. Genes encoding the different variable major proteins can be found in both "silent" and "active" environments. At the active site the gene is expressed; at the silent site the gene appears to be in storage. In variant cells, which express the new serotype-specific surface protein in a population, a gene from a silent site has replaced the previously sitting gene at the active site through transposition.