Relapsing Fever Spirochete Research Articles

Complete genome sequence of Borrelia miyamotoi strain MN18-0001, an Am-East-2 strain type isolate derived from an Ixodes scapularis tick (Minnesota).

We report the genomic sequence of the hard tick relapsing fever spirochete Borrelia miyamotoi strain MN18-0001. B. miyamotoi causes human illness and is geographically widespread in Ixodes spp. (Acari: Ixodidae) ticks. This is a chromosome- and plasmid-resolved genome assembly of an Am-East-2 strain type isolate from the midwestern United States.

Development and validation of systems for genetic manipulation of the Old World tick-borne relapsing fever spirochete, Borrelia duttonii.

Relapsing fever (RF), a vector-borne disease caused by Borrelia spp., is characterized by recurring febrile episodes due to repeated bouts of bacteremia. RF spirochetes can be geographically and phylogenetically divided into two distinct groups; Old World RF Borrelia (found in Africa, Asia, and Europe) and New World RF Borrelia (found in the Americas). While RF is a rarely reported disease in the Americas, RF is prevalent in endemic parts of Africa. Despite phylogenetic differences between Old World and New World RF Borrelia and higher incidence of disease associated with Old World RF spirochete infection, genetic manipulation has only been described in New World RF bacteria. Herein, we report the generation of genetic tools for use in the Old World RF spirochete, Borrelia duttonii. We describe methods for transformation and establish shuttle vector- and integration-based approaches for genetic complementation, creating green fluorescent protein (gfp)-expressing B. duttonii strains as a proof of principle. Allelic exchange mutagenesis was also used to inactivate a homolog of the Borrelia burgdorferi p66 gene, which encodes an important virulence factor, in B. duttonii and demonstrate that this mutant was attenuated in a murine model of RF. Finally, the B. duttonii p66 mutant was complemented using shuttle vector- and cis integration-based approaches. As expected, complemented p66 mutant strains were fully infectious, confirming that P66 is required for optimal mammalian infection. The genetic tools and techniques reported herein represent an important advancement in the study of RF Borrelia that allows for future characterization of virulence determinants and colonization factors important for the enzootic cycle of Old World RF spirochetes.

Systematic investigation of the Borrelia miyamotoi spirochetes in ticks, wildlife and domestic animal hosts in Yunnan province, Southwest China

BackgroundBorrelia miyamotoi is a spirochete species transmitted via hard ticks. Following its discovery in Japan, this pathogen has been detected around the world, and is increasingly confirmed as a human pathogen causing febrile disease, namely relapsing fever. Its presence has been confirmed in the Northeast China. However, there is little information regarding the presence of B. miyamotoi and other hard-tick-borne relapsing fever spirochetes in southern China including Yunnan province, where tick and animal species are abundant and many people both inhabit and visit for recreation. MethodsFor the present study, we collected samples of ticks, wildlife, and domestic animal hosts from different counties in Yunnan province. Nucleic acids from samples were extracted, and the presence of B. miyamotoi and other relapsing fever spirochetes was confirmed using polymerase chain reaction (PCR) for the 16S rRNA specific target gene fragment. The positive samples were then amplified for partial genome of the flaB and glpQ genes. Statistical differences in its distribution were analyzed by SPSS 20 software. Sequence of partial 16S rRNA, flaB and glpQ genome were analyzed and phylogenetic trees were constructed. ResultsA total of 8260 samples including 2304 ticks, 4120 small mammals and 1836 blood of domestic animal hosts were collected for screening for infection of B. miyamotoi and other relapsing fever spirochetes. Cattle and sheep act as the main hosts and Rhipicephalus microplus, Haemaphysalis nepalensis, H. kolonini and Ixodes ovatus were identified as the important vector host with high prevalence or wide distribution. Only one Mus caroli (mouse) and one Sorex alpinus (shrew) were confirmed positive for relapsing fever spirochetes. Evidence of vertical transmission in ticks was also confirmed. Two known strains of B. miyamotoi and one novel relapsing fever spirochetes, B. theileri-like agent, were confirmed and described with their host adaptation, mutation, and potential risk of spreading and spillover for human beings. ConclusionsOur results provide new evidence of relapsing fever spirochetes in vector and animal hosts in Yunnan province based on large sample sizes, and offer guidance on further investigation, surveillance and monitoring of this pathogen.

Multifunctional interaction of CihC/FbpC orthologs of relapsing fever spirochetes with host-derived proteins involved in adhesion, fibrinolysis, and complement evasion.

Relapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation. Binding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues. Here, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

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.

Serological evidence of louse-borne relapsing fever in northern Kenya

BackgroundTick- and louse-borne relapsing fever are highly-neglected, vector-borne diseases caused by diverse Borrelia species. Presently, there are no data available on the endemicity of tick- and louse-borne relapsing fever spirochetes in Kenya. Here, we present data of a retrospective study on the seroprevalence of louse-borne relapsing fever (LBRF) in northern Kenya. MethodsA novel immunoassay, recently established for the diagnosis of LBRF was utilized to screen 2005 blood samples collected from individuals with fever without a source in Turkana County, Kenya between May 2009 and November 2010 for anti-LBRF antibodies. ResultsOut of the 2005 sera analyzed, 287 samples (14.3 %) were considered anti-LBRF IgG positive. Subsequent analyses revealed that 87 out of 152 sera randomly selected from these 2005 samples were tested positive (57.2 %) for anti-LBRF IgM antibodies. Most of the IgG and IgM positive samples were from individuals living in northern regions of Turkana County. ConclusionOur serological finding provides strong evidence for the occurrence of LBRF in Kenya.

Composition and transstadial retention of the salivary glands in Ornithodoros hermsi (Acari: Argasidae).

Following several days of blood feeding by larval and nymphal ixodid (hard) ticks, the salivary glands degenerate and are completely replaced in the next life stage. Yet, what happens during the molt of immature argasid (soft) ticks after their rapid and small bloodmeal has remained a mystery. Multiple studies of nymphal Ornithodoros hermsi Wheeler (Acari: Argasidae) ticks infected with the relapsing fever spirochete Borrelia hermsii suggested the salivary glands in these ticks may not disintegrate after feeding. Therefore, cohorts of second-stage O. hermsi nymphs were fed and examined daily after the bloodmeal by fresh dissections and weekly by histological cross-sections of the entire tick. The composition of the salivary glands was typical for argasid ticks in having agranular (Type I) and granular (Type II) acini, the latter being surrounded by a myo-epithelial sheath. In all 197 ticks examined from 1 to 63 days after feeding, morphologically intact salivary glands were present. During apolysis, 5 ticks had extralimital clusters of granular acini adhering to otherwise intact glands. Our observations demonstrate that the salivary glands of nymphal O. hermsi do not disintegrate after feeding and new acini are produced during the molt for incorporation into the existing glands. Cumulatively, these findings suggest a fundamental difference in the transstadial development of argasid and ixodid ticks.

Experimental transmission of a novel relapsing fever group Borrelia harbored by Ornithodoros octodontus (Ixodida: Argasidae) in Chile.

Tick-borne relapsing fever spirochetes of genus Borrelia thrive in enzootic cycles involving Ornithodoros spp. (Argasidae) mainly, and rodents. The isolation of these spirochetes usually involves a murine model in which ticks are fed and the spirochetes detected in blood several days later. Such an experiment also demonstrates that a given species of tick is competent in the transmission of the bacteria. Here, soft ticks Ornithodoros octodontus were collected in Northern Chile with the objective to experimentally determine its capacity to transmit a Borrelia sp. detected in a previous study. Two Guinea pigs (Cavia porcellus) were used to feed nymphs and adults of O. octodontus and the spirochetes in blood were inspected by dark-field microscopy and nested PCR. Although spirochetes were not seen in blood, DNA was detected in only one animal 11 days after the ticks were fed. Genetic sequences of Borrelia flaB, clpX, pepX, recG, rplB, and uvrA genes retrieved from DNA extraction of positive blood were employed to construct two phylogenetic analyses. On the one hand, the flaB tree showed the Borrelia sp. transmitted by O. octodontus clustering with Borrelia sp. Alcohuaz, which was previously detected in that same tick species. On the other hand, concatenated clpX-pepX-recG-rplB-uvrA demonstrated that the characterized spirochete branches together with "Candidatus Borrelia caatinga", a recently discovered species from Brazil. Based on the genetic profile presented in this study, the name "Candidatus Borrelia octodonta" is proposed for the species transmitted by O. octodontus. The fact that spirochetes were not observed in blood of guinea pigs, may reflect the occurrence of low spirochetemia, which could be explained because the susceptibility of infection varies depending on the rodent species that is used in experimental models. Although the vertebrate reservoir of "Ca. Borrelia octodonta" is still unknown, Octodon degus, a rodent species that is commonly parasitized by O. octodontus, should be a future target to elucidate this issue.

Borrelia turicatae from Ticks in Peridomestic Setting, Camayeca, Mexico.

We conducted surveillance studies in Sinaloa, Mexico, to determine the circulation of tick-borne relapsing fever spirochetes. We collected argasid ticks from a home in the village of Camayeca and isolated spirochetes. Genomic analysis indicated that Borrelia turicatae infection is a threat to those living in resource-limited settings.

Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II.

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined. Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined. Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86-95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3' end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined. Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

Autogenous reproduction by Ornithodoros turicata (Ixodida: Argasidae) females and vertical transmission of the tick-borne pathogen Borrelia turicatae (Spirochaetales: Borreliaceae).

Previous research has implicated Ornithodoros ticks, including Ornithodoros turicata, as long-term reservoirs of relapsing fever (RF) spirochetes. Considering the tick's long lifespan and their efficiency in maintaining and transferring spirochetes within the population, the infection could persist in a given enzootic focus for decades. However, little is known about the relative importance of horizontal and vertical transmission routes in the persistence and evolution of RF Borrelia. Our observations on the reproductive biology of O. turicata in the absence of vertebrate hosts indicate an additional mechanism by which Borrelia turicatae can be maintained in the environment. This work establishes the foundation for studying O. turicata reproduction and spirochete-vector interactions, which will aid in devising control measures for Ornithodoros ticks and RF spirochetes.

Comparative genomics analysis of three conserved plasmid families in the Western Hemisphere soft tick-borne relapsing fever borreliae provides insight into variation in genome structure and antigenic variation systems.

Borrelia spirochetes, causative agents of Lyme disease and relapsing fever (RF), have uniquely complex genomes, consisting of a linear chromosome and both circular and linear plasmids. The plasmids harbor genes important for the vector-host life cycle of these tick-borne bacteria. The role of plasmids from Lyme disease causing spirochetes is more refined compared to RF Borrelia because of limited plasmid-resolved genome assemblies for the latter. We recently addressed this limitation and found that three linear plasmid families (F6, F27, and F28) were syntenic across all the RF Borrelia species that we examined. Given this conservation, we further investigated the three plasmid families. The F6 family, also known as the megaplasmid, contained regions of repetitive DNA. The F27 was the smallest, encoding genes with unknown function. The F28 family encoded the putative expression locus for antigenic variation in all species except Borrelia hermsii and Borrelia anserina. Taken together, this work provides a foundation for future investigations to identify essential plasmid-localized genes that drive the vector-host life cycle of RF Borrelia. IMPORTANCE Borrelia spp. spirochetes are arthropod-borne bacteria found globally that infect humans and other vertebrates. RF borreliae are understudied and misdiagnosed pathogens because of the vague clinical presentation of disease and the elusive feeding behavior of argasid ticks. Consequently, genomics resources for RF spirochetes have been limited. Analyses of Borrelia plasmids have been challenging because they are often highly fragmented and unassembled in most available genome assemblies. By utilizing Oxford Nanopore Technologies, we recently generated plasmid-resolved genome assemblies for seven Borrelia spp. found in the Western Hemisphere. This current study is an in-depth investigation into the linear plasmids that were conserved and syntenic across species. We identified differences in genome structure and, importantly, in antigenic variation systems between species. This work is an important step in identifying crucial plasmid-localized genetic elements essential for the life cycle of RF spirochetes.

New records of Ornithodoros turicata (Ixodida: Argasidae) in rural and urban sites in the Mexican states of Aguascalientes and Zacatecas indicate the potential for tick-borne relapsing fever.

Soft ticks from the Ornithodoros genus are vectors of relapsing fever (RF) spirochetes around the world. In Mexico, they were originally described in the 19th century. However, few recent surveillance studies have been conducted in Mexico, and regions where RF spirochetes circulate remain vague. Here, the presence of soft ticks in populated areas was assessed in two sites from the Mexican states of Aguascalientes and Zacatecas. Argasidae ticks were collected, identified by morphology and mitochondrial 16S rDNA gene sequencing, and tested for RF borreliae. The specimens in both sites were identified as Ornithodoros turicata but no RF spirochetes were detected. These findings emphasize the need to update the distribution of these ticks in multiple regions of Mexico and to determine the circulation of RF borreliosis in humans and domestic animals.

Description of a new Pavlovskyella species (Acari: Argasidae) from Chile.

Soft ticks (Argasidae) of the Pavlovskyella Pospelova-Shtrom subgenus are important vectors of relapsing fever spirochetes, which are agents of disease globally. South American representatives of the Pavlovskyella subgenus include 3 species: Ornithodoros (Pavlovskyella) brasiliensis Aragão, Ornithodoros (Pavlovskyella) furcosus Neumann, and Ornithodoros (Pavlovskyella) rostratus Aragão. Here, we describe a fourth species based on morphological and mitogenomic evidence of ticks collected in burrows of unknown hosts in central Chile. The larva of the new species separates from other South American soft ticks by the following combination of characters: 13 pairs of dorsolateral setae, dorsal plate hexagonal, hypostome blunt with denticles from apex almost to the base. Adults of this new species lack cheeks, possess a dorsoventral groove, and have humps, similar to O. (P.) brasiliensis; however, they lack bulging structures on the flanks of idiosoma. Moreover, females and males differ from O. (P.) rostratus by having 3 humps instead of spurs in tarsi I and from O. (P.) furcosus because of their smaller size and thinner anterior lip of the genital aperture in females. The phylogenetic analysis performed with mitogenomes of the Argasidae family depicts the new Pavlovskyella species from Chile in a monophyletic clade with other South American species in the subgenus, confirming a regional group.

Comparison of salivary gland and midgut microbiome in the soft ticks Ornithodoros erraticus and Ornithodoros moubata.

Ornithodoros erraticus and Ornithodoros moubata are the main vectors of African swine fever virus (ASFV) and the human relapsing fever spirochetes Borrelia hispanica and Borrelia crocidurae in the Mediterranean region and Borrelia duttoni in continental Africa. Manipulation of the tick microbiome has been shown to reduce vector fitness and competence in tick vectors, suggesting that the identification of key microbial players associated with tick tissues can inform interventions such as anti-microbiota vaccines to block pathogen development in the midgut and/or salivary glands. In this study, we analyzed and compared the microbiome of the salivary glands and midgut of O. erraticus and O. moubata. For the taxonomic and functional characterization of the tissue-specific microbiome, we used 16S rRNA amplicon sequencing and prediction of metabolic profiles using PICRUSt2. Co-occurrence networks were built to characterize the community assembly and identify keystone taxa in each tick species. Our results revealed differences in the composition, diversity, and assembly of the bacterial microbiome of salivary glands and midgut within each tick species, but differences were more noticeable in O. moubata. Differences were also found in the microbiome of each tissue, salivary gland and midgut, between species. However, the 'Core Association Networks (CAN)' analysis revealed conserved patterns of interacting taxa in tissues within and between tick species. Different keystone taxa were identified in O. erraticus and O. moubata tissues, but Muribaculaceae and Alistipes were found as keystone taxa in the salivary glands of both tick species which justifies their use as anti-microbiota vaccine candidates to alter the microbiome and reduce tick fitness and/or block pathogen transmission. The high similarity of predicted metabolic pathways profiles between tissues of the two tick species suggests that taxonomic variability of the microbiome is not associated with significant changes in microbial functional profiles. We conclude that the taxonomic structure of the microbiome in O. erraticus and O. moubata is tissue-specific, suggesting niche partitioning of bacterial communities associated to these soft ticks. However, shared keystone taxa and conserved patterns of interacting taxa between tissues and tick species suggest the presence of key microbial players that could be used as anti-microbiota vaccine candidates to affect tick physiology and/or pathogen colonization.

Genome Sequence of a Clinical Isolate of the Human Pathogenic Strain “ Candidatus Borrelia fainii” Qtaro

We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete "Candidatus Borrelia fainii" Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.

A serological assay to detect and differentiate rodent exposure to soft tick and hard tick relapsing fever infections in the United States

Human cases of relapsing fever (RF) in North America are caused primarily by Borrelia hermsii and Borrelia turicatae, which are spread by argasid (soft) ticks, and by Borrelia miyamotoi, which is transmitted by ixodid (hard) ticks. In some regions of the United States, the ranges of the hard and soft tick RF species are known to overlap; in many areas, recorded ranges of RF spirochetes overlap with Lyme disease (LD) group Borrelia spirochetes. Identification of RF clusters or cases detected in unusual geographic localities might prompt public health agencies to investigate environmental exposures, enabling prevention of additional cases through locally targeted mitigation. However, exposure risks and mitigation strategies differ among hard and soft tick RF, prompting a need for additional diagnostic strategies that differentiate hard tick from soft tick RF. We evaluated the ability of new and previously described recombinant antigens in serological assays to differentiate among prior exposures in mice to LD, soft or hard tick RF spirochetes. We extracted whole-cell protein lysates from RF Borrelia cultures and synthesized six recombinant RF antigens (Borrelia immunogenic protein A (BipA) derived from four species of RF Borrelia, glycerophosphodiester phosphodiesterase (GlpQ), and Borrelia miyamotoi membrane antigen A (BmaA)) to detect reactivity in laboratory derived (Peromyscus sp. and Mus sp.) mouse serum infected with RF and LD Borrelia species. Among 44 Borrelia exposed mouse samples tested, all five mice exposed to LD spirochetes were correctly differentiated from the 39 mice exposed to RF Borrelia using the recombinant targets. Of the 39 mice exposed to RF spirochetes, 28 were accurately categorized to species of exposure (71%). Segregation among soft tick RF species (Borrelia hermsii, Borrelia parkeri and Borrelia turicatae) was inadequate (58%) owing to observed cross-reactivity among recombinant BipA protein targets. However, among the 28 samples accurately separated to species, all were accurately assigned to soft tick or hard tick RF type. Although not adequately specific to accurately categorize exposure to soft tick RF species, the recombinant BipA protein targets from soft and hard tick RF species show utility in accurately discriminating mouse exposures to LD or RF Borrelia, and accurately segregate hard tick from soft tick RF Borrelia exposure.

Rickettsia and relapsing fever Borrelia in Alectorobius kelleyi (Ixodida: Argasidae) from peri domestic bats in the northeastern United States

The soft ticks (Argasidae) are known vectors of human and animal pathogens around the globe and are relatively understudied. Our aim was to assess the presence of Rickettsia and Borrelia bacteria in Alectorobius kelleyi (Argasidae) parasitizing synanthropic bats in the highly urbanized northeastern United States. By collaborating with parasitologists, bat scientists and wildlife rehabilitators we were successful in obtaining A. kelleyi from five states. Since Argasid larvae will attach to their hosts for many days, most A. kelleyi examined (92%) were larvae collected from sick or injured big brown bats, Eptesicus fuscus, undergoing care at rehabilitation centers. In addition, we obtained adult A. kelleyi captured in residential living areas and trapped in attics. An in-depth analysis of a A. kelleyi found to be infected with a spotted fever group Rickettsia (SFGR) revealed a dual infection with a R. belli-like taxon (ancestral group) as well as an SFGR closely related to R. peacockii, likely the same previously found in A. kelleyi from Iowa and Kansas. We found that 36% of the A. kelleyi tested carried the SFGR. Furthermore, we detected a relapsing fever spirochete, likely Candidatus Borrelia johnsonii, in 25% of the A. kelleyi from Pennsylvania. While it is unclear if these bacteria constitute a health risk to either bats or humans, our study indicates that human exposure to ectoparasites infesting peridomestic wildlife should be considered in the epidemiology of tick-borne diseases.

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure.

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations.

Borrelia miyamotoi a neglected tick-borne relapsing fever spirochete in Thailand.

Borrelia miyamotoi is a relapsing fever spirochete that shares the same vector as Lyme disease causing Borrelia. This epidemiological study of B. miyamotoi was conducted in rodent reservoirs, tick vectors and human populations simultaneously. A total of 640 rodents and 43 ticks were collected from Phop Phra district, Tak province, Thailand. The prevalence rate for all Borrelia species was 2.3% and for B. miyamotoi was 1.1% in the rodent population, while the prevalence rate was quite high in ticks collected from rodents with an infection rate of 14.5% (95% CI: 6.3-27.6%). Borrelia miyamotoi was detected in Ixodes granulatus collected from Mus caroli and Berylmys bowersi, and was also detected in several rodent species (Bandicota indica, Mus spp., and Leopoldamys sabanus) that live in a cultivated land, increasing the risk of human exposure. Phylogenetic analysis revealed that the B. miyamotoi isolates detected in rodents and I. granulatus ticks in this study were similar to isolates detected in European countries. Further investigation was conducted to determine the serological reactivity to B. miyamotoi in human samples received from Phop Phra hospital, Tak province and in rodents captured from Phop Phra district using an in-house, direct enzyme-linked immunosorbent assay (ELISA) assay with B. miyamotoi recombinant glycerophosphodiester-phosphodiesterase (rGlpQ) protein as coated antigen. The results showed that 17.9% (15/84) of human patients and 9.0% (41/456) of captured rodents had serological reactivity to B. miyamotoi rGlpQ protein in the study area. While a low level of IgG antibody titers (100-200) was observed in the majority of seroreactive samples, higher titers (400-1,600) were also detected in both humans and rodents. This study provides the first evidence of B. miyamotoi exposure in human and rodent populations in Thailand and the possible roles of local rodent species and Ixodes granulatus tick in its enzootic transmission cycle in nature.