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

Introduction: 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. Methods: 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. Results and discussion: 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.

Serological evidence of louse-borne relapsing fever in northern Kenya.

BACKGROUND: Tick- 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. METHODS: A 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. RESULTS: Out 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. CONCLUSION: Our serological finding provides strong evidence for the occurrence of LBRF in Kenya.

The global distribution and the risk prediction of relapsing fever group Borrelia: a data review with modelling analysis.

BACKGROUND: The recent discovery of emerging relapsing fever group Borrelia (RFGB) species, such as Borrelia miyamotoi, poses a growing threat to public health. However, the global distribution and associated risk burden of these species remain uncertain. We aimed to map the diversity, distribution, and potential infection risk of RFGB. METHODS: We searched PubMed, Web of Science, GenBank, CNKI, and eLibrary from Jan 1, 1874, to Dec 31, 2022, for published articles without language restriction to extract distribution data for RFGB detection in vectors, animals, and humans, and clinical information about human patients. Only articles documenting RFGB infection events were included in this study, and data for RFGB detection in vectors, animals, or humans were composed into a dataset. We used three machine learning algorithms (boosted regression trees, random forest, and least absolute shrinkage and selection operator logistic regression) to assess the environmental, ecoclimatic, biological, and socioeconomic factors associated with the occurrence of four major RFGB species: Borrelia miyamotoi, Borrelia lonestari, Borrelia crocidurae, and Borrelia hermsii; and mapped their worldwide risk level. FINDINGS: We retrieved 13 959 unique studies, among which 697 met the selection criteria and were used for data extraction. 29 RFGB species have been recorded worldwide, of which 27 have been identified from 63 tick species, 12 from 61 wild animals, and ten from domestic animals. 16 RFGB species caused human infection, with a cumulative count of 26 583 cases reported from Jan 1, 1874, to Dec 31, 2022. Borrelia recurrentis (17 084 cases) and Borrelia persica (2045 cases) accounted for the highest proportion of human infection. B miyamotoi showed the widest distribution among all RFGB, with a predicted environmentally suitable area of 6·92 million km2, followed by B lonestari (1·69 million km2), B crocidurae (1·67 million km2), and B hermsii (1·48 million km2). The habitat suitability index of vector ticks and climatic factors, such as the annual mean temperature, have the most significant effect among all predictive models for the geographical distribution of the four major RFGB species. INTERPRETATION: The predicted high-risk regions are considerably larger than in previous reports. Identification, surveillance, and diagnosis of RFGB infections should be prioritised in high-risk areas, especially within low-income regions. FUNDING: National Key Research and Development Program of China.

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 puertoricensis in opossums (Didelphis marsupialis) from Colombia.

BACKGROUND: The genus Borrelia comprises pathogenic species of bacteria that pose a significant risk to public health. Borrelia spp. are emerging or reemerging infectious agents worldwide with complex transmission cycles, and many species use rodents as vertebrate reservoir hosts. Spirochetes morphologically compatible with Borrelia have been recurrently observed in opossums; however, there is currently a lack of genetic evidence confirming infection or supporting that these marsupials are hosts of Borrelia spirochetes. METHODS: During 2017, 53 serum samples of Didelphis marsupialis from the municipality of Colosó (department of Sucre, Colombia) were collected and allocated in a serum bank. DNA extracted from the serum samples was submitted to a Borrelia genus-specific real-time PCR targeting the 16S rRNA gene. Positive samples were subsequently derived from semi-nested PCR protocols to obtain large fragments of the 16S rRNA and flaB genes. Obtained amplicons were subjected to Sanger sequencing. One positive sample was randomly selected for next-generation sequencing (NGS). Obtained reads were mapped to genomes of Borrelia spp. and sequences of two genes used in a multilocus sequence typing scheme retrieved for taxonomic assignment and phylogenetic analyses. RESULTS: Overall, 18.8% (10/53) of the samples were positive by qPCR. Of them, 80% (8/10) and 60% (6/10) were positive for the 16S rRNA and flaB genes after semi-nested PCRs, respectively. Bioinformatic analysis of one sample sequenced with NGS yielded 22 reads of genus Borrelia with different sizes. Two housekeeping genes, rplB and pyrG, were recovered. Nucleotide pairwise comparisons and phylogenetic analyses of 16S rRNA, flaB, rplB and pyrG genes showed that the Borrelia sp. found in opossums from Colosó corresponded to Borrelia puertoricensis. CONCLUSIONS: We describe the first molecular evidence to our knowledge of B. puertoricensis in Colombia, specifically in opossums, and the first detection of this spirochete in a vertebrate host since its isolation from Ornithodoros puertoricensis in Panama. This detection is also relevant because of the epidemiological importance of opossums as reservoirs of zoonotic diseases to humans.

Emerging bacterial infectious diseases/pathogens vectored by human lice.

Human lice have always been a major public health concern due to their vector capacity for louse-borne infectious diseases, like trench fever, louse-borne relapsing fever, and epidemic fever, which are caused by Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii, respectively. Those diseases are currently re-emerging in the regions of poor hygiene, social poverty, or wars with life-threatening consequences. These louse-borne diseases have also caused outbreaks among populations in jails and refugee camps. In addition, antibodies and DNAs to those pathogens have been steadily detected in homeless populations. Importantly, more bacterial pathogens have been detected in human lice, and some have been transmitted by human lice in laboratories. Here, we provide a comprehensive review and update on louse-borne infectious diseases/bacterial pathogens.

Conformational dynamics of complement protease C1r inhibitor proteins from Lyme disease- and relapsing fever-causing spirochetes.

Borrelial pathogens are vector-borne etiological agents known to cause Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind components of the human complement system to evade host immunity. One borrelial lipoprotein, BBK32, protects the Lyme disease spirochete from complement-mediated attack via an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical complement pathway, C1r. In addition, the B. miyamotoi BBK32 orthologs FbpA and FbpB also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever-causing spirochetes, remains unknown. Here, we report the crystal structure of the C-terminal domain of Borrelia hermsii FbpC to a limiting resolution of 1.5 Å. We used surface plasmon resonance and assays of complement function to demonstrate that FbpC retains potent BBK32-like anticomplement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. Taken together, these results advance our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveal a surprising plasticity in the structures of borrelial C1r inhibitors.

Quantitative Polymerase Chain Reaction from Malaria Rapid Diagnostic Tests to Detect Borrelia crocidurae, the Agent of Tick-Borne Relapsing Fever, in Febrile Patients in Senegal.

In endemic malaria areas, Plasmodium is currently diagnosed mainly through the use of rapid diagnostic tests (RDTs). However, in Senegal, many causes of fever remain unknown. Tick-borne relapsing fever, an often-neglected public health problem, is the main cause of consultation for acute febrile illness after malaria and flu in rural areas. Our objective was to test the feasibility of extracting and amplifying DNA fragments by quantitative polymerase chain reaction (qPCR) from malaria-negative RDTs for Plasmodium falciparum (malaria Neg RDTs P.f) to detect Borrelia spp. and other bacteria. Between January and December 2019, malaria Neg RDTs P.f were collected on a quarterly basis in 12 health facilities in four regions of Senegal. The DNA extracted from the malaria Neg RDTs P.f was tested using qPCR and the results were confirmed by standard PCR and sequencing. Only Borrelia crocidurae DNA was detected in 7.22% (159/2,202) of RDTs. The prevalence of B. crocidurae DNA was higher in July (16.47%, 43/261) and August (11.21%, 50/446). The annual prevalence was 9.2% (47/512) and 5.0% (12/241) in Ngayokhem and Nema-Nding, respectively, health facilities in the Fatick region. Our study confirms that B. crocidurae infection is a frequent cause of fever in Senegal, with a high prevalence of cases in health facilities in the regions of Fatick and Kaffrine. Malaria Neg RDTs P.f are potentially a good source of pathogen sampling for the molecular identification of other causes of fever of unknown origin, even in the most remote areas.

Novel Borrelia Genotypes from Brazil Indicate a New Group of Borrelia spp. Associated with South American Bats.

The bacterial genus Borrelia comprises vector-borne spirochetes that have been classified into three major groups: the relapsing fever group (RFG), the Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner sensu lato group (Bbsl), and the reptile-monotreme group (RMG). All three groups have been associated mainly with ticks and wild animals, especially rodents, birds, and reptiles. Here, we searched for Borrelia infection among 99 vampire bats [Desmodus rotundus (É. Geoffroy)] (Chiroptera: Phyllostomidae) from the Brazilian semiarid region. Through molecular investigation of bat internal organs, haplotypes of a potentially novel Borrelia organism were detected in 5% (5/99) of the bats. Borrelia DNA was detected in the liver, blood, spleen, kidney and brain, suggesting a systemic infection. Phylogenetic analyses inferred from partial sequences of the borrelial rrs and flaB genes indicated that the vampire bat-associated Borrelia sp. of this study form a monophyletic group with a newly reported Borrelia associated with a Colombia bat, distinct from the three main currently recognized groups of Borrelia spp., Bbsl, RFG, and RMG. These novel bat-associated Borrelia spp. from South America might have arisen through an independent event along the borrelial evolutionary history, since previous molecular reports of Borrelia organisms in bats or bat-associated ticks from Africa, Europe, and North America were all classified in the RFG.

Recombinant protein immunoblots for differential diagnosis of tick-borne relapsing fever and Lyme disease.

Lyme disease (LD) is caused by a group of tick-borne bacteria of the genus Borrelia termed Lyme disease Borreliae (LDB). The detection of serum antibodies to specific LDB antigens is widely used to support diagnosis of LD. Recent findings highlight a need for serological tests that can differentiate LD from tick-borne relapsing fever (TBRF) caused by a separate group of Borrelia species termed relapsing fever Borreliae. This is because LD and TBRF share some clinical symptoms and can occur in overlapping locations. The development of serological tests for TBRF is at an early stage compared with LD. This article reviews the application of line immunoblots (IBs), where recombinant proteins applied as lines on nitrocellulose membrane strips are used to detect antibodies in patient sera, for the diagnosis and differentiation of LD and TBRF.

Novel approaches for the serodiagnosis of louse-borne relapsing fever.

Louse-borne relapsing fever (LBRF) caused by B. recurrentis is a poverty-related and neglected infectious disease with an endemic focus in the Horn of Africa. Re-emergence of the disease occurred in Europe during the refugee crisis in 2015 and sporadic outbreaks were frequently reported in Eastern Africa where poor settings lack affordable diagnostics. Currently, there are no validated in vitro assays available for the serodiagnosis of LBRF. The aim of this study was to develop novel and reliable immunoassays by investigating clinically suspected and culture-confirmed serum samples from LBRF patients and a broad panel of serum samples from patients with other spirochetal, bacterial, and parasitic diseases. We identified two immunoreactive antigens (complement-inhibiting protein CihC and the glycerophosphodiester phosphodiesterase GlpQ of B. recurrentis) as the most promising target candidates leading to the evaluation of two immunoassays (line immunoblot and ELISA) for IgM and IgG. To optimize the IgM immunoassay, we conducted a bioinformatic approach to localize the relevant immunogenic regions within CihC. By utilizing a N-terminal CihC fragment, the sensitivity and specificity of both immunoassays (CihC and GlpQ) were high (IgM: sensitivity 100%, specificity of 89.9%, IgG: sensitivity 100%, specificity 99.2%). In conclusion, our findings indicate the diagnostic potential of CihC and GlpQ as valuable markers for the serodiagnosis of LBRF even at early time points of infection. Here, we provide strong evidence for the utilization of these immunoassays as reliable tools in clinical practice.

Molecular Characterization of a Novel Relapsing Fever Borrelia Species from the Desert Cottontail (Sylvilagus audubonii) in New Mexico, USA.

The Borrelia genus comprises vector-borne, spirochete bacteria infecting vertebrates worldwide. We characterized a novel relapsing fever Borrelia species from a desert cottontail (Syvilagus audubonii) from New Mexico, US, using an established multilocus sequence analysis approach. Phylogenetic analysis of the flagellin gene (flaB) and four other protein-coding loci (clpX, pepX, recG, rplB) grouped the novel Borrelia species with hard tick relapsing fever borreliae Borrelia lonestari, Borrelia theileri, and Borrelia miyamotoi. The identity of the vectors and other vertebrate hosts, geographic distribution, and zoonotic potential of this novel Borrelia species deserve further investigation.

Infection and seroprevalence of Borrelia persica in domestic cats and dogs in Israel.

BACKGROUND: Relapsing fever borreliosis is an infectious disease caused by bacteria of the genus Borrelia, inflicting recurrent episodes of fever and spirochetemia in humans. Borrelia persica, the causative agent of relapsing fever in Israel, is prevalent over a broad geographic area that extends from India to Egypt. It is transmitted by the soft tick Ornithodoros tholozani and causes disease in humans as well as domestic cats and dogs. The goal of this study was to survey domestic dogs and cats in Israel for infection with B. persica. METHODS: Blood, sera and demographic and clinical data were collected from dogs and cats brought for veterinary care in central Israel. PCR followed by DNA sequencing was used to detect B. persica DNA in blood samples, and an enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies reactive with B. persica antigens in sera from the same animals. This is the first serological survey of B. persica in dogs and the first survey for antibodies reactive with a relapsing fever Borrelia sp. in cats globally. RESULTS: Four of the 208 dogs (1.9%) and three of 103 cats (2.9%) sampled were positive by PCR for B. persica DNA, and 24 dogs (11.5%) and 18 cats (17.5%) were seropositive for B. persica antigen by ELISA. The ratio between PCR-positivity and seropositivity in both the dog and cat populations was 1:6. All four PCR-positive dogs and two of three PCR-positive cats were seronegative, suggesting a probable recent infection. Thrombocytopenia showed significant association with seropositivity in dogs (P = 0.003). In cats, anemia had a significant association with seropositivity (P = 0.0001), and thrombocytopenia was associated with the combined prevalence of seropositivity or PCR-positivity (P = 0.022). CONCLUSIONS: Borrelia persica infection is more prevalent and widespread in domestic canine and feline populations in Israel than previously thought. Dogs and cats may play a role as reservoirs and sentinels for human infection. Precautions should be taken to prevent transfusion-transmitted infection between blood donor and recipient animals.

Characterization of Immunological Responses to Borrelia Immunogenic Protein A (BipA), a Species-Specific Antigen for North American Tick-Borne Relapsing Fever.

Tick-borne relapsing fever (TBRF) is a neglected vector-borne bacterial disease distributed worldwide. Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii are three argasid-borne TBRF species previously implicated in human disease in North America. TBRF is likely underdiagnosed due to its nonspecific symptoms and poorly developed diagnostic tests. Studies suggest that the Borrelia immunogenic protein A (BipA) is specific to TBRF Borrelia but heterogenic between species. In this study, we hypothesized that antibody responses generated to BipA are specific to the North American TBRF species infecting a given animal. To test this, we characterized the expression and localization of native BipA in North American species of TBRF Borrelia. We also infected mice by needle inoculation or tick bite with B. turicatae, B. hermsii, or B. parkeri and evaluated serum sample reactivity to recombinant BipA (rBipA) that was produced from each species. Furthermore, serum samples from nonhuman primates and domestic dogs experimentally infected with B. turicatae were assessed. Lastly, we tested human Lyme disease (LD) serum samples to determine potential cross-reactivity to rBipA generated from B. turicatae, B. parkeri, and B. hermsii. Our findings indicate that rBipA has the potential to distinguish between infections of LD- and TBRF-causing spirochetes and that antibody responses were more robust toward the Borrelia species causing infection. This work further supports that rBipA can likely distinguish between B. turicatae, B. hermsii, and B. parkeri infections in mice, canines, and nonhuman primates. IMPORTANCEBorrelia species transmitted by soft or hard ticks cause tick-borne relapsing fever (TBRF). This is a debilitating disease distributed worldwide but is likely underdiagnosed or misdiagnosed as Lyme disease due to poorly developed diagnostic tests. Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii are three TBRF species previously implicated in human disease in North America. Commonly used diagnostic methods do not identify the species causing infection. In this study, we evaluated the potential of recombinant Borrelia immunogenic protein A (rBipA) as a diagnostic antigen capable of distinguishing between infections of TBRF Borrelia species. We show that serum from mice, canines, and nonhuman primates infected with B. turicatae, B. parkeri, or B. hermsii react more strongly to the rBipA from the species causing infection. Furthermore, sera from Lyme disease patients failed to cross-react with our rBipA proteins, indicating the potential to use rBipA as a species-specific diagnostic antigen for TBRF.

A Comparative Study of Body Lice and Bed Bugs Reveals Factors Potentially Involved in Differential Vector Competence for the Relapsing Fever Spirochete Borrelia recurrentis.

Borrelia recurrentis is the causative agent of louse-borne relapsing fever and the only Borrelia species transmitted by an insect rather than a tick vector. While bed bugs (Cimex lectularius L.) are not established vectors of any human pathogens, a recent study reported that they may be competent vectors of B. recurrentis. However, many aspects of infection and transmission remain unclear in this possible secondary vector. Here, we carried out several quantitative laboratory studies to gain a better understanding of the host suitability of bed bugs relative to the established body louse vector as well as the factors that may affect the ability of bed bugs to transmit the pathogen. We fed bed bugs B. recurrentis and estimated the level and duration of infection in the hemolymph using live imaging. We performed quantitative PCR (qPCR) to examine whole-body spirochete levels and the occurrence of vertical transmission to progeny. We also developed an assay to compare the amounts of force required to release infectious hemolymph from recently engorged bed bugs and body lice. Finally, we analyzed humoral antibacterial activity in the hemolymph, hemolymph pH, and hemocyte activity in both insect species. Our results confirm that within 24 h of ingestion, B. recurrentis can penetrate the midgut epithelium of bed bugs and enter the hemolymph, overcoming a major host barrier, as in body lice. Once in the hemolymph, spirochetes remain visible for at least 4 days. Moreover, we show that bed bugs are more physically susceptible to crushing than body lice, suggesting that crushing is a feasible route for the natural dissemination of B. recurrentis from the hemolymph of bed bugs, as for body lice. Nonetheless, our data also indicate that bed bugs are suboptimal hosts for B. recurrentis, as the bacterium does not appear to proliferate to high levels or stably colonize the hemolymph and exhibits pleomorphism in this environment. In particular, our data suggest that hemolymph pH and unique cellular immune responses, rather than humoral effectors, may be involved in limiting spirochete survival in bed bugs. Notably, we document the formation of extracellular DNA traps by bed bug hemocytes for the first time. For these reasons, while bed bugs may be capable of limited transmission given their ecology, vector competence is probably minimal relative to body lice. Additional mechanistic studies of human pathogen infection of bed bugs may provide much-needed insight into the biological factors that restrict their ability to act as vectors and may reveal novel mechanisms of immunity.

The arginine deaminase system plays distinct roles in Borrelia burgdorferi and Borrelia hermsii.

Borrelia species are amino acid auxotrophs that utilize di- and tri- peptides obtained through their oligopeptide transport system to supply amino acids for replicative growth during their enzootic cycles. However, Borrelia species from both the Lyme disease (LD) and relapsing fever (RF) groups harbor an amino acid transport and catabolism system, the Arginine Deiminase System (ADI), that could potentially augment intracellular L-arginine required for growth. RF spirochetes contain a "complete", four gene ADI (arcA, B, D, and C) while LD spirochetes harbor arcA, B, and sometimes D but lack arcC (encoding carbamate kinase). In this study, we evaluated the role of the ADI system in bacterial survival and virulence and discovered important differences in RF and LD ADIs. Both in vitro and in a murine model of infection, B. hermsii cells significantly reduced extracellular L-arginine levels and that reduction was dependent on arginine deiminase expression. Conversely, B. burgdorferi did not reduce the concentration of L-arginine during in vitro growth experiments nor during infection of the mammalian host, suggesting a fundamental difference in the ability to directly utilize L-arginine compared to B. hermsii. Further experiments using a panel of mutants generated in both B. burgdorferi and B. hermsii, identified important differences in growth characteristics and ADI transcription and protein expression. We also found that the ADI system plays a key role in blood and spleen colonization in RF spirochetes. In this study we have identified divergent metabolic strategies in two closely related human pathogens, that ultimately impacts the host-pathogen interface during infection.

Tick borne relapsing fever - a systematic review and analysis of the literature.

Tick borne relapsing fever (TBRF) is a zoonosis caused by various Borrelia species transmitted to humans by both soft-bodied and (more recently recognized) hard-bodied ticks. In recent years, molecular diagnostic techniques have allowed to extend our knowledge on the global epidemiological picture of this neglected disease. Nevertheless, due to the patchy occurrence of the disease and the lack of large clinical studies, the knowledge on several clinical aspects of the disease remains limited. In order to shed light on some of these aspects, we have systematically reviewed the literature on TBRF and summarized the existing data on epidemiology and clinical aspects of the disease. Publications were identified by using a predefined search strategy on electronic databases and a subsequent review of the reference lists of the obtained publications. All publications reporting patients with a confirmed diagnosis of TBRF published in English, French, Italian, German, and Hungarian were included. Maps showing the epidemiogeographic mosaic of the different TBRF Borrelia species were compiled and data on clinical aspects of TBRF were analysed. The epidemiogeographic mosaic of TBRF is complex and still continues to evolve. Ticks harbouring TBRF Borrelia have been reported worldwide, with the exception of Antarctica and Australia. Although only molecular diagnostic methods allow for species identification, microscopy remains the diagnostic gold standard in most clinical settings. The most suggestive symptom in TBRF is the eponymous relapsing fever (present in 100% of the cases). Thrombocytopenia is the most suggestive laboratory finding in TBRF. Neurological complications are frequent in TBRF. Treatment is with beta-lactams, tetracyclines or macrolids. The risk of Jarisch-Herxheimer reaction (JHR) appears to be lower in TBRF (19.3%) compared to louse-borne relapsing fever (LBRF) (55.8%). The overall case fatality rate of TBRF (6.5%) and LBRF (4-10.2%) appears to not differ. Unlike LBRF, where perinatal fatalities are primarily attributable to abortion, TBRF-related perinatal fatalities appear to primarily affect newborns.

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.

Isolation and genetic characterization of a relapsing fever spirochete isolated from Ornithodoros puertoricensis collected in central Panama.

Tick-borne relapsing fever (TBRF) spirochetes are likely an overlooked cause of disease in Latin America. In Panama, the pathogens were first reported to cause human disease in the early 1900s. Recent collections of Ornithodoros puertoricensis from human dwellings in Panama prompted our interest to determine whether spirochetes still circulate in the country. Ornithodoros puertoricensis ticks were collected at field sites around the City of Panama. In the laboratory, the ticks were determined to be infected with TBRF spirochetes by transmission to mice, and we report the laboratory isolation and genetic characterization of a species of TBRF spirochete from Panama. Since this was the first isolation of a species of TBRF spirochete from Central America, we propose to designate the bacteria as Borrelia puertoricensis sp. nov. This is consistent with TBRF spirochete species nomenclature from North America that are designated after their tick vector. These findings warrant further investigations to assess the threat B. puertoricensis sp. nov. may impose on human health.

Surveillance of Borrelia miyamotoi-carrying ticks and genomic analysis of isolates in Inner Mongolia, China.

BACKGROUND: Borrelia miyamotoi is a newly described relapsing fever spirochete transmitted by ixodid tick species. Little is known about the prevalence of B. miyamotoi infections in humans and ticks in Inner Mongolia, China. Therefore, we investigated the prevalence of B. miyamotoi in Ixodes persulcatus ticks, and we aimed to isolateB. miyamotoi from I. persulcatus from four regions of Greater Khingan, Inner Mongolia, China. METHODS: From May to June each year during the period 2016-2019, host-seeking adult I. persulcatus ticks were collected from vegetation. Genomic DNA was prepared from half of each tick body for PCR template, and the remaining half was used to cultivate B. miyamotoi in BSK-M medium. We employed quantitative real-time PCR (qPCR) to detect Borrelia DNA in the ticks and to calculate the prevalence of B. miyamotoi and infections with other borreliae. For characterization of the isolated B. miyamotoi, we performed draft genome sequencing and multilocus sequencing analysis (MLSA). RESULTS: A total of 2656 adult I. persulcatus ticks were collected. The overall prevalence of relapsing fever (RF) borreliae in ticks was 5.0% (134/2656) and that of Lyme disease (LD) borreliae was 43.8% (1164/2656). Co-infection with RF and LD borreliae was observed in 63 ticks (2.4%). Ticks that were positive for RF borreliae by qPCR were subjected to glycerophosphodiester diester phosphodiesterase gene (glpQ) PCR amplification and sequencing, through which we identified the RF borrelia specimens as B. miyamotoi. Furthermore, the B. miyamotoi strain Hetao-1 was isolated from I. persulcatus, and a draft genome sequence was obtained from the isolate. Sequencing determined the strain Hetao-1 genome to be approximately 906.1 kbp in length (28.9% average GC content), and MLSA identified the strain as ST633, which has previously been reported in Japan and Mongolia. CONCLUSION: We detected B. miyamotoi from I. persulcatus ticks collected in Inner Mongolia, and successfully isolated a B. miyamotoi strain. To our knowledge, this is the first study to culture a B. miyamotoi isolate from China. The data on the prevalence of B. miyamotoi and other borreliae in I. persulcatus ticks will be fundamental for future epidemiological studies of B. miyamotoi disease in Inner Mongolia.