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.

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

IMPORTANCE: 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.

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.

Comparative genomics of the Western Hemisphere soft tick-borne relapsing fever borreliae highlights extensive plasmid diversity.

BACKGROUND: Tick-borne relapsing fever (TBRF) is a globally prevalent, yet under-studied vector-borne disease transmitted by soft and hard bodied ticks. While soft TBRF (sTBRF) spirochetes have been described for over a century, our understanding of the molecular mechanisms facilitating vector and host adaptation is poorly understood. This is due to the complexity of their small (~ 1.5 Mb) but fragmented genomes that typically consist of a linear chromosome and both linear and circular plasmids. A majority of sTBRF spirochete genomes' plasmid sequences are either missing or are deposited as unassembled sequences. Consequently, our goal was to generate complete, plasmid-resolved genomes for a comparative analysis of sTBRF species of the Western Hemisphere. RESULTS: Utilizing a Borrelia specific pipeline, genomes of sTBRF spirochetes from the Western Hemisphere were sequenced and assembled using a combination of short- and long-read sequencing technologies. Included in the analysis were the two recently isolated species from Central and South America, Borrelia puertoricensis n. sp. and Borrelia venezuelensis, respectively. Plasmid analyses identified diverse sequences that clustered plasmids into 30 families; however, only three families were conserved and syntenic across all species. We also compared two species, B. venezuelensis and Borrelia turicatae, which were isolated ~ 6,800 km apart and from different tick vector species but were previously reported to be genetically similar. CONCLUSIONS: To truly understand the biological differences observed between species of TBRF spirochetes, complete chromosome and plasmid sequences are needed. This comparative genomic analysis highlights high chromosomal synteny across the species yet diverse plasmid composition. This was particularly true for B. turicatae and B. venezuelensis, which had high average nucleotide identity yet extensive plasmid diversity. These findings are foundational for future endeavors to evaluate the role of plasmids in vector and host adaptation.

Isolation and Molecular Characterization of Tick-Borne Relapsing Fever Borrelia Infecting Ornithodoros (Pavlovskyella) verrucosus Ticks Collected in Ukraine.

BACKGROUND: Tick-borne relapsing fever (TBRF) is a neglected zoonotic bacterial disease known to occur on 5 continents. We report a laboratory-acquired case of TBRF caused by Borrelia caucasica, which is endemic in Ukraine and transmitted by Ornithodoros verrucosus ticks. METHODS: We isolated spirochetes and characterized them by partially sequencing the 16s ribosomal ribonucleic acid (rrs), flagellin (flaB), and deoxyribonucleic acid gyrase (gyrB) genes and conducting a phylogenetic analysis. RESULTS: These analyses revealed a close relationship of Ukrainian spirochetes with the Asian TBRF species, Borrelia persica. The taxonomic and nomenclature problems related to insufficient knowledge on the spirochetes and their vectors in the region are discussed. CONCLUSIONS: Although these findings enhance our understanding of species identities for TBRF Borrelia in Eurasia, further work is required to address the neglected status of TBRF in this part of the world. Public health practitioners should consider TBRF and include the disease into differential diagnosis of febrile illnesses with unknown etiology.

Immunological Responses to the Relapsing Fever Spirochete Borrelia turicatae in Infected Rhesus Macaques: Implications for Pathogenesis and Diagnosis.

The global public health impact of relapsing fever (RF) spirochetosis is significant, since the pathogens exist on five of seven continents. The hallmark sign of infection is episodic fever and the greatest threat is to the unborn. With the goal of better understanding the specificity of B-cell responses and the role of immune responses in pathogenicity, we infected rhesus macaques with Borrelia turicatae (a new world RF spirochete species) by tick bite and monitored the immune responses generated in response to the pathogen. Specifically, we evaluated inflammatory mediator induction by the pathogen, host antibody responses to specific antigens, and peripheral lymphocyte population dynamics. Our results indicate that B. turicatae elicits from peripheral blood cells key inflammatory response mediators (interleukin-1ß and tumor necrosis factor alpha), which are associated with preterm abortion. Moreover, a global decline in peripheral B-cell populations was observed in all animals at 14 days postinfection. Serological responses were also evaluated to assess the antigenicity of three surface proteins: BipA, BrpA, and Bta112. Interestingly, a distinction was observed between antibodies generated in nonhuman primates and mice. Our results provide support for the nonhuman primate model not only in studies of prenatal pathogenesis but also for diagnostic and vaccine antigen identification and testing.

Detection of Tickborne Relapsing Fever Spirochete, Austin, Texas, USA.

In March 2017, a patient became febrile within 4 days after visiting a rustic conference center in Austin, Texas, USA, where Austin Public Health suspected an outbreak of tickborne relapsing fever a month earlier. Evaluation of a patient blood smear and molecular diagnostic assays identified Borrelia turicatae as the causative agent. We could not gain access to the property to collect ticks. Thus, we focused efforts at a nearby public park, <1 mile from the suspected exposure site. We trapped Ornithodoros turicata ticks from 2 locations in the park, and laboratory evaluation resulted in cultivation of 3 B. turicatae isolates. Multilocus sequencing of 3 chromosomal loci (flaB, rrs, and gyrB) indicated that the isolates were identical to those of B. turicatae 91E135 (a tick isolate) and BTE5EL (a human isolate). We identified the endemicity of O. turicata ticks and likely emergence of B. turicatae in this city.

Vector Competence of Geographical Populations of Ornithodoros turicata for the Tick-Borne Relapsing Fever Spirochete Borrelia turicatae.

Vector competence refers to the ability of an arthropod to acquire, maintain, and successfully transmit a microbial pathogen. Tick-borne relapsing fever (TBRF) spirochetes are globally distributed pathogens, and most species are transmitted by argasid ticks of the genus Ornithodoros. A defining characteristic in vector competence is an apparent specificity of a species of TBRF spirochete to a given tick species. In arid regions of the southern United States, Borrelia turicatae is the primary cause of TBRF. Interestingly, there are two populations of the tick vector distributed throughout this region. Ornithodoros turicata is a western population that ranges from California to Texas. There is a gap through Louisiana, Mississippi, and Alabama where the tick has not been identified. An isolated eastern population exists in Florida and was designated a subspecies, O. turicata americanus. A knowledge gap that exists is the poor understanding of vector competence between western and eastern populations of ticks for B. turicatae. In this study, we generated uninfected colonies of O. turicata that originated in Texas and Kansas and of O. turicataamericanus. B. turicatae acquisition, maintenance through the molt, and subsequent transmission were evaluated. Our findings revealed significant differences in murine infection after feeding infected O. turicata and O. turicataamericanus ticks on the animals. Interestingly, the salivary glands of both tick populations were colonized with B. turicatae to similar densities. Our results suggest that the salivary glands of the tick colonies assessed in this study impact vector competence of the evaluated B. turicatae isolates.IMPORTANCE Several knowledge gaps exist in the vector competence of various geographical populations of O. turicata that transmit B. turicatae A western population of this tick is distributed from California to Texas, and an eastern population exists in Florida. Utilizing western and eastern populations of the vector, we studied acquisition and transmission of two B. turicatae isolates. Regardless of the isolate used, infection frequencies were poor in mice after the eastern population feeding on them. Since salivary gland colonization is essential for B. turicatae transmission, these tissues were further evaluated. Interestingly, the salivary glands from the two populations were similarly colonized with B. turicatae. These findings suggest the role of tick saliva in the establishment of infection and that the salivary glands may be a bottleneck for successful transmission.

Imaging of Borrelia turicatae Producing the Green Fluorescent Protein Reveals Persistent Colonization of the Ornithodoros turicata Midgut and Salivary Glands from Nymphal Acquisition through Transmission.

Relapsing fever (RF) spirochetes colonize and are transmitted to mammals primarily by Ornithodoros ticks, and little is known regarding the pathogen's life cycle in the vector. To further understand vector colonization and transmission of RF spirochetes, Borrelia turicatae expressing a green fluorescent protein (GFP) marker (B. turicatae-gfp) was generated. The transformants were evaluated during the tick-mammal infectious cycle, from the third nymphal instar to adult stage. B. turicatae-gfp remained viable for at least 18 months in starved fourth-stage nymphal ticks, and the studies indicated that spirochete populations persistently colonized the tick midgut and salivary glands. Our generation of B. turicatae-gfp also revealed that within the salivary glands, spirochetes are localized in the ducts and lumen of acini, and after tick feeding, the tissues remained populated with spirochetes. The B. turicatae-gfp generated in this study is an important tool to further understand and define the mechanisms of vector colonization and transmission.IMPORTANCE In order to interrupt the infectious cycle of tick-borne relapsing fever spirochetes, it is important to enhance our understanding of vector colonization and transmission. Toward this, we generated a strain of Borrelia turicatae that constitutively produced the green fluorescent protein, and we evaluated fluorescing spirochetes during the entire infectious cycle. We determined that the midgut and salivary glands of Ornithodoros turicata ticks maintain the pathogens throughout the vector's life cycle and remain colonized with the spirochetes for at least 18 months. We also determined that the tick's salivary glands were not depleted after a transmission blood feeding. These findings set the framework to further understand the mechanisms of midgut and salivary gland colonization.

Syntaxin 31 functions in Glycine max resistance to the plant parasitic nematode Heterodera glycines.

A Glycine max syntaxin 31 homolog (Gm-SYP38) was identified as being expressed in nematode-induced feeding structures known as syncytia undergoing an incompatible interaction with the plant parasitic nematode Heterodera glycines. The observed Gm-SYP38 expression was consistent with prior gene expression analyses that identified the alpha soluble NSF attachment protein (Gm-α-SNAP) resistance gene because homologs of these genes physically interact and function together in other genetic systems. Syntaxin 31 is a protein that resides on the cis face of the Golgi apparatus and binds α-SNAP-like proteins, but has no known role in resistance. Experiments presented here show Gm-α-SNAP overexpression induces Gm-SYP38 transcription. Overexpression of Gm-SYP38 rescues G. max [Williams 82/PI 518671], genetically rhg1 (-/-), by suppressing H. glycines parasitism. In contrast, Gm-SYP38 RNAi in the rhg1 (+/+) genotype G. max [Peking/PI 548402] increases susceptibility. Gm-α-SNAP and Gm-SYP38 overexpression induce the transcriptional activity of the cytoplasmic receptor-like kinase BOTRYTIS INDUCED KINASE 1 (Gm-BIK1-6) which is a family of defense proteins known to anchor to membranes through a 5' MGXXXS/T(R) N-myristoylation sequence. Gm-BIK1-6 had been identified previously by RNA-seq experiments as expressed in syncytia undergoing an incompatible reaction. Gm-BIK1-6 overexpression rescues the resistant phenotype. In contrast, Gm-BIK1-6 RNAi increases parasitism. The analysis demonstrates a role for syntaxin 31-like genes in resistance that until now was not known.

Vertical transmission of Borrelia turicatae (Spirochaetales: Borreliaceae ) by autogenously reproducing Ornithodoros turicata (Ixodida: Argasidae) female naturally infected with the spirochetes.

Ornithodoros turicata is a vector of relapsing fever (RF) spirochetes in North America and transmits Borrelia turicatae to a variety of vertebrate hosts. The remarkably long lifespan of O. turicata and its ability to maintain spirochetes horizontally (between life stages) and vertically to progeny promotes the perpetuation of B. turicatae in nature. Nevertheless, the reproductive biology of O. turicata is poorly understood. In this report, we collected ticks from a park within a neighborhood of Austin, Texas. They were reared to adulthood and male ticks were individually housed with females. We observed autogenous reproduction by the ticks and further investigated vertical transmission of B. turicatae by quantifying filial infection rates in a cohort of progeny ticks. These results indicate that O. turicata transovarially transmits B. turicatae during autogenous reproduction and further signify the tick as a natural reservoir of the spirochetes. Importance: 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 B. turicata 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.

Characterization of the arthropod associated lipoprotein (Alp) in the tick-mammalian transmission cycle of Borrelia turicatae.

Pathogenic species of Borrelia are etiological agents of tick-borne relapsing fever (TBRF). Most species of TBRF Borrelia are transmitted by argasid ticks, and persistent colonization of the salivary glands is vital for spirochete transmission. This is due to the fast-feeding dynamics of the vector. However, the molecular mechanisms leading to vector colonization by the spirochete and their transmission to the vertebrate host remain vague. Previous work in Borrelia hermsii identified the arthropod associated lipoprotein (Alp) as being produced by spirochetes colonizing tick salivary glands. Upon transmission to mice, alp expression was down-regulated and the protein was undetectable in B. hermsii infecting mouse blood. Furthermore, Alp has homologs in multiple TBRF Borrelia species including Borrelia turicatae, Borrelia duttonii, and Borrelia recurrentis. To further evaluate the role of Alp in tick colonization and transmission, the gene was deleted in B. turicatae and the mutant's phenotype was evaluated. Our findings indicate that Alp is dispensable for colonization of the tick salivary glands and for the establishment of infection in laboratory mice.

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.

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.

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.

The impact of in vitro cultivation on the natural life cycle of the tick-borne relapsing fever spirochete Borrelia turicatae.

Tick-borne relapsing fever is an infectious disease caused by Borrelia species and are primarily transmitted by Ornithodoros ticks. Prior work indicated that in vitro cultivated spirochetes remain infectious to mice by needle inoculation; however, the impact of laboratory propagation on the pathogens natural life cycle has not been determined. Our current study assessed the effect of serial cultivation on the natural tick-mammalian transmission cycle. First, we evaluated genomic DNA profiles from B. turicatae grown to 30, 60, 120, and 300 generations, and these spirochetes were used to needle inoculate mice. Uninfected nymphal ticks were fed on these mice and acquisition, transstadial maintenance, and subsequent transmission after tick bite was determined. Infection frequencies in mice that were fed upon by ticks colonized with B. turicatae grown to 30, 60, and 120 generations were 100%, 100%, and 30%, respectively. Successful infection of mice by tick feeding was not detected after 120 generations. Quantifying B. turicatae in tick tissues indicated that by 300 generations they no longer colonized the vector. The results indicate that in vitro cultivation significantly affects the establishment of tick colonization and murine infection. This work provides a foundation for the identification of essential genetic elements in the tick-mammalian infectious cycle.

Humoral immune response of pigs, Sus scrofa domesticus, upon repeated exposure to blood-feeding by Ornithodoros turicata Duges (Ixodida: Argasidae).

BACKGROUND: Ornithodoros turicata is an important vector of both human and veterinary pathogens. One primary concern is the global spread of African swine fever virus and the risk of its re-emergence in the Americas through potential transmission by O. turicata to domestic pigs and feral swine. Moreover, in Texas, African warthogs were introduced into the state for hunting purposes and evidence exists that they are reproducing and have spread to three counties in the state. Consequently, it is imperative to develop strategies to evaluate exposure of feral pigs and African warthogs to O. turicata. RESULTS: We report the development of an animal model to evaluate serological responses of pigs to O. turicata salivary proteins after three exposures to tick feeding. Serological responses were assessed for ~ 120 days by enzyme-linked immunosorbent assay and immunoblotting using salivary gland extracts from O. turicata. CONCLUSIONS: Our findings indicate that domestic pigs seroconverted to O. turicata salivary antigens that is foundational toward the development of a diagnostic assay to improve soft tick surveillance efforts.

Differential Expression of Putative Ornithodoros turicata Defensins Mediated by Tick Feeding.

Additional research on soft ticks in the family Argasidae is needed to bridge the knowledge gap relative to hard ticks of the family Ixodidae; especially, the molecular mechanisms of Ornithodoros biology. Ornithodoros species are vectors of human and animal pathogens that include tick-borne relapsing fever spirochetes and African swine fever virus. Soft tick vector-pathogen interactions involving components of the tick immune response are not understood. Ticks utilize a basic innate immune system consisting of recognition factors and cellular and humoral responses to produce antimicrobial peptides, like defensins. In the present study, we identified and characterized the first putative defensins of Ornithodoros turicata, an argasid tick found primarily in the southwestern United States and regions of Latin America. Four genes (otdA, otdB, otdC, and otdD) were identified through sequencing and their predicted amino acid sequences contained motifs characteristic of arthropod defensins. A phylogenetic analysis grouped these four genes with arthropod defensins, and computational structural analyses further supported the identification. Since pathogens transmitted by O. turicata colonize both the midgut and salivary glands, expression patterns of the putative defensins were determined in these tissues 1 week post engorgement and after molting. Defensin genes up-regulated in the tick midgut 1 week post blood feeding were otdA and otdC, while otdD was up-regulated in the midgut of post-molt ticks. Moreover, otdB and otdD were also up-regulated in the salivary glands of flat post-molt ticks, while otdC was up-regulated within 1 week post blood-feeding. This work is foundational toward additional studies to determine mechanisms of vector competence and pathogen transmission from O. turicata.