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.

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.

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.

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.

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.

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 a uniquely complex genome consisting of a linear chromosome and circular and linear plasmids. The plasmids harbor genes important for the vector-host life cycle of these tick-borne bacteria. The role of Lyme disease causing Borrelia plasmids is more refined compared to RF spirochetes because of limited plasmid-resolved genomes for RF spirochetes. We recently addressed this limitation and found that three linear plasmid families (F6, F27, and F28) were syntenic across all species. 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 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 and 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. By utilizing Oxford Nanopore Technologies, we recently generated plasmid-resolved genomes for seven Borrelia spp. found in the Western Hemisphere. This current study is a more in-depth investigation into the linear plasmids that were conserved and syntenic across species. This analysis determined differences in genome structure and, importantly, in antigenic variation systems between species. This work is an important step in identifying crucial plasmid-borne genetic elements essential for the life cycle of RF spirochetes.

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.

Case Report: Exposure to Relapsing Fever Group Borreliae in Patients with Undifferentiated Febrile Illness in Mexico.

Relapsing fever (RF) borreliosis is a neglected disease in Mexico. A retrospective serological survey using diagnostic antigens GlpQ and BipA from Borrelia turicatae was performed to evaluate human exposure to RF borreliae. Seventy serum samples were used from a cohort of patients with undifferentiated febrile illness in Mexico. Four samples were positive to GlpQ and three to BipA. Results indicate that RF borreliae continue to circulate in regions of Mexico and pose a risk to human health.

Amplification and sequencing of entire tick mitochondrial genomes for a phylogenomic analysis.

The mitochondrial genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be cost-prohibitive at scale. To address this issue, we developed a cost-effective approach to amplify and sequence the whole mitogenome of individual tick specimens. Using two different primer sites, this approach generated two full-length mitogenome amplicons that were sequenced using the Oxford Nanopore Technologies' Mk1B sequencer. We used this approach to generate 85 individual tick mitogenomes from samples comprised of the three tick families, 11 genera, and 57 species. Twenty-six of these species did not have a complete mitogenome available on GenBank prior to this work. We benchmarked the accuracy of this approach using a subset of samples that had been previously sequenced by low-coverage Illumina genome skimming. We found our assemblies were comparable or exceeded the Illumina method, achieving a median sequence concordance of 99.98%. We further analyzed our mitogenome dataset in a mitophylogenomic analysis in the context of all three tick families. We were able to sequence 72 samples in one run and achieved a cost/sample of ~ $10 USD. This cost-effective strategy is applicable for sample identification, taxonomy, systematics, and population genetics for not only ticks but likely other metazoans; thus, making mitogenome sequencing equitable for the wider scientific community.

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.

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.

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.

Modification of the multiplex plasmid PCR assay for Borrelia miyamotoi strain LB-2001 based on the complete genome sequence reflecting genomic rearrangements differing from strain CT13-2396.

The genome of Borrelia spp. consists of an approximate 1 megabase chromosome and multiple linear and circular plasmids. We previously described a multiplex PCR assay to detect plasmids in the North American Borrelia miyamotoi strains LB-2001 and CT13-2396. The primer pair sets specific for each plasmid were derived from the genome sequence for B. miyamotoi strain CT13-2396, because the LB-2001 complete sequence had not been generated. The recent completion of the LB-2001 genome sequence revealed a distinct number of plasmids (n = 12) that differed from CT13-2396 (n = 14). Notable was a 97-kilobase plasmid in LB-2001, not present in CT13-2396, that appeared to be a rearrangement of the circular plasmids of strain CT13-2396. Strain LB-2001 contained two plasmids, cp30-2 and cp24, that were not annotated for strain CT13-2396. Therefore, we re-evaluated the original CT13-2396-derived multiplex PCR primer pairs and determined their location in the LB-2001 plasmids. We modified the original multiplex plasmid PCR assay for strain LB-2001 to include cp30-2 and cp24. We also determined which LB-2001 plasmids corresponded to the amplicons generated from the original CT13-2396 primer sets. These observations provide a more precise plasmid profile based on the multiplex PCR assay and reflect the complexity of gene rearrangements that occur in B. miyamotoi strains isolated from the same geographic region.

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.

The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology.

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole-genome sequencing was performed on cdaA mutants, and single-nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium Borrelia burgdorferi Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.