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.

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

[This corrects the article DOI: 10.1371/journal.ppat.1010370.].

Inactivation of genes for antigenic variation in the relapsing fever spirochete Borrelia hermsii reduces infectivity in mice and transmission by ticks.

Borrelia hermsii, a causative agent of relapsing fever of humans in western North America, is maintained in enzootic cycles that include small mammals and the tick vector Ornithodoros hermsi. In mammals, the spirochetes repeatedly evade the host's acquired immune response by undergoing antigenic variation of the variable major proteins (Vmps) produced on their outer surface. This mechanism prolongs spirochete circulation in blood, which increases the potential for acquisition by fast-feeding ticks and therefore perpetuation of the spirochete in nature. Antigenic variation also underlies the relapsing disease observed when humans are infected. However, most spirochetes switch off the bloodstream Vmp and produce a different outer surface protein, the variable tick protein (Vtp), during persistent infection in the tick salivary glands. Thus the production of Vmps in mammalian blood versus Vtp in ticks is a dominant feature of the spirochete's alternating life cycle. We constructed two mutants, one which was unable to produce a Vmp and the other was unable to produce Vtp. The mutant lacking a Vmp constitutively produced Vtp, was attenuated in mice, produced lower cell densities in blood, and was unable to relapse in animals after its initial spirochetemia. This mutant also colonized ticks and was infectious by tick-bite, but remained attenuated compared to wild-type and reconstituted spirochetes. The mutant lacking Vtp also colonized ticks but produced neither Vtp nor a Vmp in tick salivary glands, which rendered the spirochete noninfectious by tick bite. Thus the ability of B. hermsii to produce Vmps prolonged its survival in blood, while the synthesis of Vtp was essential for mammalian infection by the bite of its tick vector.

Tickborne relapsing fever, Bitterroot Valley, Montana, USA.

In July 2013, a resident of the Bitterroot Valley in western Montana, USA, contracted tickborne relapsing fever caused by an infection with the spirochete Borrelia hermsii. The patient's travel history and activities before onset of illness indicated a possible exposure on his residential property on the eastern side of the valley. An onsite investigation of the potential exposure site found the vector, Ornithodoros hermsi ticks, and 1 chipmunk infected with spirochetes, which on the basis of multilocus sequence typing were identical to the spirochete isolated from the patient. Field studies in other locations found additional serologic evidence and an infected tick that demonstrated a wider distribution of spirochetes circulating among the small mammal populations. Our study demonstrates that this area of Montana represents a previously unrecognized focus of relapsing fever and poses a risk for persons of acquiring this tickborne disease.

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

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

Large linear plasmids of Borrelia species that cause relapsing fever.

Borrelia species of relapsing fever (RF) and Lyme disease (LD) lineages have linear chromosomes and both linear and circular plasmids. Unique to RF species, and little characterized to date, are large linear plasmids of ∼160 kb, or ∼10% of the genome. By a combination of Sanger and next-generation methods, we determined the sequences of large linear plasmids of two New World species: Borrelia hermsii, to completion of its 174-kb length, and B. turicatae, partially to 114 kb of its 150 kb. These sequences were then compared to corresponding sequences of the Old World species B. duttonii and B. recurrentis and to plasmid sequences of LD Borrelia species. The large plasmids were largely colinear, except for their left ends, about 27 kb of which was inverted in New World species. Approximately 60% of the B. hermsii lp174 plasmid sequence was repetitive for 6 types of sequence, and half of its open reading frames encoded hypothetical proteins not discernibly similar to proteins in the database. The central ∼25 kb of all 4 linear plasmids was syntenic for orthologous genes for plasmid maintenance or partitioning in Borrelia species. Of all the sequenced linear and circular plasmids in Borrelia species, the large plasmid's putative partition/replication genes were most similar to those of the 54-kb linear plasmids of LD species. Further evidence for shared ancestry was the observation that two of the hypothetical proteins were predicted to be structurally similar to the LD species' CspA proteins, which are encoded on the 54-kb plasmids.

Borrelia hermsii acquisition order in superinfected ticks determines transmission efficiency.

Multilocus sequence typing of Borrelia hermsii isolates reveals its divergence into two major genomic groups (GG), but no differences in transmission efficiency or host pathogenicity are associated with these genotypes. To compare GGI and GGII in the tick-host infection cycle, we first determined if spirochetes from the two groups could superinfect the tick vector Ornithodoros hermsi. We infected mice with isolates from each group and fed ticks sequentially on these mice. We then fed the infected ticks on naive mice and measured GGI and GGII spirochete densities in vector and host, using quantitative PCR of genotype-specific chromosomal DNA sequences. Sequential feedings resulted in dual tick infections, showing that GGI or GGII primary acquisition did not block superinfection by a secondary agent. On transmission to naive mice at short intervals after acquisition, ticks with primary GGI and secondary GGII spirochete infections caused mixed GGI and GGII infections in mice. However, ticks with primary GGII and secondary GGI spirochete infections caused only GGII infections with all isolate pairs examined. At longer intervals after acquisition, the exclusion of GGI by GGII spirochetes declined and cotransmission predominated. We then examined GGI and GGII spirochetemia in mice following single inoculation and coinoculation by needle and found that GGI spirochete densities were reduced on multiple days when coinoculated with GGII. These findings indicate that dual GGI-GGII spirochete infections can persist in ticks and that transmission to a vertebrate host is dependent on the order of tick acquisition and the interval between acquisition and transmission events.

Kinetics of tick infection by the relapsing fever spirochete Borrelia hermsii acquired through artificial membrane feeding chambers.

The relapsing fever agent Borrelia hermsii is transmitted by the tick Ornithodoros hermsi. To study the B. hermsii-tick interactions required for pathogen acquisition and transmission we developed an artificial membrane feeding system for O. hermsi nymphs and adults that results in a high percentage of engorgement. This system provides the nutritional requirements necessary for the tick to develop, mate, and produce viable eggs. By inoculating the blood with B. hermsii, we were able to obtain infected ticks for quantitative studies on pathogen acquisition and persistence. These ticks subsequently transmitted the spirochetes to mice, validating this system for both acquisition and transmission studies. Using this feeding method, a mutant of the antigenic variation locus of B. hermsii (Vmp-) that is incapable of persisting in mice was acquired by ticks at equivalent densities as the wild-type. Furthermore, Vmp is not required for persistence in the tick, as the mutant and wild-type strains are maintained at similar numbers after ecdysis and subsequent feeding. These results support the theory that Vmp is an adaptation for mammalian infection but unnecessary for survival within the tick. Interestingly, B. hermsii numbers severely declined after acquisition, though these ticks still transmitted the infection to mice. This procedure reduces animal use and provides a safe, highly controlled and well-contained alternative method for feeding and maintaining O. hermsi colonies. Importantly, this system permits quantitative studies with B. hermsii strains through ingestion during the blood meal, and thus more closely recapitulates pathogen acquisition in nature than other artificial systems.

Cotransmission of divergent relapsing fever spirochetes by artificially infected Ornithodoros hermsi.

The soft tick Ornithodoros hermsi, which ranges in specific arboreal zones of western North America, acts as a vector for the relapsing fever spirochete Borrelia hermsii. Two genomic groups (genomic group I [GGI] and GGII) of B. hermsii are differentiated by multilocus sequence typing yet are codistributed in much of the vector's range. To test whether the tick vector can be infected via immersion, noninfected, colony-derived O. hermsi larvae were exposed to reduced-humidity conditions before immersion in culture suspensions of several GGI and GGII isolates. We tested for spirochetes in ticks by immunofluorescence microscopy and in mouse blood by quantitative PCR of the vtp locus to differentiate spirochete genotypes. The immersed larval ticks were capable of spirochete transmission to mice at the first nymphal feeding. Tick infection with mixed cultures of isolates DAH (vtp-6) (GGI) and MTW-2 (vtp-5) (GGII) resulted in ticks that caused spirochetemias in mice consisting of MTW-2 or both DAH and MTW-2. These findings show that this soft tick species can acquire B. hermsii by immersion in spirochete suspensions, that GGI and GGII isolates can coinfect the tick vector by this method, and that these spirochetes can be cotransmitted to a rodent host.

Transovarial Transmission of Borrelia hermsii by Its Tick Vector and Reservoir Host Ornithodoros hermsi.

Transovarial passage of relapsing fever spirochetes (Borrelia species) by infected female argasid ticks to their progeny is a widespread phenomenon. Yet this form of vertical inheritance has been considered rare for the North American tick Ornithodoros hermsi infected with Borrelia hermsii. A laboratory colony of O. hermsi was established from a single infected female and two infected males that produced a population of ticks with a high prevalence of transovarial transmission based on infection assays of single and pooled ticks feeding on mice and immunofluorescence microscopy of eggs and larvae. Thirty-eight of forty-five (84.4%) larval cohorts (groups of larvae originating from the same egg clutch) transmitted B. hermsii to mice over four and a half years, and one hundred and three single and one hundred and fifty-three pooled nymphal and adult ticks transmitted spirochetes during two hundred and fourteen of two hundred and fifty-six (83.6%) feedings on mice over seven and a half years. The perpetuation of B. hermsii for many years by infected ticks only (without acquisition of spirochetes from vertebrate hosts) demonstrates the reservoir competence of O. hermsi. B. hermsii produced the variable tick protein in eggs and unfed larvae infected by transovarial transmission, leading to speculation of the possible steps in the evolution of borreliae from a tick-borne symbiont to a tick-transmitted parasite of vertebrates.

Detection of Lassa virus, Mali.

To determine whether Lassa virus was circulating in southern Mali, we tested samples from small mammals from 3 villages, including Soromba, where in 2009 a British citizen probably contracted a lethal Lassa virus infection. We report the isolation and genetic characterization of Lassa virus from an area previously unknown for Lassa fever.

Bloodmeal size and spirochete acquisition of Ornithodoros hermsi (Acari: Argasidae) during feeding.

Ornithodoros hermsi Wheeler (Acari: Argasidae) is the vector of Borrelia hermsii, the primary cause of tick-borne relapsing fever in North America. This tick is one of the smallest Ornithodoros species involved with the biological transmission of spirochetes; yet, the amount of blood ingested while feeding is unknown. Therefore, we determined the amount of blood O. hermsi ingested during a bloodmeal to establish its potential for spirochete acquisition while feeding on an infected host. Ticks at different developmental stages were weighed before and after feeding and the volume of blood ingested was calculated. Females ingested the most blood, averaging approximately 15 microl per meal, but late-stage nymphs took in the most blood in proportion to unfed body weight. A cohort of nymphs was weighed three more times during the 48 h after feeding, which demonstrated that O. hermsi may have excreted coxal fluid ranging from 24 - 36% of the bloodmeal weight. We also developed a quantitative polymerase chain reaction method to determine the number of spirochetes ingested and maintained within the ticks after feeding. The density of spirochetes in ticks having just engorged was slightly less than in the host's blood. In the first 5 d after feeding, the number of spirochetes within the ticks declined from the number initially ingested but then remained constant through 15 d. These observations establish a basis for future studies to determine the minimum number of spirochetes required in the host's blood to allow O. hermsi to become persistently infected and transmit during subsequent bloodmeals.

Transgenic functional complementation with a transmission -associated protein restores spirochete infectivity by tick bite.

The relapsing fever spirochete Borrelia hermsii and the Lyme disease spirochete Borrelia burgdorferi sensu stricto each produces an abundant, orthologous, outer membrane protein, Vtp and OspC, respectively, when transmitted by tick bite. Gene inactivation studies have shown that both proteins are essential for spirochete infectivity when transmitted by their respective tick vectors. Therefore, we transformed a vtp-minus mutant of B. hermsii with ospC from B. burgdorferi and examined the behavior of this transgenic spirochete in its soft tick vector Ornithodoros hermsi. IFA staining indicated up to 97.8 % of the transgenic B. hermsii upregulated OspC in the ticks' salivary glands compared to no more than 12.8 % in the midgut, similar to our previous findings with wild-type B. hermsii producing Vtp. Transformation with ospC also restored B. hermsii infectivity to mice when fed upon by infected ticks. Previous sequence analysis of Vtp for 79 isolates and DNA samples of B. hermsii in our laboratory showed this protein is highly polymorphic with 9 divergent amino acid types, yet strikingly the signal peptide is identical among all samples and the same for all OspC signal peptides for B. burgdorferi and related species examined to date. Searches in multiple genome sequences for other species of relapsing fever spirochetes failed to find the same signal peptide sequence to help identify potential transmission-associated proteins. However, some candidate signal peptides with highly similar sequences were found and worthy of future efforts with other species. While OspC of B. burgdorferi restored infectivity to a Vtp-minus mutant of B. hermsii, the functions of these proteins are not known. Our results should stimulate investigators to search for orthologous transmission-associated proteins in other tick-borne spirochetes to better understand how this group of pathogens has coevolved with diverse tick vectors.

Tick-borne relapsing fever and Borrelia hermsii, Los Angeles County, California, USA.

The primary cause of tick-borne relapsing fever in western North America is Borrelia hermsii, a rodent-associated spirochete transmitted by the fast-feeding soft tick Ornithodoros hermsi. We describe a patient who had an illness consistent with relapsing fever after exposure in the mountains near Los Angeles, California, USA. The patient's convalescent-phase serum was seropositive for B. hermsii but negative for several other vector-borne bacterial pathogens. Investigations at the exposure site showed the presence of O. hermsi ticks infected with B. hermsii and the presence of rodents that were seropositive for the spirochete. We determined that this tick-borne disease is endemic to the San Gabriel Mountains near the greater Los Angeles metropolitan area.

A system for site-specific genetic manipulation of the relapsing fever spirochete Borrelia hermsii.

The lack of a system for genetic manipulation has hindered studies on the molecular pathogenesis of relapsing fever Borrelia. The focus of this chapter is to describe selectable markers, manipulation strategies, and methods to electro-transform and clone wild-type infectious Borrelia hermsii. Preliminary studies suggest that the variable tick protein (Vtp) of B. hermsii is involved in tick-to-mammal transmission. To address this hypothesis, we have developed a system for genetic manipulation and have constructed clones of a Vtp mutant and an isogenic reconstituted strain. The methods described here are applicable for the inactivation of other loci in B. hermsii and should be adaptable for other species of relapsing fever spirochetes.

Colony formation in solid medium by the relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae.

Relapsing fever (RF) in North America is caused primarily by the spirochete Borrelia hermsii and is associated with the bite of its tick vector Ornithodoros hermsi. Although this spirochete was known long before the discovery of the Lyme disease (LD) spirochete, Borrelia burgdorferi, basic methods to facilitate the study of B. hermsii have lagged behind. One important technique to expedite the study of the molecular biology and pathogenesis of B. hermsii would be a reliable method to grow and clone these bacteria in solid medium, which we now describe. We have defined the solidifying agent, plating temperature, oxygen concentration, and pH for the efficient plating of two species of RF spirochetes, B. hermsii and Borrelia turicatae. Importantly, this technique allowed us to successfully isolate virulent, clonal cell lines of spirochetes, and to enumerate and isolate viable B. hermsii from infected mouse blood and tick tissues. Our results also demonstrate the value of testing a range of several environmental variables to increase the efficiency of bacterial isolation, which may be helpful for researchers working on other prokaryotes that are intractable for in vitro growth.

Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism.

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

Host associations and genomic diversity of Borrelia hermsii in an endemic focus of tick-borne relapsing fever in western North America.

BACKGROUND: An unrecognized focus of tick-borne relapsing fever caused by Borrelia hermsii was identified in 2002 when five people became infected on Wild Horse Island in Flathead Lake, Montana. The terrestrial small mammal community on the island is composed primarily of pine squirrels (Tamiasciurus hudsonicus) and deer mice (Peromyscus maniculatus), neither of which was known as a natural host for the spirochete. Thus a 3-year study was performed to identify small mammals as hosts for B. hermsii. METHODS: Small mammals were captured alive on two island and three mainland sites, blood samples were collected and examined for spirochetes, and serological tests performed to detect anti-B. hermsii antibodies. Ornithodoros hermsi ticks were collected and fed on laboratory mice to assess infection. Genomic DNA samples from spirochetes isolated from infected mammals and ticks were analyzed by multilocus sequence typing. RESULTS: Eighteen pine squirrels and one deer mouse had detectable spirochetemias when captured, from which 12 isolates of B. hermsii were established. Most pine squirrels were seropositive, and the five species of sciurids combined had a significantly higher prevalence of seropositive animals than did the other six small mammal species captured. The greater diversity of small mammals on the mainland in contrast to the islands demonstrated that other species in addition to pine squirrels were also involved in the maintenance of B. hermsii at Flathead Lake. Ornithodoros hermsi ticks produced an additional 12 isolates of B. hermsii and multilocus sequence typing identified both genomic groups of B. hermsii described previously, and identified a new genomic subdivision. Experimental infections of deer mice with two strains of B. hermsii demonstrated that these animals were susceptible to infection with spirochetes belonging to Genomic Group II but not Genomic Group I. CONCLUSIONS: Pine squirrels are the primary hosts for the maintenance of B. hermsii on the islands in Flathead Lake, however serological evidence showed that numerous additional species are also involved on the mainland. Future studies testing the susceptibility of several small mammal species to infection with different genetic types of B. hermsii will help define their role as hosts in this and other endemic foci.

Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection.

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3' end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe's surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

Vaccination with the variable tick protein of the relapsing fever spirochete Borrelia hermsii protects mice from infection by tick-bite.

BACKGROUND: Tick-borne relapsing fevers of humans are caused by spirochetes that must adapt to both warm-blooded vertebrates and cold-blooded ticks. In western North America, most human cases of relapsing fever are caused by Borrelia hermsii, which cycles in nature between its tick vector Ornithodoros hermsi and small mammals such as tree squirrels and chipmunks. These spirochetes alter their outer surface by switching off one of the bloodstream-associated variable major proteins (Vmps) they produce in mammals, and replacing it with the variable tick protein (Vtp) following their acquisition by ticks. Based on this reversion to Vtp in ticks, we produced experimental vaccines comprised on this protein and tested them in mice challenged by infected ticks. METHODS: The vtp gene from two isolates of B. hermsii that encoded antigenically distinct types of proteins were cloned, expressed, and the recombinant Vtp proteins were purified and used to vaccinate mice. Ornithodoros hermsi ticks that were infected with one of the two strains of B. hermsii from which the vtp gene originated were used to challenge mice that received one of the two Vtp vaccines or only adjuvant. Mice were then followed for infection and seroconversion. RESULTS: The Vtp vaccines produced protective immune responses in mice challenged with O. hermsi ticks infected with B. hermsii. However, polymorphism in Vtp resulted in mice being protected only from the spirochete strain that produced the same Vtp used in the vaccine; mice challenged with spirochetes producing the antigenically different Vtp than the vaccine succumbed to infection. CONCLUSIONS: We demonstrate that by having knowledge of the phenotypic changes made by B. hermsii as the spirochetes are acquired by ticks from infected mammals, an effective vaccine was developed that protected mice when challenged with infected ticks. However, the Vtp vaccines only protected mice from infection when challenged with that strain producing the identical Vtp. A vaccine containing multiple Vtp types may have promise as an oral vaccine for wild mammals if applied to geographic settings such as small islands where the mammal diversity is low and the Vtp types in the B. hermsii population are defined.