Seroprevalence for the tick-borne relapsing fever spirochete Borrelia turicatae among small and medium sized mammals of Texas

Background In low elevation arid regions throughout the southern United States, Borrelia turicatae is the principal agent of tick-borne relapsing fever. However, endemic foci and the vertebrate hosts involved in the ecology of B. turicatae remain undefined. Experimental infection studies suggest that small and medium sized mammals likely maintain B. turicatae in nature, while the tick vector is a long-lived reservoir. Methodology/principal findings Serum samples from wild caught rodents, raccoons, and wild and domestic canids from 23 counties in Texas were screened for prior exposure to B. turicatae. Serological assays were performed using B. turicatae protein lysates and recombinant Borrelia immunogenic protein A (rBipA), a diagnostic protein that is unique to RF spirochetes and may be a species-specific antigen. Conclusions/significance Serological responses to B. turicatae were detected from 24 coyotes, one gray fox, two raccoons, and one rodent from six counties in Texas. These studies indicate that wild canids and raccoons were exposed to B. turicatae and are likely involved in the pathogen’s ecology. Additionally, more work should focus on evaluating rodent exposure to B. turicatae and the role of these small mammals in the pathogen’s maintenance in nature.


Introduction
Tick-borne relapsing fever (RF) is primarily caused by spirochetes in the genus Borrelia and the pathogens are transmitted when infected Ornithodoros ticks feed on a competent vertebrate host. In the United States and Mexico, there is an association between Ornithodoros ticks and RF spirochete species where Ornithodoros hermsi, Ornithodoros parkeri, Ornithodoros turicata, and Ornithodoros talaje transmit Borrelia hermsii, Borrelia parkeri, Borrelia turicatae, and Borrelia mazzottii, respectively [1]. Furthermore, these tick species involved in human disease are distributed in varying ecological niches. For example, the ecology of O. hermsi is associated with coniferous forests at elevations above 900 meters throughout the western United States and Canada [2][3][4][5][6]. Ornithodoros parkeri has also been collected in semi-arid regions of the western United States at elevations from sea level to over 2,000 meters [7,8]. Ornithodoros turicata is found in arid regions of Mexico, the mid-and southwestern United States from California to Texas, and a population exists in Florida [9][10][11]. The ecology of O. talaje overlaps that of O. turicata and collections have occurred in Mexico and in Texas [1,12,13]. Of the Ornithodoros species that transmit RF spirochetes, O. turicata and O. talaje are currently the only ones known in Texas, yet there are few records of O. talaje collections and B. mazzottii has not been isolated in the laboratory.
The biology of both RF spirochetes and their tick vector have posed challenges in defining the pathogens' ecology. Ornithodoros species are rapid feeding ticks that reside in cavities including wood crevices, dens, nests, and karst formations [6,7,9,14]. Thus, the vector is rarely found attached on the vertebrate host. Moreover, in the vertebrate host, spirochetes replicate in the blood reaching densities of~1 x 10 7 bacteria per ml before being cleared by an antibody mediated response [15]. The pathogens undergo antigenic variation and subsequently repopulate the blood [15]. This dynamic between antigenic variation and the host antibody response can continue for two to three months in a competent host [11,16]. The cyclic nature of RF spirochetes within a competent host poses challenges when attempting to directly detect the pathogens in the blood of wild caught animals because there are quiescent periods when the spirochetes are undetectable. Since RF spirochetes induce a robust IgG response [17][18][19], serological surveillance is a practical approach toward defining the pathogens' ecology given the temporal persistence of generated antibodies in the host's blood.
The ecology of B. turicatae is poorly defined and in this current study we utilized a diagnostic antigen, the Borrelia immunogenic protein A (BipA), which has been used to assess canine, rodent, and human exposure to the pathogen [17,20]. Aside from the closely related B. parkeri, BipA is highly variable between species of RF spirochetes [20]. Moreover, a BipA homologue has not been identified in other viral, parasitic, or bacterial pathogens [17]. Utilizing this diagnostic antigen, we evaluated the exposure of wild and domestic canids, raccoons, and rodents to B. turicatae. Serum samples were collected from 23 counties in Texas and screened against B. turicatae protein lysates and recombinant BipA (rBipA). Most rodents were also identified to species by morphology and molecular sequencing of the cytochrome B gene. Our findings indicate that Canis latrans (coyote), Urocyon cinereoargenteus (gray fox), Procyon lotor (raccoon), and Peromyscus leucopus (white-footed mouse) may be vertebrate hosts for B. turicatae in nature.

Animal trapping
Animal samplings occurred between 2005 and 2018. Rodents were captured alive using Sherman live traps (H.B. Sherman Traps, Tallahassee, FL). Traps were placed in and around houses, barns, and fields in the late afternoon and baited with dried oats. The following morning traps were collected, and the animals processed. Animals were euthanized by inhalation of isoflurane and exsanguinated by cardiac puncture. A drop of blood was placed on a microscope slide and the presence of spirochetes was evaluated by dark field microscopy. Peripheral blood smears were also made on microscope slides. The remaining blood was centrifuged at 1,000 x g and serum separated from the blood clot. Animals were evaluated for argasid ticks.
Shelter dogs and wild canids and raccoons were also sampled. Serum samples from stray domestic dogs located in Brownsville, TX were collected, as previously described [21]. Coyote, gray fox, and raccoon serum samples were collected as part of the Texas Department of State Health Services rabies surveillance program. The animals were captured in Tomahawk traps, terminally sampled, and serum samples stored at -20˚C.

Mammalian identification
Canids and raccoons were identified to the species level using morphological characteristics. Rodents were identified by morphological characteristics and molecular analysis of the cytB gene. For rodent morphological characteristics, body weights were recorded with Pesola spring scales (PESOLA SG, Baar, Switzerland), gender determined, and body and tail measurements recorded. Photographs of each animal were obtained for future reference. For molecular analysis, a 3-mm tissue biopsy was collected from each animal, stored in 90% ethanol, and DNA extracted using the DNeasy Blood and Tissue kit (Qiagen Sciences, Inc., Germantown, MD). PCR was performed using forward (5'-CCATGAGGACAAATATCCTTCTGAGGG-3') and reverse (5'-GCCCTCAGAAGGATATTGTCCTCATGG-3') primers for cytB, and sequencing performed as previously described [19,22]. Sequences were assembled into overlapping contiguous DNA segments (contigs) using Vector NTI 11.0 software (ThermoFisher Scientific, Waltham, MA). Contigs were evaluated using BLASTn on NCBI.

Serological assays
Immunoblotting was performed to evaluate seroconversion against B. turicatae protein lysates and rBipA, as previously described [17]. Briefly, protein lysates from 1 x 10 7 B. turicatae spirochetes and 1 µg of rBipA were loaded into the wells of Mini-PROTEAN TGX precast gels (Bio-Rad, Hercules, CA). Gels were run for 1.5 hours and proteins were transferred onto Immobilon PVDF membranes (Millipore, Billerica, MA). Membranes were blocked overnight with Tropix Iblock (Thermo Fisher Scientific, Waltham, MA) and then probed for one hour at room temperature with serum samples diluted 1:200. The secondary molecule was HRP-conjugated protein G (Thermo Fisher Scientific, Waltham, MA) for canids and rodents at a 1:4,000 dilution. Raccoon serum samples were probed with a goat anti-raccoon IgG-HRP conjugated antibody (Alpha Diagnostics Intl. Inc., San Antonio, TX) at a 1:4,000 dilution. The substrate used to detect binding was Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK). A sample was considered positive for B. turicatae if we detected reactivity to at least five proteins in the B. turicatae protein lysate and rBipA.

Ecoregion mapping
For visualizing the ecoregions of Texas we obtained a shapefile from the United States Environmental Protection Agency, which included the following 12 ecoregions: Arizona/New Mexico Mountain, Central Great Plains, Chihuahua Deserts, Cross Timbers, East Central Texas Plains, Edwards Plateau, High Plains, South Central Plains, Southern Texas Plains, Southwestern Tablelands, Texas Blackland Prairies, and Western Gulf Coastal Plains [23]. These ecoregions were defined based upon several biotic and abiotic factors such as climate, vegetation, soil type, geology, land use, wildlife, and hydrology [23]. This shapefile was then imported into ArcMap and we overlaid each county where collections occurred in Texas noting the taxa group (coyote = C, Dog = D, gray fox = GF, raccoons = RA, and rodents = R) and the number of collections (Fig 1).

Statistics
Statistical analysis was performed using R 3.3.1 (R foundation for Statistical Computing, Vienna, Austria, https://www.R-project.org/). The 95% confidence intervals (CI) were determined for each group of samples tested that had at least one positive sample, using the proportions test. A binomial distribution was assumed with determining CI.

Results
One to four field sites were sampled in 23 counties of Texas between 2005 and 2018. Sites included private property that was accessible through the Texas Ecolab Program and Texas Parks and Wildlife Management Areas. Counties where samples were collected were within the following Texas ecoregions: Central Great Plains, Chihuahuan desert, Cross Timber, East Central Texas Plains, Edwards Plateau, South Central Plains, Southern Texas Plains, Southwestern Tablelands, Texas Blackland Prairies, and Western Gulf Coastal Plains (Fig 1). A total of 463 canids were sampled and included 185 shelter dogs (Canis lupus familiaris), 220 coyotes (C. latrans) and 58 gray foxes (U. cinereoargenteus) (Fig 1). Serum samples were also collected from 25 raccoons (P. lotor) and 263 rodents (Fig 1). Argasid ticks were not detected on the animals.
Animals were considered susceptible to infection by B. turicatae based on serological reactivity to at least five bands in B. turicatae protein lysates and rBipA. Assessing serological responses of canids (Fig 2) indicated a total seroprevalence of 5.4% (CI = 3.6-8.0%) ( Table 1). None of the 185 shelter dogs that were screened had a detectable antibody response in the diagnostic assay, while 10.9% and 1.7% (CI = 7.3-16.0% and 0.09-10.5%) seroprevalence was detected in coyotes and gray fox, respectively. Webb County had the highest number of seropositive coyotes with a total of 10 animals. Presidio and Zapata County each had five seropositive animals, while Dimmit County had four. In the animals exposed to B. turicatae, a gender difference was not detected. In El Paso County, there was a single juvenile male gray fox that was seropositive, resulting in 1.7% (CI = 0.7-53.3%) prevalence among gray fox.

Discussion
In this study, we began to define the ecology of B. turicatae in Texas by assessing serological responses as an indicator of host competency. While RF spirochete infections can persist for several months in a competent host [11], the pathogens' life cycle is recurrent and direct detection of infection can be challenging because of the brevity of time when spirochetes are detectable in the blood. To circumvent this, we indirectly detected exposure to B. turicatae by Tick-borne relapsing fever seroprevalence in Texas assessing the vertebrate antibody response. Our findings indicate that wild canids are likely a host for B. turicatae in west Texas. These studies were also the first known serological evaluation of rodents and raccoons to B. turicatae and provided verification that exposure is occurring in this tick-host-pathogen relationship.
Rodents and insectivores are known reservoir hosts for at least two species of RF spirochete [3,19,24,25], but the role of these small mammals in the ecology of B. turicatae is vague. In high elevation regions of the Western United States, sciurid rodents are the primary vertebrate host for B. hermsii, while the pathogens have also been detected in Neotoma macrotis [3,24]. In regions of western Africa, Borrelia crocidurae is maintained in Mastomys and Crocidura species [19]. Previous tick transmission studies of B. turicatae to laboratory mice suggest that wild rodents may be susceptible to infection [17,26,27], and the identified seropositive P. leucopus from this current study indicated that white-footed mice are a potential competent host. However, we sampled the field site where this animal was collected three more times from 2012 to 2014 and failed to identify other positive rodents. Additional studies are needed to investigate the life cycle of B. turicatae in rodents to determine whether the pathogen attains densities in the animals that will facilitate spirochete acquisition and colonization of the tick vector.
There is mounting evidence that canids likely support the maintenance and dissemination of B. turicatae in nature. For example, the competency of domestic canines for B. turicatae was demonstrated as nearly half of the B. turicatae isolates have originated from sick dogs [28]. Moreover, successful infection of B. turicatae to a laboratory dog by tick bite suggested that the spirochetes attained sufficient densities in the blood to infect ticks [17]. In this current report, B. turicatae positive coyote and gray fox serum samples originated from Dimmit, Presidio, Webb, Zapata, and El Paso County, all of which border Mexico. With the broad home range of Tick-borne relapsing fever seroprevalence in Texas El Paso (11) (Table 2). One female and one male, both adults, were considered positive for B. turicatae protein lysate and rBipA (Fig 2). Both raccoons were sampled in a residential area, and a total seroprevalence of 8% (CI = 1.4-27.5%) was detected. coyotes, these mammals are likely circulating B. turicatae between the United States and Mexico.
Coyotes possess highly organized social systems even in urban settings and are classified as transient or resident based on their territorial range [29,30]. Transient coyotes are typically solitary subordinate young adults with a home range of 40 km 2 to 395 km 2 . Resident coyotes have a home range of 8 km 2 to 29 km 2 and are part of the larger pack that include breeders, juveniles, and pups [30]. Coyote dens are often found in or around urban settings and with the expansion of these areas in Mexico and the United States, coyotes and humans are commonly in contact [30,31].
A knowledge gap in the ecology of B. turicatae is a poor understanding regarding the dissemination of the vector in nature. Ornithodoros turicata are rapid feeders, completing a bloodmeal within five to 60 minutes after attachment [26]. However, it is unclear whether some proportion of ticks remain on the wild vertebrate host after engorgement, either attached or unattached, allowing for increased dissemination. Interestingly, we have collected engorged Carios kelleyi nymphs, which are rapid feeding argasid ticks of bats [9]. This suggests that some argasid species may remain on the vertebrate host for a duration of time after feeding. Population genetic studies are needed to evaluate the genetic diversity between O. turicata populations at different spatial scales collected in the United States, to estimate dissemination patterns of both vector and pathogen.
A limitation of our study is the likely circulation of additional uncharacterized RF spirochete species in the southern United States. While BipA is highly divergent between most species of RF spirochete and the recombinant protein can discriminate between B. hermsii and B. turicatae infections [17], additional work is needed to obtain novel spirochete species circulating in nature. For example, Ornithidoros talaje was recently collected in Texas [1], and while we failed to detect Borrelia DNA in these the ticks, the circulation of Borrelia mazzottii in the state exists. In 1955, B. mazzottii was reported to be transmissible by O. talaje ticks that were collected in northern Mexico [12], but since then reports of the disease have been absent. Recently, RF spirochetes were detected in a blood smear of a sick patient in Sonora, Mexico, but the species was unidentified [32]. Furthermore, Candidatus Borrelia texasensis was initially isolated in medium from an adult ixodid tick, Dermacentor variabilis, which was feeding on a coyote collected in Webb County, Texas [33]. The spirochete was initially cultured and grouped with RF spirochetes, but Lin and colleagues were unable to revive frozen stocks and an isolate does not exist. While it is unclear whether coyotes are a competent host for Candidatus Borrelia texasensis, the findings suggest that the mammals may be exposed to additional species of RF spirochete.
We recommend increased surveillance of small and medium sized mammals within metropolitan areas of Texas. San Antonio, Austin, and Dallas, Texas are in the top 11 most populated cities in the United States, are rapidly expanding, and evidence indicates that B. turicatae may be emerging in these areas. In 2017 there was an outbreak of TBRF among conference attendees in Austin, Texas [34], which is located in Travis County. This outbreak was in a densely populated area of the city and B. turicatae infected ticks were collected from rodent dens at a public park near the conference site. In our current report, there was little overlap between the Texas ecoregions that were sampled for the different vertebrate species (Table 1), and only three rodents were collected in Travis County. Future studies should focus on small and medium sized vertebrate sampling in regions where B. turicatae is emerging, and investigate host competence for the pathogen. As these studies are conducted, a refined understanding of Tick-borne relapsing fever seroprevalence in Texas the vertebrate hosts that support the ecology of B. turicatae will be attained, and surveillance and countermeasures can be implemented to improve public health.