Rickettsia rickettsii subsp. californica subsp. nov., the etiologic agent of Pacific Coast tick fever.

The etiologic agent of Pacific Coast tick fever (PCTF), a moderately severe tickborne illness that resembles Rocky Mountain spotted fever (RMSF), was first isolated in 1966 from specimens of Dermacentor occidentalis (the Pacific Coast tick) obtained in California. For several decades, this bacterium was identified ambiguously as the unclassified spotted fever group Rickettsia species 364-D, Rickettsia 364, or Rickettsia philipii. However, none of these epithets satisfied criteria of formal bacterial nomenclature. Data developed from mouse serotyping studies performed 45 years ago, and multi-locus sequence typing several decades later, indicated that this bacterium was similar to, but distinct from isolates of Rickettsia rickettsii, the etiological agent of RMSF. We applied an integrative taxonomic approach, combining phenotypic, ecological, and clinical data with whole genome sequencing of 11 contemporary isolates of this pathogen to identify it as a distinct subspecies of R. rickettsii, and propose the name Rickettsia rickettsii subsp. californica subsp. nov.

Borrelia maritima sp. nov., a novel species of the Borrelia burgdorferi sensu lato complex, occupying a basal position to North American species.

Borrelia species are vector-borne parasitic bacteria with unusual, highly fragmented genomes that include a linear chromosome and linear as well as circular plasmids that differ numerically between and within various species. Strain CA690T, which was cultivated from a questing Ixodes spinipalpis nymph in the San Francisco Bay area, CA, was determined to be genetically distinct from all other described species belonging to the Borrelia burgdorferi sensu lato complex. The genome, including plasmids, was assembled using a hybrid assembly of short Illumina reads and long reads obtained via Oxford Nanopore Technology. We found that strain CA690T has a main linear chromosome containing 902176 bp with a blast identity ≤91 % compared with other Borrelia species chromosomes and five linear and two circular plasmids. A phylogeny based on 37 single-copy genes of the main linear chromosome and rooted with the relapsing fever species Borrelia duttonii strain Ly revealed that strain CA690T had a sister-group relationship with, and occupied a basal position to, species occurring in North America. We propose to name this species Borrelia maritima sp. nov. The type strain, CA690T, has been deposited in two national culture collections, DSMZ (=107169) and ATCC (=TSD-160).

Recurrent evolution of host and vector association in bacteria of the Borrelia burgdorferi sensu lato species complex.

BACKGROUND: The Borrelia burgdorferi sensu lato (s.l.) species complex consists of tick-transmitted bacteria and currently comprises approximately 20 named and proposed genospecies some of which are known to cause Lyme Borreliosis. Species have been defined via genetic distances and ecological niches they occupy. Understanding the evolutionary relationship of species of the complex is fundamental to explaining patterns of speciation. This in turn forms a crucial basis to frame testable hypotheses concerning the underlying processes including host and vector adaptations. RESULTS: Illumina Technology was used to obtain genome-wide sequence data for 93 strains of 14 named genospecies of the B. burgdorferi species complex and genomic data already published for 18 additional strain (including one new species) was added. Phylogenetic reconstruction based on 114 orthologous single copy genes shows that the genospecies represent clearly distinguishable taxa with recent and still ongoing speciation events apparent in Europe and Asia. The position of Borrelia species in the phylogeny is consistent with host associations constituting a major driver for speciation. Interestingly, the data also demonstrate that vector associations are an additional driver for diversification in this tick-borne species complex. This is particularly obvious in B. bavariensis, a rodent adapted species that has diverged from the bird-associated B. garinii most likely in Asia. It now consists of two populations one of which most probably invaded Europe following adaptation to a new vector (Ixodes ricinus) and currently expands its distribution range. CONCLUSIONS: The results imply that genotypes/species with novel properties regarding host or vector associations have evolved recurrently during the history of the species complex and may emerge at any time. We suggest that the finding of vector associations as a driver for diversification may be a general pattern for tick-borne pathogens. The core genome analysis presented here provides an important source for investigations of the underlying mechanisms of speciation in tick-borne pathogens.

Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov. prevail in diverse enzootic transmission cycles.

Two species of the genus Borrelia, Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov., were first described by Postic and co-workers on the basis of genetic analyses of several loci. Multilocus sequence analysis of eight housekeeping loci confirmed that these two Borrelia genomospecies are distinct members of the Borrelia burgdorferi sensu lato complex. B. bissettiae sp. nov. was initially described in transmission cycles involving Neotoma fuscipes wood rats and Ixodes pacificus ticks in California, and Neotoma mexicana and Ixodes spinipalpis in Colorado. The preferred host of B. californiensis sp. nov. appears to be the California kangaroo rat, Dipodomys californicus; Ixodes jellisoni, I. spinipalipis and I. pacificus ticks are naturally infected with it. Thus, the ecological associations of the two genomospecies and their genetic distance from all other known Borrelia genomospecies species justify their description as separate genomospecies: B. bissettiae sp. nov. (type strain DN127T = DSM 17990T = CIP 109136T) and B. californiensis (type strain CA446T = DSM 17989T = ATCC BAA-2689T).

Borrelia kurtenbachii sp. nov., a widely distributed member of the Borrelia burgdorferi sensu lato species complex in North America.

Lyme borreliosis group spirochaetes are parasitic bacteria transmitted by vector ticks of the genus Ixodes and distributed mainly between 40° and 60° northern latitudes. Since Borrelia burgdorferi sensu stricto (hereinafter, B. burgdorferi) was described in the north-eastern USA during the early 1980s, an increasing diversity has been noted within the species complex. Here, we describe a novel genomic species, Borrelia kurtenbachii sp. nov. (type strain 25015(T) = ATCC BAA-2495(T) =  DSM 26572(T)), that is prevalent in transmission cycles among vector ticks and reservoir hosts in North America. Confirmation of the presence of this species in Europe awaits further investigation.

Rickettsia species identified in adult, host-seeking Dermacentor occidentalis (Acari: Ixodidae) from Baja California, Mexico, and Oregon and Washington, United States.

The Pacific Coast tick (Dermacentor occidentalis Marx, 1892) is a frequently encountered and commonly reported human-biting tick species that has been recorded from most of California and parts of southwestern Oregon, southcentral Washington, and northwestern Mexico. Although previous investigators have surveyed populations of D. occidentalis for the presence of Rickettsia species across several regions of California, populations of this tick have not been surveyed heretofore for rickettsiae from Baja California, Oregon, or Washington. We evaluated 1,367 host-seeking, D. occidentalis adults collected from 2015 to 2022 by flagging vegetation at multiple sites in Baja California, Mexico, and Oregon and Washington, United States, using genus- and species-specific assays for spotted fever group rickettsiae. DNA of Rickettsia 364D, R. bellii, and R. tillamookensis was not detected in specimens from these regions. DNA of R. rhipicephali was detected in D. occidentalis specimens obtained from Ensenada Municipality in Baja California and southwestern Oregon, but not from Washington. All ompA sequences of R. rhipichephali that were amplified from individual ticks in southwestern Oregon were represented by a single genotype. DNA of the Ixodes pacificus rickettsial endosymbiont was amplified from specimens collected in southwestern Oregon and Klickitat County, Washington; to the best of our knowledge, this Rickettsia species has never been identified in D. occidentalis. Collectively, these data are consistent with a relatively recent introduction of Pacific Coast ticks in the northernmost extension of its recognized range.

Detection of Borrelia burgdorferi sensu stricto ospC alleles associated with human lyme borreliosis worldwide in non-human-biting tick Ixodes affinis and rodent hosts in Southeastern United States.

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in European and North American regions where Lyme disease is endemic and where it is not endemic revealed a close relatedness of geographically distinct populations. ospC alleles A, B, and L were detected on both continents in vectors and hosts, including humans. Six ospC alleles, A, B, L, Q, R, and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3, and M, were identified in the far-western United States. Four ospC alleles, B, G, H, and L, were abundant in the southeastern United States. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern United States with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in European and North American regions where the disease is endemic were detected in B. burgdorferi strains isolated from the non-human-biting tick Ixodes affinis and rodent hosts in the southeastern United States. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern United States, a finding that confirms the hypothesis of transoceanic migration of Borrelia species.

Recovery of western black-legged tick and vertebrate populations after a destructive wildfire in an intensively-studied woodland in northern California.

Despite increasing severity and frequency of wildfires, knowledge about how fire impacts the ecology of tick-borne pathogens is limited. In 2018, the River Fire burned a forest in the far-western U.S.A. where the ecology of tick-borne pathogens had been studied for decades. Forest structure, avifauna, large and small mammals, lizards, ticks, and tick-borne pathogens (Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi) were assessed after the wildfire in 2019 and 2020. Burning reduced canopy cover and eliminated the layer of thick leaf litter that hosted free-living ticks, which over time was replaced by forbs and grasses. Tick abundance and the vertebrate host community changed dramatically. Avian species adapted to cavity nesting became most prevalent, while the number of foliage-foraging species increased by 83% as vegetation regenerated. Nine mammalian species were observed on camera traps, including sentinel (black-tailed jackrabbits) and reservoir hosts (western gray squirrels) of B. burgdorferi. One Peromyscus sp. mouse was captured in 2019 but by 2020, numbers were rebounding (n=37), although tick infestations on rodents remained sparse (0.2/rodent). However, western fence lizards (n=19) hosted 8.6 ticks on average in 2020. Assays for pathogens found no B. miyamotoi in either questing or host-feeding ticks, A. phagocytophilum DNA in 4% (1/23) in 2019, and 17% (29/173) in 2020 for questing and host-feeding ticks combined, and B. burgdorferi DNA in just 1% of all ticks collected in 2020 (2/173). We conclude that a moderately severe wildfire can have dramatic impacts on the ecology of tick-borne pathogens, with changes posited to continue for multiple years.

Two boundaries separate Borrelia burgdorferi populations in North America.

Understanding the spread of infectious diseases is crucial for implementing effective control measures. For this, it is important to obtain information on the contemporary population structure of a disease agent and to infer the evolutionary processes that may have shaped it. Here, we investigate on a continental scale the population structure of Borrelia burgdorferi, the causative agent of Lyme borreliosis (LB), a tick-borne disease, in North America. We test the hypothesis that the observed population structure is congruent with recent population expansions and that these were preceded by bottlenecks mostly likely caused by the near extirpation in the 1900s of hosts required for sustaining tick populations. Multilocus sequence typing and complementary population analytical tools were used to evaluate B. burgdorferi samples collected in the Northeastern, Upper Midwestern, and Far-Western United States and Canada. The spatial distribution of sequence types (STs) and inferred population boundaries suggest that the current populations are geographically separated. One major population boundary separated western B. burgdorferi populations transmitted by Ixodes pacificus in California from Eastern populations transmitted by I. scapularis; the other divided Midwestern and Northeastern populations. However, populations from all three regions were genetically closely related. Together, our findings suggest that although the contemporary populations of North American B. burgdorferi now comprise three geographically separated subpopulations with no or limited gene flow among them, they arose from a common ancestral population. A comparative analysis of the B. burgdorferi outer surface protein C (ospC) gene revealed novel linkages and provides additional insights into the genetic characteristics of strains.

Detection and Isolation of Rickettsia tillamookensis (Rickettsiales: Rickettsiaceae) From Ixodes pacificus (Acari: Ixodidae) From Multiple Regions of California.

The western black-legged tick (Ixodes pacificus) is the most frequently identified human-biting tick species in the western United States and the principal vector of at least three recognized bacterial pathogens of humans. A potentially pathogenic Rickettsia species, first described in 1978 and recently characterized as a novel transitional group agent designated as Rickettsia tillamookensis, also exists among populations of I. pacificus, although the distribution and frequency of this agent are poorly known. We evaluated DNA extracts from 348 host-seeking I. pacificus nymphs collected from 9 locations in five California counties, and from 916 I. pacificus adults collected from 24 locations in 13 counties, by using a real-time PCR designed specifically to detect DNA of R. tillamookensis. DNA of R. tillamookensis was detected in 10 (2.9%) nymphs (95% CI: 1.6-5.2%) and 17 (1.9%) adults (95% CI: 1.2-3.0%) from 11 counties of northern California. Although site-specific infection rates varied greatly, frequencies of infection remained consistently low when aggregated by stage, sex, habitat type, or geographical region. Four novel isolates of R. tillamookensis were cultivated in Vero E6 cells from individual adult ticks collected from Alameda, Nevada, and Yolo counties. Four historical isolates, serotyped previously as 'Tillamook-like' strains over 40 yr ago, were revived from long-term storage in liquid nitrogen and confirmed subsequently by molecular methods as isolates of R. tillamookensis. The potential public health impact of R. tillamookensis requires further investigation.

Genetic diversity of Borrelia burgdorferi and detection of B. bissettii-like DNA in serum of north-coastal California residents.

In North America, Lyme borreliosis (LB) is a tick-borne disease caused by infection with the spirochete Borrelia burgdorferi. We studied the genetic diversity of LB spirochetes in north-coastal California residents. Spirochete DNA was detected in 23.7% (27/114) of the study subjects using a PCR protocol optimized for increased sensitivity in human sera. Californians were most commonly infected with B. burgdorferi ospC genotype A, a globally widespread spirochete associated with high virulence in LB patients. Sequence analysis of rrf-rrl and p66 loci in 11% (3/27) of the PCR-positive study subjects revealed evidence of infection with an organism closely related to B. bissettii. This spirochete, heretofore associated with LB only in Europe, is widely distributed among ticks and wildlife in North America. Further molecular testing of sera from residents in areas where LB is endemic is warranted to enhance our understanding of the geographic distribution and frequency of occurrence of B. bissettii-like infections.

Impact of the experimental removal of lizards on Lyme disease risk.

The distribution of vector meals in the host community is an important element of understanding and predicting vector-borne disease risk. Lizards (such as the western fence lizard; Sceloporus occidentalis) play a unique role in Lyme disease ecology in the far-western United States. Lizards rather than mammals serve as the blood meal hosts for a large fraction of larval and nymphal western black-legged ticks (Ixodes pacificus--the vector for Lyme disease in that region) but are not competent reservoirs for the pathogen, Borrelia burgdorferi. Prior studies have suggested that the net effect of lizards is to reduce risk of human exposure to Lyme disease, a hypothesis that we tested experimentally. Following experimental removal of lizards, we documented incomplete host switching by larval ticks (5.19%) from lizards to other hosts. Larval tick burdens increased on woodrats, a competent reservoir, but not on deer mice, a less competent pathogen reservoir. However, most larvae failed to find an alternate host. This resulted in significantly lower densities of nymphal ticks the following year. Unexpectedly, the removal of reservoir-incompetent lizards did not cause an increase in nymphal tick infection prevalence. The net result of lizard removal was a decrease in the density of infected nymphal ticks, and therefore a decreased risk to humans of Lyme disease. Our results indicate that an incompetent reservoir for a pathogen may, in fact, increase disease risk through the maintenance of higher vector density and therefore, higher density of infected vectors.

Effects of an invasive forest pathogen on abundance of ticks and their vertebrate hosts in a California Lyme disease focus.

Invasive species, including pathogens, can have important effects on local ecosystems, including indirect consequences on native species. This study focuses on the effects of an invasive plant pathogen on a vertebrate community and Ixodes pacificus, the vector of the Lyme disease pathogen (Borrelia burgdorferi) in California. Phytophthora ramorum, the causative agent of sudden oak death, is a non-native pathogen killing trees in California and Oregon. We conducted a multi-year study using a gradient of SOD-caused disturbance to assess the impact on the dusky-footed woodrat (Neotoma fuscipes) and the deer mouse (Peromyscus maniculatus), two reservoir hosts of B. burgdorferi, as well as the impact on the Columbian black-tailed deer (Odocoileus hemionus columbianus) and the western fence lizard (Sceloporus occidentalis), both of which are important hosts for I. pacificus but are not pathogen reservoirs. Abundances of P. maniculatus and S. occidentalis were positively correlated with greater SOD disturbance, whereas N. fuscipes abundance was negatively correlated. We did not find a change in space use by O. hemionus. Our data show that SOD has a positive impact on the density of nymphal ticks, which is expected to increase the risk of human exposure to Lyme disease all else being equal. A positive correlation between SOD disturbance and the density of nymphal ticks was expected given increased abundances of two important hosts: deer mice and western fence lizards. However, further research is needed to integrate the direct effects of SOD on ticks, for example via altered abiotic conditions with host-mediated indirect effects.

Prevalence of Babesia canis DNA in Ixodes ricinus ticks collected in forest and urban ecosystems in west-central Poland.

Babesia canis, a widely distributed European tick-borne protozoan haemoparasite, causes canine babesiosis, the most important tick-borne disease afflicting dogs worldwide. The meadow tick, Dermacentor reticulatus, is considered to be the primary vector of this parasite in central Europe. Females of the more broadly distributed and medically important castor bean tick, Ixodes ricinus, also commonly feed upon dogs, but their role in the enzootic transmission cycle of B. canis is unclear. Here, we screened 1,598 host-seeking I. ricinus ticks collected from two different ecosystems, forest stands vs. urban recreational forests, for the presence of B. canis DNA. Ticks were sampled during their two seasonal peaks of activity, spring (May/June) and late summer (September). Babesia species were identified by amplification and sequencing of a hypervariable 18S rRNA gene fragment. Babesia canis was the only piroplasm detected in 13% of 200 larvae and 8.2% of 324 nymphs in the forest ecosystems. In urban recreational areas, B. canis DNA was found in 1.5% of 460 nymphs, 3.5% of 289 females and 3.2% of 280 males. Additionally, three samples, including one female, one male, and one nymph, were co-infected with B. venatorum and one nymph with B. divergens or B. capreoli. Our findings implicate that B. canis can be transmitted transovarially and maintained transstadially within populations of I. ricinus, but the vector competence of I. ricinus for transmitting B. canis remains to be investigated.

Modeling future climate suitability for the western blacklegged tick, Ixodes pacificus, in California with an emphasis on land access and ownership.

In the western United States, Ixodes pacificus Cooley & Kohls (Acari: Ixodidae) is the primary vector of the agents causing Lyme disease and granulocytic anaplasmosis in humans. The geographic distribution of the tick is associated with climatic variables that include temperature, precipitation, and humidity, and biotic factors such as the spatial distribution of its primary vertebrate hosts. Here, we explore (1) how climate change may alter the geographic distribution of I. pacificus in California, USA, during the 21st century, and (2) the spatial overlap among predicted changes in tick habitat suitability, land access, and ownership. Maps of potential future suitability for I. pacificus were generated by applying climate-based species distribution models to a multi-model ensemble of climate change projections for the Representative Concentration Pathway (RCP) 4.5 (moderate emission) and 8.5 (high emission) scenarios for two future periods: mid-century (2026-2045) and end-of-century (2086-2099). Areas climatically-suitable for I. pacificus are projected to expand by 23% (mid-century RCP 4.5) to 86% (end-of-century RCP 8.5) across California, compared to the historical period (1980-2014), with future estimates of total suitable land area ranging from about 88 to 133 thousand km2, or up to about a third of California. Regions projected to have the largest area increases in suitability by end-of-century are in northwestern California and the south central and southern coastal ranges. Over a third of the future suitable habitat is on lands currently designated as open access (i.e. publicly available), and by 2100, the amount of these lands that are suitable habitat for I. pacificus is projected to more than double under the most extreme emissions scenario (from ~23,000 to >51,000 km2). Of this area, most is federally-owned (>45,000 km2). By the end of the century, 26% of all federal land in the state is predicted to be suitable habitat for I. pacificus. The resulting maps may facilitate regional planning and preparedness by informing public health and vector control decision-makers.

Community ecology and disease risk: lizards, squirrels, and the Lyme disease spirochete in California, USA.

Vector-borne zoonotic diseases are often maintained in complex transmission cycles involving multiple vertebrate hosts and their arthropod vectors. In the state of California, U.S.A., the spirochete Borrelia burgdorferi, which causes Lyme disease, is transmitted between vertebrate hosts by the western black-legged tick, Ixodes pacificus. Several mammalian species serve as reservoir hosts of the spirochete, but levels of tick infestation, reservoir competence, and Borrelia-infection prevalence vary widely among such hosts. Here, we model the host (lizards, Peromyscus mice, Californian meadow voles, dusky-footed wood rats, and western gray squirrels), vector, and pathogen community of oak woodlands in northwestern California to determine the relative importance of different tick hosts. Observed infection prevalence of B. burgdorferi in host-seeking I. pacificus nymphs was 1.8-5.3%, and our host-community model estimated an infection prevalence of 1.6-2.2%. The western gray squirrel (Sciurus griseus) was the only source of infected nymphs. Lizards, which are refractory to Borrelia infection, are important in feeding subadult ticks but reduce disease risk (nymphal infection prevalence). Species identity is therefore critical in understanding and determining the local disease ecology.

Population structure of the lyme borreliosis spirochete Borrelia burgdorferi in the western black-legged tick (Ixodes pacificus) in Northern California.

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.

Feeding preferences of the immature stages of three western north American ixodid ticks (Acari) for avian, reptilian, or rodent hosts.

Larval and nymphal Ixodes pacificus Cooley and Kohls, I. (Ixodes) jellisoni Cooley and Kohls, and Dermacentor occidentalis Marx were tested for host preference when simultaneously presented with a deer mouse (Peromyscus maniculatus Wagner), California kangaroo rat (Dipodomys californicus Merriam), western fence lizard (Sceloporus occidentalis Baird and Girard), and California towhee (Pipilo crissalis Vigors) in an experimental apparatus. Differences were observed in the preferences among the three species and between life stages. More larvae of all species approached and contacted hosts than did nymphs. Subadult I. pacificus entered all host-containing chambers in the highest numbers and remained on lizards most often after contact. Subadult I. jellisoni entered and remained in the chambers containing kangaroo rats, while rejecting mice, lizards, and birds as hosts. Subadult D. occidentalis most frequently entered rodent-containing chambers and contacted these hosts. After overnight exposure to all nonavian hosts, only I. pacificus parasitized and fed successfully on all three animals. I. jellisoni fed only on kangaroo rats and D. occidentalis fed only on rodents. Molting success ranged from approximately 66 to 95% among tick species and stages. We concluded that, under laboratory conditions, I. pacificus larvae and nymphs prefer western fence lizards, but also will parasitize rodents. Dermacentor occidentalis immatures use deer mice and kangaroo rats similarly, whereas I. jellisoni subadults exclusively parasitize kangaroo rats. California towhees are considerably less attractive as hosts for these three ticks. These host preferences are consistent with what is known about the natural feeding habits of all three ticks.

Phylogeography of Borrelia spirochetes in Ixodes pacificus and Ixodes spinipalpis ticks highlights differential acarological risk of tick-borne disease transmission in northern versus southern California.

The common human-biting tick, Ixodes pacificus, is the primary vector of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (ss) in western North America and has been found to harbor other closely-related spirochetes in the Borrelia burgdorferi sensu lato (sl) complex. Between 2008-2015, 11,066 adult and 3,815 nymphal I. pacificus and five adult and 144 nymphal Ixodes spinpalpis, a commonly collected wildlife tick, were collected from 42 California counties. Borrelia burgdorferi sl was detected in 1.2% and 3.8% I. pacificus adults and nymphs, respectively. Results from this study indicate genetic diversity and geographic structure of B. burgdorferi sl in California I. pacificus ticks, by sequence comparison of the16S rRNA gene, with B. burgdorferi ss, the agent of Lyme disease, found only in I. pacificus collected from the north and central coastal and Sierra Nevada foothill regions; B. burgdorferi ss was not detected in ticks tested from southern California. In contrast, Borrelia bissettiae, a member of the B. burgdorferi sl complex, was detected in both I. pacificus and I. spinipalpis, in the coastal region of both northern and southern California, but was absent from ticks in the Sierra Nevada foothills. In a similar pattern to B. bissettiae, Borrelia americana (a member of the B. burgdorferi sl complex) was detected in a single adult I. pacificus from the north coast and two I. spinipalpis nymphs from south-coastal California. This study highlights that the geographic area of Lyme disease acarological risk in California is the north-central and Sierra Nevada foothill regions of the state with little to no risk in the southern regions of the state.