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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., (Published by Oxford University Press on behalf of Entomological Society of America 2022.)

Background and Aims: Outbreaks of severe and chronic tick-borne diseases (TBDs) are on the rise. This is through the transmission of infectious disease agents to humans during tick feeding. The transmission rate and extent of microbial exchange, however, vary based on the tick microbiome composition. While select microbes are determined to be members of the normal tick microbiome and others are clearly recognized mammalian and/or avian pathogens, the status of many other members of the tick microbiota with respect to human and alternate host pathogenesis remains unclear. Moreover, the species-level 16S microbiome of prominent TBD vectors, including Ixodes pacificus , have not been extensively studied. To elucidate the I. pacificus microbiome composition, we performed a pan-domain species-specific characterization of the bacterial microbiome on adult I. pacificus ticks collected from two regional parks within Western California. Our methods provide for characterizing nuances within cohort microbiomes and their relationships to geo-locale of origin, surrounding fauna, and prevalences of known and suspected pathogens in relation to current TBD epidemiological zones. Methods: Ninety-two adult I. pacificus bacterial microbiomes were characterized using a high-fidelity, pan-domain, species-specific, full-length 16S rRNA amplification method using circular consensus sequencing performed on the Pacific Biosciences Sequel platform. Data analyses were performed with the MCSMRT data analysis package and database. Results: The species-specific I. pacificus microbiome composition illustrates a complex assortment of microflora, including over 900 eubacterial species with high taxonomic diversity, which was revealed to vary by sex and geo-locale, though the use of full-length 16S gene sequencing. The TBD-associated pathogens, such as Borrelia burgdorferi , Anaplasma phagocytophilum , and Rickettsia monacensis , were identified along with a host of bacteria previously unassociated with ticks. Conclusion: Species-level taxonomic classification of the I. pacificus microbiome revealed that full-length bacterial 16S gene sequencing is required for the granularity to elucidate the microbial diversity within and among ticks based on geo-locale.

In California, the western blacklegged tick, Ixodes pacificus Cooley and Kohls, is the principal vector of the Borrelia burgdorferi sensu lato (sl) complex (Spirochaetales: Spirochaetaceae, Johnson et al.), which includes the causative agent of Lyme disease (B. burgdorferi sensu stricto). Ixodes pacificus nymphs were sampled from 2015 to 2017 at one Sierra Nevada foothill site to evaluate our efficiency in collecting this life stage, characterize nymphal seasonality, and identify environmental factors affecting their abundance and infection with B. burgdorferi sl. To assess sampling success, we compared the density and prevalence of I. pacificus nymphs flagged from four questing substrates (logs, rocks, tree trunks, leaf litter). Habitat characteristics (e.g., canopy cover, tree species) were recorded for each sample, and temperature and relative humidity were measured hourly at one location. Generalized linear mixed models were used to assess environmental factors associated with I. pacificus abundance and B. burgdorferi sl infection. In total, 2,033 substrates were sampled, resulting in the collection of 742 I. pacificus nymphs. Seasonal abundance of nymphs was bimodal with peak activity occurring from late March through April and a secondary peak in June. Substrate type, collection year, month, and canopy cover were all significant predictors of nymphal density and prevalence. Logs, rocks, and tree trunks had significantly greater nymphal densities and prevalences than leaf litter. Cumulative annual vapor pressure deficit was the only significant climatic predictor of overall nymphal I. pacificus density and prevalence. No associations were observed between the presence of B. burgdorferi sl in nymphs and environmental variables., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

Tick-borne diseases in California include Lyme disease (caused by Borrelia burgdorferi), infections with Borrelia miyamotoi, and human granulocytic anaplasmosis (caused by Anaplasma phagocytophilum). We surveyed multiple sites and habitats (woodland, grassland, and coastal chaparral) in California to describe spatial patterns of tick-borne pathogen prevalence in western black-legged ticks (Ixodes pacificus). We found that several species of Borrelia- B. burgdorferi, Borrelia americana, and Borrelia bissettiae-were observed in habitats, such as coastal chaparral, that do not harbor obvious reservoir host candidates. Describing tick-borne pathogen prevalence is strongly influenced by the scale of surveillance: aggregating data from individual sites to match jurisdictional boundaries (e.g., county or state) can lower the reported infection prevalence. Considering multiple pathogen species in the same habitat allows a more cohesive interpretation of local pathogen occurrence. IMPORTANCE Understanding the local host ecology and prevalence of zoonotic diseases is vital for public health. Using tick-borne diseases in California, we show that there is often a bias to our understanding and that studies tend to focus on particular habitats, e.g., Lyme disease in oak woodlands. Other habitats may harbor a surprising diversity of tick-borne pathogens but have been neglected, e.g., coastal chaparral. Explaining pathogen prevalence requires descriptions of data on a local scale; otherwise, aggregating the data can misrepresent the local dynamics of tick-borne diseases.

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 CA690 T , 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 CA690 T 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 CA690 T 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, CA690 T , has been deposited in two national culture collections, DSMZ (=107169) and ATCC (=TSD-160).

A rickettsial isolate was obtained from a partially engorged Ixodes pacificus female, which was collected from Humboldt County, California. The isolate was provisionally named Rickettsia endosymbiont Ixodes pacificus (REIP). The REIP isolate displayed the highest nucleotide sequence identity to Rickettsia species phylotype G021 in I. pacificus (99%, 99%, and 100% for ompA, 16S rRNA, and gltA, respectively), a bacterium that was previously identified in I. pacifiucs by PCR. Analysis of sequences from complete opening frames of five genes, 16S rRNA, gltA, ompA, ompB, and sca4, provided inference to the bacteria's classification among other Rickettsia species. The REIP isolate displayed 99.8%, 99.4%, 99.2%, 99.5%, and 99.6% nucleotide sequence identity for 16S rRNA, gltA, ompA, ompB, and sca4 gene, respectively, with genes of 'R. monacensis' str. IrR/Munich, indicating the REIP isolate is closely related to 'R. monacensis'. Our suggestion was further supported by phylogenetic analysis using concatenated sequences of 16S rRNA, gltA, ompA, ompB, and sca4 genes, concatenated sequences of dksA-xerC, mppA-purC, and rpmE-tRNA fMet intergenic spacer regions. Both phylogenetic trees implied that the REIP isolate is most closely related to 'R. monacensis' str. IrR/Munich. We propose the bacterium be considered as 'Rickettsia monacensis' str. Humboldt for its closest phylogenetic relative (=DSM 103975 T = ATCC TSD-94 T)., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

From July 2006 through August 2017, a passive surveillance study of Ixodes ticks submitted from California, Oregon, and Washington was conducted by the TickReport program at the University of Massachusetts, Amherst. In total, 549 human-biting Ixodes ticks were submitted comprising both endemic and nonendemic species. We found that 430 endemic ticks were from 3 Ixodes species: Ixodes pacificus, Ixodes spinipalpis, and Ixodes angustus, whereas Ixodes scapularis (n = 111) was the most common species among the 119 nonendemic ticks. The submission peak for nymphal I. pacificus and I. spinipalpis was June, while submission peak for adult I. pacificus and nymphal I. angustus was April and September, respectively. Endemic ticks commonly attached to the lower extremities of their victims, and individuals younger than 9 years old were frequently bitten. The infection prevalence of Borrelia burgdorferi sensu lato, Borrelia miyamotoi, and Anaplasma phagocytophilum in I. pacificus ticks was 1.31%, 1.05%, and 0.52%, respectively, and the prevalence of B. burgdorferi s. l. and A. phagocytophilum in I. spinipalpis ticks was 14.29% and 10.71%, respectively. Furthermore, two species within the B. burgdorferi s. l. complex were detected in West Coast ticks: B. burgdorferi sensu stricto and Borrelia lanei. I. spinipalpis had the highest Borrelia prevalence among endemic ticks, and it was caused exclusively by B. lanei. Borrelia mayonii, Babesia microti, and Ehrlichia muris-like agent were not detected in these endemic ticks. In this study, we show that many nonendemic Ixodes ticks (119/549) are most likely acquired from travel to a different geographic region. We report cases of conventionally recognized nonhuman feeders (I. spinipalpis and I. angustus) parasitizing humans. The highest pathogen prevalence in I. spinipalpis may indicate a larger public health threat than previously thought, and the enzootic life cycle and pathogenicity of B. lanei warrant further study.

Borrelia miyamotoi is an increasingly recognized human pathogen transmitted by Ixodes ticks in the Northern Hemisphere. In North America, infection prevalences of B. miyamotoi are characteristically low (<10%) in Ixodes scapularis (Say; Acari: Ixodidae) and Ixodes pacificus (Cooley & Kohls; Acari: Ixodidae), both of which readily bite humans. We tested 3,255 host-seeking I. pacificus nymphs collected in 2004 from 79 sites throughout Mendocino County in north-coastal California for presence of B. miyamotoi. The collection sites represented a variety of forest types ranging from hot, dry oak woodlands in the southeast, to coastal redwoods in the west, and Ponderosa pine and Douglas fir-dominated areas in the northern part of the county. We found that B. miyamotoi was geographically widespread, but infected I. pacificus nymphs infrequently (cumulative prevalence of 1.4%). Infection prevalence was not significantly associated with geographic region or woodland type, and neither density of host-seeking nymphs, nor infection with Borrelia burgdorferi sensu stricto was associated with B. miyamotoi infection status in individual ticks. Because B. burgdorferi prevalence at the same sites was previously associated with woodland type and nymphal density, our results suggest that despite sharing a common vector, the primary modes of enzootic maintenance for the two pathogens are likely different.

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called 'Candidatus Cryptoplasma californiense'. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.

Vector-borne pathogens are transmitted between vertebrate hosts and arthropod vectors, two immensely different environments for the pathogen. There is further differentiation among vertebrate hosts that often have complex, species-specific immunological responses to the pathogen. All this presents a heterogeneous environmental and immunological landscape with possible consequences on the population genetic structure of the pathogen. We evaluated the differential genetic diversity of the Lyme disease pathogen, Borrelia burgdorferi, in its vector, the western black-legged tick (Ixodes pacificus), and in its mammal host community using the 5S-23S rRNA intergenic spacer region. We found differences in haplotype distribution of B. burgdorferi in tick populations from two counties in California as well as between a sympatric tick and vertebrate host community. In addition, we found that three closely related haplotypes consistently occurred in high frequency in all sample types. Lastly, our study found lower species diversity of the B. burgdorferi species complex, known as B. burgdorferi sensu lato, in small mammal hosts versus the tick populations in a sympatric study area., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

We investigated the involvement of birds in the ecology of the western black-legged tick, Ixodes pacificus, and its associated zoonotic bacteria, Borrelia burgdorferi and Anaplasma phagocytophilum, at two interior coast-range study sites in northern California. Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis (GA), and B. burgdorferi s.s., the agent of Lyme disease (LD), are tick-borne pathogens that are well established in California. We screened blood and ticks from 349 individual birds in 48 species collected in 2011 and 2012 using pathogen-specific PCR. A total of 617 immature I. pacificus was collected with almost three times as many larvae than nymphs. There were 7.5 times more I. pacificus at the Napa County site compared to the Yolo County site. Two of 74 (3%) nymphal pools from an Oregon junco (Junco hyemalis) and a hermit thrush (Catharus guttatus) and 4 individual larvae (all from Oregon juncos) were PCR-positive for B. burgdorferi. Blood samples from a golden-crowned sparrow (Zonotrichia atricapilla) and a European starling (Sturnus vulgaris) were positive for A. phagocytophilum DNA at very low levels. Birds that forage on ground or bark and nest on the ground, as well as some migratory species, are at an increased risk for acquiring I. pacificus. Our findings show that birds contribute to the ecologies of LD and GA in California by serving as a blood-meal source, feeding and transporting immature I. pacificus, and sometimes as a source of Borrelia infection., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Vector and host abundance affect infection transmission rates, prevalence, and persistence in communities. Biological diversity in hosts and vectors may provide "rescue" hosts which buffer against pathogen extinction and "dilution" hosts which reduce the force of infection in communities. Anaplasma phagocytophilum is a tick-transmitted zoonotic pathogen that circulates in small mammal and tick communities characterized by varying levels of biological diversity. We examined the prevalence of A. phagocytophilum in Ixodes spp. ticks in 11 communities in northern and central California. A total of 1020 ticks of 8 species was evaluated. Five percent of ticks (5 species) were PCR-positive, with the highest prevalence (6-7%) in I. pacificus and I. ochotonae. In most species, adults had a higher prevalence than nymphs or larvae. PCR prevalence varied between 0% and 40% across sites; the infection probability in ticks increased with infestation load and prevalence in small mammals, but not tick species richness, diversity, evenness, or small mammal species richness. No particular tick species was likely to "rescue" infection in the community; rather the risk of A. phagocytophilum infection is related to exposure to particular tick species and life stages, and overall tick abundance., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The emerging tick-borne pathogen Anaplasma phagocytophilum infects humans, domestic animals, and wildlife throughout the Holarctic. In the western US, the ecology of A. phagocytophilum is particularly complex, with multiple pathogen strains, tick vectors, and reservoir hosts. A recent phylogenetic analysis of A. phagocytophilum strains isolated from various small mammal hosts in California documented distinct clustering of woodrat strains separate from sciurid (chipmunk and squirrel) strains. Here, we identified strains of A. phagocytophilum in various Ixodes tick species in California and related these genotypes to those found among reservoir and clinical hosts from the same areas. The sequences from all of the nidicolous (nest-dwelling) Ixodes ticks grouped within a clade that also contained all of the woodrat-origin A. phagocytophilum strains. Two of the I. pacificus sequences were also grouped within this woodrat clade, while the remaining five belonged to a less genetically diverse clade that included several sciurid-origin strains as well as a dog, a horse, and a human strain. By comparing A. phagocytophilum strains from multiple sources concurrently, we were able to gain a clearer picture of how A. phagocytophilum strains in the western US are partitioned, which hosts and vectors are most likely to be infected with a particular strain, and which tick species and reservoir hosts pose the greatest health risk to humans and domestic animals., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Little is known about the eco-epidemiology of Lyme disease in southern California, a region where the incidence is much lower than it is in northern California. Here, we sought to discover the previously unknown microhabitats of nymphs of the primary vector, the western black-legged tick (Ixodes pacificus), in 3 moderately to heavily-utilized state parks in the Santa Monica Mountains in Los Angeles County; to elucidate the seasonal distribution and abundance of adults of I. pacificus and another human-biter, the Pacific Coast tick (Dermacentor occidentalis); and to determine what Lyme-disease or relapsing-fever group borreliae are present in questing nymphs or adult ticks. I. pacificus nymphs were collected infrequently at various times of day in 2 chaparral or 7 woodland litter areas by dragging (combined mean=0.4 nymphs per hour). The western fence lizard (Sceloporus occidentalis) was a choice sentinel animal for detecting the presence of I. pacificus nymphs (and larvae) in diverse biotopes even when dragging litter in them was fruitless. The abundance and seasonality of I. pacificus and D. occidentalis adults resembled what had been documented previously for these ticks in northern California. Overall, zero of 27 free-living and 118 lizard-infesting I. pacificus nymphs, 7 (0.29%) of 2392 I. pacificus adults and 2 (0.22%) of 896 D. occidentalis adults were infected with Borrelia burgdorferi sensu lato (Bb sl), but none of them harbored B. miyamotoi, a relapsing-fever group spirochete implicated recently as a zoonotic pathogen in Russia. Borrelia americana and the human pathogen B. burgdorferi sensu stricto were each detected in one (0.04%), and uncharacterized Bb sl in 5 adult I. pacificus (0.21%) that clustered with B. americana. Both PCR-positive D. occidentalis adults contained B. burgdorferi sensu stricto. We conclude that the acarologic risk of being bitten by a B. burgdorferi sensu lato-infected ixodid tick in the habitats studied is slight, which offers a plausible explanation for the low reported incidence of Lyme disease in south coastal California., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The western black-legged tick, Ixodes pacificus Cooley and Kohls, commonly bites humans in the far western U.S. In addition to transmitting Lyme borreliosis and anaplasmosis, it is a host of nonpathogenic bacteria as well as some of unknown pathogenicity. In this study, we report the detection, prevalence, and burden of 2 rickettsial phylotypes with unknown pathogenicity in I. pacificus ticks from 6 California counties using real-time quantitative PCR with phylotype-specific primers and probes. Prevalence of rickettsial phylotypes G021 and G022 from 247 I. pacificus ticks was 100% and 2.0%, respectively. The median burden of phylotype G021 was 7.3 per tick cell, whereas the burden of phylotype G022 was 0.8 per tick cell. The burden of phylotype G021 significantly differed between collection sites and between vegetation habitats. Ticks collected from the coastal sage scrub habitat of southern California had a lower burden of phylotype G021 when compared to central California oak woodland, northern California oak woodland, and mixed evergreen and ponderosa pine-oak habitats of northern California. No significant correlation was found between the burden of the phylotype G021 in the presence and absence of the phylotype G022 in I. pacificus, suggesting that the presence of these Rickettsia species do not interfere with each other in I. pacificus., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Global changes such as deforestation, climate change, and invasive species have the potential to greatly alter zoonotic disease systems through impacts on biodiversity. This study examined the impact of the invasive pathogen that causes sudden oak death (SOD) on the ecology of Lyme disease in California. The Lyme disease bacterium, Borrelia burgdorferi, is maintained in the far western United States by a suite of animal reservoirs including the dusky-footed woodrat (Neotoma fuscipes) and deer mouse (Peromyscus maniculatus), and is transmitted by the western black-legged tick (Ixodes pacificus). Other vertebrates, such as the western fence lizard (Sceloporus occidentalis), are important tick hosts but are not reservoirs of the pathogen. Previous work found that higher levels of SOD are correlated with greater abundance of P. maniculatus and S. occidentalis and lower N. fuscipes abundance. Here we model the contribution of these tick hosts to Lyme disease risk and also evaluate the potential impact of SOD on infection prevalence of the tick vector. By empirically parameterizing a static model with field and laboratory data on tick hosts, we predict that SOD reduces an important index of disease risk, nymphal infection prevalence, leading to a reduction in Lyme disease risk in certain coastal woodlands. Direct observational analysis of the impact of SOD on nymphal infection prevalence supports these model results. This study underscores the important direct and indirect impacts of invasive plant pathogens on biodiversity, the transmission cycles of zoonotic diseases, and ultimately human health.

Risk of exposure to nymphal Ixodes pacificus Cooley and Kohls ticks was investigated at 7 picnic areas in Tilden Regional Park, a heavily used recreation area of over 2000 acres in northwestern California, east of San Francisco Bay. Wooden picnic tables, tree trunks, logs, leaf litter, surrounding vegetation, and rock walls were checked for ticks using standard 1-m(2) flannel tick flags at biweekly intervals from March to August 2008. Results indicate that nymphal I. pacificus were commonly found on wooden picnic tables and other wooden materials, such as tree trunks and logs, at an equal proportion to those found in leaf litter. Nymphal I. pacificus in picnic areas peaked in April, with a secondary peak in early June. Five of 170 (2.9%) nymphal I. pacificus collected at picnic sites were positive for Borrelia spirochetes, of which 3 (1.8%) were identified as B. burgdorferi sensu stricto using molecular techniques. In addition, a nymphal I. auritulus collected from a rock wall in a picnic area tested positive for a mixture of B. burgdorferi and B. bissettii; this tick species feeds exclusively on birds. This study indicates a moderate risk of acquiring a nymphal tick at Tilden Park picnic areas, but due to the low B. burgdorferi infection prevalence, the risk of acquiring Lyme disease appears to be low., (Published by Elsevier GmbH.)

We amplified 16S rRNA, gltA, and ompA genes from Ixodes pacificus by polymerase chain reaction. Sequencing, BLAST analysis, and phylogenetic constructions indicated that two Rickettsia phylotypes are present in I. pacificus. While phylotype G021 has high homology to Ixodes scapularis endosymbiotic Rickettsia, phylotype G022 is a deeply branched novel spotted fever group Rickettsia.

Larval and nymphal western blacklegged tick, Ixodes pacificus Cooley & Kohls (Acari: Ixodidae), were collected from birds, rodents, and lizards at Quail Ridge Reserve located in Napa County in northwestern California. Species from three vertebrate classes were sampled simultaneously from two transects during two consecutive spring seasons. Feeding larval and nymphal ticks were removed and preserved for counting, examination and testing for the presence of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner. Mean infestations with I. pacificus subadults on lizards were 10.0, on birds 2.9, and on rodents 1.3. I. pacificus larvae (204) collected from 10 avian species and (215) collected from two rodent species were tested for the presence of B. burgdorferi s.s. via real-time polymerase chain reaction. Three B. burgdorferi-infected larvae were taken from two Junco hyemalis and two infected larvae from one Neotoma fuscipes Baird. This is the detection of B. burgdorferi ss in an Ixodes pacificus larvae feeding on a Junco hyemalis L., [corrected] in western North America.

Granulocytic anaplasmosis (GA) is a tick-borne emerging infectious disease caused by the bacterium Anaplasma phagocytophilum. From fall 2005 to spring 2007, A. phagocytophilum infection prevalence in small mammals and tick abundance were monitored at 4 study sites in coastal California. The abundance of different life stages of questing Ixodes pacificus ticks fluctuated seasonally with the number of adults peaking December to February, nymphs peaking May to July, and larvae peaking April to June. Numerous Ixodes tick species were found attached to dusky-footed woodrats (Neotoma fuscipes), chimunks (Tamias spp.), and deer mice (Peromyscus maniculatus); however, attached tick larvae on all 3 rodent species were primarily I. pacificus, attached nymphs were primarily I. angustus, and adults were either I. ochotonae, I. spinipalpis, or I. woodi. A. phagocytophilum DNA was detected by PCR in 2.2% (n=275, 95% C.I.=0.09-4.9) of sampled ticks. The overall A. phagocytophilum seroprevalence among small mammals was 7.4% (n=654, 95% C.I.=5.5-9.7) while 7.2% (n=125, 95% C.I.=3.5-13.4) of the animals were found to be PCR-positive. Seropositive animals included woodrats, chipmunks, and deer mice, although only woodrats and chipmunks had PCR-detectable infections. Seroprevalence varied temporally among species with the majority of exposed deer mice detected in fall 2006 and the majority of exposed woodrats and chipmunks identified in spring 2007. This study highlights the importance of multiple-year monitoring of both vectors and wildlife hosts in order to better understand the complex ecology of A. phagocytophilum and other related tick-borne disease agents., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Ixodes spp. tick-borne zoonotic diseases are present across the Holarctic in humans, domestic animals, and wildlife. Small mammals are reservoirs for the rickettsial pathogen Anaplasma phagocytophilum and tick vectors may include catholic-feeding bridge vectors as well as host-specialist or nidicolous ticks. Far western North American communities in which A. phagocytophilum is maintained are complex ecologically, with multiple reservoir host and tick species, multiple strains of the bacterial pathogen A. phagocytophilum and differences in dynamics of hosts and vectors across heterogeneous landscapes. We evaluated sites in northern California in order to identify primarily nidicolous ticks and the hosts they infest. A total of 667 ticks was found in 11 study sites, including 288 on flags and 379 attached to small mammals. Larvae were over-represented among attached ticks and adults on flags. The most abundant species was I. pacificus. Two-hundred fourteen nidicolous ticks were found, most abundantly I. angustus and I. spinipalpis. All adult I. ochotonae, I. auritulus, I. angustus, I. jellisoni, and I. woodi were female, while the male:female ratio of I. spinipalpis was 1.2:1 and 1:1 for I. pacificus. The greatest number of ticks was obtained from Tamias ochrogenys, Peromyscus spp., and Neotoma fuscipes. Of 234 small mammal individuals that were infested with Ixodes spp., only 81 (34.6%) were infested with I. pacificus. The remaining infested small mammals hosted nidicolous tick species. Eight ticks were PCR-positive, including 6 I. pacificus (one adult, one larva, and 6 nymphs), and 2 adult I. ochotonae and high PCR prevalences of 18% and 9% were detected in woodrats and chipmunks, respectively. Nymphal I. angustus ticks were active year-long with a possible increase in August while larval activity was only observed in December and spring months and adults only during spring and fall. Overall, we show high tick species richness and year-round high levels of infestation on rodents by several different nidicolous ticks in areas where A. phagocytophilum is enzootic, including on reported reservoir species., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The abiotic and biotic factors that govern the spatial distribution of Lyme disease vectors are poorly understood. This study addressed the influence of abiotic and biotic environmental variables on Ixodes pacificus Cooley & Kohls (Acari:Ixodidae) nymphs, because it is the primary vector of Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwaldt & Brenner in the far-western United States. Three metrics of Lyme disease risk were evaluated: the density of nymphs, the density of infected nymphs, and the nymphal infection prevalence. This study sampled randomly located plots in oak (Quercus spp.) woodland habitat in Sonoma County, CA. Each plot was drag-sampled for nymphal ticks and tested for B. burgdorferi infection. Path analysis was used to evaluate the direct and indirect relationship between topographic, forest structure and microclimatic variables on ticks. Significant negative correlations were found between maximum temperature in the dry season and the density of infected ticks in 2006 and tick density in 2007, but we did not find a significant relationship with nymphal infection prevalence in either year. Tick density and infected tick density had an indirect, positive correlation with elevation, mediated through temperature. This study found that in certain years but not others, temperature maxima in the dry season may constrain the density and density of infected I. pacificus nymphs. In other years, biotic or stochastic factors may play a more important role in determining tick density.

Previous studies revealed that the Pacific Coast tick (Dermacentor occidentalis) is infected occasionally with the agents of Lyme disease (Borrelia burgdorferi) or human granulocytic anaplasmosis (Anaplasma phagocytophilum) and that it is an inefficient experimental vector of B. burgdorferi. The relationship of the pajahuello tick (Ornithodoros coriaceus) to each of these bacterial zoonotic agents has not been reported. The primary bridging vector of both bacterial zoonotic agents to humans is the western black-legged tick (Ixodes pacificus). Because of the spatial and temporal overlap of D. occidentalis and O. coriaceus populations with those of I. pacificus in natural foci of B. burgdorferi and A. phagocytophilum in northwestern California, we conducted field and laboratory studies to determine if the Pacific Coast tick or the pajahuello tick potentially may serve as secondary vectors of either bacterium. Our findings reconfirmed that wild-caught D. occidentalis ticks are infected infrequently with B. burgdorferi or A. phagocytophilum, but some adult ticks from dense woodlands or chaparral were found to contain 2 important veterinary pathogens for the first time (Anaplasma bovis, A. ovis). The high prevalence of A. bovis infection (4.3%, n=185 ticks) within chaparral-derived ticks suggests that D. occidentalis could be an efficient vector of this rickettsia. Experimental attempts to transmit borreliae or Anaplasma spp. that may have been present in >100 wild-caught D. occidentalis adults to naïve rabbits were unsuccessful. Anaplasma spp. were not detected in O. coriaceus, but one (4.3%) of 23 nymphs was infected with B. bissettii. This finding and an antecedent report of a B. burgdorferi-like spirochete from the same tick species demonstrate that O. coriaceus sometimes acquires and transstadially passes Lyme disease group spirochetes. I. pacificus nymphs inhabiting a woodland nidus of B. burgdorferi and A. phagocytophilum had a 5-fold higher prevalence of borreliae than adult ticks from the same generational cohort. In contrast to the results of preceding studies carried out at the same site, none of the nymphal or adult ticks was PCR-positive for A. phagocytophilum. This suggests that the distribution of this rickettsia is highly focal or variable from year-to-year within this particular woodland., (© 2010 Elsevier GmbH. All rights reserved.)

In the far-western United States, the nymphal stage of the western black-legged tick, Ixodes pacificus, has been implicated as the primary vector to humans of Borrelia burgdorferi sensu stricto (hereinafter referred to as B. burgdorferi), the causative agent of Lyme borreliosis in North America. In the present study, we sought to determine if infection prevalence with B. burgdorferi in I. pacificus nymphs and the density of infected nymphs differ between dense-woodland types within Mendocino County, California, and to develop and evaluate a spatially-explicit model for density of infected nymphs in dense woodlands within this high-incidence area for Lyme borreliosis. In total, 4.9% (264) of 5431 I. pacificus nymphs tested for the presence of B. burgdorferi were infected. Among the 78 sampling sites, infection prevalence ranged from 0 to 22% and density of infected nymphs from 0 to 2.04 per 100 m(2). Infection prevalence was highest in woodlands dominated by hardwoods (6.2%) and lowest for redwood (1.9%) and coastal pine (0%). Density of infected nymphs also was higher in hardwood-dominated woodlands than in conifer-dominated ones that included redwood or pine. Our spatial risk model, which yielded an overall accuracy of 85%, indicated that warmer areas with less variation between maximum and minimum monthly water vapor in the air were more likely to include woodlands with elevated acarological risk of exposure to infected nymphs. We found that 37% of dense woodlands in the county were predicted to pose an elevated risk of exposure to infected nymphs, and that 94% of the dense-woodland areas that were predicted to harbor elevated densities of infected nymphs were located on privately-owned land., (Published by Elsevier GmbH.)

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.

The nymph of the western black-legged tick (Ixodes pacificus) is an important bridging vector of the Lyme disease spirochete (Borrelia burgdorferi) to humans in the far-western United States. The previously unknown dispersal capabilities of this life stage were studied in relation to logs, tree trunks, and adjacent leaf-litter areas in a mixed hardwood forest using mark-release-recapture methods. In two spatially and temporally well-spaced trials involving logs, the estimated mean distances that nymphs dispersed ranged from approximately 0.04 to 0.20 m/day on logs vs 0.11 to 0.72 m/day in litter. Prior to recapture in either trial and within the confines of the sampling grids, the greatest estimated dispersal distances by individual nymphs released on logs, and in litter 0.5 m or 1.5 m from logs, were 2.4, 3.0, and 3.0 m, respectively. Nymphs released on logs or litter tended to remain within the same biotopes in which they were freed while host-seeking. In two simultaneous trials involving trunks spaced close-at-hand, nymphs released at the trunk/litter interface on all four aspects collectively dispersed a mean of 0.353 m/day on trunks vs 0.175 m/day in litter. In either trial, the greatest distances that recaptured nymphs climbed trunks, or dispersed in litter in an encircling 3-m grid, were 1.55 m and 2.97 m, respectively. Nymphs ascending trunks did not exhibit a preference for any one aspect, and the B. burgdorferi-infection prevalences in nymphs that climbed trunks (3.2-4.0%) did not differ significantly from those that moved horizontally into litter (10.5-17.6%). We conclude that I. pacificus nymphs use an ambush host-seeking strategy; that they disperse slowly in all biotopes studied; that they usually continue to host-seek in or on whatever substratum they access initially; and that B. burgdorferi-infected nymphs are as likely to move horizontally as vertically when offered a choice.

Analysis of borrelia isolates collected from ticks, birds, and rodents from the southeastern United States revealed the presence of well-established populations of Borrelia burgdorferi sensu stricto, Borrelia bissettii, Borrelia carolinensis, and Borrelia sp. nov. Multilocus sequence analysis of five genomic loci from seven samples representing Borrelia sp. nov. isolated from nymphal Ixodes minor collected in South Carolina showed their close relatedness to California strains known as genomospecies 1 and separation from any other known species of the B. burgdorferi sensu lato complex. One nucleotide difference in the size of the 5S-23S intergenic spacer region, one substitution in 16S rRNA gene signature nucleotides, and silent nucleotide substitutions in sequences of the gene encoding flagellin and the gene p66 clearly separate Borrelia sp. nov. isolates from South Carolina into two subgroups. The sequences of isolates of each subgroup share the same restriction fragment length polymorphism patterns of the 5S-23S intergenic spacer region and contain unique signature nucleotides in the 16S rRNA gene. We propose that seven Borrelia sp. nov. isolates from South Carolina and two California isolates designated as genomospecies 1 comprise a single species, which we name Borrelia americana sp. nov. The currently recognized geographic distribution of B. americana is South Carolina and California. All strains are associated with Ixodes pacificus or Ixodes minor and their rodent and bird hosts.

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.

Granulocytic anaplasmosis (GA) is a potentially fatal tick-borne rickettsial disease that occurs sporadically in the far western United States. We evaluated the prevalence of Anaplasma phagocytophilum in multiple species of lizards and snakes from enzootic sites in northern California, described the infestation prevalence of its tick vector Ixodes pacificus on reptiles, and conducted an experimental challenge of western fence lizards (Sceloporus occidentalis) and Pacific gopher snakes (Pituophis catenifer) with A. phagocytophilum delivered via needle inoculation or tick bite. Both serologically and polymerase-chain reaction (PCR)-positive lizards (seroprevalence = 10.8%, PCR prevalence = 10.2%) and snakes (seroprevalence = 5.8%, PCR prevalence = 11.7%) were detected among wild-caught animals. A DNA sequence of the A. phagocytophilum groESL gene from a PCR-positive snake was 100% homologous to that of the human-derived A. phagocytophilum. Experimental attempts to infect naïve animals were unsuccessful for snakes (n = 2), but 1 of 12 lizards became infected for 1 wk only by tick bite. Xenodiagnostic I. pacificus larvae that fed on a PCR-positive lizard did not acquire or transmit rickettsiae. Our findings suggest that lizards and snakes are exposed to A. phagocytophilum by infected ticks, but that they do not serve as primary reservoir hosts of this rickettsia.

This study was undertaken to determine which rodent species serve as primary reservoirs for the Lyme disease spirochete Borrelia burgdorferi in commonly occurring woodland types in inland areas of northwestern California, and to examine whether chaparral or grassland serve as source habitats for dispersal of B. burgdorferi- or B. bissettii-infected rodents into adjacent woodlands. The western gray squirrel (Sciurus griseus) was commonly infected with B. burgdorferi in oak woodlands, whereas examination of 30 dusky-footed woodrats (Neotoma fuscipes) and 280 Peromyscus spp. mice from 13 widely spaced Mendocino County woodlands during 2002 and 2003 yielded only one infected woodrat and one infected deer mouse (P. maniculatus). These data suggest that western gray squirrels account for the majority of production by rodents of fed Ixodes pacificus larvae infected with B. burgdorferi in the woodlands sampled. Infections with B. burgdorferi also were rare in woodrats (0/47, 0/3) and mice (3/66, 1/6) captured in chaparral and grassland, respectively, and therefore these habitats are unlikely sources for dispersal of this spirochete into adjacent woodlands. On the other hand, B. bissettii was commonly detected in both woodrats (22/47) and mice (15/66) in chaparral. We conclude that the data from this and previous studies in northwestern California are suggestive of a pattern where inland oak-woodland habitats harbor a B. burgdorferi transmission cycle driven primarily by I. pacificus and western gray squirrels, whereas chaparral habitats contain a B. bissettii transmission cycle perpetuated largely by I. spinipalpis, woodrats and Peromyscus mice. The dominant role of western gray squirrels as reservoirs of B. burgdorferi in certain woodlands offers intriguing opportunities for preventing Lyme disease by targeting these animals by means of either host-targeted acaricides or oral vaccination against B. burgdorferi.

A total of 2,121 small mammals in California were assessed for Anaplasma phagocytophilum from 2006 through 2008. Odds ratios were >1 for 4 sciurids species and dusky-footed woodrats. High seroprevalence was observed in northern sites. Ten tick species were identified. Heavily infested rodent species included meadow voles, woodrats, deer mice, and redwood chipmunks.

Anaplasma phagocytophilum is a zoonotic tick-borne rickettsial pathogen that causes granulocytic anaplasmosis (GA) in humans, horses, and dogs. In California, dusky-footed woodrats (Neotoma fuscipes) are a putative reservoir host, and Ixodes pacificus is a vector for transmission from rodents to humans, dogs, and horses. Cases are clustered in coastal and Sierra Nevada foothill regions, but not necessarily in proximity to infected woodrats. This study was designed to compare exposures and active infections of A. phagocytophilum in multiple rodents at a fine spatial scale in a hyperenzootic area and to evaluate the spatial clustering of infections. Of 331 rodents, the seroprevalence was 14.5%, with 60% in tree squirrels (Sciurus griseus and Tamiasciurus douglasii), 29% in woodrats, 14% in flying squirrels (Glaucomys sabrinus), and 5% in chipmunks (Tamias senex). No seropositive ground squirrels (Spermophilus beecheyi) were detected. The seroprevalence was significantly higher west of the Trinity River (23.1%) than east (11.8%) of the river. One Douglas squirrel and one western gray squirrel were polymerase chain reaction (PCR) positive. There was more spatial clustering among seropositive animals compared with all animals tested across the spatial scales evaluated, and this clustering was significantly greater than expected by chance alone. A significant cluster of 24 seropositive animals was found west of the Trinity River, with a population of 56 animals considered within the 50% population-at-risk, and a radius of 362.8 meters. The diversity of cricetine and sciurid rodents infected suggests that squirrels and chipmunks may be underappreciated contributors to A. phagocytophilum ecology in the western United States. The spatial clustering of exposed animals suggests interesting underlying spatially heterogeneous environmental variables that could facilitate the persistence of A. phagocytophilum in nature.

Ixodes pacificus, particularly the nymphal life stage, is the primary vector to humans of the Lyme disease agent Borrelia burgdorferi in California. During 2004, we collected I. pacificus nymphs from 78 woodland sites in ecologically diverse Mendocino County, which has a moderately high incidence of Lyme disease. Within this county, nymphal density was elevated in forested areas with a growing degree day range of 2,600-3,000 (10 degrees C base). Using a geographic information systems approach, we identified all areas in California sharing these environmental characteristics and thus projected to pose high acarologic risk of exposure to host-seeking nymphal ticks. Such areas were most commonly detected in the northwestern part of the state and along the Sierra Nevada foothills in the northeast, but the analysis also identified isolated areas with high acarologic risk in southern California. This mirrors the spatial distribution of endemic Lyme disease during 1993-2005; most cases occurred in counties to the northwest (58%) or northeast (26%), whereas fewer cases were reported from southern California (16%). Southern zip-codes from which Lyme disease cases had been reported were commonly located in close proximity to areas with high projected acarologic risk. Overall, Lyme disease incidence in zip code areas containing habitat with high projected acarologic risk was 10-fold higher than in zip code areas lacking such habitat and 27 times higher than for zip code areas without this habitat type within 50 km. A comparison of spatial Lyme disease incidence patterns based on county versus zip code units showed that calculating and displaying disease incidence at the zip code scale is a useful method to detect small, isolated areas with elevated disease risk that otherwise may go undetected.

Lyme borreliosis is associated with several genospecies of Borrelia burgdorferi sensu lato (s.l.) (Spirochaetales), but human disease has been associated only with Borrelia burgdorferi sensu stricto (s.s.) Johnson, Schmid, Hyde, Steigerwalt & Brenner in the western United States. Restriction fragment length polymorphism (RFLP) analysis of rrf-rrl amplicons from 124 tick and mammalian isolates from various habitats yielded 13 RFLP patterns. Of these patterns, six were patterns previously associated either with Borrelia bissettii Postic, Marti Ras, Lane, Hendson & Baranton or Borrelia burgdorferi s.s., and the remaining seven patterns belonged to diverse and previously uncharacterized Borrelia spp. Uncharacterized Borrelia spp. were cultured most frequently from Ixodes spinipalpis Hadwen & Nuttall and California kangaroo rats, Dipodomys californicus Merriam, inhabiting grasslands, and B. bissettii from I. spinipalpis and dusky-footed woodrats, Neotoma fuscipes Baird, associated with oak woodlands or chaparral. B. burgdorferi s.s. typically was isolated from host-seeking Ixodes pacificus Cooley & Kohls collected in dense oak woodlands, woodland-grass, or redwood forests. Although some isolates of B. burgdorferi s.s. were cultured from woodrats, there was no clear association of this human pathogen with any vertebrate host. These findings, along with recent evidence indicating that the western gray squirrel, Sciurus griseus Ord, may be an important reservoir of B. burgdorferi s.s. in Californian oak woodlands, suggest that our earlier hypothesis implicating an enzootic cycle involving woodrats and I. spinipalpis is insufficient to account for observed patterns of infection in nature.

Ixodes pacificus nymphs are the primary vectors to humans of Borrelia burgdorferi, the etiologic agent of Lyme disease, in California. We used a supervised classification model, based on remote sensing (RS) data from multi-seasonal Landsat TM 5 images, to identify the key habitat in Mendocino County where humans are exposed to I. pacificus nymphs (woodlands carpeted with leaf litter). The model, based on the normalized difference vegetation index (NDVI), brightness, and wetness, separated the nymphal risk habitat (52.6% of the county) from other habitat types with > 93% user and producer accuracies. Next, we determined the density of questing nymphs in 62 woodland-leaf areas located throughout Mendocino County and created forward-stepwise regression models explaining the variation in nymphal density based on traits attainable by a lay-person in the field (e.g., tree species present, deer signs; r(2) = 0.43, P < 0.0001), or geographic information systems (GIS)/RS-based environmental data (r(2) = 0.50, P < 0.0001). The GIS/RS model, using July NDVI, November greenness, a coastal influence category, May solar insolation, November hours of sunlight, and dominant hydrologic grouping as input variables, was 22% more accurate in predicting nymphal density at 16 validation sites (r(2) = 0.72) than the field-derived data model (r(2) = 0.50). The habitat classification and GIS/RS models were combined to create a continuous nymphal density surface for the entirety of Mendocino County. This risk surface showed that 11.9% of the county was classified as habitat posing at least moderate risk of human exposure to nymphs (> 6.4 nymphs per 100 m(2)). Furthermore, high-risk areas (> 10.5 nymphs per 100 m(2); 1.7% of the county) tended to cluster in the central interior and most heavily populated region of Mendocino County, but were rare in the proximity of coastal population centers.

Presence of Bartonella DNA was explored in 168 questing adult Ixodes pacificus ticks from Santa Cruz County, California. Bartonella henselae type I DNA was amplified from 11 ticks (6.55%); previously, two (1.19%) were found to be infected with Borrelia burgdorferi and five (2.98%) with Anaplasma phagocytophilum. Detection of B. henselae was not dependent on co-infection. The present study offers additional evidence that Ixodes spp. ticks may act as hosts and possibly vectors for B. henselae.

Birds from 45 species were sampled during three spring seasons from an isolated canyon on the Sutter Buttes in California for the presence of subadult stages of Ixodes pacificus Cooley & Kohls, and for infection with Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner. These birds were found to have an infestation prevalence of 45%, a density of 1.7 ticks per bird, and an intensity of 3.8 ticks per infested bird. There was a significant difference in the I. pacificus infestations between canopy and ground-dwelling birds. Birds also demonstrated an overall infection with B. burgdorferi of 6.4% with significant difference between bird species. Amplification and subsequent sequencing of the 23s-5s rRNA intergenic spacer region of the Borrelia genome from one bird, a hermit thrush, Catharus guttatus (Nuttall), showed that the infection in this bird was caused by B. burgdorferi sensu stricto; the first such finding in a bird from the far west. Our results suggest that birds play a role in the distribution and maintenance of I. pacificus, and possibly of B. burgdoferi, at the Sutter Buttes, CA.

We investigated whether host-seeking nymphs and adults of the western blacklegged tick, Ixodes pacificus Cooley & Kohls, the primary vector of Lyme disease spirochetes in far-western North America, are infected naturally with relapsing-fever group spirochetes in Mendocino County, California. Relapsing-fever group borreliae were detected in four (1.7%) of 234 nymphal and two (0.7%) of 282 adult host-seeking I. pacificus ticks by polymerase chain reaction and sequence analysis of the 16S rRNA and flagellin genes, respectively, exhibiting 99 and 98.5% sequence homology to Borrelia miyamotoi Fukunaga. Phylogenetic analysis based on these two genes revealed that the borreliae detected in these ticks belong to the relapsing-fever group and that these are closely related to, if not identical with, B. miyamotoi.

Two forms of tick-borne leukocytotropic rickettsioses have been recognized in California since the mid-1990s: human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis and human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum. Between 1997 and 1999, two cases of HME and four cases of HGA were diagnosed in residents of southern Humboldt County, California. Environmental followup at case-patients' residences revealed dense populations of Ixodes pacificus ticks, particularly in grassy roadside areas. PCR evidence of A. phagocytophilum was detected in approximately 2.0% of I. pacificus; E. chaffeensis was not detected in any of 625 ticks tested. Serologic antibody to A. phagocytophilum was detected in two of 54 participants in a community epidemiologic study; one of these also had antibody to E. chaffeensis. Over 85% of study participants reported finding a tick on themselves in the preceding 12 mo. Residents of southern Humboldt County are at significant risk of tick bites and should take appropriate prevention measures to avoid infection with rickettsia and other tick-transmitted pathogens.

Ixodes pacificus is the main tick vector for transmission of Anaplasma phagocytophilum and Borrelia burgdorferi to large vertebrates in California. The present study was undertaken in I. pacificus-infested counties in California to examine spatial and temporal relationships among A. phagocytophilum and B. burgdorferi-exposed coyotes with vegetation type and climate. The overall A. phagocytophilum and B. burgdorferi seroprevalences were 39.5% (N=215) and 18.9% (N=148), respectively, with no association with sex. PCR for A. phagocytophilum and B. burgdorferi was negative in all blood and kidney samples. Increased seroprevalence was a positive function of rainfall. Ehrlichial seropositivity was increased in blue-oak foothill pine, montane hardwood, and redwood vegetation regions, and decreased in coastal sagebrush and cropland. Increased exposure to B. burgdorferi occurred in blue oak woodland.

In California, Ixodes pacificus Cooley & Kohls nymphs have been implicated as the primary bridging vectors to humans of the spirochetal bacterium causing Lyme disease (Borrelia burgdorferi). Because the nymphs typically do not ascend emergent vegetation, risk of human exposure is minimal in grasslands, chaparral, and woodland-grass. Instead, woodlands with a ground cover dominated by leaf litter (hereinafter referred to as woodland-leaf) have emerged as a primary risk habitat for exposure to B. burgdorferi-infected nymphs. As a means of differentiating woodland-leaf habitats from others with minimal risk (e.g., chaparral, grassland, and woodland-grass), we constructed a maximum likelihood model of these habitat types within a 7,711-ha area in southeastern Mendocino County based on the normalized difference vegetation index derived from Landsat 5 Thematic Mapper imagery (based on a 30 by 30-m pixel size) over four seasons. The overall accuracy of the model to discriminate woodland-leaf, woodland-grass, open grassland, and chaparral was 83.85% (Kappa coefficient of 0.78). Validation of the accuracy of the model to classify woodland-leaf yielded high values both for producer accuracy (93.33% of validated woodland-leaf pixels correctly classified by the model) and user accuracy (96.55% of model-classified validation pixels correctly categorized as woodland-leaf). Woodland-leaf habitats were found to be highly aggregated within the examined area. In conclusion, our model successfully used remotely sensed data as a predictor of habitats where humans are at risk for Lyme disease in the far-western United States.

The primary aims of this study were to quantify the density of Ixodes pacificus Cooley and Kohls nymphs and adults of the same generational cohort collected within a single year in six oak or madrone leaf litter habitats and to compare the prevalence of infection with Borrelia burgdorferi sensu lato (s.l.) in adults originating from nymphal cohorts with a low (<1%) versus high (>10%) infection prevalence. Because adult densities were very low both in and adjacent to several sites, direct comparisons of infection prevalence between nymphs and adults were possible only for two sites. Mean density in these sites decreased from 11.95/100 m2 for nymphs to 0/100 m2 for adults in leaf litter, and infection prevalence with B. burgdorferi s.l. was four-fold higher in nymphs (7.4%) versus adults (1.6%) of the same generational cohort collected in ecotones bordering the leaf litter areas. Assuming a density of adults in leaf litter of 0.04/100 m2 (mean for all six examined sites) and an infection prevalence similar to that found in adults collected from litter ecotones, the risk of encountering infected ticks in leaf litter decreased >1,000-fold from the nymphal to adult stage. Regardless of site-specific infection prevalence in the nymphal stage (n = 2 sites; 0.7 versus 14%), the infection prevalence for the adults of the same generational cohort was similarly low (1.5-1.6%). Peak densities of adult I. pacificus were 0-0.1/100 m2 in leaf litter, 0-6.5/100 m2 in ecotonal grasslands, and 2.0-39.0/100 m2 in ecotonal chaparral. Despite more intensive sampling efforts in leaf litter, the vast majority of the 282 adults collected came from grass or chaparral ecotones (98.9%, n = 279) rather than leaf litter (1.1%, n = 3). The study yielded eight B. burgdorferi s.l.-infected adults; four of these carried B. burgdorferi sensu stricto Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, and the remaining four were infected with currently undescribed B. burgdorferi s.l. spirochetes. This is the first study comparing both density of I. pacificus nymphs and adults and prevalence of infection with B. burdorferi s.l in these ticks within the same generational cohort and sampling area.

We assessed the effects of sympatric (occupying the same or overlapping geographic areas) and allopatric (occurring in separate geographic areas) isolates of Anaplasma phagocytophilum on the survival of Ixodes scapularis Say larvae that were derived from ticks collected in Bridgeport, CT. Seven isolates of A. phagocytophilum, originating from different geographic regions of the United States, were tested: four isolates from the northeast (Bridgeport, Dawson, Gaillard, and NY-8), two from the Midwest (Webster and Sp-Is), and one from California (MRK). BALB/c mice were infected with each of the seven isolates via exposure to infected I. scapularis nymphs, whereas uninfected nymphs fed upon control mice. Both infected and control mice were infested with uninfected larvae at 1, 2, 3, 4, 6, and 9 wk after nymphal infestation. The molting success in cohorts of infected and uninfected ticks was calculated as the percentage of larvae successfully molting into nymphal stage, and the prevalence of infection in molted nymphs was determined by polymerase chain reaction. In ticks that became infected with the Bridgeport or Sp-Is isolates, the molting success decreased with an increase in the prevalence of infection. Ticks that fed upon mice infected with six allopatric isolates (Dawson, Gaillard, NY-8, Sp-Is, Webster, and MRK) showed significantly lower levels of survival than those fed upon control mice, regardless of the prevalence of infection, whereas in ticks fed upon mice infected with a sympatric isolate (Bridgeport), the overall molting success was similar to the control. Thus, some but not all of the A. phagocytophilum isolates have adverse effects on ticks. Ticks exposed to harmful isolates may experience higher levels of bacterial metabolism, and/or reduced quality of their blood meal, thereby reducing their survival. Noted differences between isolates may be due to the origin of a particular isolate and/or the degree of coadaptation between the pathogen and its vector on the population level.

Over a 5-year period (1997-2001) the population densities of Ixodes pacificus Cooley & Kohls (Acari: Ixodidae) nymphs infected with spirochaetes of Borrelia burgdorferi sensu lato (s.l.) were evaluated in areas of 2000 ha at two localities (CHR, nine sites; HREC, seven sites) 25 km apart in Mendocino County, north-western California. The 5-year median density of infected nymphs was significantly higher at CHR than at HREC (0.51 vs. 0.09 per 100 m(2) and site-specific yearly densities exceeding one infected nymph per 100 m2 were 10-fold more likely to occur at CHR than at HREC. The importance of long-term data in acarologic risk assessment was demonstrated by significantly higher median yearly densities of infected nymphs at CHR from 1997 to 1999, whereas both areas had similar densities during 2000-2001. Overall, the causative agent of Lyme borreliosis in North America, B. burgdorferi Johnson et al. sensu stricto (s.s.) accounted for 76% of 46 genetically characterized B. burgdorferi s.l. infections from I. pacificus nymphs. Tremendous variability in acarologic risk was recorded within both areas: yearly densities of infected nymphs varied 11-97-fold between sites at CHR and 8-30-fold at HREC. Part of this variation could be explained by environmental traits, most notably deer usage. However, correlations between environmental factors and density of infected nymphs (for CHR and HREC combined) did not necessarily apply when these areas were considered separately. Thus, a Lyme borreliosis ecology model developed in one of these areas needs testing in the other area.

Epidemiological evidence suggests that the nymph of the western black-legged tick, Ixodes pacificus Cooley and Kohls, is the primary vector of the Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, and Brenner, to humans in northwestern California. In spring 2002, six different human behaviors were evaluated as potential risk factors for acquiring I. pacificus nymphs in a deciduous woodland in Mendocino County, California. Also, the prevalence of B. burgdorferi sensu lato (s.l.) and the causative agents of human granulocytic (Anaplasma phagocytophilum [Foggie] Dumler, Barbet, Bekker, Dasch, Palmer, Ray, Rikihisa, and Rurangirwa) and monocytic ehrlichioses (Ehrlichia chaffeensis Anderson, Dawson, Jones, and Wilson) was determined in nymphs that had been collected from subjects or by dragging leaf litter. Activities involving a considerable degree of contact with wood resulted in greater acquisition of nymphs than those involving exposure solely to leaf litter. Time-adjusted tick-acquisition rates demonstrated that sitting on logs was the riskiest behavior, followed, in descending rank, by gathering wood, sitting against trees, walking, stirring and sitting on leaf litter, and just sitting on leaf litter. The number of ticks acquired appeared to be unrelated to the type of footwear worn (hiking boots, hiking sandals, or running shoes). Overall, 3.4% (n = 234) of the nymphs were infected with A. phagocytophilum, 3.9% (n = 181) with B. burgdorferi s.l., and none (n = 234) with E. chaffeensis. Of 13 nymphs infected with either A. phagocytophilum or B. burgdorferi s.l., 2 (15.4%) were coinfected with both bacteria, as were 1.3% of 158 nymphs obtained from leaf litter, the first report of coinfection in this life stage of I. pacificus. Four unattached, infected nymphs were removed from subjects, including two acquired while sitting on logs that contained A. phagocytophilum, another with the same bacterium obtained while walking, and one acquired while gathering wood that was infected with B. burgdorferi s.l. Despite the use of extreme personal preventive measures by both subjects, two attached, uninfected nymphs were removed from one of them > or = 1-2 d postexposure. The public health implications of these findings are discussed.

During the spring and early summer of 2002, we examined the relative importance of Borrelia-refractory lizards (Sceloporus occidentalis, Elgaria spp.) versus potential Borrelia burgdorferi sensu lato (s.l.)-reservoirs (rodents) as hosts for Ixodes pacificus immatures in 14 woodland areas (six oak, five mixed oak/Douglas fir, and three redwood/tanoak areas) distributed throughout Mendocino County, California. Lizards were estimated to serve as hosts for 93-98% of all larvae and > or =99.6% of all nymphs infesting lizards or rodents in oak woodlands and oak/Douglas fir sites in the southern part of the county. In redwood/tanoak woodlands and oak/Douglas fir sites in northern Mendocino County, the contribution of rodents to larval feedings reached 36-69% but lizards still accounted for 94-100% of nymphal bloodmeals. From late April to mid-June, I. pacificus larvae were recovered from 95 to 96% of lizards and dusky-footed woodrats (Neotoma fuscipes) and from 59% of Peromyscus spp. mice. In contrast, 99% of lizards but few woodrats (15%) and none of the mice were infested by nymphs. Comparisons of tick loads for 19 lizard-Peromyscus spp. mouse pairings, where the lizard and mouse were captured within 10m of each other, revealed that the lizards harbored 36 times more larvae and >190 times more nymphs than the mice. In oak woodlands, loads of I. pacificus larvae decreased from late April/early May to late June for S. occidentalis lizards but increased for Peromyscus spp. mice. We conclude that the relative utilization of Borrelia-refractory lizards, as compared to rodents, by I. pacificus larvae was far higher in dry oak woodlands than in moister habitats such as redwood/tanoak and oak/Douglas fir woodlands in northern Mendocino County. Non-lizard-infesting potential enzootic vectors of B. burgdorferi s.l. (I. angustus and I. spinipalpis) were recorded from rodents in three of six oak woodland areas, two of five oak/Douglas fir woodland areas, and two of three redwood/tanoak woodland areas.

Dusky-footed wood rats (Neotoma fuscipes Baird) and two species of Peromyscus mice (P. maniculatus Wagner and P. truei Shufeldt) were collected over a 16-month period from three sites in Sonoma County, California. Blood was collected from 93 wood rats and 177 mice and serum or plasma was tested for seroreactivity with Ehrlichia phagocytophila sensu lato (also known as the human granulocytic ehrlichiosis agent). Thirty-five (37.6%) wood rats and 15 (8.5%) mice were seropositive. Positive Neotoma serology by site ranged from 9.4% to 62.1%. Polymerase chain reaction (PCR) testing for the Ehrlichia groESL heat shock operon was performed on all the seropositive and selected seronegative wood rats; 24 (68.6%) seropositive animals were PCR positive. Two seroconversions and no seroreversions were detected among 18 of the seropositive wood rats that were recaptured and tested multiple times (range = 2-6). Fourteen (77.8%) of the 18 were also PCR positive with six of these positive at every testing point (range = 2-6). One wood rat remained serologically and PCR positive in six specimens collected over a 14-month period. One male of 84 questing adult Ixodes pacificus Cooley & Kohls collected was PCR-positive for E. phagocytophila. Borrelia burgdorferi, the agent of Lyme disease, was cultured from ear punch biopsies from six of seven E. phagocytophila seropositive and one of four seronegative wood rats.

An acarologic study was conducted in a semirural community in northern California to determine the relative abundance of, and the prevalence of infection with, three emerging bacterial pathogens in the western black-legged tick (Ixodes pacificus). These included the agents causing Lyme disease (Borrelia burgdorferi), human granulocytic ehrlichiosis [Ehrlichia phagocytophila (formerly Ehrlichia equi)], and human monocytic ehrlichiosis (Ehrlichia chaffeensis). The study area in Sonoma County consisted of two properties each with four residents and an uninhabited adjacent comparison area. Six of the eight residents had been either physician-diagnosed or serodiagnosed previously with Lyme disease, and, of these, one also had been serodiagnosed with human monocytic ehrlichiosis. Direct immunofluorescent/culture assays and bacterial species-specific polymerase chain reaction assays were used to test whole ticks individually for presence of B. burgdorferi and Ehrlichia spp., respectively. Overall, 6.5% of the nymphal (n = 589) and 1.6% of the adult ticks (n = 318) from the same generational cohort were found to contain B. burgdorferi. In contrast, none of 465 nymphs and 9.9% of 202 adults were infected with E. phagocytophila. Excised tissues from another 95 adult ticks yielded a comparable E. phagocytophila infection prevalence of 13.7%. E. chaffeensis was not detected in either nymphal or adult ticks. Using a combination of culture and polymerase chain reaction assays, coinfection of I. pacificus adults with B. burgdorferi and E. phagocytophila was demonstrated for the first time. The marked disparity in the infection prevalence of these pathogens in nymphal and adult ticks suggests that their maintenance cycles are inherently different.

Ticks are the vectors of many zoonotic diseases in the United States, including Lyme disease, human monocytic and granulocytic ehrlichioses, and Rocky Mountain spotted fever. Most known Bartonella species are arthropod borne. Therefore, it is important to determine if some Bartonella species, which are emerging pathogens, could be carried or transmitted by ticks. In this study, adult Ixodes pacificus ticks were collected by flagging vegetation in three sites in Santa Clara County, Calif. PCR-restriction fragment length polymorphism and partial sequencing of 273 bp of the gltA gene were applied for Bartonella identification. Twenty-nine (19.2%) of 151 individually tested ticks were PCR positive for Bartonella. Male ticks were more likely to be infected with Bartonella than female ticks (26 versus 12%, P = 0.05). None of the nine ticks collected at Baird Ranch was PCR positive for Bartonella. However, 7 (50%) of 14 ticks from Red Fern Ranch and 22 (17%) of 128 ticks from the Windy Hill Open Space Reserve were infected with Bartonella. In these infected ticks, molecular analysis showed a variety of Bartonella strains, which were closely related to a cattle Bartonella strain and to several known human-pathogenic Bartonella species and subspecies: Bartonella henselae, B. quintana, B. washoensis, and B. vinsonii subsp. berkhoffii. These findings indicate that I. pacificus ticks may play an important role in Bartonella transmission among animals and humans.