Ticks and Tick-borne Diseases 5 (2014) 951–961

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis

Original article

Remarkable diversity of tick or mammalian-associated Borreliae in the metropolitan San Francisco Bay Area, California Natalia Fedorova a,b,∗ , Joyce E. Kleinjan b , David James b , Lucia T. Hui b , Hans Peeters c , Robert S. Lane a a

Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA Alameda County Vector Control Services District, Alameda, CA 94502, USA c Sunol, CA 94586, USA b

a r t i c l e

i n f o

Article history: Received 12 February 2014 Received in revised form 20 July 2014 Accepted 21 July 2014 Available online 14 August 2014 Keywords: Borrelia burgdorferi sensu lato osp C Borrelia miyamotoi Ixodes Rodents

a b s t r a c t The diversity of Lyme disease (LD) and relapsing fever (RF)-group spirochetes in the metropolitan San Francisco Bay area in northern California is poorly understood. We tested Ixodes pacificus, I. spinipalpis, and small mammals for presence of borreliae in Alameda County in the eastern portion of San Francisco Bay between 2009 and 2012. Analyses of 218 Borrelia burgdorferi sensu lato (Bb sl) culture or DNA isolates recovered from host-seeking I. pacificus ticks revealed that the human pathogen Bb sensu stricto (hereinafter, B. burgdorferi) had the broadest habitat distribution followed by B. bissettii. Three other North American Bb sl spirochetes, B. americana, B. californiensis and B. genomospecies 2, also were detected at lower prevalence. OspC genotyping of the resultant 167 B. burgdorferi isolates revealed six ospC alleles (A, D, E3, F, H and K) in I. pacificus. A novel spirochete belonging to the Eurasian Bb sl complex, designated CA690, was found in a questing I. spinipalpis nymph. Borrelia miyamotoi, a relapsing-fever (RF) group spirochete recently implicated as a human pathogen, was detected in 24 I. pacificus. Three rodent species were infected with Bb sl: the fox squirrel (Sciurus niger) with B. burgdorferi, and the dusky-footed wood rat (Neotoma fuscipes) and roof rat (Rattus rattus) with B. bissettii. Another spirochete that clustered phylogenetically with the Spanish R57 Borrelia sp. in a clade distinct from both the LD and RF groups infected some of the roof rats. Together, eight borrelial genospecies were detected in ticks or small mammals from a single Californian county, two of which were related phylogenetically to European spirochetes. © 2014 Elsevier GmbH. All rights reserved.

Introduction The western blacklegged tick, Ixodes pacificus, is widely distributed in the far-western United States, particularly in California. It is the primary vector of the human pathogen Borrelia burgdorferi sensu stricto (hereinafter, B. burgdorferi) in this region (Burgdorfer et al., 1985; Clover and Lane, 1995). The number of confirmed human cases of Lyme disease (LD) reported annually in California is low and most cases are contracted in the less populous northwestern counties where the incidence locally can be high (Lane et al., 1992). In that region, the infection prevalence of I. pacificus nymphs with B. burgdorferi sometimes approaches that of Ixodes scapularis nymphs in highly endemic areas of the northeastern United States (Lane et al., 1992; Eisen et al., 2003a, 2010).

∗ Corresponding author at: Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA. Tel.: +1 5107178387. E-mail address: [email protected] (N. Fedorova). http://dx.doi.org/10.1016/j.ttbdis.2014.07.015 1877-959X/© 2014 Elsevier GmbH. All rights reserved.

A few studies in the San Francisco Bay Area have yielded information about the spatial and temporal variability of borrelial infected-I. pacificus ticks (Kramer and Beesley, 1993; Li et al., 2000; Padgett and Bonilla, 2011; Swei et al., 2011; Salkeld et al., 2014), but none of them characterized B. burgdorferi populations intraspecifically. The ospC gene, which encodes a highly polymorphic outer surface lipoprotein crucial for initial infection of the mammalian host (Tilly et al., 2006), has been used commonly for B. burgdorferi strain typing. The relationship between nymphal abundance, prevalence of pathogens and incidence of human cases of LD varies geographically partly because of differences in B. burgdorferi genotypic frequency (Pepin et al., 2012). B. burgdorferi exhibits substantial diversity. More than 25 major ospC genotypes have been identified so far, 17 of which were recorded in I. scapularis nymphs from the northeastern United States (Travinsky et al., 2010). Nine genotypes have been associated with disseminated infections in humans with genotypes A, B, I and K considered to be highly invasive (Wormser et al., 2008; Dykhuizen et al., 2008). In northwestern California, 12 ospC alleles were detected in I. pacificus nymphs from dense woodlands (Girard et al., 2009).


N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961

Genotypes H3 and E3 were the most abundant and A, D, F, G and H likewise occurred at high frequencies. Only ospC genotype A has been found in I. pacificus adults from southern California (Lane et al., 2013). The population structure of B. burgdorferi in I. pacificus from other parts of California, a large and ecologically diverse state, is unknown. The relapsing-fever (RF) group spirochete, Borrelia miyamotoi, has been detected in Ixodes spp. ticks worldwide, and recently was incriminated as a human pathogen for the first time in Russia, Europe and the United States (Platonov et al., 2011; Houvius et al., 2013; Krause et al., 2013; Gugliotta et al., 2013). In the northeastern United States, B. burgdorferi and B. miyamotoi infect white-footed mice (Peromyscus leucopus) and other vertebrates and are transmitted among them by I. scapularis (Barbour et al., 2009). In California, B. miyamotoi has been detected in low percentages of I. pacificus ticks (Mun et al., 2006; Padgett and Bonilla, 2011; Salkeld et al., 2014), but its primary reservoir host(s) in the Far West has (have) not been identified. Borrelia spp. diversity in wildlife has been studied with regard to the ongoing spread of Lyme borreliosis in North America (Scott et al., 2010a; Hamer et al., 2010; Fleer et al., 2011). The western gray squirrel (Sciurus griseus), the dusky-footed wood rat (Neotoma fuscipes), and the California kangaroo rat (Dipodomys californicus) are reservoir hosts of Bb sl in northwestern California (Lane and Brown, 1991; Brown and Lane, 1992; Lane et al., 1999; Brown et al., 2006). The western gray squirrel is the primary reservoir host of B. burgdorferi in certain dense woodlands (Lane et al., 2005; Salkeld et al., 2008). Native animals in peri-urban and urban areas co-exist with introduced species including the fox squirrel (Sciurus niger) and the roof rat (Rattus rattus). Borreliae were detected in those species previously in California (Peavey et al., 1997; Salkeld et al., 2008), but their role in spreading pathogens is not fully understood. Here, we sought to determine the diversity of Borrelia spp. in host-seeking I. pacificus and small mammals in several habitat types in Alameda County, San Francisco Bay area, California, and to ascertain the frequency distribution of ospC genotypes among isolates of B. burgdorferi.

Materials and methods The study areas Alameda County comprises 2100 km2 along the eastern margin of San Francisco Bay. This climatically and ecologically diverse county was populated by approximately 1.56 million residents in 2012, making it the seventh most populous county out of 58 in the state. The low mountain ranges and hills (elevations range up to 500 m), part of the Pacific Coast Ranges, separate the coastal area characterized by a maritime Mediterranean climate from inland areas with a continental-type Mediterranean climate. Overall, ambient temperature averages 15.1 ◦ C, and the mean annual rainfall is 58.9 cm (http://www.usa.com/alameda-county-caweather.htm). The habitats form a heterogeneous mixture of chaparral, grassland and woodlands, as well as agricultural lands and residential developments. Vegetative details of the surveillance sites were classified according to Sawyer and Keeler-Wolf (1995) and included Annual grasses and forbs, Blue oak (Quercus douglasii), California bay (Umbellularia californica), Coast live oak (Quercus agrifolia), California sycamore (Platanus racemosa), Coast redwood (Sequoia sempervirens) and Valley oak (Quercus lobata) alliances. Collection sites sampled in the cool, moist maritime region incorporated the Coast redwood alliance and evergreen California bay and Coast live oak woodland alliances. The warmer and drier inland sites were distinguished by deciduous oaks,

and included the Blue oak, Valley oak and California sycamore woodland alliances. The annual grasses and forbs alliance was distributed throughout the county. Here, we combine the habitats that were characterized by presence of evergreen oak or deciduous oak species. Geographic coordinates of the sites were recorded using a handheld Global Positioning System receiver Trimble Geo XT (Trimble corp., Sunnyvale, CA). Tick sampling Seventy-one sites were sampled for presence of host-seeking ticks as part of the Alameda County Vector Control Services District surveillance program between 2009 and 2012. The principal criterion used for site selection was potential public exposure to vector ticks, and thus included parks and native habitat adjoining urban developments. Host-seeking Ixodes spp. adults and nymphs were collected at 63 and 43 sites, respectively, in recreational and peri-urban areas of Castro Valley, Fremont, Hayward, Livermore, Oakland, Pleasanton, and Sunol. The numbers of sampling occasions per site varied, and sites found to have Borrelia-infected ticks were screened monthly thereafter during the peak tick activity period for at least two years. Nymphs were collected from leaf-litter areas bordering trails or creek banks by dragging a 1.25 × 1.00-m2 piece of corduroy from March to November. Questing adults were sampled by flagging a 1.0 × 1.0 m2 flannel-cloth over low vegetation from November to May. The cloth was examined frequently for presence of ticks. Sampling was conducted between 10:00 and 16:00 h for about 1-h per sampling occasion. Adult and nymphal ticks were kept alive at 10–15 ◦ C and 95% relative humidity prior to species identification and testing. Small mammal sampling Small mammals were captured at a residential property in Sunol in an area of high tick abundance, at five sites in Oakland and at one site in Berkeley from 2008 to 2012 as part of ongoing studies at the Museum of Vertebrate Zoology, University of California, Berkeley, CA (Conroy et al., 2012) or the Department of Environmental Science, Policy and Management at the University of California, Berkeley, CA. Most mammals were inspected for ticks, and 2-millimeter ear-punch biopsies (EPB) usually were excised from both pinnae, and then placed in culture medium for isolation attempts or stored in 95% ethanol for DNA extraction. Capture and handling of wildlife was carried out in compliance with the California Department of Fish and Wildlife regulations. Culturing spirochetes Live ticks were vortexed for 30 s in 3% hydrogen peroxide, rinsed in 70% ethanol and washed twice in sterile phosphatebuffered saline solution. The midgut diverticula were macerated and cultured in 1 ml of BSK-H medium supplemented with 12% heat-treated rabbit sera and 2% fetal-bovine sera. EPB from mammalian ears were cleaned with 10% povidone iodine, 70% ethanol and 3% hydrogen peroxide, and then placed in BSK-H medium for culturing spirochetes. Cultures were incubated at 34 ◦ C and examined for spirochetes using dark-field microscopy at magnifications of 100× and 400× two to four days later. In 2009, all ticks were examined by placing their tissues in BSK-H medium. Similarly, most ticks collected between 2010 and 2012 were cultured, though about one-sixth of them were tested by polymerase chain reaction (PCR). The direct DNA extraction method was used as a supporting method: 1) to process sets of ticks from selected sites where unexpected high infection prevalence was observed by the culturing technique; 2) as a backup method if culturing was not feasible.

N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961

DNA extraction and PCR Genomic DNA from cultured and uncultivable isolates, ticks and mammalian EPB was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol (Girard et al., 2009). All DNA samples were screened initially with a nested PCR targeting the non-coding 5S-23S rRNA intergenic spacer (IGS) of Bb sl (Lane et al., 2004). A nested ospC PCR (Bunikis et al., 2004) was used to amplify a partial ospC gene of B. burgdorferi. Additionally, DNA extracts were tested with the Borrelia genus-specific nested 16S-23S rRNA IGS assay (Bunikis et al., 2004). Cycling conditions for the 5S-23S IGS, 16S-23S IGS, and ospC PCR protocols were described in detail previously (Girard et al., 2011). To characterize the novel Bb sl isolate CA690, eight housekeeping genes (clpA, ATP-dependent Clp protease subunit A gene; clpX, ATP-dependent Clp protease subunit X gene; nifS, aminotransferase gene; pepX, dipeptidyl aminopeptidase gene; pyrG, CTP synthase; recG, DNA recombinase gene; rplB, 50S ribosomal protein L2 gene; uvrA, excinuclease ABC subunit A) were amplified following the multilocus sequence typing (MLST) scheme by nested or semi-nested PCR as described previously (Margos et al., 2008) with minor modifications. Two additional PCR protocols were employed for characterizing the unusual isolates obtained from rodents. A 1360-bp region of the 16S rRNA (Guner et al., 2003) and a 243-bp fragment of the groEL gene (Gil et al., 2005) were amplified. Amplitaq DNA polymerase (Life Technologies, Grand Island, NY) was used for all amplifications. Multiple negative (deionized water) and positive controls (B. burgdorferi CA4) were included with each PCR run. Care was taken to prevent cross-contamination including implementing recommendations by Kwok and Higuchi (1989). All PCR products were electrophoresed with 1.5% agarose gels, stained with ethidium bromide, and visualized by UV transillumination. Products from positive samples were purified using the QIAquick PCR purification Kit (Qiagen, Valencia, CA). Sequencing was performed at the University of California, Berkeley, DNA Sequencing Facility using forward and reverse primers. Sequence analysis Sequences were assembled and manually edited using Sequencher 4.5 (Gene Codes Corp., Ann Arbor, MI). Contigs having more than one nucleotide at the same position in forward and reverse directions were considered to represent mixed infections. Each sequence was compared to the fragments of the same loci from various borrelial genospecies available in the GenBank database utilizing the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/). The criterion for inclusion within a given Borrelia species or ospC type was a similarity of ≥99%. Additionally, sequences of the individual housekeeping genes were compared to the MLST database (http://borrelia.mlst.net). For phylogenetic analysis, sequences were aligned with the sequences selected from the GenBank or MLST database using ClustalX v1.83.1 and edited using Mesquite (http://mesquiteproject.org). For isolate CA690, the sequences of eight housekeeping gene fragments were concatenated producing a 4788-bp sequence. The analysis was carried out according to the best-fit nucleotide substitution model as selected by ModelTest v3.7 (Posada and Crandall, 1998) based on the Akaike Information Criteria using the FindModel program (http://www.lanl.gov). The GTR +  was chosen as a nucleotide substitution model. Bayesian methods implemented in the MrBayes program version v3.1.2 (Ronquist et al., 2012) were used to create phylogenetic trees and assess statistical support clades. 50% majority-rule consensus trees were obtained from four converged chains after 10,000,000 generations of Markov Chain Monte Carlo simulations with the first 2500 sample trees discarded.


DNA sequences have been deposited in GenBank for the 16S23S rRNA IGS locus of B. miyamotoi (KF957669), the 16S rRNA locus of the novel Borrelia sp. organism from R. rattus (KF957670KF957671), and the clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA loci of the novel CA690 Bb sl spirochete (KF939517-KF939524). The sequences of the 8 housekeeping genes for isolate CA690 (ST 551) also are available on the MLST website http://borrelia.mlst.net hosted at Imperial College London, U.K. Statistical analysis Fisher’s exact test (two-tailed) was used to compare the frequency distributions of ospC genotypes in ticks and small mammals. Effects were considered to be significant at a level of P ≤ 0.05. Results In total, 5960 I. pacificus and 40 I. spinipalpis were tested for presence of Borrelia spp. by cultivation in BSK-H medium (5154 I. pacificus, 37 I. spinipalpis) or by PCR using DNA extracted from ticks (806 I. pacificus, 3 I. spinipalpis). Bb sl in I. pacificus Among 2890 I. pacificus nymphs tested countywide, 189 (6.5%) were positive for Bb sl (Table 1). Infected nymphs were detected at 23 (53.5%) sites and 3 locations yielded an infection prevalence of more than 16% that was detected by two methods (culturing and direct DNA extraction). Three hot spots were identified: one in the Oakland Hills area, another in the Sunol area and a third in the Sunol Regional Wilderness (Fig. 1). Overall, B. burgdorferi and B. bissettii were the most common spirochetes detected in nymphs: 76.7% of all positive nymphs contained B. burgdorferi and 19.6% harbored B. bissettii (Table 2). Although B. burgdorferi occurred in all plant alliances surveyed that produced positive nymphs, 95.7% of all B. burgdorferi-infected nymphs were found in inland sites composed of Valley oak, Blue oak or California sycamore alliances. In contrast, 70.8% of all B. bissettii-infected ticks were isolated from nymphs collected in the California bay and Coast live oak alliances of the maritime zone. Among 3070 I. pacificus adults tested, 29 (0.9%) were infected with Bb sl (Table 1). Infections in 79.3% and 10.3% of the positive adults were characterized as B. burgdorferi and B. bissettii, respectively (Table 2). All 812 I. pacificus adults collected from the Annual grasses and forbs alliance in both the inland and maritime zones were negative for Bb sl. Frequency distribution of ospC genotypes in I. pacificus OspC DNA was amplified from 144 B. burgdorferi-infected I. pacificus nymphs and 23 adults. Six ospC alleles were detected (Table 3). The same ones found in I. pacificus nymphs also were detected in adults. B. burgdorferi genotype H (45.8%) was encountered most frequently in nymphs, followed by F (27.1%) and A (18.8%). This distribution was influenced heavily by B. burgdorferi populations in the Sunol-area hot spots because 87.5% of infected nymphs were collected there. The most common ospC genotype in I. pacificus adults was F (30.4%), followed by types A and H (21.8%). OspC genotypes K, D and E3 were recorded in nymphs and adult ticks at low prevalence. RF-group spirochetes in I. pacificus B. miyamotoi was detected in 11 (0.4%) of 2890 nymphs and 13 (0.4%) of 3070 adult ticks (Table 1). Most of the B. miyamotoiinfected ticks (10 nymphs, 9 adults) came from evergreen habitats


N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961

Table 1 Prevalence of Borrelia burgdorgeri sensu lato and B. miyamotoi in host-seeking I. pacificus ticks by habitat type, Alameda County, California, 2009–2012. Habitat type (number of sites)

Nymphs Maritime areas California Bay and Coast Live Oak Alliances (21) Redwood Alliance (5) Inland areas Valley Oak, Blue Oak and California Sycamore Alliances (17) Total (43 sites) Adults Maritime areas California Bay and Coast Live Oak Alliances (24) Annual Grasses and Forbs Alliance (9) Inland areas Valley Oak, Blue Oak and California Sycamore Alliances (21) Annual Grasses and Forbs Alliance (9) Total (63 sites): a

Number of ticks tested

Number (%) of ticks infected with different Borrelia species B. burgdorferi s. l.

B. miyamotoi

1522 234

48 (3.2)a 3 (1.3)

10 (0.7)a 0

1134 2890

138 (12.2) 189 (6.5)

1 (0.1) 11 (0.4)

1194 645

15 (1.3)a 0

6 (0.5)a 0

1064 167 3070

14 (1.3) 0 29 (0.9)

7 (0.7) 0 13 (0.4)

Two nymphs and one adult were co-infected with B. burgdorferi s. l. and B. miyamotoi.

in the maritime region indicating that this spirochete is more focal in distribution than B. burgdorferi. All 24 sequences of the partial 16S-23S rRNA IGS were identical to each other. They were compared to sequences from Asia (strain HT31, Japan), Europe (strain 203, Sweden), and North America (strains 2225 and C5N52, Connecticut; strain TB006, Tennessee; strain K07-557, Michigan; strain

NT178 and NT154, California). The 464-bp sequences of B. miyamotoi from I. pacificus in the present study showed ≥99.8% homology to the Californian strains, 91.3–91.8% homology to the eastern and midwestern North American strains, and 90.0 and 93.3% homology to the Asian strain and the European strain, respectively. The North American strains formed a single clade, though the northeastern

Fig. 1. Map of Alameda County, California, USA, showing site-specific B. burgdorferi sensu lato infection prevalence in I. pacificus nymphs. If less than 20 nymphs were collected, the Bb sl infection prevalence is not shown; those locations are marked by a dark triangle.

N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961


Table 2 Borrelia burgdorferi sensu lato genospecies detected in I. pacificus ticks by habitat type, Alameda County, California, 2009–2012. Number of ticks

Habitat type (number of sites with Bb sl infected ticks/Number of sites)

Number (%) of B. burgdorferi sensu lato spirochetes by genospecies

B. burgdorferi Nymphs Maritime areas California Bay and Coast Live Oak Alliances (11/23) Redwood Alliance (3/5) Inland areas Valley Oak, Blue Oak and California Sycamore Alliances (9/15) Total

B. bissettii

B. americana

B. californiensis

B. genomosp. 2

Mixed Bb sl

34 (70.8) 0

0 0

0 0

1 (2.1) 0

3 (6.3) 0

48 3

10 (20.8) 3 (100)


132 (95.7)

3 (2.2)


1 (0.1)


2 (1.4)


145 (76.7)

37 (19.6)


1 (0.5)

1 (0.5)

5 (2.6)


11 (73.3)

3 (20.0)




1 (6.7)


12 (85.7)


1 (7.1)



1 (7.1)


23 (79.3)

3 (10.3)

1 (3.4)



2 (6.9)

Adults Maritime areas California Bay and Coast Live Oak Alliances (9/24) Inland areas Valley Oak, Blue Oak and California Sycamore Alliances (6/21) Total

B. miyamotoi HT31, Japan B. miyamotoi HT24, Japan B. miyamotoi 203, Sweden B. miyamotoi 2225, CT, USA 1.00

B. miyamotoi TB006, TN, USA B. miyamotoi K07-557, MI, USA

B. miyamotoi C5N52, CT, USA


B. miyamotoi NT178, Mendocino Co., CA, USA 0.83 0.91 1.00

B. miyamotoi CA770, Alameda Co., CA, USA B. miyamotoi CA751, Alameda Co., CA, USA B. miyamotoi NT154, Mendocino Co., CA, USA B. lonestari B. hermsii


B. turicatae

0.1 Fig. 2. Phylogenetic tree of Borrelia miyamotoi (AY363703 [HT31], AY363704 [HT24], AY363705 [203], AY363706 [2225], HQ658901 [TB006], GU993308 [K07-557], AY374140 [C5N52], KF957668 [NT178], KF957667 [NT154], KF957669 [CA770]), B. lonestari (AY363707), B. hermsii (AY515267), and B. turicatae (AY526494) based on 16S-23S rRNA intergenic spacer fragment. The 50% majority rule consensus tree was determined by Bayesian analysis. Values at nodes refer to Bayesian posterior probabilities. The scale bar corresponds to 10% divergence.

Table 3 OspC genotypes identified in B. burgdorferi-infected I. pacificus ticks collected in Alameda County, California, 2009–2012. Allele A D E3 F H K Mixed Total

Number of nymphs with allele (frequency, %)

Number of adults with allele (frequency, %)

27 (18.8) 1 (0.1) 2 (0.1) 39 (27.1) 66 (45.8) 1 (0.1) 8 (5.6)

5 (21.8) 2 (8.7) 1 (4.3) 7 (30.4) 5 (21.8) 2 (8.7) 1 (4.3)

144 (100)

23 (100)

and midwestern strains clustered separately from those in the Far West (Fig. 2). Borrelia infections in I. spinipalpis ticks Forty I. spinipalpis ticks were collected at 11 sites, 37.5% (14 nymphs, 1 adult) of which originated from one maritime-zone location in the Oakland Hills. One adult and four nymphs were positive for Bb sl infection. B. bissettii was the most prevalent genospecies in

I. spinipalpis (four out of five). A culture isolate, designated CA690, had a unique sequence. Additional amplifying and sequencing of the 16S-23S rRNA IGS and 16S rRNA gene fragments identified its distant genetic relationship to all borrelial genospecies found before in California (data not shown). Isolate CA690 grew well in BSK-H medium and exhibited typical helical morphology associated with Bb sl spirochetes. Genetic characterization of isolate CA690 The sequences of eight chromosomally located housekeeping genes clpA, clpX, nifS, pepX, pyrG, recG, rplB, uvrA of isolate CA690 were novel compared to the sequences available in the MLST and GenBank databases. The sequences of individual loci showed less than 94.0% similarity to sequences deposited in the MLST database that currently houses data for more than 1300 Borrelia strains worldwide. The housekeeping gene sequences from CA690 were aligned with the sequences of 19 strains present in the MLST database. Those strains represent 18 Borrelia genospecies plus B. genomospecies 2: B. afzelii (PKo), B. americana (SCW30e), B. andersonii (21123), B. bavariensis (PFek), B. bissettii (CA128), B. burgdorferi (B31), B. californiensis (CA443), B. carolinensis (SCJ1), B. garinii (20047), B. japonica (tonbetsu lo 7), B. kurtenbachii (25015),


N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961

B. s

Detection of a novel Borrelia sp. similar to the Spanish R57 isolate




e ica nia on p sita . turdi a j u l . B B B.


ta B.

B. valaisiana

1.0 0 .54







B. spielman

B. a fzeli i


1.0 1.0

1.0 sis B. californien 1.0 0.60 1.0 nbachii B. kurte nensis i l i o r ti B. ca set bis na B. ca eri m a B.

ii nuk e ngtz B. ya


B. bavariensis B. g arin ii


B. b u

B. a n



rgdo rfer i




0.02 Fig. 3. Phylogenetic analysis of Bb sl isolates including the novel CA690 isolate. The tree was generated using concatenated sequences of 8 chromosomally located housekeeping gene fragments. The 50% majority rule consensus tree was determined by Bayesian analysis. Values at nodes refer to Bayesian posterior probabilities. The scale bar corresponds to 2% divergence.

B. lusitaniae (PoHL1), B. sinica (CMN3), B. spielmanii (A14S), B. tanukii (HK501), B. turdi (Ya501), B. valaisiana (VS116), B. yangtze (QLM4P1) and B. genomospecies 2 (CA2). The pairwise genetic similarities of CA690 and 19 strains were less than 92.4%. A phylogenetic analysis of concatenated sequences revealed that isolate CA690 clustered with Eurasian Bb sl spirochetes while forming a separate clade (Fig. 3).

An unusual spirochete was isolated from 43.5% (10/23) of the roof rats caught at two locations in the Oakland Hills. The organism manifested typical Borrelia morphology and translational movements when viewed by dark-field microscopy and was vital for about 2 to 3 days, but attempts to grow it in BSK-H medium were unsuccessful. To determine its taxonomic status, additional PCR assays were performed to amplify genetic markers commonly used for borrelial strain identification: two rRNA intergenic spacers and the 16S rRNA gene. The 5S-23S rRNA IGS PCR was unsuccessful. The 16S-23S IGS rRNA PCR produced an ∼1200-bp amplicon that did not reveal close matches to sequences published in GenBank. The 16S rRNA PCR amplified a ∼1300-bp fragment that was genetically similar (>98.0%) to the R57 organism detected in wood mice (Apodemus sylvaticus) and bank voles (Clethrionomys glareolus) in Spain (Gil et al., 2005). Similarly, amplification of the groEL gene fragment revealed a relationship to the R57 spirochete. The phylogenetic analysis of the 1245-bp partial 16S rRNA gene that included sequences representing species of the LD and RF groups and the R57 strain demonstrated that the novel spirochetes from roof rats and the R57 isolate shared a common ancestor and formed a distinct, strongly supported clade (Fig. 4). Frequency distribution of ospC genotypes in B. burdorferi-infected fox squirrels and I. pacificus nymphs The diversity and frequency distribution of ospC genotypes found in I. pacificus nymphs collected in the Sunol area were compared with what was found in B. burgdorferi-infected fox squirrels from the same locality. Although all ospC major groups detected in squirrels were present in ticks, genotype A was found in 26.7% of infected nymphs but not in fox squirrels (Table 5). On the other hand, genotype E3 occurred in 29.4% of infected squirrels but only in 0.1% of infected nymphs. Two alleles, F and H, were common in I. pacificus nymphs and fox squirrels, albeit at dissimilar frequencies.

Borrelia spp. in small mammals Discussion A total of 101 animals belonging to eight species was tested for presence of borreliae (Table 4). Few ticks were found attached to them because the mammals were not collected during the principal activity period of I. pacificus subadults. Bb sl infections were found in EPB excised from three rodent species, i.e., fox squirrel, duskyfooted wood rat and roof rat. B. burgdorferi was detected in 44.2% (19/43) of fox squirrels. B. bissettii was identified in 23.1% (3/13) of wood rats and 13.1% (3/23) of roof rats.

Prevalence of Bb sl in I. pacificus This study represents the first intensive effort to identify borrelial genospecies and the genotypes of B. burgdorferi in host-seeking populations of I. pacificus in ecologically diverse AC. Previous smallscale studies (1986–2007) in AC established that Bb sl occurred in I. pacificus in regional parks and that the risk of acquiring LD

Table 4 Prevalence of Borrelia spp. in small mammals collected from Alameda County, California, 2008–2012. Species

Sciurus niger Neotoma fuscipes Peromyscus maniculatus Peromyscus truei Rattus norvegicus Rattus rattus Scapanus latimanus Sorex ornatus Total a b

Bbss, B. burgdorferi sensu stricto. Bbis, B. bissettii.

Common name

Fox squirrel Dusky-footed wood rat Deer mouse Pinyon mouse Norway rat Roof rat Broad-footed mole Ornate shrew

Number tested

43 13 2 13 1 23 1 5 101

Number (%) of mammals infected with Borrelia spp. Borrelia burgdorferi group

Relapsing fever group



B. miyamotoi


19 (44.2) 0 0 0 0 0 0 0

0 3 (23.1) 0 0 0 3 (13.1) 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 10 (43.5) 0 0





N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961 1.00


B. sinica CNM3 (AB022101) B. japonica HO14 (L46696)


B. bissettii DN127 (AJ224141)


B. burgdorferi B31 (U03396) B. lusitaniae PotiB2 (X98228) 1.00

B. garinii PBi (X85199) B. afzelii DK4 (X85194)

0.83 0.50

B. valaisiana CKA4a (AB022143) B. tanukii Hk501 (D67023)


B. miyamotoi HT31 (D45192)

0.99 0.88

B. lonestari Texas20 (U23211) B.coriaceae Co53 (U42286)

0.61 1.00

B. hispanica UESV/246 (U42294) B. hermsii HS1 (U42292) B. parkeri M3001 (U42296)

CA684 (KF957672) 1.00

CA682 (KF957670) CA681


ALEPB216 (KF957671)

R 57 (AY626138)


Fig. 4. Phylogenetic tree based on partial 16S rRNA gene sequences of selected Borrelia genospecies including the R57-like spirochete from Rattus rattus (USA), the R57 spirochete (Spain), and strains of LD- and RF-group spirochetes. Values at nodes refer to Bayesian posterior probabilities. The scale bar corresponds to 2% divergence.

at those sites was low (Li et al., 2000; http://www.cdph.ca.gov/ HealthInfo/discond/Documents/TestingResultsforLymeDisease Agent.pdf). In the current study, nearly 6000 individually tested ticks from 71 sites yielded an unanticipated wealth of new information about the diversity and spatial distribution of tick-associated borreliae. To our knowledge, no other county in the United States has yielded such a remarkable biodiversity of Borrelia spp. from ticks and small mammals. The nymphal Bb sl infection prevalence in woodland sites ranged from 1.0% to 24.0%, which is comparable to those in highly endemic areas of northwestern California (Tälleklint-Eisen and Lane, 1999; Eisen et al., 2004b, 2010). Three hot spots had high infection prevalence ranging from 16 to 24%. It is not clear what factors contributed to such large percentages of infected nymphs at those locations. In Mendocino County, a spatially explicit model of the density of infected nymphs in woodlands suggested that site-specific meteorological variables are better predictors of the infected nymphs than woodland types (Eisen et al., 2010). However, the roles played by reservoir and non-reservoir hosts in determining the local infection prevalence in ticks were not evaluated. In about one-half of our surveillance sites, both I. pacificus nymphs and adults were collected, though the sites producing the

highest nymphal abundance often did not yield many adult ticks. These findings mirror those reported earlier from northern and southern California (Clover and Lane, 1995; Eisen et al., 2004a; Lane et al., 2013). The overall Bb sl infection prevalence in adult ticks in AC was similar to that reported for adult ticks from northwestern California (1–3%) (e.g., Burgdorfer et al., 1985; Lane and Lavoie, 1988; Eisen et al., 2004a). Diversity of Borreliae in I. pacificus The Bb sl complex consists of 20 confirmed or proposed species (Margos et al., 2011; Ivanova et al., 2013). Of these, B. americana, B. bissettii, B. burgdorferi, B. californiensis and B. genomospecies 2, occur in California (Burgdorfer et al., 1985; Brown et al., 1992, 2006; Postic et al., 1998, 2007). Each of them was detected in I. pacificus in AC. This high Bb sl diversity differs from that in southern California where only B. americana and B. burgdorferi have been reported from I. pacificus (Meyers et al., 1992; Rudenko et al., 2009; Lane et al., 2013). The genospecies with the broadest geographical and habitat distributions in AC was B. burgdorferi – about three-quarters of all positive ticks contained it. In Mendocino County, nymphs inhabiting dense woodlands had a similar ratio of B. burgdorferi

Table 5 Comparison of ospC genotypes detected in B. burgdorferi-infected fox squirrels versus host-seeking I. pacificus nymphs from Sunol, Alameda County, California, 2009–2012. Allele A D E3 F H Mixed Total

Number of alleles in squirrels (frequency, %)

Number of alleles in I. pacificus nymphs (frequency, %)

Statistical comparison (P)

0 1 (5.9) 5 (29.4) 4 (17.6) 7 (41.2) 1 (5.9)

24 (26.7) 1 (0.1) 1 (0.1) 38 (42.2) 20 (22.2) 6 (6.7)

0.041 0.307 90% of the B. burgdorferi strains found in nymphal or adult tick populations. Because the impact of an ospC genotype on human health is the result of its invasiveness and the frequency with which humans are exposed to it, the three locations where more than 15% of nymphs were infected are high-risk areas. Identifying and then monitoring high-risk areas is what drives tick-surveillance and educational programs. The multi-system clinical features of LD are attributable to differences in pathogenicity of various Borrelia species. B. bissettii causes clinical LD in central and southern Europe (Rudenko et al., 2008; Hulinska et al., 2009) and B. bissettii-like spirochetes infect people in northwestern California (Girard et al., 2011). Those findings combined with the discovery of a moderately-high B. bissettii infection prevalence in I. pacificus nymphs at some localities in the maritime region of AC should prompt county and state health officials to educate health-care providers that spirochetes besides B. burgdorferi occasionally may infect Californians in that region. In that regard, several patients in Florida and Georgia presenting with Lyme disease-like illnesses recently were discovered to be infected with either B. americana or B. andersonii (Clark et al., 2013). Prior


to this breakthrough, the only LD spirochete in the United States considered to cause frank illness was B. burgdorferi. Conclusions This, the first large-scale survey of Bb sl and B. miyamotoi populations in rural and peri-urban areas of AC, demonstrated that Ixodes spp. ticks and small mammals harbor an extraordinary biodiversity of Borrelia spp. All six borrelial species found previously in I. pacificus ticks statewide were detected in ticks from AC. In addition, we discovered two novel spirochetes, one in an I. spinipalpis tick and the other in roof rats. These novel spirochetes were phylogenetically related to European borreliae. Although the Bb sl infection prevalence in I. pacificus nymphs varied from 0 to 24%, a few hotspots were identified in which more than 15% of the ticks were infected. Individuals recreating or working in those specific habitats in spring or summer would be at elevated risk of encountering a spirocheteinfected tick. Furthermore, ospC genotyping revealed six alleles (A, D, E3, F, H and K) in B. burgdorferi-infected I. pacificus ticks, and 93% of those ticks contained genotypes causing disseminated illness in people. Acknowledgments This research was made possible by generous funding provided by the Alameda County Vector Control Services District, California, USA. We thank M. Yung for assistance with preparing the map; E. Omi-Olsen for preparation of mammalian-tissue samples for testing purposes; staff members of the University and Jepson Herbaria of the University of California at Berkeley for identifying plant species; and Dr. G. Margos for support with the MLST database. References Barandika, J.F., Hurtado, A., Garcia-Esteban, C., Gil, H., Escudero, R., Barral, M., Jado, I., Juste, R.A., Anda, P., Garcia-Perez, A.L., 2007. Tick-borne zoonotic bacteria in wild and domestic small mammals in northern Spain. Appl. Environ. Microbiol. 73, 6166–6171. Barbour, A.G., Bunikis, J., Travinsky, B., Gatewood Hoen, A., Diuk-Wasser, M.A., Fish, D., Tsao, J.I., 2009. Niche partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector and mammalian reservoir species. Am. J. Trop. Med. Hyg. 81, 1120–1131. Burgdorfer, W., Lane, R.S., Barbour, A.G., Gresbrink, R.A., Anderson, J.R., 1985. The western black-legged tick, Ixodes pacificus: a vector of Borrelia burgdorferi. Am. J. Trop. Med. Hyg. 34, 925–930. Brisson, D., Dykhuizen, D.E., 2004. OspC diversity in Borrelia burgdorferi: different hosts are different niches. Genetics 168, 713–722. Brisson, D., Baxamusa, N., Schwartz, I., Wormser, G.P., 2011. Biodiversity of Borrelia burgdorferi strains in tissues of Lyme disease patients. PLoS One 6, e22926, http://dx.doi.org/10.1371/journal.pone.0022926. Brown, R.N., Lane, R.S., 1992. Lyme disease in California: a novel enzootic transmission cycle of Borrelia burgdorferi. Science 256, 1439–1442. Brown, R.N., Peot, M.A., Lane, R.S., 2006. Sylvatic maintenance of Borrelia burgdorferi (Spirochaetales) in Northern California: untangling the web of transmission. J. Med. Entomol. 43, 743–751. Bunikis, J., Garpmo, U., Tsao, J., Berglund, J., Fish, D., Barbour, A.G., 2004. Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe. Microbiology 150, 1741–1755. Burkot, T.R., Maupin, G.O., Schneider, B.S., Denatale, C., Happ, C.M., Rutherford, J.S., Zeidner, N.S., 2001. Use of a sentinel host system to study the questing behavior of Ixodes spinipalpis and its role in the transmission of Borrelia bissettii, human granulocytic Ehrlichiosis, and Babesia microti. Am. J. Trop. Med. Hyg. 65, 293–299. Chowdri, H.R., Gugliotta, J.L., Berardi, V.P., Goethert, H.K., Molloy, P.J., Sterling, S.L., Telford, S.R., 2013. Borrelia miyamotoi infection presenting as human granulocytic anaplasmosis: a case report. Ann. Intern. Med. 159, 21–27. Clark, K.L., Leydet, B., Hartman, S., 2013. Lyme Borreliosis in human patients in Florida and Georgia, USA. Int. J. Med. Sci. 10, 915–931. Clover, J.R., Lane, R.S., 1995. Evidence implicating nymphal Ixodes pacificus (Acari: Ixodidae) in the epidemiology of Lyme disease in California. Am. J. Trop. Med. Hyg. 53, 237–240. Conroy, C.J., Rowe, C.K., Rowe, K.M.C., Kamath, P.L., Aplin, K.P., Hui, L., James, D.K., Moritz, C., Patton, J.L., 2012. Cryptic genetic diversity in Rattus of the San Francisco Bay region, California. BioInvasions 15, 741–758.


N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961

Cosson, J., Michelet, L., Chotte, J., LeNaour, E., Cote, M., Devillers, E., Poulle, M., Huet, D., Gala, M., Geller, J., Moutailler, S., Vayssier-Taussat, M., 2014. Genetic characterization of the human relapsing fever spirochete Borrelia miyamotoi in vectors and animal reservoirs of Lyme disease spirochetes in France. Parasit Vectors 7, http://dx.doi.org/10.1186/1756-3305-7-233. Dykhuizen, D.E., Brisson, D., Sandigursky, S., Wormser, G.P., Nowakowski, J., Nadelman, R.B., Schwartz, I., 2008. The propensity of different Borrelia burgdorferi sensu stricto genotypes to cause disseminated infections in humans. Am. J. Trop. Med. Hyg. 78, 806–810. Earnhart, C.G., Buckles, E.L., Dumler, J.S., Marconi, R.T., 2005. Demonstration of OspC type diversity in invasive human Lyme disease isolates and identification of previously uncharacterized epitopes that define the specificity of the OspC murine antibody response. Infect. Immun. 73, 7869–7877. Eisen, R.J., Eisen, L., Castro, M.B., Lane, R.S., 2003a. Environmentally related variability in risk of exposure to Lyme disease spirochetes in northern California: effect of climatic conditions and habitat type. Environ. Entomol. 32, 1010–1018. Eisen, L., Dolan, M.C., Piesman, J., Lane, R.S., 2003b. Vector competence of Ixodes pacificus and I. spinipalpis (Acari: Ixodidae), and reservoir competence of the dusky-footed woodrat (Neotoma fuscipes) and the deer mouse (Peromyscus maniculatus), for Borrelia bissettii. J. Med. Entomol. 40, 311–320. Eisen, R.J., Mun, J., Eisen, L., Lane, R.S., 2004a. Life stage-related differences in density of questing ticks and infection with Borrelia burgdorferi sensu lato within a single cohort of Ixodes pacificus (Acari: Ixodidae). J. Med. Entomol. 41, 768–773. Eisen, L., Eisen, R.J., Chang, C.C., Mun, J., Lane, R.S., 2004b. Acarological risk of exposure to Borrelia burgdorferi spirochaetes: long-term evaluations in northwestern California, with implications for Lyme borreliosis risk-assessment models. Med. Veterin. Entomol. 18 (3), 8–49. Eisen, L., Eisen, R.J., Lane, R.S., 2004c. The roles of birds, lizards, and rodents as hosts for the western black-legged tick Ixodes pacificus. J. Vector Ecol. 29, 295–308. Eisen, L., Eisen, R.J., Mun, J., Salkeld, D.J., Lane, R.S., 2009. Transmission cycles of Borrelia burgdorferi and B. bissettii in relation to habitat type in northwestern California. J. Vector Ecol. 34, 81–91. Eisen, R.J., Eisen, L., Girard, Y.A., Fedorova, N., Mun, J., Slikas, B., Leonhard, S., Kitron, U., Lane, R.S., 2010. A spatially-explicit model of acarological risk of exposure to Borrelia burgdorferi-infected Ixodes pacificus nymphs in northwestern California based on woodland type, temperature, and water vapor. Ticks Tick-Borne Dis. 1, 35–43. Fleer, K.A., Foley, P., Calder, L., Foley, J.E., 2011. Arthropod vector and vector-borne pathogens in Yosemite National Park. J. Med. Entomol. 48, 101–110. Fraenkel, C.J., Garpmo, U., Berglund, J., 2002. Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks. J. Clin. Microbiol. 40, 3308–3312. Fukunaga, M., Takahashi, Y., Tsuruta, Y., Matsushita, O., Ralph, D., McClelland, M., Nakao, M., 1995. Genetic and phenotypic analysis of Borrelia miyamoto sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector of Lyme disease in Japan. Int. J. Syst. Bacteriol. 45, 804–810. Furman, D.P., Loomis, E.C., 1984. The ticks of California. Bulletin of the California Insect Survey, Vol. 25. University of California Press, Berkeley, CA. Geller, J., Nazarova, L., Katargina, O., Jarvekulg, L., Fomenko, N., Golovljova, I., 2012. Detection and genetic characterization of relapsing fever spirochete Borrelia miyamotoi in Estonian ticks. PLoS One 7 (12), e51914. Gil, H., Barral, M., Escudero, R., Garcia-Perez, A.L., Anda, P., 2005. Identification of a new species among small mammals in areas of northern Spain where Lyme disease is endemic. Appl. Environ. Microbiol. 71, 1336–1345. Girard, Y.G., Travinsky, B., Schotthoefer, A., Fedorova, N., Eisen, R.J., Eisen, L., Barbour, A.G., Lane, R.S., 2009. Population structure of the Lyme borreliosis spirochete Borrelia burgdorferi in the western black-legged tick (Ixodes pacificus) in northern California. Appl. Environ. Microbiol. 75, 7243–7252. Girard, Y.G., Fedorova, N., Lane, R.S., 2011. Genetic diversity of Borrelia burgdorferi and detection of B. bissettii-like DNA in serum of north-coastal California residents. J. Clin. Microbiol. 49, 945–954. Gugliotta, J.L., Goethert, H.K., Berardi, V.P., Telford, S.R., 2013. Meningoencephalitis from Borrelia miyamotoi in an immunocompromised patient. N. Engl. J. Med. 368, 240–245. Guner, E.S., Hashimoto, N., Takada, N., Kaneda, K., Imai, Y., Masuzawa, T., 2003. First isolation and characterization of Borrelia burgdorferi sensu lato strains from Ixodes ricinus ticks in Turkey. J. Med. Microbiol. 52, 807–813. Hamer, S.A., Tsao, J.I., Walker, E.D., Hickling, G.J., 2010. Invasion of the Lyme disease vector Ixodes scapularis: implications for Borrelia burgdorferi endemicity. EcoHealth 7, 47–63. Hamer, S.A., Hickling, G.J., Keith, R., Sidge, J.L., Walker, E.D., Tsao, J.I., 2012. Associations of passerine birds, rabbits, and ticks with Borrelia miyamotoi and Borrelia andersonii in Michigan, U. S. A. Parasit. Vectors 5, 231. Houvius, J.W.R., de Wever, B., Sohne, M.C., Brouwer, M.C., Coumou, J., Wagemakers, A., Oei, A., Knol, H., Narasimhan, S., Hodiamont, C.J., Jahfari, S., Pals, S.T., Horlings, H.M., Fikrig, E., Sprong, H., van Oers, M.H., 2013. A case of meningoencephalitis by the relapsing fever spirochete Borrelia miyamotoi in Europe. Lancet 382, 658. Hulinska, D., Votypka, J., Vanousova, D., Hercogova, J., Hulinsky, V., Drevova, H., Kurzova, Z., Uherkova, L., 2009. Identification of Anaplasma phagocytophillum and Borrelia burgdorferi sensu lato in patients with erythema migrans. Folia Microbiol. 54, 246–256. ˜ D., Murúa, R., Moreno, C.X., Hernández, Ivanova, L.B., Tomova, A., González-Acuna, C., Cabello, J., Cabello, C., Daniels, T.J., Godfrey, H.P., Cabello, F.C., 2013. Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this species in the Southern Hemisphere. Environ. Microbiol. [Epub ahead of print].

Jameson Jr., E.W., Peeters, H.J., 2004. Mammals of California. University of California Press, Berkeley, CA. King, J.L., Sue, M.C., Muchlinski, A.E., 2010. Distribution of the Eastern Fox Squirrel (Sciurus niger) in Southern California. The Southwestern Naturalist 55, 42–49. Kramer, V.L., Beesley, C., 1993. Temporal and spatial distribution of Ixodes pacificus and Dermacentor occidentalis (Acari: Ixodidae) and prevalence of Borrelia burdorferi in Contra Costa County, California. J. Med. Entomol. 30, 549–554. Krause, P.J., Narasimhan, S., Wormser, G.P., Rollend, L., Fikrig, E., Lepore, T., Barbour, A., Fish, D., 2013. Human Borrelia miyamotoi Infection in the United States. N. Engl. J. Med. 368, 291–293. Kurtenbach, K., De Michelis, S., Etti, S., Schäfer, S.M., Sewell, H.S., Brade, V., Kraiczy, P., 2002. Host association of Borrelia burgdorferi sensu lato – the key role of host complement. Trends Microbiol. 10, 74–79. Kwok, S., Higuchi, R., 1989. Avoiding false positives with PCR. Nature 339, 237–238. Lane, R.S., Lavoie, P.E., 1988. Lyme borreliosis in California: acarological, clinical, and epidemiological studies. Ann. N.Y. Acad. Sci. 539, 192–203. Lane, R.S., Brown, R.N., 1991. Wood rats and kangaroo rats: potential reservoirs of the Lyme disease spirochetes in California. J. Med. Entomol. 28, 299–302. Lane, R.S., Manweiler, S.A., Stubbs, H.A., Lennette, E.T., Madigan, J.E., Lavoie, P.E., 1992. Risk factors for Lyme disease in a small rural community in Northern California. Am. J. Epidemiol. 136, 1358–1368. Lane, R.S., Peavey, C.A., Padgett, K.A., Hendson, M., 1999. Life history of Ixodes (Ixodes) jellisoni (Arcari: Ixodidae) and its vector competence for Borrelia burgdorferi sensu lato. J. Med. Entomol. 36, 329–340. Lane, R.S., Steinlein, D.B., Mun, J., 2004. Human behaviors elevating exposure to Ixodes pacificus (Acari: Ixodidae) nymphs and their associated bacterial zoonotic agents in a hardwood forest. J. Med. Entomol. 41, 239–248. Lane, R.S., Mun, J., Eisen, R.J., Eisen, L., 2005. Western gray squirrel (Rodentia: Sciuridae): a primary reservoir host of Borrelia burgdorferi in Californian oak woodlands? J. Med. Entomol. 42, 388–396. Lane, R.S., Fedorova, N., Kleinjan, J.E., Maxwell, M., 2013. Eco-epidemiological factors contributing to the low risk of human exposure to ixodid tick-borne borreliae in southern California, USA. Ticks Tick Borne Dis. 4, 377–385. Li, X., Peavy, C.A., Lane, R.S., 2000. Density and spatial distribution of Ixodes pacificus (Acari: Ixodae) in two recreational areas in North coastal California. Am. J. Trop. Med. Hyg. 62, 415–422. Lin, T., Oliver, J.H., Gao, L., Kollars Jr., T.M., Clark, K.L., 2001. Genetic heterogeneity of Borrelia burgdorferi sensu lato in the southern United States based on restriction fragment length polymorphism and sequence analysis. J. Clin. Microbiol. 39, 2500–2507. Margos, G., Gatewood, A.G., Aanensen, D.M., Hanincova, K., Terekhova, D., Vollmer, S.A., Cornet, M., Piesman, J., Donaghy, M., Bormane, A., Hurn, K., 2008. MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc. Natl. Acad. Sci. U.S.A. 105, 8730–8735. Margos, G., Vollmer, S.A., Cornet, M., Garnier, M., Fingerle, V., Wilske, B., Bormane, A., Vitorino, L., Collares-Pereira, M., Drancourt, M., Kurtenbach, K., 2009. A new Borrelia species defined by multilocus sequence analysis of housekeeping genes. Appl. Environ. Microbiol. 75, 5410–5416. Margos, G., Hojgaard, A., Lane, R.S., Cornet, M., Fingerle, V., Rudenko, N., Ogden, N., Aanensen, D.M., Fish, D., Piesman, J., 2010. Multilocus sequence analysis of Borrelia bissettii strains from North America reveals a new Borrelia species Borrelia kurtenbachii. Ticks Tick Borne Dis. 1, 151–158. Margos, G., Vollmer, S.A., Ogden, N.H., Fish, D., 2011. Population genetics, taxonomy, phylogeny and evolution of Borrelia burgdorferi sensu lato. Infect. Genet. Evol. 11, 1545–1563. Meyers, H.B., Moore, D.F., Gellert, G., Euler, G.L., Prendergast, T.J., Badri, M., Webb, J.P., Fogarty, C.L., 1992. Isolation of Borrelia burgdorferi from ticks in southern California. West J. Med. 157 (4), 455–456. Mun, J., Eisen, R.J., Eisen, L., Lane, R.S., 2006. Detection of a Borrelia miyamotoi sensu lato relapsing-fever group spirochete from Ixodes pacificus in California. J. Med. Entomol. 43, 120–123. Norris, D.E., Klompen, J.S., Keirans, J.E., Lane, R.S., Piesman, J., Black, W.C.T., 1997. Taxonomic status of Ixodes neotomae and I. spinipalpis (Acari: Ixodidae) based on mitochondrial DNA evidence. J. Med. Entomol. 34, 696–703. Padgett, K.A., Bonilla, D.L., 2011. Novel exposure sites for nymphal Ixodes pacificus within picnic areas. Ticks Tick-Borne Dis. 2, 191–195. Peavey, C.A., Lane, R.S., Kleinjan, J.E., 1997. Role of small mammals in the ecology of Borrelia burgdorferi in a peri-urban park in north coastal California. Exp. Appl. Acarol. 21, 569–584. Pepin, K.M., Eisen, R.J., Mead, P.S., Piesman, J., Fish, D., Hoen, A.G., Barbour, A.G., Hamer, S., Diuk-Wasser, M.A., 2012. Geographic variation in the relationship between human Lyme disease incidence and density of infected host-seeking Ixodes scapularis nymphs in the Eastern United States. Am. J. Trop. Med. Hyg. 86, 1062–1071. Platonov, A.E., Karan, L.S., Kolyasnikova, N.M., Makhneva, N.A., Toporkova, M.G., Maleev, V.V., Fish, D., Krause, P.J., 2011. Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg. Infect. Dis. 17, 1816–1823. Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Postic, D., Ras, N.M., Lane, R.S., Hendson, M., Baranton, G., 1998. Expanded diversity among Californian Borrelia isolates and description of Borrelia bissettiii sp. nov. (formerly Borrelia group DN127). J. Clin. Microbiol. 36, 3497–3504. Postic, D., Garnier, M., Baranton, G., 2007. Multilocus sequence analysis of atypical Borrelia burgdorferi sensu lato isolates – description of Borrelia californiensis sp. nov., and genomospecies 1 and 2. Int. J. Med. Microbiol. 297, 263–271.

N. Fedorova et al. / Ticks and Tick-borne Diseases 5 (2014) 951–961 Richter, D., Schlee, D.B., Matuschka, F.R., 2003. Relapsing fever-like spirochetes infecting European vector tick of Lyme disease agent. Emerg. Infect. Dis. 9, 697–701. Ronquist, F., Teslenko, M., Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. System. Biol. 61, 539–542. Rudenko, N., Golovchenko, M., Mokracek, A., Piskunova, N., Ruzek, D., Mallatova, N., Grubhoffer, L., 2008. Detection of Borrelia bissettii in cardiac valve tissue of a patient with endocarditis and aortic valve stenosis in the Czech Republic. J. Clin. Microbiol. 46, 3540–3543. Rudenko, N., Golovchenko, M., Lin, T., Gao, L., Grubhoffer, L., Oliver Jr., H.J., 2009. Delineation of a new species of the Borrelia burgdorferi sensu lato complex, Borrelia americana sp. nov. J. Clin. Microbiol. 47, 3875–3880. Salkeld, D.J., Leonhard, S., Girard, Y.A., Hahn, N., Mun, J., Padgett, K.A., Lane, R.S., 2008. Identifying the reservoir hosts of the Lyme disease spirochete Borrelia burgdorferi in California: the role of the western gray squirrel (Sciurus griseus). Am. J. Trop. Med. Hyg 79, 535–540. Salkeld, D.J., Cinkovich, S., Nieto, N.C., 2014. Tick-borne pathogens in Northwestern California, USA. Emerg. Infect. Dis. 20, 493–494. Sawyer, J.O., Keeler-Wolf, T., 1995. A Manual of California Vegetation. Native Plant Society, Sacramento, CA. Scoles, G.A., Papero, M., Beati, L., Fish, D., 2001. A relapsing fever group spirochete transmitted by Ixodes scapularis ticks. Vector Borne Zoonotic. Dis. 1, 21–34. Scott, J.D., Lee, M.K., Fernando, K., Durden, L.A., Jorgensen, D.R., Mak, S., Morshed, M.G., 2010a. Detection of Lyme disease spirochete, Borrelia burgdorferi sensu


lato, including three novel genotypes in ticks (Acari: Ixodidae) collected from songbirds (Passeriformes) across Canada. J. Vector. Ecol. 35, 124–139. Scott, M.C., Rosen, M.E., Hamer, S.A., Baker, E., Edwards, H., Crowder, C., Tsao, J.I., Hickling, G.J., 2010b. High-prevalence Borrelia miyamotoi infection among wild turkeys (Meleagris gallopavo) in Tennessee. J. Med. Entomol. 47, 1238–1242. Stein, B.A., Kutner, L.S., Adams, J.S., 2000. Precious Heritage: The Status of Biodiversity in the United States. The Nature Conservancy and Association for Biodiversity Information. Oxford University Press, Inc, New York, New York. Swei, A., Ostfeld, R.S., Lane, R.S., Briggs, C.J., 2011. Impact of the experimental removal of lizards on Lyme disease risk. Proc. Biol. Sci., Epub 2011, February 16. Tälleklint-Eisen, L., Lane, R.S., 1999. Variation in the density of questing Ixodes pacificus (Acari: Ixodidae) nymphs infected with Borrelia burgdorferi at different spatial scales in California. J. Parasitol. 85, 824–831. Tilly, K., Krum, J.G., Bestor, A., Jewett, M.W., Grimm, D., Bueschel, D., Byram, R., Dorward, D., Vanraden, M.J., Stewart, P., Rosa, P., 2006. Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect. Immun. 74, 3554–3564. Travinsky, B., Bunikis, J., Barbour, A.G., 2010. Geographic differences in genetic locus linkages for Borrelia burgdorferi. Emerg. Infect. Dis. 16, 1147–1150. Vredevoe, L.K., Stevens, J.R., Schneider, B.S., 2004. Detection and characterization of Borrelia bissettii in rodents from the central California coast. J. Med. Entomol. 41, 736–745. Wormser, G.P., Brisson, D., Liveris, D., Hanincova, K., Sandigursky, S., Nowakowski, J., Nadelman, R.B., Ludin, S., Schwartz, I., 2008. Borrelia burgdorferi genotype predicts the capacity for hematogenous dissemination during early Lyme disease. J. Infect. Dis. 198, 1358–1364.

Remarkable diversity of tick or mammalian-associated Borreliae in the metropolitan San Francisco Bay Area, California.

The diversity of Lyme disease (LD) and relapsing fever (RF)-group spirochetes in the metropolitan San Francisco Bay area in northern California is poo...
2MB Sizes 0 Downloads 3 Views