Tick-borne pathogens in northwestern California, USA.

To the Editor: In northwestern California, USA, the western black-legged tick, Ixodes pacificus, is a known vector of Borrelia burgdorferi, the spirochete that causes Lyme disease. B. miyamotoi, which is more closely related to spirochetes that cause relapsing fever, has also been detected in 2 locations in California (,) and has recently been implicated as a human pathogen in the northeastern United States (,). Other studies may have unintentionally included B. miyamotoi infections among measures of B. burgdorferi if the diagnostics were for spirochetes (e.g., direct fluorescent antibody tests or dark-field microscopy) or genetically targeted for Borrelia spp. ().

To investigate Borrelia spp. ecology in California, we collected adult I. pacificus ticks by dragging a 1-m2 white flannel blanket along vegetation and/or leaf litter in 12 recreational areas in the San Francisco Bay area during January–May 2012 (Table). Habitat varied from chaparral and grassland to coastal live oak woodland. Ticks were pooled for examination by quantitative PCR (qPCR) for the presence of Borrelia spp. We interpreted the prevalence of Borrelia spp. from positive pools as the minimum infection prevalence (i.e., assuming 1 positive tick/positive pool). DNA was extracted from ticks by using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s protocols and then stored at −20°C until use. DNA was analyzed by qPCR, with use of primer and fluorescent hybridization probes previously developed to differentiate Borrelia spp. spirochetes (). To identify the Borrelia spp. genotype, we attempted to sequence the 16S–23S (rrs-rrlA) intergenic spacer of each sample positive by qPCR (). The nested PCR product was further purified by using the QIAquick Kit (QIAGEN) and then sequenced (Environmental Genetics and Genomics Laboratory, Northern Arizona University, Flagstaff, AZ, USA; www.enggen.nau.edu/dna.html) by using capillary Sanger sequencing on an ABI 3730 sequencer (Life Technologies, Grand Island, NY, USA). BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare each sequence to other Borrelia spp. sequences available from GenBank.

Table

Borrelia spp. infection prevalence among adult Ixodes pacificus ticks in northwestern California, USA, January–May 2012*

Location, County (reference)	No. Borrelia spp. ticks infected/total (%)
	B. burgdorferi sensu stricto	B. burgdorferi sensu lato	B. miyamotoi	Unsequenced species	All species
Jasper Ridge Biologic Preserve, San Mateo				1/32 (3.1)	1/32 (3.1)
Pulgas Ridge OSP, San Mateo				2/118 (1.7)	2/118 (1.7)
Thornewood OSP, San Mateo†	1/156 (0.6)	2/156 (1.3)	2/156 (1.3)	4/156 (2.6)	9/156 (5.8)
Thornewood OSP, San Mateo‡					0/9 (0)
Windy Hill OSP, San Mateo†	2/120 (1.7)			1/120 (0.8)	3/120 (2.5)
Windy Hill OSP, San Mateo§	2/122 (1.6)	3/122 (2.5)	1/122 (0.8)	2/122 (1.6)	8/122 (6.6)
Wunderlich County Park, San Mateo					0/15 (0)
Foothills Park, Santa Clara					0/13 (0)
Henry W. Coe State Park, Santa Clara			3/132 (2.3)		3/132 (2.3)
Monte Bello OSP, Santa Clara	1/46 (2.2)			1/46 (2.2)	2/46 (4.3)
Sanborn County Park, Santa Clara			4/53 (7.5)		4/53 (7.5)
Sierra Azul OSP, Santa Clara			2/112 (1.8)		2/112 (1.8)
Los Trancos OSP, San Mateo and Santa Clara			1/58 (1.7)	1/58 (1.7)	2/58 (3.4)
Castle Rock State Park, Santa Cruz		1/51 (2.0)		2/51 (3.9)	3/51 (5.8)
Castle Rock State Park, Santa Cruz (6)					13/264 (4.9)
Tilden Regional Park, Contra Costa (2)	1/814 (0.1)		4/814 (0.5)		5/814 (0.6)
China Camp State Park, Marin		1/143 (0.7)	1/143 (0.7)	2/143 (1.4)	4/143 (2.8)
Hopland Research and Extension Center, Mendocino (1,7)	4/282 (1.4)		2/282 (0.7)
Total (this study)	6/1,108 (0.5)	7/1,108 (0.6)	14/1,108 (1.3)	16/1,108 (1.4)	43/1,108 (3.6)

*Data are from this study and from previously published research (indicated by reference no.). OSP, open space preserve.
†Woodland.
‡Redwood.
§Chaparral/grassland.

From a total of 1,180 adult ticks, we found 43 samples positive for Borrelia spp., resulting in a minimum infection prevalence of 3.6% (Table). We obtained intergenic spacer sequence data for 27 of the positive samples; 6 samples were B. burgdorferi sensu stricto, 7 were B. burgdorferi sensu lato (both on the basis of alignments of 816 bp), and 14 were B. miyamotoi (on the basis of alignments of 503 bp). The B. miyamotoi sequences for our samples from California and those for isolates from the eastern United States () and Japan () formed a monophyletic clade that was oriented as a sister clade to the 3 Borrelia spp. that cause tick-borne relapsing fever in the United States (B. hermsii, B. turicatae, and B. parkeri).

We found borreliae-infected adult I. pacificus ticks at all 12 sites from which tick sample sizes exceeded 30. When the presence of B. burgdorferi sensu stricto or B. burgdorferi sensu lato was detected (4/12 sites each), prevalence was 0.6%–2.2% and 0.7%–2.5%, respectively. B. miyamotoi was detected at 7/12 sites, and prevalence ranged from 0.7% to 7.5%. A previous survey of B. burgdorferi in nearby Santa Cruz County recreational areas reported an infection prevalence of ≈6% among adult I. pacificus ticks (); the study did not, however, differentiate between Borrelia spp. and therefore may have included B. miyamotoi among its prevalence measures (). In our study, B. burgdorferi was found more frequently in woodland habitats, but it was also detected in a grassland–chaparral habitat several hundred meters from the nearest woodland. We did not detect B. bissettii, a species recently implicated as a human pathogen in Mendocino County, California (). The high level of habitat variation in northwestern California presents a varied risk for Borrelia-associated tick-borne disease in humans because of diverse variations in vertebrate reservoir ecology, tick abundance, and human exposure to ticks. This variation emphasizes the need to understand the local epidemiology and ecology of a disease.

In adult I. pacificus ticks in the San Francisco Bay area, B. miyamotoi is as abundant as its congener B. burgdorferi. Human disease caused by B. miyamotoi infection has not been reported in California, and transmission efficiency of B. miyamotoi by I. pacificus ticks is unknown. However, it is possible that B. miyamotoi infections in ticks and humans have not been accurately diagnosed. We advocate for increased scrutiny of the eco-epidemiology of B. miyamotoi in human, tick, and possible vertebrate host populations in northwestern California.