Distribution and Diversity of Borrelia burgdorferi Sensu Lato Group Bacteria in Sciurids of California

Abstract

California has a remarkable diversity of squirrel and chipmunk species (sciurids), and five named and several unnamed genospecies in the Borrelia burgdorferi sensu lato group (BBSL) of bacteria as well, many of which utilize sciurids as reservoirs. We investigated the prevalence, spatial distribution, and diversity of BBSL in sciurids of California by literature search, PCR of 585 ear tissue samples from 15 sciurid species prospectively collected across 19 California counties, and DNA sequencing when possible. Seven publications documented BBSL infections in western gray squirrels (Sciurus griseus), fox squirrels (Sciurus niger), eastern gray squirrels (Sciurus carolinensis), Douglas squirrels (Tamiasciurus douglasii), and redwood chipmunks (Tamias ochrogenys) in northern California. Prospective sampling added new BBSL infection records for long-eared chipmunks (Tamias quadrimaculatus), Allen's chipmunks (Tamias senex), and Siskiyou chipmunks (Tamias siskiyou). Infection was detected in the Mendocino, North Coast, West Sierra, and Central Valley regions of California. The overall PCR prevalence was 9.4% (n = 585), and exceeded 40% (n = 84) in Mendocino and farther north along the Pacific coast. Redwood (40.7%, n = 81) and Siskiyou (22.2%, n = 18) chipmunks had the highest prevalence of BBSL infection. BBSL infections were associated with arboreal and semiarboreal sciurid species and species occurring in conifer forests. Western gray squirrels and Allen's chipmunks in Humboldt County and redwood chipmunks in Mendocino County were infected with B. burgdorferi sensu stricto, while we identified Borrelia bissettiae in Douglas squirrels and Siskiyou chipmunks in Humboldt and Del Norte Counties. This indicates that further study of sciurids can aid in describing the ecology of BBSL in California.

Introduction

California hosts 30 species of sciurids (family Sciuridae) throughout the state and across many habitat types (Hall and Kelson 1981, Hartman 1999), including species of tree squirrels, flying squirrels, ground squirrels, chipmunks, and marmots. Sciurid ectoparasites include ticks, fleas, and lice, and some sciurid species are reservoirs for important zoonoses, including the Lyme disease-causing agent, Borrelia burgdorferi sensu stricto, in North America and Europe (Piesman and Gray 1994, Craine et al. 1997, Slajchert et al. 1997, DeNatale et al. 2002, Salkeld et al. 2008, Smith et al. 2010), Borrelia hermsii (Fritz et al. 2013), the agent of tick-borne relapsing fever, and Anaplasma spp. (Nieto et al. 2010, Foley and Nieto 2011), among other diseases.

The ability to act as a reservoir for B. burgdorferi, and the large geographic ranges of some sciurids, make the family potentially important to the ecology of the pathogen. However, we expect differences among the sciurids across California in their status as reservoirs for B. burgdorferi for a number of reasons. Some have minimal range overlap with the main tick vector Ixodes pacificus. Even among species that overlap in distribution with the pathogen and tick, fine scale differences in habitat use, such as feeding on the forest floor versus canopy, could account for differential contributions to maintaining the pathogen. Moreover, there are surely inherent differences in susceptibility and tolerance to infection across members of the family.

In eastern North America, the primary reservoir for B. burgdorferi sensu stricto (BBSS) is the white-footed mouse (Peromyscus leucopus), with eastern chipmunks (Tamias striatus) serving as a secondary vertebrate reservoir species (Keesing et al. 2012). In contrast, other Peromyscus species in California are rarely infected with BBSS. Rather, western gray (Sciurus griseus) and nonnative eastern gray (Sciurus carolinensis) and fox (Sciurus niger) squirrels are important vertebrate reservoirs, with other rodent species important locally (Salkeld et al. 2008, Eisen et al. 2009, Nieto et al. 2010). However, BBSS is only one member of a larger group of related pathogens, designated Borrelia burgdorferi sensu lato (BBSL), which in California also includes B. americana, B. bissettiae, B. californiensis, B. carolinensis, and several unnamed Borrelia “genospecies” (Eisen and Lane 2002). Often, investigators have not performed additional testing on PCR- or culture-positive wildlife to identify the particular genospecies.

Previous studies examining the distribution of Borrelia spp. in California have typically been geographically restricted or they have focused on sigmodontine rodents such as woodrats (Neotoma spp.) or mice (Peromyscus spp.) as hosts for Borrelia spp. (Brown and Lane 1992, Schwan et al. 1993, Brown and Lane 1994, Vredevoe et al. 2004, Salkeld et al. 2014). In addition, investigators have not routinely performed DNA sequence analysis required to identify Borrelia genospecies. We suspected that the diversity of Borrelia will be reflected in the diversity of sciurids of California. Identifying which sciurid species are hosts to BBSL, where particular host-pathogen strain pairs occur, and which environmental and behavioral factors may make sciurids susceptible to BBSL is important to understanding this pathogen in California.

This study aims to provide insight into the diversity, distribution, and prevalence of BBSL in sciurids of California and identify risk factors to sciurid hosts through the compilation of data from past studies from the literature and prospective sampling and testing.

Materials and Methods

Data were collected from literature by online searching PubMed (NCBI, ncbi.nlm.nih.gov) and Wildlife & Ecology Studies Worldwide (EBSCO, ebscohost.com) databases using the key search terms “Borrelia” combined with “California,” “squirrel,” “chipmunk,” “marmot,” and Latin names for sciurid species occurring in California. We also comprehensively searched the literature cited sections of all articles we found.

For prospective sampling, sciurids were opportunistically live-trapped in Sherman traps (HB Sherman, Tallahassee, FL) and Tomahawk traps (Tomahawk Live Trap, Hazlehurst, WI) at sites in 18 California counties (Alpine, El Dorado, Humboldt, Inyo, Lake, Lassen, Los Angeles, Madera, Marin, Mariposa, Mendocino, Mono, Napa, San Bernardino, Santa Cruz, Solano, Tuolumne, and Yolo) between September 2004 and March 2014 from multiple studies with mildly variant trapping methods (Nieto et al. 2010, Foley and Nieto 2011, Foley and Piovia-Scott 2014). Although ticks were collected at many sites, methods were variable and these data are not analyzed in this study. Additional postmortem samples were provided by a collaborator from Sacramento County (fox squirrels) and from the UC Davis Museum of Wildlife and Fish Biology collected by the U.S. Forest Service in the Sierra Nevada in 2012 and 2013.

Most samples were accompanied by metadata, which included date collected, and location, typically indicated by GPS coordinate. Work was performed in compliance with the UC Davis or Humboldt State IACUC and under valid scientific collecting permits from the California Department of Fish and Wildlife, the U.S. Forest Service, and landowners where applicable.

DNA was extracted from 25 mg of ear tissue using a kit (Qiagen Blood and Tissue Purification kit; Qiagen, Valencia, CA) following the manufacturer's instructions. We used real-time PCR (Barbour et al. 1996, Barbour et al. 2009) run in a combined thermocycler/fluorometer (ABI Prism 7700; Applied Biosystems, Foster City, CA) to detect Borrelia spp. DNA in the sample. Water negative controls were included in each run, while positive controls consisted of DNA from cultured BBSS. Samples were considered positive if the cycle threshold (CT) <40 and there was a characteristic amplification curve.

To identify genospecies, real-time PCR-positive samples were evaluated using conventional PCR for the flagellin gene as described previously (Rudenko et al. 2009a, Rudenko et al. 2011), modified to use GoTaq Green Master Mix (Promega, Madison, WI). PCR products were run on a 1% agarose gel stained with Gelstar (Lonza, Rockland, ME) and evaluated under UV transillumination. Amplicons were prepared using a kit (QIAquick Gel Extraction Kit; Qiagen) and then sequenced in an ABI 3730 sequencer (Davis Sequencing, CA). Results were examined for accuracy of base determination and end-read errors were trimmed, and we compared sequences to those in the GenBank database using the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information, Bethesda, MD). BBSL DNA sequences from this study were submitted to GenBank with the following accession numbers: KY635947 and KY700689-KY700697.

Presence of BBSL was mapped by county using ArcMap V10.3 (ESRI, Redlands, CA). For analysis, counties with positive samples were grouped by region (North Coast was Del Norte and Humboldt Counties; Mendocino was Mendocino County; Central Valley was Sacramento, Yolo, and Solano Counties; and West Sierra was Tuolumne and El Dorado Counties). Using GPS locations, we determined vegetation community and dominant vegetation type for each sample for which GPS data were available by extracting from the FRAP Vegetation GIS data set (2015 FVEG, frap.fire.ca.gov, accessed December 1, 2016). Elevation was determined for each sample location in Google Earth v7.1.8 (Google, San Francisco, CA) and was then assigned into low <1000 meters, medium 1000–2000 meters, or high >2000 meters elevation classes.

The date of collection of each sample was categorized by seasons, which were defined as Winter (December–February), Spring (March–May), Summer (June–August), and Fall (September–November). Hosts were classified by how they use space, with categories Ground, consisting of antelope ground squirrels (Ammospermophilus leucurus), golden-mantled ground squirrels (Callospermophilus lateralis), and California ground squirrels (Otospermophilus beecheyi); Semiarboreal which had all the chipmunks; and Arboreal with northern flying squirrels (Glaucomys sabrinus), Sciurus spp., and Douglas squirrels. Host species were also classified by sex (male or female) and age class (adult or juvenile).

Data were analyzed with the statistical package “R” (R-Development Core Team, www.r-project.org) with a cutoff of p < 0.05 and a confidence interval (95% CI) not containing 1 for inferring statistical significance. Associations between BBSL infection and the above variables were analyzed using logistic regression. Effect size was determined using an r ² calculated utilizing the Rsq function in package binomTools (Haubo et al. 2011) available in program R.

Results

Seven publications documented B. burgdorferi infection in five sciurid species in California (Table 1), including western gray squirrels (Lane et al. 2005, Salkeld et al. 2008, Eisen et al. 2009, Leonhard et al. 2010, Nieto et al. 2010), fox squirrels (Salkeld et al. 2008, Fedorova et al. 2014), eastern gray squirrels (Nieto et al. 2010), Douglas squirrels (Nieto et al. 2010), and redwood chipmunks (Foley and Nieto 2011). From these publications, when a genospecies was identified, BBSS was the only genospecies detected in sciurids (Lane et al. 2005, Salkeld et al. 2008, Eisen et al. 2009, Leonhard et al. 2010, Fedorova et al. 2014); however, Lane et al. (Lane et al. 2005) identified Borrelia bissettiae and an uncharacterized BBSL genospecies in ticks removed from western gray squirrels. All BBSL detections occurred in 11 northern California counties on the west coast or in the Sierra Nevada foothills (Fig. 1).

FIG. 1.

Map of California counties and regions from which sciurids were tested for Borrelia spp. infection and were either found positive (dark gray) or negative (light gray). Represented data are from prospective sampling or reports from the literature, as described in the text.

Table 1.

Prevalence of Borrelia burgdorferi Sensu Lato in Sciurids in California Documented in Published Literature and Through Prospective Sampling

	Prevalence (no. of animals tested)
Host species	Literature search	This study
Yellow-pine chipmunk	—	0.0 (59)
Least chipmunk	—	0.0 (4)
Redwood chipmunk	0.0 (3)^a,+, 24.7 (73)^b	40.7 (81)
Long-eared chipmunk	—	2.9 (70)
Allen's chipmunk	0.0 (3)^a,+	6.9 (102)
Siskiyou chipmunk	—	22.2 (18)
Lodgepole chipmunk	—	0.0 (10)
Unknown chipmunk	—	6.2 (16)
Northern flying squirrel	0.0 (2)^c, 0 (1)^a	0.0 (47)
Eastern gray squirrel	11.1 (9)^c	0.0 (20)
Western gray squirrel	80.0 (10)^d, 29.7 (222)^e, 0.0 (1)^a, 75.0 (4)^d,++, 37.8 (37)^c	12.9 (31)
Fox squirrel	0.0 (1)^c, 44.2 (43)^f	3.2 (31)
Douglas squirrel	50.0 (4)^c	5.0 (60)
White-tailed antelope squirrel	—	0.0 (1)
California ground squirrel	—	0.0 (30)
Golden-mantled ground squirrel	—	0.0 (5)

Prospective sampling occurred between 2004 and 2014.

Eisen et al. (2009 ⁺).

Foley and Nieto (2011).

Nieto et al. (2010).

Lane et al. (2005).

Salkeld et al. (2008).

Fedorova et al. (2014).

Chipmunk species ambiguously identified in publication; Redwood or Allen's chipmunk.

Prevalence based on number of natural infections before experimental infection.

From prospective sampling, a total of 585 sciurids were examined from study sites in nine distinct regions: of these, there were 55 PCR-positive samples in seven species with the total real-time PCR prevalence of BBSL in sciurids being 9.4% (CI = 7.2–12.1%, n = 585, Table 2). Among BBSL PCR-positive sciurids, the mean CT was 36.2 (SE = 0.47). Of the 55 PCR-positive samples, Flagellin was successfully sequenced from 11 samples, all from the North Coast and Mendocino regions (Table 2 and Fig. 1). B. bissettiae was identified from Siskiyou chipmunks and Douglas squirrels and BBSS was identified from redwood and Allen's chipmunks. In ranked order by prevalence, PCR-positive species were redwood chipmunks (40.7%, CI = 30.1–52.2%, n = 81), Siskiyou chipmunks (22.2%, CI = 7.4–48.1%, n = 18), western gray squirrels (12.9%, CI = 4.2–30.8%, n = 31), Allen's chipmunks (6.9%, CI = 3.0–14.1%, n = 102), Douglas squirrels (5.0%, CI = 1.3–14.8%, n = 60), fox squirrels (3.2%, CI = 0.2–18.5%, n = 31), and long-eared chipmunks (Tamias quadrimaculatus) (2.9%, CI = 0.5–10.9%, n = 70).

Table 2.

Regions, Host Species, Vegetation Communities, and Dominant Vegetation Types in California Where Borrelia burgdorferi Sensu Lato Infections Were Detected by Real-Time PCR

Factor Region	Detail Host species	No. of animals tested (No. of PCR positive)	Prevalence (95% CI)	r²	OR (CI)	p	BBSL genospecies (n)
Central coast		9 (0)	0.0 (0.0–37.1)	—	—	—
	Eastern gray squirrel	7 (0)	—	—	—	—
	Western gray squirrel	2 (0)	—	—	—	—
Central Valley		60 (2)	3.3 (0.6–12.5)	0.005	3.7 (0.5–27.7)	0.2
	Eastern gray squirrel	13 (0)	—	—	—	—
	Western gray squirrel	10 (1)	10.0 (0.5–45.9)	—	—	—
	Fox squirrel	31 (1)	3.2 (0.2–18.5)	—	—	—
	California ground squirrel	6 (0)	—	—	—	—
East Sierra		26 (0)	0.0 (0.0–16.0)	—	—	—
	California ground squirrel	2 (0)	—	—	—	—
	Yellow-pine chipmunk	19 (0)	—	—	—	—
	Lodge-pole chipmunk	2 (0)	—	—	—	—
	Unknown chipmunk	3 (0)	—	—	—	—
Mendocino		84 (34)	40.5 (30.1–51.8)	0.189	72.4 (16.3–320.7)	8.8 × 10⁻⁹
	Western gray squirrel	3 (1)	33.3 (1.8–87.5)	—	—	—
	Redwood chipmunk	81 (33)	40.7 (30.1–52.2)	—	—	—	Borrelia burgdorferi sensu stricto (2)
Mojave		1 (0)	0.0 (0.0–94.5)	—	—	—
	White-tailed antelope squirrel	1 (0)	—	—	—	—
North coast		144 (17)	11.8 (7.2–18.5)	0.002	14.3 (3.1–64.7)	0.0004
	Northern flying squirrel	6 (0)	—	—	—	—
	Western gray squirrel	6 (2)	33.3 (6.0–75.9)	—	—	—
	Douglas squirrel	35 (3)	8.6 (2.2–24.2)	—	—	—	Borrelia bissettiae (1)
	Allen's chipmunk	78 (7)	9.0 (4.0–18.2)	—	—	—	B. burgdorferi sensu stricto (4)
	Siskiyou chipmunk	18 (4)	22.2 (7.4–48.1)	—	—	—	B. bissettiae (4)
	Unknown chipmunk	1 (1)	—	—	—	—
North Sierra		28 (0)	0.0 (0.0–15.0)	—	—	—
	Golden-mantled ground squirrel	1 (0)	—	—	—	—
	Yellow-pine chipmunk	9 (0)	—	—	—	—
	Long-eared chipmunk	1 (0)	—	—	—	—
	Allen's chipmunk	17 (0)	—	—	—	—
Southern California		9 (0)	0.0 (0.0–37.1)	—	—	—
	Western gray squirrel	9 (0)	—	—	—	—
West Sierra		215 (2)	0.9 (0.2–3.7)	0.051	Referent	5.0 × 10⁻¹¹
	Northern flying squirrel	41 (0)	—	—	—	—
	Western gray squirrel	1 (0)	—	—	—	—
	California ground squirrel	21 (0)	—	—	—	—
	Golden-mantled ground squirrel	4 (0)	—	—	—	—
	Douglas squirrel	19 (0)	—	—	—	—
	Yellow-pine chipmunk	31 (0)	—	—	—	—
	Least chipmunk	4 (0)	—	—	—	—
	Long-eared chipmunk	67 (2)	3.0 (0.5–11.3)	—	—	—
	Allen's chipmunk	7 (0)	—	—	—	—
	Lodgepole chipmunk	8 (0)	—	—	—	—
	Unknown chipmunk	12 (0)	—	—	—	—

Vegetation community	Dominant vegetation
Agriculture		6 (0)	0.0 (0.0–48.3)	—	—	—
	Agriculture	6 (0)	—	—	—	—
Conifer Forest		429 (46)	10.7 (8.0–14.1)	0.183	7.1 (0.9–54.5)	0.1
	Douglas Fir	46 (7)	15.2 (6.8–29.5)	0.003	11.2 (2.2–58.1)	0.003	B. bissettiae (2), B. burgdorferi sensu stricto (3)
	Jeffrey Pine	81 (0)	—	—	—	—
	Mixed conifer	127 (2)	1.6 (0.3–6.1)	0.037	Referent	—
	Mixed conifer–hardwood	13 (1)	7.7 (0.4–37.9)	0	5.2 (0.4–64.9)	0.2
	Montane hardwood	21 (0)	—	—	—	—
	Pine	7 (0)	—	—	—	—
	Red fir	4 (0)	—	—	—	—
	Redwood	115 (36)	31.3 (23.2–40.7)	0.162	28.5 (6.5–125.3)	6.1 × 10⁻⁶	B. bissettiae (2), B. burgdorferi sensu stricto (2)
	White fir	15 (0)	—	—	—	—
Desert		1 (0)	0.0 (0.0–94.5)	—	—	—
	Riparian woodland	1 (0)	—	—	—	—
Hardwood Forest		25 (2)	8.0 (1.4–27.5)	0.012	5.1 (0.4–62.4)	0.1905
	Montane hardwood	22 (2)	9.1 (1.6–30.6)	—	—	—	B. bissettiae (1)
	Montane riparian	3 (0)	—	—	—	—
Shrub		3 (1)	33.3 (1.8–87.5)	0.25	29.5 (1.2–703.5)	0.0329
	Coastal scrub	2 (1)	50.0 (9.5–90.5)	—	—	—
	Sagebrush	1 (0)	—	—	—	—
Urban		60 (1)	1.7 (0.1–10.1)	NA	Referent	5.3 × 10⁻⁵
	Golf course	30 (1)	3.3 (0.2–19.1)	—	—	—
	Urban	30 (0)	—	—	—	—

Host space use	Host species
Semiarboreal	—	360 (47)	13.1 (9.8–17.1)	0.025	3.4 (1.5–7.5)	0.002	B. bissettiae (4), B. burgdorferi sensu stricto (6)
	Yellow-pine chipmunk	59 (0)	0.0 (0.0–7.6)	—	—	—
	Least chipmunk	4 (0)	0.0 (0.0–60.4)	—	—	—
	Redwood chipmunk	81 (33)	40.7 (30.1–52.2)	—	—	—	B. burgdorferi sensu stricto (2)
	Long-eared chipmunk	70 (2)	2.9 (0.5–10.9)	—	—	—
	Allen's chipmunk	102 (7)	6.9 (3.0–14.1)	—	—	—	B. burgdorferi sensu stricto (4)
	Siskiyou chipmunk	18 (4)	22.2 (7.4–48.1)	—	—	—	B. bissettiae (4)
	Lodgepole chipmunk	10 (0)	0.0 (0.0–34.5)	—	—	—
	Unknown chipmunk	16 (1)	6.2 (0.3–32.3)	—	—	—
Arboreal	—	189 (8)	4.2 (2.0–8.5)	0.015	Referent	2.0 × 10⁻¹⁶	B. bissettiae (1)
	Northern flying squirrel	47 (0)	0.0 (0.0–9.4)	—	—	—
	Eastern gray squirrel	20 (0)	0.0 (0.0–20.0)	—	—	—
	Western gray squirrel	31 (4)	12.9 (4.2–30.8)	—	—	—
	Fox squirrel	31 (1)	3.2 (0.2–18.5)	—	—	—
	Douglas squirrel	60 (3)	5.0 (1.3–14.8)	—	—	—	B. bissettiae (1)
Ground	—	36 (0)	0.0 (0.0–12.0)	0.007	NA	0.983
	White-tailed antelope squirrel	1 (0)	0.0 (0.0–94.5)	—	—	—
	California ground squirrel	30 (0)	0.0 (0.0–14.1)	—	—	—
	Golden-mantled ground squirrel	5 (0)	0.0 (0.0–53.7)	—	—	—

Season
Spring	—	80 (25)	31.3 (21.6–42.7)	0.093	21.7 (8.8–53.5)	9.6 × 10⁻¹²
Summer	—	341 (7)	2.1 (0.9–4.4)	NA	Referent	2.0 × 10⁻¹⁶
Fall	—	136 (18)	13.2 (8.2–20.4)	0.006	7.3 (2.9–18.2)	1.5 × 10⁻⁵
Winter	—	15 (3)	20.0 (5.3–48.6)	0.004	11.9 (2.7–53.5)	0.001

Sampling occurred between 2004 and 2014.

BBSL, B. burgdorferi sensu lato group; CI, 95%confidence interval; n, number of individual hosts sequenced to genospecies; OR, odds ratio.

There were no PCR-positive antelope, California or golden mantled ground squirrels, northern flying squirrels, eastern gray squirrels, or least (T. minimus), yellow pine (T. amoenus), or lodgepole (T. speciosus) chipmunks. Because the presence of host species is so closely associated with habitat factors, we performed logistic regression analysis only for the latter risk factor.

No PCR-positive samples were recovered from the Mojave Desert, Central Coast, East Sierra, North Sierra, or Southern California. Prevalence ranged from the lowest nonzero prevalence in the West Sierra region of 0.9% (n = 215), where PCR-positive sciurids were represented by long-eared chipmunks, to a high of 40.5% (n = 84) in the Mendocino region comprising redwood chipmunks and western gray squirrels (Table 2). Differences in prevalence among regions were significant: when compared to the lowest nonzero region (West Sierra), the odds of sciurids being infected with BBSL were highest in the Mendocino region (odds ratio [OR] = 72.4, p = 8.8 × 10⁻⁹), followed by the North Coast region (OR = 14.3, p = 0.0004), and lowest in the Central Valley region (OR = 3.7, p = 0.2).

PCR-positive sciurids occurred in the Central Valley near Vacaville, CA (western gray squirrel, 10.0%, n = 10, Fig. 1), and at golf courses near Sacramento, CA (Fox squirrel, 3.2%, n = 31, Fig. 1). Outside of these Central Valley samples, positive samples were collected from conifer forests (n = 46), hardwood forests (n = 2), and shrub areas (n = 1, Table 2). Across different conifer vegetation subtypes, prevalence was significantly highest in redwood habitat (31.3%, CI = 23.2–40.7%, n = 115) with the lowest nonzero prevalence in mixed-conifer habitat (1.6%, CI = 0.3–6.1%, Table 2, p = 9.979 × 10⁻¹⁴, n = 127). Compared to mixed-conifer habitat (referent group, r ² = 0.037), the odds of sciurids being infected with BBSL were highest in Douglas fir habitat (OR = 11.2, CI = 2.2–58.1, r ² = 0.003) followed by redwood habitat (OR = 28.5, CI = 6.5–125.3, r ² = 0.162) and mixed conifer-hardwood habitat (OR = 5.2, CI = 0.4–64.9, r ² = 0.000).

Differences in prevalence based on typical host space use were significant (p = 0.0005), with a 13.1% prevalence in semiarboreal dwelling species (OR = 3.4%, CI = 1.5–7.5%, r ² = 0.025, p = 0.002), followed by 4.2% in arboreal species (referent group, r ² = 0.015). No ground dwelling species tested positive. The highest seasonal prevalence occurred in Spring (31.3%, CI = 21.6–42.7%, OR = 21.7%, p = 9.64 × 10⁻¹², n = 80), followed by Winter (20.0%, CI = 5.3–48.6%, OR = 11.9%, p = 0.001, n = 15) and Fall (13.2%, CI = 8.2–20.4%, OR = 2.9%, p = 1.47 × 10⁻⁵, n = 136), and Summer having the lowest prevalence (2.1%, CI = 0.9–4.4%, OR = referent group, p = 2.0 × 10⁻¹⁶, n = 341). No difference in prevalence was observed between males and females, between adults and juveniles, or between elevation classes.

Discussion

This study presents insights into the potential role of sciurids in the ecology and distribution of Borrelia spp., which is often difficult to discern because of lack of identification of test-positive results to genospecies. In this study, we focused on a diverse group of rodents, the family Sciuridae in California, containing multiple species known to be reservoirs for some Borrelia genospecies. As in previous studies (Lane et al. 2005, Salkeld et al. 2008, Eisen et al. 2009, Leonhard et al. 2010, Nieto et al. 2010, Salkeld and Lane 2010, Foley and Nieto 2011), we found Borrelia spp. in forested coastal areas of Humboldt and Mendocino counties. We performed much more comprehensive testing in the Central Valley and much of the Sierra Nevada than previous studies.

Compared to the 0.0% prevalence of BBSL reported in previous studies (Salkeld et al. 2008), we detected a higher prevalence (3.3%) in the Central Valley. In addition, we detected lower prevalence in the Sierra Nevada (0.0–0.9%) and along the central coast (0.0%) compared to previous studies, 20.0% and 15.4–44.2%, respectively (Salkeld et al. 2008, Fedorova et al. 2014). In general, conifer forests had higher prevalence of BBSL than other vegetation communities; particularly, redwood habitat had the highest prevalence of all conifer forest types. Test-positive rodents continued to be present where human risk is high based on passive reporting of human cases and studies of tick infection rates (Eisen et al. 2009, Padgett et al. 2014, Salkeld et al. 2015, Centers for Disease Control and Prevention, 2015).

We documented BBSL infections in seven sciurid species, including three (long-eared, Allen's, and Siskiyou chipmunks) in which BBSL had not been previously documented. This is the first report of B. bissettiae in sciurids from California (in Siskiyou chipmunks and Douglas squirrels), although it was detected in ticks removed from squirrels (Lane et al. 2005) as well as in chipmunks in Colorado (DeNatale et al. 2002). In contrast to a previous 7% prevalence of B. burgdorferi infection in eastern gray squirrels in California (Salkeld et al. 2008), our 22 samples of this species were PCR negative. The sciurid species infected with BBSL in this study included arboreal squirrels and semiarboreal chipmunks.

Tree squirrels and many chipmunks spend time foraging and conducting other activities on the ground where they may encounter ticks. Some species of chipmunks utilize a ground-based nest before utilizing a tree-cavity nest to raise pups (Broadbooks 1974). Nymphal I. pacificus ticks have been documented on tree trunks up to about a meter above ground (Lane et al. 2009), but have not been documented in canopies. It is remarkable that arboreal and semiarboreal sciurids had higher infection risk than ground-dwelling species, even though ground-dwelling species would reasonably be more exposed to ticks. This highlights the need to better understand the role of range overlap between host and vectors, host behavior, and the innate susceptibility. Understanding the complement system's reaction to B. burgdorferi (Oosting et al. 2014) in different hosts may help explain differential host susceptibility and whether ground-dwelling sciurids in California might be resistant to infection.

It was interesting that we found only two of the known genospecies of Borrelia in California–BBSS, the major North American cause of Lyme disease, and B. bissettiae, which was associated with Lyme borreliosis in people from the Czech Republic (Rudenko et al. 2009b). It is unfortunate that either weak positive real-time test results or lack of sample precluded obtaining sequence data for numerous specimens. Notably, the overall CT, an indicator of bacterial load, was relatively high. Some of the weaker positive samples could have been false positive, supporting our use of the data not to overinterpret prevalence, but rather to focus on identifying Borrelia genospecies. This inability to sequence some of our samples limited our ability to analyze the diversity of BBSL genospecies in the state.

One possible location we might expect other genospecies to occur might be remote or isolated areas, as we have seen with B. carolinensis in an extremely isolated vole species (Microtus californicus scirpensis) in the Mojave Desert (Ott-Conn 2014). We did not have samples from sciurid species that occur in isolation (e.g., Mount Pinos lodgepole chipmunk, Tamias speciosus callipeplus), but future studies on BBSL diversity should target these geographically isolated host species, which could potentially coevolve with geographically restricted and unique strains of B. burgdorferi.

Increased host diversity is commonly associated with an increase in microbe diversity (Gavish et al. 2014). The spatial scale of our study is relatively coarse and we acknowledge that grouping samples from multiple years by site is less preferable than if there had been systematic sampling across all sites and years. Because of this, our data are best used to highlight general patterns among regions rather than highly specific differences in site. The regions may differ in prevalence because of environmental factors and host species assemblages, including whether the behavior of a potential host promotes contact with ticks. Along the coast, moisture and temperature are known to be important factors for survival of ticks that vector BBSL in California (Nieto et al. 2010, Stephenson et al. 2016). Conifer forests are characteristically cool, and redwood in particular offers a moist environment. Several sciurids are reservoirs for BBSL, although additional work to assess reservoir competence for some of the species we show, in this study, to be infected is needed to more fully understand their role in maintaining or amplifying infections in nature.

Footnotes

Acknowledgments

We thank U.S. Forest Service, U.S. Bureau of Land Management, the Hoopa Tribe, CalFire, California Department of Parks and Recreation, U.S. National Park Service, the U.C. Reserve System, U.C. Davis, U.S. Department of Agriculture, and Shoshone Village for access to study areas, samples, and logistical support. Greg Hacker and Rick Brown (Humboldt State University), and the small mammal crew from Green Diamond Resource Company helped in the collection of samples. Funding for this research was provided, in part, by the Center for Vector-borne Disease, the Marin Rod and Gun Club, and the Stockton Sportsman Club.

Author Disclosure Statement

No competing financial interests exist.

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