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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Ticks Tick Borne Dis. 2010 Dec 1;1(4):159–167. doi: 10.1016/j.ttbdis.2010.09.004

Differences in prevalence of Borrelia burgdorferi and Anaplasma spp. infection among host-seeking Dermacentor occidentalis, Ixodes pacificus, and Ornithodoros coriaceus ticks in northwestern California

Robert S Lane a,1,*, Jeomhee Mun b,1, Miguel A Peribáñez c, Natalia Fedorova a
PMCID: PMC3044915  NIHMSID: NIHMS253657  PMID: 21359090

Abstract

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

Keywords: Borrelia burgdorferi, Anaplasma spp., Dermacentor occidentalis, Ixodes pacificus, Ornithodoros coriaceus

Introduction

In northwestern California, certain subtypes of dense woodlands carpeted with leaf or fir-needle litter have been identified as principal foci for ticks and vertebrates that host the bacteria that cause Lyme disease (Borrelia burgdorferi) or human granulocytic anaplasmosis (Anaplasma phagocytophilum) (e.g., Clover and Lane, 1995; Eisen et al., 2003, 2004a, 2006, 2010; Foley et al., 2008, 2009; Girard et al., 2009; Lane et al., 2001, 2007; Tälleklint-Eisen and Lane, 1999). The primary vectors of B. burgdorferi and A. phagocytophilum to humans in this region are nymphs or adult females of the western black-legged tick (Ixodes pacificus) (Barlough et al., 1997; Burgdorfer et al., 1985; Clover and Lane, 1995; Kramer et al., 1997; Lane et al., 2007; Richter et al., 1995), but it is the nymphal stage that predominates in certain woodlands with relative densities sometimes reaching 32–56 ticks per 100 m2 (Lane et al., 2004; Tälleklint-Eisen and Lane, 2000).

Two other human-biting ticks that occur in Californian dense woodlands are the Pacific Coast tick (Dermacentor occidentalis) and the pajahuello tick (Ornithodoros coriaceus). Dermacentor occidentalis has been found infected naturally with a diverse suite of microorganisms including several well-known zoonotic pathogens: Colorado tick fever virus, A. phagocytophilum, B. burgdorferi, Coxiella burnetii, Ehrlichia chaffeensis, and Francisella tularensis (Enright et al., 1971; Holden et al., 2003; Kohls, 1955; Lane and Lavoie, 1988; Parker et al., 1929). Additionally, an unclassified rickettsia of the spotted fever group, designated 364D and associated with D. occidentalis, has been implicated as a cause of a Rocky Mountain spotted fever-like illness (Lane et al., 1981a; Philip et al., 1981) and, more recently, of an eschar-associated illness (Shapiro et al., 2010). O. coriaceus is a notorious human-biter known for causing systemic allergic reactions in some individuals and, in California during the early 20th Century, its bite reportedly was “…more feared than the rattlesnake by the natives…” (Herms, 1923). This tick is the primary vector of the relapsing-fever group spirochete Borrelia coriaceae (Johnson et al., 1987; Lane et al., 1985a), the etiologic agent of Q fever (C. burnetii) has been isolated from it (Enright et al., 1971), and it has been incriminated as a vector of a novel alpha-proteobacterium that is believed to be the cause of epizootic bovine abortion (King et al., 2005). Because of the spatial and temporal overlap of D. occidentalis and O. coriaceus populations with those of I. pacificus in woodland foci of Lyme disease and human granulocytic anaplasmosis, we initially sought to determine the relative abundance of host-seeking D. occidentalis versus O. coriaceus ticks in dense woodlands and their potential involvement as secondary vectors of either bacterium. We subsequently assessed the relative abundance of D. occidentalis adults in dense woodlands versus adjacent chaparral habitats, and their prevalence of infection with and capacity to experimentally transmit naturally acquired borreliae or Anaplasma spp. For comparative purposes, we also evaluated the relative abundance and prevalence of borrelial and Anaplasma infection in D. occidentalis nymphs and adults versus those of I. pacificus nymphs and adults from a dense woodland known to harbor both B. burgdorferi and A. phagocytophilum. The results of these multi-year studies are the subject of this report.

Materials and methods

The study area

This study was conducted in 8 dense woodlands located at the University of California Hopland Research and Extension Center (HREC) in Lake and Mendocino counties (39°0´35.0˝N, 123°4´35.9˝W), northwestern California, from 2003 to 2005 and in 2009 and 2010. The woodlands were composed of oaks (Quercus spp.) and Pacific madrone (Arbutus menziesii) with an understory of leaf litter and herbaceous vegetation as described earlier (Eisen et al., 2004b, 2009; Lane et al., 2004, 2007). The Beasley, Hunt Club, James II, and Maude sites were bordered primarily by chaparral, whereas the Cell Tower, Parson, Pepperwood, and Tank sites were bounded by grassland (Eisen et al., 2009). The chaparral was comprised of chamise (Adenostoma fasciculatum), oaks (Quercus spp.), California lilac (Ceanothus spp.), and other species. The patch-sizes that were sampled ranged from <0.40 ha (e.g., Hunt Club, Maude) to approximately 6.3 ha (James II). Further details of the James-II site, which was the only woodland site sampled for presence of ticks during all 5 years, have been published previously (Lane et al., 2004, 2005, 2007).

CO2-baited pitfall trapping, 2003

To determine the relative abundance of O. coriaceus nymphs and adults and of D. occidentalis adults by pitfall trapping in all 8 dense woodlands, 10 white enamelware pans (18×29×5 cm) were sunk 5 cm into the ground at approximately 10-m intervals on 12 sampling occasions spanning 13 June to 7 August. Each trap within the resultant ≈90-m transect-line was baited with a chunk of dry ice. The dry ice was elevated about 2 cm above the bottom of the pan on 2 wooden strips. The pans were examined for presence of ticks after one and 2 h of operation, and trapping was discontinued thereafter. All ticks found were preserved in 95% ethanol for later identification and testing by polymerase chain reaction (PCR). Two transects were set in 7 of the sites, whereas 5 transects were set in the James-II because it is a well-defined focus of both B. burgdorferi and A. phagocytophilum (Lane et al., 2004, 2005, 2007).

Flagging woodlands and chaparral, 2004 and 2005

Since few D. occidentalis adults were collected in all 8 dense woodlands in 2003 by pitfall trapping, preliminary sampling was carried out in the James-II woodland (one hour) on 26 May and adjoining chaparral (2 hours) on 27 May 2004 to determine if enough adult ticks could be taken by flagging these habitats to enable a more systematic investigation. In 2005, 4 dense woodlands bordered by chaparral (Beasley, Hunt Club, James II, Maude) were flagged on 5 occasions between 12 May and 4 June, as were their adjoining patches of chaparral on 4 occasions between 12 and 26 May. Sampling was conducted either during the morning (≈0800 to 1040 h) or mid- to late afternoon (1540 to 2045 h). Low numbers of I. pacificus adults also were collected from both habitat types, but those data are not presented here because our specific aim was to determine if D. occidentalis adults may serve as potential secondary vectors of B. burgdorferi or Anaplasma spp.

Flagging/dragging woodland, 2009 and 2010

The James-II woodland site was sampled on 5 occasions by flagging or dragging so that we could make a direct comparison of the relative abundance of, and the B. burgdorferi- versus Anaplasma spp. infection prevalences in, host-seeking D. occidentalis adults and nymphs with those of the primary vector, I. pacificus. In 2009, D. occidentalis and I. pacificus adults were collected by flagging between ≈0800 and 1030 h on 24 May and 7 June, I. pacificus nymphs by dragging between 1150 and 1435 h on 24 May, and D. occidentalis and I. pacificus nymphs by dragging between 0715 and 0930 h on 19 July. In 2010, I. pacificus and a few D. occidentalis adults were flagged between 1255 and 1530 h on 16 and 31 January. The latter sampling events were conducted to coincide more closely with the winter activity peak of I. pacificus adults at the HREC (Lane, 1990).

Attempts to culture B. burgdorferi

Because the 2004 pilot study produced several B. burgdorferi-infected D. occidentalis adults from the James-II woodland, we tried to isolate spirochetes from adult ticks flagged at the same site in 2005. Thirty-five host-seeking D. occidentalis adults (15 males, 20 females) collected on 12 or 25 May were surface-sterilized by vortexing them for 30 s with ≈5 ml each of hydrogen peroxide, 70% ethanol, and sterile phosphate-buffered saline. Next, ticks were dissected and their unminced tissues placed into 1.5-ml of BSK-H culture medium containing 50 µl of rifampicin in an Eppendorf tube. The cultures were incubated at 34.5°C and examined for presence of spirochetes by dark-field microscopy 6 times for one month at 400×.

Transmission trial

In an attempt to experimentally transmit either B. burgdorferi or Anaplasma spp. from potentially infected, wild-caught D. occidentalis adults to New Zealand white rabbits, 14 or 15 pairs of male and female ticks collected at the James-II site in 2005 were put on each of 4 naïve rabbits inside specially constructed tick-feeding capsules. Each rabbit was bled before tick exposure, and at 2, 4, and 6 weeks post-infestation. The whole-blood samples were frozen at −80°C until they were tested by PCR.

DNA extraction, and pathogen detection in ticks, cultures and rabbit blood

All biological specimens were tested individually for presence of B. burgdorferi sensu lato (s.l.) and Anaplasma spp. DNA was extracted from ticks or whole blood following previously published protocols (Lane et al., 2004, 2005). In 2005, not all D. occidentalis adults were tested by PCR to keep the sample sizes manageable, i.e., usually to minima of ≈30–50 individuals per site when large samples of ticks were obtained by flagging. Also, we used 140 ticks for the borrelial isolation attempts and the B. burgdorferi and Anaplasma spp. transmission trial. Details of the PCR assays and sequencing analysis followed those of Lane et al. (2004, 2005). Ear-punch biopsies from wild-caught western gray squirrels that had been infected with either B. burgdorferi or A. phagocytophilum, or B. burgdorferi isolate CA4 and A. phagocytophilum strain MRK, were employed as positive controls from 2003 to 2005 and 2009–2010, respectively. PCR water was employed as a negative control with each run. B. burgdorferi s.l. infection was assessed with a nested PCR format that specifically targets the 5S-23S rRNA spacer region (Lane et al., 2004). Anaplasma spp. infection was evaluated by targeting the 16S rRNA gene (Massung et al., 1998). Products from positive samples were purified using either the QIAquick PCR Purification Kit or QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Positive amplicons were sequenced on an ABI 3700 Sequencer (Applied Biosystems, Foster City, CA) or at the University of California, Berkeley, DNA Sequencing Facility using internal PCR primers. The contigs were compared with sequences available in GenBank using the software Sequencher 4 or 4.6 (Gene Code, Ann Arbor, MI).

Statistical analyses

In numerous pair-wise comparisons, McNemar’s test with continuity correction was used to determine the significant values for the prevalence of B. burgdorferi versus Anaplasma spp. infection in D. occidentalis adults within sites, and B. burgdorferi or Anaplasma spp. infection in adult ticks between sites, in either chaparral or woodland in 2004 and 2005. Likewise, the same test was used to compare the prevalence of the same bacteria in D. occidentalis or I. pacificus by life stage during each sampling occasion in 2009 or 2010; the prevalence of B. burgdorferi infection in I. pacificus ticks from the same generational cohort (spring 2009 nymphs versus winter 2009–2010 adults); the prevalence of B. burgdorferi infection in D. occidentalis versus I. pacificus nymphs in summer 2009; and the prevalence of Anaplasma spp. in D. occidentalis adults versus I. pacificus adults in spring 2009. No attempt was made to adjust for multiple statistical tests. The 5% level of probability was set for rejecting the null hypothesis, and only statistically significant probability values are presented below. Differences in the bacterial prevalence data for the D. occidentalis adults used during the transmission trial (Table 5) were not analyzed statistically because each group of those ticks had fed upon the same individual animals.

Table 5.

Results of experimental attempts to infect New Zealand white rabbits with B. burgdorferi or Anaplasma spp. by feeding potentially infected, wild-caught D. occidentalis adults on them.

Rabbit number Number of ticks
put on rabbita 		Number of replete ticks tested/
number PCR-positive (%) for:		 PCR test results of rabbit bloodb for:
B. burgdorferi Anaplasma spp. B. burgdorferi Anaplasma spp.
838 14m, 14f 0/22 0/22 negative negative
839 14m, 14f 0/27 0/27 negative negative
840 15m, 15f 0/27 1/27 (3.7)c negative negative
841 15m, 15f 0/29 2/29 (6.9)d negative negative
Total 58m, 58f 0/105 3/105 (2.9)
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a

m, male; f, female.

b

PCR was performed on blood specimens taken before and at 2, 4, and 6 weeks after rabbits had been infested with ticks.

c

Sequenced as A. bovis.

d

Both positive amplicons sequenced as A. ovis.

Results

CO2-baited pitfall trapping, 2003

In total, 190 traps yielded 34 O. coriaceus nymphs or adults and 12 D. occidentalis adults (Table 1); 79% of the O. coriaceus and 75% of the D. occidentalis ticks were collected at the James-II woodland. The remaining O. coriaceus were captured at one of the other woodlands bordered by chaparral (Beasley), whereas the D. occidentalis adults were caught at 2 sites bordered either by chaparral (Hunt Club) or grassland (Tank). The percentage of traps that produced ≥1 tick was similar for both tick species (7.4%, O. coriaceus; 4.7%, D. occidentalis), but the mean number of ticks collected per trap was 3 times higher for O. coriaceus than it was for D. occidentalis (Table 1).

Table 1.

Number of O. coriaceus and D. occidentalis ticks collected in carbon dioxide-baited pitfall traps in 8 dense woodlands bordered by chaparral or grassland, Hopland area, California, 13 June to 7 August 2003.

Woodland sitea Number of
traps set		 Number of ticks collected by
speciesb (stagec)		 Percentage of traps
yielding ≥1 tick		 Mean number of ticks/trap ±
SD
O-cor D-occ O-cor D-occ O-cor D-occ
Beasley 20 7 (n) 0 5.0 – 0.35±1.56 –
Hunt Club 20 0 1 (f) – 5.0 – 0.05±0.22
James II 50 27 (23n, 2m, 2f) 9 (3m, 6f) 26.0 12.0 0.54±1.51 0.18±0.63
Maude 20 0 0 – – – –
Cell Tower, Parson, Pepperwood, Tank 80 0 2 (m, f) – 2.5 – 0.03±0.17
Total 190 34 (30n, 2m, 2f) 12 (4m, 8f) 7.4 4.7 0.18±0.95 0.06±0.35
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a

Beasley, Hunt Club, James II and Maude were bordered by chaparral, the remaining four sites by grassland. Data for the grass-bordered sites were pooled because they yielded only two ticks.

b

O-cor, Ornithodoros coriaceus, D-occ, Dermacentor occidentalis.

c

n, nymph; m, male; f, female.

All individuals of both tick species were PCR-negative for Anaplasma spp., but one of 23 O. coriaceus nymphs from the James II produced a B. burgdorferi s.l.-positive PCR product that sequenced as B. bissettii. Although the 12 D. occidentalis adults also were tested for B. burgdorferi s.l., the results are not included here because of suspected contamination.

Flagging woodlands and chaparral, 2004–2005

Preliminary sampling of the adult D. occidentalis populations in the James-II woodland (one hour) and its adjoining chaparral (two hours) in 2004 produced 30 and 29 adults, respectively. Of these, 13.3% (4/30) from the woodland were infected with B. burgdorferi, but not with Anaplasma spp., whereas 3.4% (1/29) of ticks from chaparral were infected with A. phagocytophilum, but not with Borrelia spp.

These findings spurred a more in-depth examination of D. occidentalis as a potential vector of Borrelia and Anaplasma spp. the following year. In total, 587 adult ticks were flagged from 4 woodlands and their adjacent patches of chaparral and, of these, one-half were tested for presence of these bacteria (Tables 2 and 3). The relative abundance of ticks was 5 times greater in chaparral than in woodland (56.4 versus 11.2 ticks per h, Tables 2 and 3).

Table 2.

Relative abundance of D. occidentalis adults collected by flagging low vegetation in 4 dense woodlands, Hopland area, California, 12 May to 4 June 2005, and prevalence of infection with B. burgdorferi or Anaplasma spp. in 43% of those ticks.

Site (na) Number of ticks
collected by sexb 		Mean number
ticks per hour (range)		 Number of ticks PCR-positive/number tested
(percentage positive)		 Species of
Anaplasmac
B. burgdorferi Anaplasma spp.
Beasley (2) 49 (21m, 28f) 24.5 (12–37) 0/46 0/46 –
Hunt Club (3.75) 43 (21m, 22f) 11.5 (8–17)d 0/43 1/43 (2.3) Ao
James II (17) 203 (99m, 104f) 11.9 (3–21) 0/33 1/33 (3.0) Ab
Maude (4.5) 10 (6m, 4f) 2.2 (0–4) 0/10 1/10 (10.0) Ab
Total (27.25) 305 (147m, 158f) 11.2 (0–37) 0/132 3/132 (2.3) 2 Ab, 1 Ao
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a

Number of hours spent flagging at each site.

b

m, male; f, female.

c

Determined by sequencing analysis; Ab, A. bovis; Ao, A. ovis.

d

Number of ticks for one sample extrapolated from 0.75 hour.

Table 3.

Relative abundance of D. occidentalis adults collected by flagging chaparral abutting 4 dense woodlands, Hopland area, California, 12 to 26 May 2005, and prevalence of infection with B. burgdorferi or Anaplasma spp. in >50% of those ticks.

Site (na) Number of ticks
collected by sexb 		Number of ticks positive/number tested
(percentage positive)		 Species of
Anaplasmac
B. burgdorferi Anaplasma spp.
Beasley (2) 86 (47m, 39f) 0/41 2/41 (4.9) 2 Ab
Hunt Club (1) 77 (38m, 39f) 1/39 (2.6) 1/39 (2.6) Ao
James II (1) 38 (20m, 18f) 0/38 0/38 –
Maude (1) 81 (40m, 41f) 0/38 6/38 (15.8) 6 Ab
Total (5) 282 (145m, 137f) 1/156 (0.6) 9/156 (5.8) 8 Ab, 1 Ao
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a

Number of hours spent flagging each site.

b

m, male; f, female.

b

Determined by sequencing analysis; Ab, A. bovis; Ao, A. ovis.

In dense woodlands, B. burgdorferi was detected in zero, and Anaplasma spp. in 3 (2.3%) of 132 ticks. Sequencing revealed that 2 of the positive ticks were infected with A. bovis and one with A. ovis (Table 2). In chaparral, B. burgdorferi was detected in one (0.6%), and Anaplasma spp. in 9 (5.8%), of 156 ticks (p=0.026). Among the 4 individual sites, only Maude yielded a statistically significant pair-wise difference in B. burgdorferi versus Anaplasma spp. prevalence (p=0.041). Sequencing disclosed that 8 of the positive amplicons were A. bovis and one was A. ovis (Table 3).

Flagging/dragging woodland, 2009–2010

In spring 2009, more than twice as many D. occidentalis adults as compared with I. pacificus adults, and 21 times as many I. pacificus nymphs versus adult ticks were collected per hour (Table 4). Additionally, 1.6 times more D. occidentalis nymphs as compared with I. pacificus nymphs were caught in summer. In winter 2009–2010, the relative abundance of D. occidentalis and I. pacificus adults were 1/15th and 2.6 times greater than what they were in spring 2009, respectively.

Table 4.

Relative abundance of, and prevalence of B. burgdorferi or Anaplasma spp. infection in, D. occidentalis versus I. pacificus nymphs and adults collected by flagging low vegetation (adults) or dragging the forest floora (nymphs) in the James-II woodland, Hopland area, California, 2009–2010.

Tick species by
stage (nb)		 Season and yearc Number of ticks
collectedd 		Mean number
ticks per hour ± S.D.
(range)		 Number of ticks PCR-positive/number tested
(percentage positive)
B. burgdorferi Anaplasma spp.
D. occidentalis
  Adults (8) spring 2009 46 (19m, 27f) 5.6 ± 5.8 (1–19) 0/46 1/46 (2.2)e
  Adults (8) winter 2009–10 3 (1m, 2f) 0.4 ± 0.5 (0–1) 0/3 0/3
  Nymphs (4) summer 2009 39 9.8 ± 11.3 (0–20) 1/38 (2.6) 0/38
I. pacificus
  Adults (8) spring 2009 20 (13m, 7f) 2.5 ± 2.6 (0–8) 0/20 0/20
  Adults (8) winter 2009–10 51 (25m, 26f) 6.4 ± 4.4 (2–14) 1/51 (2.0) 0/51
  Nymphs (4) spring 2009 423 105.8 ± 54.8
(48–156)		 5/50 (10.0) 0/50
  Nymphs (4) summer 2009 24 6.0 ± 6.7
(2–16)		 2/24 (8.3) 0/24
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a

Primarily leaf litter, though wood (e.g., logs) occasionally was sampled.

b

Number of hours spent dragging or flagging.

c

Sampling for D. occidentalis and I. pacificus adults was performed on 24 May and 7 June 2009 and on 16 and 31 January 2010; for D. occidentalis nymphs on 19 July 2009; and for I. pacificus nymphs on 24 May and 19 July 2009. The collection and test results for the D. occidentalis adults in spring 2009 and for the I. pacificus adults in winter 2009–2010 were combined because the 2 sampling occasions during each of those seasons were separated by only 2 weeks.

d

m, male; f, female.

e

Sequenced as A. bovis.

None of the D. occidentalis or I. pacificus adults was PCR-positive for borreliae, whereas 2.2% of the D. occidentalis adults contained A. bovis in spring 2009. The Anaplasma spp. prevalence in D. occidentalis did not differ significantly from the zero prevalence for the 20 I. pacificus adults collected simultaneously. Among the nymphs, 2.6% of the D. occidentalis and 8.3–10.0% of the I. pacificus were infected with B. burgdorferi in spring or summer 2009. The prevalence of spirochete infection in the D. occidentalis nymphs (2.6%) versus that in the I. pacificus nymphs (8.3%) collected in summer did not differ significantly, nor did the prevalence in I. pacificus nymphs (10.0%, spring 2009) and adult ticks (2.0%, winter 2009–2010) from the same generational cohort (Table 4).

Attempts to culture B. burgdorferi

Borreliae were not cultured from the tissues of 35 D. occidentalis adults that had been placed in BSK-H medium.

Transmission trial

Four rabbits were exposed to the feeding activities of either 28 or 30 D. occidentalis, half of which were females (Table 5). Blood samples taken from each rabbit before and at 2, 4, or 6 weeks post-tick exposure were PCR-negative for both B. burgdorferi or Anaplasma spp. The number of fed or partially-fed ticks recovered from each rabbit ranged from 22 to 29. All 105 ticks were PCR-negative for B. burgdorferi, but one male tick (3.7%) from rabbit 840 was positive for A. bovis and 2 male ticks (6.9%) from rabbit 841 were infected with A. ovis.

Discussion

CO2 trapping studies, 2003

The use of carbon dioxide as a host-seeking attractant is the only method known to be suitable for efficiently collecting the soft tick O. coriaceus (Garcia, 1962; Hokama and Howarth, 1977). In the present study, nearly 200 h of C02-baited, pitfall-trapping in 8 dense woodlands yielded few O. coriaceus or D. occidentalis ticks, which suggests that oak-dominated dense woodlands are not a preferred habitat for either tick species. In preceding investigations aimed at determining the vector specificity of ticks for B. burgdorferi at the HREC, basically the same CO2-baited pitfall-trapping procedure was used to sample populations of O. coriaceus and, coincidentally, D. occidentalis (Lane and Manweiler, 1988; R.S. Lane, unpubl. data). In 1984, for example, 40 trapping sessions were conducted in each habitat type on 8 dates spanning 29 June to 6 September. The mean numbers of O. coriaceus nymphs/adults collected per trapping period in chaparral (8.05) and woodland-grass (2.28) were 45 and 13 times greater than the mean number (0.18) caught in dense woodlands in 2003 (R.S. Lane, unpubl. data; present study). Although the chaparral/woodland-grass collections were made 19 years earlier, we nevertheless posit that woodland-grass and particularly chaparral are more favored habitats than dense woodlands for the pajahuello tick in the Hopland area.

Moreover, the unpublished 1984 data for D. occidentalis from chaparral (mean, 0.13 ticks per trapping session) and woodland-grass (mean, 0.00) mirror the 2003 dense-woodland data (mean, 0.06) from the current study. Similarly, Garcia (1962) noted that CO2 trapping yielded much lower frequencies of D. occidentalis adults than those of O. coriaceus in chaparral-covered foothills in the San Francisco Bay region. Our findings and those of Garcia (1962) demonstrate that CO2 trapping is ineffective for determining the relative abundance of the Pacific Coast tick in these habitat types.

Flagging woodlands and chaparral, 2004–2005

Not surprisingly, flagging produced many more D. occidentalis adults per timed-sampling effort than pitfall trapping in dense woodlands. The actual area covered by flagging on an hourly basis was orders of magnitude greater than pitfall trapping, the effective radius of CO2 trapping for collecting D. occidentalis adults was just 4 m in chaparral (Lane et al., 1985b), and some of the D. occidentalis adults attracted to pitfall traps escaped (Hokama and Howarth, 1977; R.S. Lane, pers. observations). In 2005, a mean of 11.2 D. occidentalis adults was flagged hourly from the 4 dense woodlands sampled, which was higher than the mean numbers of D. occidentalis ticks typically flagged per hour (≤10.4) in northern California, except from chaparral (Lane, 1992; Lane and Lavoie, 1988; present study).

Flagging woodland, 2009–2010

In spring 2009, the relative abundance of D. occidentalis adults in the James II was one-half what it was in spring 2005 but 15 times greater than it was in January 2010. At the HREC, D. occidentalis adults are active in chaparral during winter and spring with a peak in March (Lane, 1990). If the seasonal peak activity period of D. occidentalis adults is similar in dense woodlands and chaparral, we likely missed the acme of host-seeking in both habitat types and in all years by having sampled for adult Pacific Coast ticks in January, May, and June. The collection of 9.8 D. occidentalis nymphs per hour by dragging the forest floor in mid-July is the highest relative abundance of host-seeking D. occidentalis nymphs ever recorded from any habitat. Previously, Castro et al. (1997) collected 23 Dermacentor spp. nymphs from 3 (chaparral, Douglas fir, oak woodland) of 4 habitats sampled semi-monthly by flagging over a one-year period in neighboring Sonoma County, California. Twenty (87%) of the nymphs were flagged from chaparral. Subsequently, Castro et al. (2001; M.B. Castro, pers. comm.) collected an average of 0.4 D. occidentalis nymphs per hour by flagging leaf litter in 2 oak-woodland habitats (designated sites B and C) in the same county.

The overall hourly means recorded for I. pacificus adults from the James-II site on paired dates in spring 2009 (i.e., 2.5) and winter 2009–2010 (6.4) are on the low side compared with previous studies from other habitats in which up to an average of 106.5 adult ticks were collected per hour (Lane, 1992; Lane and Lavoie, 1988). This probably represents a true difference in the size of adult-tick populations present in dense woodlands versus the other habitat types, though it may reflect the months or time-of-day when samples were taken or a dearth of suitable vegetative perches for host seeking in the James-II woodland. At the HREC, I. pacificus adults are active in chaparral from January through May, with a pronounced peak in early March and a slight peak in November (Lane, 1990). Since we sampled specifically for I. pacificus adults in January, we may have inadvertently bypassed the annual peak-activity period for this tick, too. As anticipated (Lane et al., 2004), the James-II population of I. pacificus nymphs was high (106 ticks per h) in late May, low in mid-July when its seasonal activity period was winding down, and inactive in January.

Prevalence of B. burgdorferi s.l. in ticks

In northwestern California, host-seeking D. occidentalis or O. coriaceus have been evaluated before as possible secondary vectors of B. burgdorferi, but to our knowledge not in dense woodlands carpeted with leaf litter (e.g., Barlough et al., 1997; Kramer et al., 1999; Lane, 1992, 1996; Lane and Lavoie, 1988; Lane and Manweiler, 1988; Lane et al., 1985a, 2004, 2005; Nicholson et al., 1999). Among hundreds of host-seeking D. occidentalis adults collected from chaparral, grassland, or woodland-grass in several studies (reviewed by Lane, 1996), only 0.8% (2/253) D. occidentalis adults flagged from chaparral at the HREC tested positive for B. burgdorferi with monoclonal antibodies by indirect immunofluorescence (Lane and Lavoie, 1988).

Overall, 0.9% (5/536) of the D. occidentalis adults and 2.6% (1/38) of the nymphs we collected from woodlands or chaparral in all years except 2003 were found to be infected with B. burgdorferi by 3 methods (culture, PCR alone, or PCR post-feeding on rabbits). Similarly, 0.8% (3/353) of D. occidentalis adults collected from recreational areas in central coastal California were PCR positive for B. burgdorferi (Holden et al., 2003). By comparison, 9.5% (7/74) of the I. pacificus nymphs and 1.4% (1/71) of the adults collected during spring, summer, or winter in 2009/2010 were PCR positive for B. burgdorferi.

We conclude that D. occidentalis larvae or nymphs occasionally acquire and transstadially pass B. burgdorferi after having fed on reservoir hosts in dense woodlands (Lane et al., 2005; Salkeld et al., 2008). For example, one of 5 D. occidentalis larvae removed from western gray squirrels at the HREC was infected with B. burgdorferi (Lane et al., 2005). The viability, infectivity, and transmissibility of B. burgdorferi in or by D. occidentalis adults remain unknown because all ticks used in isolation attempts (n=35) or recovered from rabbits (n=105) during the transmission trial were not infected with borreliae. However, several vector competence studies, and a seroepidemiologic investigation using anti-arthropod saliva antibodies as a biologic marker for tick exposure in a rural community at high risk for Lyme disease, demonstrated that this tick is an inefficient experimental or natural vector of B. burgdorferi (Brown and Lane, 1992; Lane et al., 1994, 1999; Li and Lane, 1996).

Although the pajahuello tick (O. coriaceus) is the primary vector of the relapsing-fever group spirochete B. coriaceae, we report here for the first time the detection of a Lyme disease group spirochete (B. bissettii) in this human-biter or, for that matter, in any Ornithodoros species, by PCR. The infected nymph was collected in the James-II woodland, a highly enzootic site for the human pathogen B. burgdorferi (Lane et al., 2007). This woodland, and 3 others we investigated (Beasley, Hunt Club, Maude), are bordered largely by chaparral wherein exists a B. bissettii transmission cycle driven primarily by I. spinipalpis ticks, dusky-footed wood rats (Neotoma fuscipes), and Peromyscus spp. mice (Eisen et al., 2009). Likewise, host-seeking I. pacificus nymphs inhabiting such woodlands at the HREC and elsewhere in Mendocino County occasionally are infected with B. bissettii, as was an I. pacificus larva removed from a western gray squirrel in the James-II woodland (Eisen et al., 2004a, 2009; Lane et al., 2005).

In an earlier study at the HREC, tissue smears prepared from 5.6% (1/18) spirochete-infected O. coriaceus ticks collected from either chaparral or woodland–grass habitats reacted with a monoclonal antibody (H5332) believed to be specific for B. burgdorferi (Lane and Manweiler, 1988). Thus, O. coriaceus occasionally acquires and maintains B. burgdorferi s.l., and therefore this tick cannot be ruled out as a potential secondary vector of Lyme disease group spirochetes pending the results of experimental transmission studies.

To our knowledge, the only other soft ticks that have been implicated in the ecology or epidemiology of B. burgdorferi s.l. are either bat- or bird-feeding parasites in the genus Argas (A. reflexus, A. vespertilionis) (Genchi et al., 1989; Hubbard et al., 1998; Stanek and Simeoni, 1989). In Europe, circumstantial evidence suggests that the pigeon tick (A. reflexus) occasionally may transmit B. burgdorferi s.l. to people inside buildings where pigeons roost (Genchi et al., 1989; Stanek and Simeoni, 1989).

I. pacificus nymphs inhabiting the James-II woodland had a 5-fold higher prevalence of borreliae (10%, n=50, spring 2009) than did adult ticks (2%, n=51, winter 2009–2010) from the same generational cohort. Similarly, the combined infection prevalence with B. burgdorferi s.l. was 5-fold higher in nymphs (7.4%) than in adult ticks (1.6%) from the same generational cohort in 2 woodlands at the HREC (Eisen et al., 2004c). The adult ticks were collected in ecotones bordering the leaf-litter areas. Reservoir-incompetent and spirochete-cleansing western fence lizards are primary hosts of I. pacificus immatures in certain woodlands including the James II, which presumably accounts for much of the reduction in B. burgdorferi infection prevalence from the nymphal to adult-tick stage (Eisen et al., 2004b; Lane et al., 2007).

Prevalence of A. phagocytophilum in ticks

A. phagocytophilum was not detected in the 34 O. coriaceus or 12 D. occidentalis collected from dense woodlands in 2003, but the following year one (3.4%) of 29 D. occidentalis adults from chaparral and none of 30 adults from woodland were infected with this rickettsia during the pilot study. In 2005, none of 288 D. occidentalis adults from chaparral or dense woodland was infected, nor were 105 adult ticks from the James II that had been put on rabbits. In 2009–2010, 49 D. occidentalis adults likewise were PCR-negative for A. phagocytophilum. Hence, just one (0.2%) of 513 D. occidentalis adults tested from both habitats in all years contained A. phagocytophilum. In central coastal California, A. phagocytophilum was detected in a slightly higher percentage (1.1%, 4/353) of host-seeking D. occidentalis adults (Holden et al., 2003). In other Californian studies involving lower sample sizes, host-seeking D. occidentalis adults invariably were PCR-negative for A. phagocytophilum [e.g., Barlough et al., 1997 (n=57 ticks); Kramer et al., 1999 (n=162); Nicholson et al., 1999 (n=9)].

Taken together, these findings, like those presented above for B. burgdorferi, suggest that D. occidentalis is not a primary vector of A. phagocytophilum, and its involvement in the transmission cycles of both bacteria is tangential to that of I. pacificus (Lane et al., 2007; present study). Although all 145 I. pacificus nymphs or adults tested from the James II during 2009 or 2010 were PCR-negative for A. phagocytophilum, an earlier study carried out there revealed that up to 15.6% of the I. pacificus nymphs from various biotopes (e.g., logs) are infected with it in certain years (Lane et al., 2007). This finding suggests that year-to-year transmission cycles of A. phagocytophilum are either highly focal or variable within this particular woodland.

Prevalence of A. bovis and A. ovis in ticks

Our detection of A. bovis and A. ovis in D. occidentalis ticks are the first such associations for these veterinary pathogens. Prior to 2003, A. bovis had been recognized primarily as a parasite of cattle and buffalo in Africa, the Middle East, and South America, and A. ovis as a parasite of domestic sheep and goats worldwide and bighorn sheep in North America (Davidson and Goff, 2001; Goethert and Telford, 2003; Goff et al., 1993; Uilenberg, 1997). Since then, the known geographic and vertebrate host/vector tick distributions of A. bovis or closely related rickettsiae have expanded to include cottontail rabbits (Sylvilagus floridanus), rabbit ticks (Haemaphysalis leporispalustris), and Ixodes spp. in North America (Goethert and Telford, 2003), Haemaphysalis ticks from Thailand (Parola et al., 2003), Korea (Kim et al., 2003), and Russia (Shpynov et al., 2006), and cattle, sika deer (Cervus nippon nippon, C. nippon yesoensis), and Haemaphysalis ticks from Japan (Kawahara et al., 2006; Jilintai et al., 2009).

Similarly, A. ovis has been detected recently in an increasing number of wild ruminants around the globe (e.g., de la Fuente et al., 2007, 2008; Haigh et al., 2008; Yabsley et al., 2005). Neither rickettsia has been considered to be a zoonotic pathogen, but Chochlakis et al. (2010) presented intriguing molecular evidence that a patient from Cyprus who experienced a febrile illness accompanied by hepatosplenomegaly and lymphadenopathy was infected with A. ovis. Here, we incriminate D. occidentalis as a potentially important vector of A. bovis. The high prevalence of A. bovis infection (4.3%, n=185 ticks) within chaparral-derived ticks in 2004 and 2005 suggests that D. occidentalis could be an efficient vector of this rickettsia. The occasional presence of this rickettsia in Pacific Coast ticks in adjacent woodlands may represent spill-over from reservoir hosts and vector ticks that predominate in chaparral. In the northeastern and southern United States, lagomorphs (cottontails, Sylvilagus spp.; black-tailed jack rabbits, Lepus californicus) and the ubiquitous rabbit tick H. leporispalustris have been implicated in the enzootiology of A. bovis (Goethert and Telford, 2003; Yabsley et al., 2006). Although the enzootic cycle of A. bovis awaits clarification in the Far West, brush rabbits (Sylvilagus bachmani) and black-tailed jack rabbits are resident at the HREC, they overlap spatially in chaparral, and they are infested by both H. leporispalustris and D. occidentalis (Furman and Loomis, 1984; Lane and Burgdorfer, 1988; Lane et al., 1981b).

D. occidentalis also is the most likely vector of A. ovis in northwestern California given earlier experimental data and our molecular evidence demonstrating that this tick can transmit A. ovis intrastadially (D. Stiller, unpublished, cited by Davidson and Goff, 2001) and transstadially (present study), respectively. Previously, D. hunteri, a parasite mainly of bighorn sheep (Ovis canadensis) and occasionally of mule deer (Odocoileus hemionus), was the suspected vector of A. ovis in the western United States (Stiller et al., 1999; Yabsley et al., 2005). A. ovis has been detected in mule deer in the western United States (de la Fuente et al., 2007; Yabsley et al., 2005), but collection records of D. hunteri ticks from California are restricted to the arid southeastern region inhabited by desert bighorn sheep (Furman and Loomis, 1984). At the HREC, the only ungulates present are domestic sheep and a subspecies of mule deer (Columbian black-tailed deer, O. hemionus columbianus). D. occidentalis seldom parasitizes sheep, but the adult ticks abundantly infest Columbian black-tailed deer (Furman and Loomis, 1984; Westrom, 1975). This cervid reportedly is susceptible to infection with A. marginale, deer exposed to this rickettsia develop long-term rickettsemias without manifesting clinical illness, and naturally infected D. occidentalis adults can transmit A. marginale to cattle (Davidson and Goff, 2001; Howarth and Roby, 1972; Howarth et al., 1969).

A caveat concerning Anaplasma studies conducted before the 1990s was that calf inoculation, Giemsa-stained blood smears, and serologic methods were used to diagnose such infections in wild ungulates (Howarth et al., 1969; Kuttler, 1984) because DNA probes and monoclonal antibodies had not been developed yet. Accordingly, some of the Anaplasma infections attributed to A. marginale in deer in California (e.g., Howarth and Roby, 1972; Howarth et al., 1969) may have represented other Anaplasma spp. Indeed, A. bovis and A. ovis infections were not detected in wild deer until recently (de la Fuente et al., 2007, 2008; Haigh et al., 2008; Kawahara et al., 2006; Yabsley et al., 2005). In California, the diagnostic dilemma is obfuscated further by the presence of 2 additional Anaplasma spp. in deer, i.e., A. phagocytophilum (reported as Ehrlichia equi) and Anaplasma sp. (reported as Ehrlichia-like sp. of white-tailed deer) (Foley et al., 1998).

Transmission trial

Our initial attempts to experimentally transmit Anaplasma spp. and B. burgdorferi from potentially infected, wild-caught D. occidentalis adults to 4 naïve rabbits were unsuccessful. Three adult male ticks recovered from 2 of the rabbits were PCR-positive for either A. bovis or A. ovis, whereas none of the ticks was discovered to contain B. burgdorferi. Our findings suggest that the New Zealand white rabbit may not be susceptible to infection with either A. bovis or A. ovis, or if rabbits develop a rickettsemia, it may be transient, of low titer, or occur more than 6 weeks after the attachment of an infectious tick. D. occidentalis is a competent experimental vector of the bovine and deer rickettsia A. marginale (referenced by Davidson and Goff, 2001), and males of the Rocky Mountain wood tick (Dermacentor andersoni) are primary vectors of A. marginale to cattle. Moreover, D. andersoni males become persistently infected, feed intermittently, freely transfer among cattle, and therefore can transmit the infection to more than one animal (Kocan et al., 1992a, 1992b). A similar infection cycle has been described for A. ovis in D. andersoni males (Kocan and Stiller, 1992). We chose rabbits instead of mice as our laboratory animal model because D. occidentalis adults rarely attach to wild rodents, but they readily feed upon rabbits both in the field and laboratory (Lane and Burgdorfer, 1988; Casher et al., 2002; present study).

Conclusions

We and other researchers have demonstrated that in dense woodlands in northwestern California, D. occidentalis occasionally acquires and maintains B. burgdorferi and A. phagocytophilum, and that O. coriaceus sometimes acquires and transstadially passes B. burgdorferi s.l. Experimental proof that these ticks are efficient vectors of either agent is lacking, however. On the other hand, D. occidentalis adults, particularly in chaparral, may be primary vectors of the veterinary pathogens A. bovis and A. ovis. The 5-fold greater prevalence of B. burgdorferi infection in I. pacificus nymphs as compared with the adults from the same generational cohort in 2009–2010 reconfirms antecedent observations, but the absence of A. phagocytophilum in the nymphs contravenes recent findings from the same woodland suggesting that nidi of this bacterium are either highly focal or variable from year to year.

Acknowledgements

We thank J.E. Kleinjan, E. Omi-Olsen, and M.I. Yasuda for technical assistance, J.E. Foley for providing A. phagocytophilum strain MRK, and M.B. Castro and K.M. Kocan for helpful discussions and relevant literature. This research was supported by grant AI22501 from the National Institutes of Health (NIH) to R.S.L., by a grant from the Sec. De Estado de Universidades e Investigacion del MEC to M.A.P., and by the use of the Louisiana State University Museum of Natural Science sequencing facility. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Footnotes

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References

  1. Barlough JE, Madigan JE, Kramer VL, Clover JR, Hui LT, Webb JP, Vredevoe LK. Ehrlichia phagocytophila genogroup rickettsiae in ixodid ticks from California collected in 1995 and 1996. J. Clin. Microbiol. 1997;35:2018–2021. doi: 10.1128/jcm.35.8.2018-2021.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown RN, Lane RS. Lyme disease in California: a novel enzootic transmission cycle of Borrelia burgdorferi. Science. 1992;256:1439–1442. doi: 10.1126/science.1604318. [DOI] [PubMed] [Google Scholar]
  3. Burgdorfer W, Lane RS, Barbour AG, Gresbrink RA, Anderson JR. The western black-legged tick, Ixodes pacificus: a vector of Borrelia burgdorferi. Am. J. Trop. Med. Hyg. 1985;34:925–930. doi: 10.4269/ajtmh.1985.34.925. [DOI] [PubMed] [Google Scholar]
  4. Casher L, Lane R, Barrett R, Eisen L. Relative importance of lizards and mammals as hosts for ixodid ticks in northern California. Exp. Appl. Acarol. 2002;26:127–143. doi: 10.1023/a:1020911306291. [DOI] [PubMed] [Google Scholar]
  5. Castro MB, Hui LT, Keith RD, Dill CH. The distribution of ticks in four distinct habitats in a nature preserve in Sonoma County, California. Proc. Mosq. Vector Control Assoc. Calif. 1997;65:42–47. [Google Scholar]
  6. Castro MB, Nicholson WL, Kramer VL, Childs JE. Persistent infection in Neotoma fuscipes (Muridae: Sigmodontinae) with Ehrlichia phagocytophila sensu lato. Am. J. Trop. Med. Hyg. 2001;65:261–267. doi: 10.4269/ajtmh.2001.65.261. [DOI] [PubMed] [Google Scholar]
  7. Chochlakis D, Ioannou I, Tselentis Y, Psaroulaki A. Human anaplasmosis and Anaplasma ovis variant. Emerg. Infect. Dis. 2010;16:1031–1032. doi: 10.3201/eid1606.090175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clover JR, Lane RS. Evidence implicating nymphal Ixodes pacificus (Acari: Ixodidae) in the epidemiology of Lyme disease in California. Am. J. Trop. Med. Hyg. 1995;53:237–240. doi: 10.4269/ajtmh.1995.53.237. [DOI] [PubMed] [Google Scholar]
  9. Davidson WR, Goff WL. Anaplasmosis. In: Williams ES, Barker IK, editors. Infectious Diseases of Wild Mammals. third ed. Ames, Iowa: Iowa State Press; 2001. pp. 455–466. [Google Scholar]
  10. de la Fuente J, Atkinson MW, Naranjo V, Fernández de Mera IG, Mangold AJ, Keating KA, Kocan KM. Sequence analaysis of the msp4 gene of Anaplasma ovis strains. Vet. Microbiol. 2007;119:375–381. doi: 10.1016/j.vetmic.2006.09.011. [DOI] [PubMed] [Google Scholar]
  11. de La Fuente J, Ruiz-Fons F, Naranjo V, Torina A, Rodríguez O, Gortázar C. Evidence of Anaplasma infections in European roe deer (Capreolus capreolus) from southern Spain. Res. Vet. Sci. 2008;84:382–386. doi: 10.1016/j.rvsc.2007.05.018. [DOI] [PubMed] [Google Scholar]
  12. Eisen L, Eisen RJ, Chang C-C, Mun J, Lane RS. Acarologic risk of exposure to Borrelia burgdorferi spirochaetes: long-term evaluations in north-western California, with implications for Lyme borreliosis risk-assessment models. Med. Vet. Entomol. 2004a;18:38–49. doi: 10.1111/j.1365-2915.2004.0476.x. [DOI] [PubMed] [Google Scholar]
  13. Eisen L, Eisen RJ, Lane RS. The roles of birds, lizards, and rodents as hosts for the western black-legged tick Ixodes pacificus. J. Vector Ecol. 2004b;29:295–308. [PubMed] [Google Scholar]
  14. Eisen L, Eisen RJ, Mun J, Salkeld DJ, Lane RS. Transmission cycles of Borrelia burgdorferi and B. bissettii in relation to habitat type in northwestern California. J. Vector Ecol. 2009;34:81–91. doi: 10.3376/038.034.0110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eisen RJ, Eisen L, Castro MB, Lane RS. Environmentally related variability in risk of exposure to Lyme disease spirochetes in northern California: effect of climatic conditions and habitat type. Environ. Entomol. 2003;32:1010–1018. [Google Scholar]
  16. Eisen RJ, Eisen L, Girard YA, Fedorova N, Mun J, Slikas B, Leonhard S, Kitron U, Lane RS. 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. 2010;1:35–43. doi: 10.1016/j.ttbdis.2009.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eisen RJ, Lane RS, Fritz CL, Eisen L. Spatial patterns of Lyme disease risk in California based on disease incidence data and modeling of vector-tick exposure. Am. J. Trop. Med. Hyg. 2006;75:669–676. [PubMed] [Google Scholar]
  18. Eisen RJ, Mun J, Eisen L, Lane RS. 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. 2004c;41:768–773. doi: 10.1603/0022-2585-41.4.768. [DOI] [PubMed] [Google Scholar]
  19. Enright JB, Franti CE, Behymer DE, Longhurst WM, Dutson VJ, Wright ME. Coxiella burneti in a wildlife-livestock environment. Distribution of Q fever in wild animals. Am. J. Epidemiol. 1971;94:79–90. doi: 10.1093/oxfordjournals.aje.a121298. [DOI] [PubMed] [Google Scholar]
  20. Foley JE, Barlough JE, Kimsey RB, Madigan JE, DeRock E, Poland A. Ehrlichia spp. in cervids from California. J. Wildl. Dis. 1998;34:731–737. doi: 10.7589/0090-3558-34.4.731. [DOI] [PubMed] [Google Scholar]
  21. Foley JE, Nieto NC, Adjemian J, Dabritz H, Brown RN. Anaplasma phagocytophilum infection in small mammal hosts of Ixodes ticks, western United States. Emerg. Infect. Dis. 2008;14:1147–1150. doi: 10.3201/eid1407.071599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Foley JE, Nieto NC, Foley P. Emergence of tick-borne granulocytic anaplasmosis associated with habitat type and forest change in northern California. Am. J. Trop. Med. Hyg. 2009;81:1132–1140. doi: 10.4269/ajtmh.2009.09-0372. [DOI] [PubMed] [Google Scholar]
  23. Furman DP, Loomis EC. The ticks of California (Acari: Ixodida) Bull. Calif. Insect Surv. 1984;25:1–239. [Google Scholar]
  24. Garcia R. Carbon dioxide as an attractant for certain ticks (Acarina: Argasidae and Ixodidae) Ann. Entomol. Soc. Amer. 1962;55:605–606. [Google Scholar]
  25. Genchi C, Magnino S, Pacetti A, Simeoni J, di Sacco B, Falagiani P. Argas reflexus, possible vettore di Borrelia burgdorferi. Giornale di Malattie Infettive e Parassitarie. 1989;41:403–408. [Google Scholar]
  26. Girard YG, Travinsky B, Schotthoefer A, Fedorova N, Eisen RJ, Eisen L, Barbour AG, Lane RS. Population structure of the Lyme borreliosis spirochete Borrelia burgdorferi in the western black-legged tick (Ixodes pacificus) in northern California. Appl. Environ. Microbiol. 2009;75:7243–7252. doi: 10.1128/AEM.01704-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Goethert HK, Telford SR., III Enzootic transmission of Anaplasma bovis in Nantucket cottontail rabbits. J. Clin. Microbiol. 2003;41:3744–3747. doi: 10.1128/JCM.41.8.3744-3747.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Goff W, Stiller D, Jessup D, Msolla P, Boyce W, Foreyt W. Characterization of an Anaplasma ovis isolate from desert bighorn sheep in southern California. J. Wildl. Dis. 1993;29:540–546. doi: 10.7589/0090-3558-29.4.540. [DOI] [PubMed] [Google Scholar]
  29. Haigh JC, Gerwing V, Erdenebaatar J, Hill JE. A novel clinical syndrome and detection of Anaplasma ovis in Mongolian reindeer (Rangifer tarandus) J. Wildl. Dis. 2008;44:569–577. doi: 10.7589/0090-3558-44.3.569. [DOI] [PubMed] [Google Scholar]
  30. Herms WB. Medical and Veterinary Entomology. second ed. New York: The MacMillan Company; 1923. [Google Scholar]
  31. Hokama Y, Howarth JA. Dry-ice (C02) trap for efficient field collection of Ornithodoros coriaceus (Acarina: Argasidae) J. Med. Entomol. 1977;13:627–628. doi: 10.1093/jmedent/13.4-5.627. [DOI] [PubMed] [Google Scholar]
  32. Holden K, Boothby JT, Anand S, Massung RF. Detection of Borrelia burgdorferi, Ehrlichia chaffeensis, and Anaplasma phagocytophilum in ticks (Acari: Ixodidae) from a coastal region of California. J. Med. Entomol. 2003;40:534–539. doi: 10.1603/0022-2585-40.4.534. [DOI] [PubMed] [Google Scholar]
  33. Howarth JA, Roby TO. Transmission of anaplasmosis by field collections of Dermacentor occidentalis Marx (Acarina: Ixodidae) Proc. 76th Annu. Mtg. U.S. Anim. Health Assoc. 1972:98–102. [Google Scholar]
  34. Howarth JA, Roby TO, Amerault TE, McNeal DW. Prevalence of Anaplasma marginale infection in California deer as measured by calf inoculation and serologic techniques. Proc. 73rd Annu. Mtg. U.S. Anim. Health Assoc. 1969:136–147. [PubMed] [Google Scholar]
  35. Hubbard MJ, Baker AS, Cann KJ. Distribution of Borrelia burgdorferi s.l. spirochaete DNA in British ticks (Argasidae and Ixodidae) since the 19th Century, assessed by PCR. Med. Vet. Entomol. 1998;12:89–97. doi: 10.1046/j.1365-2915.1998.00088.x. [DOI] [PubMed] [Google Scholar]
  36. Jilintai, Seino N, Hayakawa D, Suzuki M, Hata H, Kondo S, Matsumoto K, Yokoyama N, Inokuma H. Molecular survey for Anaplasma bovis and Anaplasma phagocytophilum infection in cattle in a pastureland where sika deer appear in Hokkaido, Japan. Jpn. J. Infect. Dis. 2009;62:73–75. [PubMed] [Google Scholar]
  37. Johnson RC, Burgdorfer W, Lane RS, Barbour AG, Hayes SF, Hyde FW. Borrelia coriaceae sp. nov.: putative agent of epizootic bovine abortion. Int. J. System. Bacteriol. 1987;37:72–74. [Google Scholar]
  38. Kawahara M, Rikihisa Y, Lin Q, Isogai E, Tahara K, Itagaki A, Hiramitsu Y, Tajima T. Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Appl. Environ. Microbiol. 2006;72:1102–1109. doi: 10.1128/AEM.72.2.1102-1109.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kim C-M, Kim M-S, Park M-S, Park J-H, Chae J-S. Identification of Ehrlichia chaffeensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea. Vector-Borne Zoonotic Dis. 2003;3:17–26. doi: 10.1089/153036603765627424. [DOI] [PubMed] [Google Scholar]
  40. King DP, Chen CI, Blanchard MT, Aldridge BM, Anderson M, Walker R, Maas J, Hanks D, Hall M, Stott JL. Molecular identification of a novel deltaproteobacterium as the etiologic agent of epizootic bovine abortion (foothill abortion) J. Clin. Microbiol. 2005;43:604–609. doi: 10.1128/JCM.43.2.604-609.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kocan KM, Stiller D. Development of Anaplasma ovis (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred from infected to susceptible sheep. J. Med. Entomol. 1992;29:98–107. doi: 10.1093/jmedent/29.1.98. [DOI] [PubMed] [Google Scholar]
  42. Kocan KM, Goff WL, Stiller D, Claypool PL, Edwards W, Ewing SA, Hair JA, Barron SJ. Persistence of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred successively from infected to susceptible calves. J. Med. Entomol. 1992a;29:657–668. doi: 10.1093/jmedent/29.4.657. [DOI] [PubMed] [Google Scholar]
  43. Kocan KM, Stiller D, Goff WL, Claypool PL, Edwards W, Ewing SA, McGuire TC, Hair JA, Barron SJ. Development of Anaplasma marginale in male Dermacentor andersoni transferred from parasitemic to susceptible cattle. Am. J. Vet. Res. 1992b;53:499–507. [PubMed] [Google Scholar]
  44. Kohls GM. Colorado tick fever discovered in California. Calif. Vector Views. 1955;2:17. [Google Scholar]
  45. Kramer VL, Randolph MP, Hui LT, Irwin WE, Gutierrez AG, Vugia DJ. Detection of the agents of human ehrlichioses in ixodid ticks from California. Am. J. Trop. Med. Hyg. 1999;60:62–65. doi: 10.4269/ajtmh.1999.60.62. [DOI] [PubMed] [Google Scholar]
  46. Kuttler KL. Anaplasma infections in wild and domestic ruminants: a review. J. Wildl. Dis. 1984;20:12–20. doi: 10.7589/0090-3558-20.1.12. [DOI] [PubMed] [Google Scholar]
  47. Lane RS. Seasonal activity of two human-biting ticks. Calif. Agric. March–April. 1990:23–25. [Google Scholar]
  48. Lane RS. Borrelia burgdorferi in ticks on Angel Island, California: low risk of human infection. Bull. Soc. Vector Ecol. 1992;17:75–82. [Google Scholar]
  49. Lane RS. Risk of human exposure to vector ticks (Acari: Ixodidae) in a heavily used recreational area in northern California. Am. J. Trop. Med. Hyg. 1996;55:165–173. [PubMed] [Google Scholar]
  50. Lane RS, Burgdorfer W. Spirochetes in mammals and ticks (Acari: Ixodidae) from a focus of Lyme borreliosis in California. J. Wildl. Dis. 1988;24:1–9. doi: 10.7589/0090-3558-24.1.1. [DOI] [PubMed] [Google Scholar]
  51. Lane RS, Lavoie PE. Lyme borreliosis in California: acarological, clinical, and epidemiological studies. Ann. N.Y. Acad. Sci. 1988;539:192–203. doi: 10.1111/j.1749-6632.1988.tb31853.x. [DOI] [PubMed] [Google Scholar]
  52. Lane RS, Manweiler SA. Borrelia coriaceae in its tick vector, Ornithodoros coriaceus (Acari: Argasidae), with emphasis on transstadial and transovarial infection. J. Med. Entomol. 1988;25:172–177. doi: 10.1093/jmedent/25.3.172. [DOI] [PubMed] [Google Scholar]
  53. Lane RS, Anderson JR, Yaninek JS, Burgdorfer W. Diurnal host seeking of adult Pacific Coast ticks, Dermacentor occidentalis (Acari: Ixodidae), in relation to vegetational type, meteorological factors, and rickettsial infection rates in California, USA. J. Med. Entomol. 1985b;22:558–571. doi: 10.1093/jmedent/22.5.558. [DOI] [PubMed] [Google Scholar]
  54. Lane RS, Brown RN, Piesman J, Peavey CA. Vector competence of Ixodes pacificus and Dermacentor occidentalis (Acari: Ixodidae) for various isolates of Lyme disease spirochetes. J. Med. Entomol. 1994;31:417–424. doi: 10.1093/jmedent/31.3.417. [DOI] [PubMed] [Google Scholar]
  55. Lane RS, Burgdorfer W, Hayes SF, Barbour AG. Isolation of a spirochete from the soft tick, Ornithodoros coriaceus: a possible agent of epizootic bovine abortion. Science. 1985a;230:85–87. doi: 10.1126/science.3898367. [DOI] [PubMed] [Google Scholar]
  56. Lane RS, Emmons RW, Dondero DV, Nelson BC. Ecology of tick-borne agents in California. I. Spotted fever group rickettsiae. Am. J. Trop. Med. Hyg. 1981b;30:239–252. doi: 10.4269/ajtmh.1981.30.239. [DOI] [PubMed] [Google Scholar]
  57. Lane RS, Foley JE, Eisen L, Lennette ET, Peot MA. Acarologic risk of exposure to emerging tick-borne bacterial pathogens in a semirural community in northern California. Vector-Borne Zoonotic Dis. 2001;1:197–210. doi: 10.1089/153036601753552567. [DOI] [PubMed] [Google Scholar]
  58. Lane RS, Moss RB, Hsu Y-P, Wei T, Mesirow ML, Kuo MM. Anti-arthropod saliva antibodies among residents of a community at high risk for Lyme disease in California. Am. J. Trop. Med. Hyg. 1999;61:850–859. doi: 10.4269/ajtmh.1999.61.850. [DOI] [PubMed] [Google Scholar]
  59. Lane RS, Mun J, Eisen RJ, Eisen L. Western gray squirrel (Rodentia: Sciuridae): A primary reservoir host of Borrelia burgdorferi in Californian oak woodlands? J. Med. Entomol. 2005;42:388–396. doi: 10.1093/jmedent/42.3.388. [DOI] [PubMed] [Google Scholar]
  60. Lane RS, Mun J, Peribáñez MA, Stubbs HA. Host-seeking behavior of Ixodes pacificus (Acari: Ixodidae) nymphs in relation to environmental parameters in dense-woodland and woodland-grass habitats. J. Vector Ecol. 2007;32:342–357. doi: 10.3376/1081-1710(2007)32[342:hboipa]2.0.co;2. [DOI] [PubMed] [Google Scholar]
  61. Lane RS, Philip RN, Casper EA. Ecology of tick-borne agents in California. II. Further observations on rickettsiae. In: Burgdorfer W, Anacker RL, editors. Rickettsiae and Rickettsial Diseases. New York: Academic Press; 1981a. pp. 575–584. [Google Scholar]
  62. Lane RS, Steinlein DB, Mun J. Human behaviors elevating exposure to Ixodes pacificus (Acari: Ixodidae) nymphs and their associated bacterial zoonotic agents in a hardwood forest. J. Med. Entomol. 2004;41:239–248. doi: 10.1603/0022-2585-41.2.239. [DOI] [PubMed] [Google Scholar]
  63. Li X, Lane RS. Vector competence of ixodid ticks (Acari) for Borrelia burgdorferi as determined with a capillary-feeding technique. J. Spirochetal Tick-borne Dis. 1996;3:116–123. [Google Scholar]
  64. Massung RF, Slater K, Owens JH, Nicholson WL, Mather TN, Solberg VB, Olson JG. Nested PCR assay for detection of granulocytic ehrlichiae. J. Clin. Microbiol. 1998;36:1090–1095. doi: 10.1128/jcm.36.4.1090-1095.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Nicholson WL, Castro MB, Kramer VL, Sumner JW, Childs JE. Dusky-footed wood rats (Neotoma fuscipes) as reservoirs of granulocytic ehrlichiae (Rickettsiales: Ehrlichieae) in northern California. J. Clin. Microbiol. 1999;37:3323–3327. doi: 10.1128/jcm.37.10.3323-3327.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Parker RR, Brooks CS, Marsh H. The occurrence of Bacterium tularense in the wood tick, Dermacentor occidentalis, in California. Public Health Rep. 1929;44:1299–1300. [Google Scholar]
  67. Parola P, Cornet J-P, Sanogo YO, Miller RS, Thien HV, Gonzalez J-P, Raoult D, Telford SR, III, Wongsrichanalai C. Detection of Ehrlichia spp., Anaplasma spp., and other Eubacteria in ticks from the Thai-Myanmar border and Vietnam. J. Clin. Microbiol. 2003;41:1600–1608. doi: 10.1128/JCM.41.4.1600-1608.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Philip RN, Lane RS, Casper EA. Serotypes of tick-borne spotted fever group rickettsiae from western California. Am. J. Trop. Med. Hyg. 1981;30:722–727. doi: 10.4269/ajtmh.1981.30.722. [DOI] [PubMed] [Google Scholar]
  69. Richter PJ, Jr, Kimsey RB, Madigan JE, Barlough JE, Dumler JS, Brooks DL. Ixodes pacificus (Acari: Ixodidae) as a vector of Ehrlichia equi (Rickettsiales: Ehrlichieae) J. Med. Entomol. 1996;33:1–5. doi: 10.1093/jmedent/33.1.1. [DOI] [PubMed] [Google Scholar]
  70. Salkeld DJ, Leonhard S, Girard YA, Hahn N, Mun J, Padgett KA, Lane RS. 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. 2008;79:535–540. [PMC free article] [PubMed] [Google Scholar]
  71. Shapiro MR, Fritz CL, Tait K, Paddock CD, Nicholson WL, Abramowicz KF, Karpathy SE, Dasch GA, Sumner JW, Adem PV, Scott JJ, Padgett KA, Zaki SR, Eremeeva ME. Rickettsia 364D: a newly recognized cause of eschar-associated illness in California. Clin. Infect. Dis. 2010;50:541–548. doi: 10.1086/649926. [DOI] [PubMed] [Google Scholar]
  72. Shpynov S, Fournier P-E, Rudakov N, Tarasevich I, Raoult D. Detection of members of the genera Rickettsia, Anaplasma, and Ehrlichia in ticks collected in the Asiatic part of Russia. Ann. N.Y. Acad. Sci. 2006;1078:378–383. doi: 10.1196/annals.1374.075. [DOI] [PubMed] [Google Scholar]
  73. Stanek G, Simeoni J. Are pigeons’ ticks transmitters of Borrelia burgdorferi to humans? A preliminary report. In: Stanek G, Kristoferitsch W, Pletschette M, Barbour AG, Flamm H, editors. Lyme Borreliosis II. Zentralblatt Bakteriol. Microbiol. Hyg. Vol. 18. 1989. pp. 42–43. [Google Scholar]
  74. Stiller D, Crosbie PR, Boyce WM, Goff WL. Dermacentor hunteri (Acari: Ixodidae): an experimental vector of Anaplasma marginale and A. ovis (Rickettsiales: Anaplasmataceae) to calves and sheep. J. Med. Entomol. 1999;36:321–324. doi: 10.1093/jmedent/36.3.321. [DOI] [PubMed] [Google Scholar]
  75. Tälleklint-Eisen L, Lane RS. Variation in the density of questing Ixodes pacificus (Acari: Ixodidae) nymphs infected with Borrelia burgdorferi at different spatial scales in California. J. Parasitol. 1999;85:824–831. [PubMed] [Google Scholar]
  76. Tälleklint-Eisen L, Lane RS. Spatial and temporal variation in the density of Ixodes pacificus (Acari: Ixodidae) nymphs. Environ. Entomol. 2000;29:272–280. [Google Scholar]
  77. Uilenberg G. General review of tick-borne diseases of sheep and goats world-wide. Parassitologia. 1997;39:161–165. [PubMed] [Google Scholar]
  78. Westrom DR. Ph.D. Dissertation. Berkeley: Univ. Calif; 1975. The population dynamics and distribution of ectoparasites on black-tailed beer (Odocoileus hemionus columbianus) in Mendocino County, California; p. 184. [Google Scholar]
  79. Yabsley MJ, Davidson WR, Stallknecht DE, Varela AS, Swift PK, Devos JC, Jr, Dubay SA. Evidence of tick-borne organisms in mule deer (Odocoileus hemionus) from the western United States. Vector-Borne Zoonotic Dis. 2005;5:351–362. doi: 10.1089/vbz.2005.5.351. [DOI] [PubMed] [Google Scholar]
  80. Yabsley MJ, Romines J, Nettles VF. Detection of Babesia and Anaplasma species in rabbits from Texas and Georgia, USA. Vector-Borne Zoonotic Dis. 2006;6:7–13. doi: 10.1089/vbz.2006.6.7. [DOI] [PubMed] [Google Scholar]

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