Western Gray Squirrel (Rodentia: Sciuridae): A Primary Reservoir Host of Borrelia burgdorferi in Californian Oak Woodlands?

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1 VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION Western Gray Squirrel (Rodentia: Sciuridae): A Primary Reservoir Host of Borrelia burgdorferi in Californian Oak Woodlands? ROBERT S. LANE, 1 JEOMHEE MUN, REBECCA J. EISEN, AND LARS EISEN Division of Insect Biology, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA J. Med. Entomol. 42(3): 388Ð396 (2005) ABSTRACT In California, dense woodlands have been recognized as important biotopes where humans are exposed to the nymphal stage of the western blacklegged tick, Ixodes pacificus Cooley & Kohls, the primary vector of the Lyme disease spirochete Borrelia burgdorferi sensu stricto (s.s.), in the far-western United States. To identify the principal reservoir host(s) of this spirochete, and of closely related spirochetes in the B. burgdorferi sensu lato (s.l.) complex, in dense woodlands in Mendocino County, California, 50 species of birds and mammals, including wood rats and kangaroo rats, were evaluated as potential hosts for vector ticks and borreliae in 2002 and Although polymerase chain reaction (PCR) and sequencing analyses revealed that many vertebrate species had been exposed to one or more members of the B. burgdorferi s.l. spirochetal complex, only the western gray squirrel, Sciurus griseus, fulþlled the major criteria for a reservoir host of B. burgdorferi s.s. Ear-punch biopsies from eight of 10 squirrels collected from Þve separate woodlands were PCRpositive for B. burgdorferi s.s., 47% of I. pacificus larvae (n 64) and 31% of nymphs (n 49) removed from squirrels contained B. burgdorferi s.l., and the engorgement status of I. pacificus larvae was associated positively with acquisition of spirochetes. Overall, 83 and 100% of the amplicons sequenced from PCR-positive I. pacificus larvae and nymphs, respectively, were identiþed as B. burgdorferi s.s. Among the Þve remaining positive I. pacificus larvae, three contained B. bissettii and two had uncharacterized B. burgdorferi s.l. Borrelia burgdorferi s.s. was detected in one of Þve larvae and zero of two nymphs of the PaciÞc Coast tick, Dermacentor occidentalis Marx, that likewise had been removed from squirrels. The rickettsial agent of human anaplasmosis, Anaplasma phagocytophilum, was detected in the blood or ear biopsies of two squirrels and in one (1.6%) of 64 I. pacificus larvae and two (4.1%) of 49 nymphs obtained from squirrels. The one rickettsial-positive larva was coinfected with B. burgdorferi s.s. The apparently high reservoir potential of S. griseus for B. burgdorferi s.s., plus the fact that the geographic distribution of this squirrel coincides well with that of most reported human cases of Lyme disease in this region, indicated that it may be essential for maintaining foci of B. burgdorferi s.s. in certain types of woodlands. The Þndings with respect to A. phagocytophilum, although of less certain signiþcance, suggest that S. griseus could serve as a secondary host of this rickettsia. KEY WORDS Sciurus griseus, Borrelia burgdorferi, Anaplasma phagocytophilum, Ixodes pacificus, Dermacentor occidentalis Borrelia burgdorferi sensu stricto (s.s.), or closely related described or uncharacterized genospecies in the Lyme disease spirochetal complex, B. burgdorferi sensu lato (s.l), have been detected in or isolated from at least 32 species of birds and mammals in California. These include 11 species of rodents, two species of lagomorphs, three species of deer, and 16 species of birds (Lane and Burgdorfer 1986, 1988; Lane 1990; Lane and Brown 1991; Lane and Loye 1991; Brown and Lane 1992, 1996; Gordus and Theis 1993; Peavey et al. 1997; Wright et al. 2000; Vredevoe et al. 2004; M. A. Peot, R. N. Brown, and R.S.L., unpublished data). Of 1 Corresponding author, blane@nature.berkeley.edu these, Þve species of rodents have been implicated previously as amplifying or reservoir hosts of B. burgdorferi s.l., i.e., the dusky-footed wood rat, Neotoma fuscipes; the California kangaroo rat, Dipodomys californicus; the brush mouse, Peromyscus boylii; the deer mouse, Peromyscus maniculatus; and the piñon mouse, Peromyscus truei (Lane and Brown 1991; Brown and Lane 1992, 1996; Lane et al. 1994, 1999; Peavey and Lane 1995; Eisen et al. 2003a). Reservoir, as used here, refers to the ability of a vertebrate host to become infected with and maintain a zoonotic agent long enough to serve as a source of infection for uninfected vector ticks that feed on it. Reservoir hosts increase the number of infected ticks in a given area, and their /05/0388Ð0396$04.00/ Entomological Society of America

2 May 2005 LANE ET AL.: GRAY SQUIRRELS AND LYME DISEASE SPIROCHETES 389 infectivity may persist for life or last for only a few days (Kahl et al. 2002). In northwestern California, B. burgdorferi was thought to be maintained in an enzootic cycle involving the nonhuman-biting tick Ixodes spinipalpis Hadwen & Nuttall (formerly I. neotomae); the duskyfooted wood rat; and, within more restricted habitats, the California kangaroo rat (Brown and Lane 1992). In fact, 102 of the Þrst 105 B. burgdorferi s.l. isolates obtained from wildlife in this region were cultivated from ear-punch biopsies taken from these rodents (Brown and Lane 1992). Isolates were identiþed by their reactivities to six monoclonal antibodies by indirect immunoßuorescence and by SDS-polyacrylamide gel electrophoresis. Infected wood rats typically were associated with chaparral or woodlands, whereas infected kangaroo rats were caught in chaparral or grassland bordering chaparral (Lane and Brown 1991, Brown and Lane 1996, Lane et al. 1999). However, a recent molecular and phylogenetic analysis of isolates of B. burgdorferi s.l. from mammals revealed that only Þve (22%) of 23 isolates from duskyfooted wood rats and one (5%) of those from 20 California kangaroo rats actually were B. burgdorferi. In contrast, 18 (78%) of the isolates from wood rats were identiþed as B. bissettii, and 19 (95%) from kangaroo rats represented uncharacterized Borrelia spp. (M. A. Peot, R. N. Brown, and R.S.L., unpublished data). Recently, dense woodlands carpeted with leaf litter have been identiþed as important biotopes of, and elevated risk habitats for, human exposure to nymphs of the western blacklegged tick, Ixodes pacificus Cooley & Kohls (Clover and Lane 1995; Tälleklint- Eisen and Lane 1999, 2000; Lane et al. 2001, 2004; Eisen et al. 2003b, 2004a). This tick is the primary vector of B. burgdorferi s.s. (hereinafter referred to as B. burgdorferi unless designated otherwise) to people in the far-western United States (Burgdorfer et al. 1985, Lane and Lavoie 1988). In 2002, an initial attempt to identify a small mammalian reservoir for B. burgdorferi in dense woodlands throughout inland areas of Mendocino County, California, yielded just one infected rodent (a deer mouse) out of 201 Peromyscus spp. and 19 wood rats (N. fuscipes) examined (unpublished data). Comparably low spirochetal infection prevalences were detected in Peromyscus spp. and N. fuscipes inhabiting a mixed hardwood forest in the western Sierra Nevada foothills of northern California (Wright et al. 2000). Both studies suggested that wood rats, Peromyscus spp., and, because of their absence, kangaroo rats, are not the primary reservoirs of B. burgdorferi in certain subtypes of Californian hardwoods. In 2003, an intensive effort was made to determine the reservoir host(s) of B. burgdorferi in dense woodlands and in adjoining chaparral or grasslands in the Hopland area of southeastern Mendocino County, California (unpublished data). Concomitantly, we sought to determine the reservoir of another, albeit much less frequently recognized, tick-transmitted human pathogen within the same habitats, i.e., the rickettsia causing human granulocytic anaplasmosis (Anaplasma phagocytophilum). Besides rodents, other small mammals and birds were evaluated concurrently as potential hosts for B. burgdorferi s.l., A. phagocytophilum and ixodid ticks. Here, we report our Þndings concerning the western gray squirrel, Sciurus griseus, which was the only animal implicated as a primary reservoir host of B. burgdorferi. The apparently lesser role of this rodent in the transmission cycle of A. phagocytophilum also is discussed brießy. Materials and Methods Study Area. This investigation was conducted at the 2,168-ha University of California Hopland Research and Extension Center, which is located on the western slopes of the Mayacmas Mountains in the Russian River Valley. Rolling hills interspersed with ravines characterize the topography, which is covered principally with grassland, woodlandðgrass, dense woodland, and chaparral. The climate is Mediterranean with hot, dry summers and cool, moist winters. Western gray squirrels, hereinafter referred to as squirrels, were collected from Þve dense woodlands located at elevations ranging between 280 and 830 m. The principal tree species present in these woodlands included PaciÞc madrone, Arbutus menziesii Pursh; California black oak, Quercus kelloggii Newb.; and interior live oak, Quercus wislizenii A.DC. Collection and Processing of Samples. Ten adult squirrels (four males and six females) were collected by shooting from 16 May to 3 July Use of squirrels in this research was reviewed and approved by the Animal Care and Use Committee at the University of California at Berkeley and by the California Department of Fish and Game. Squirrels were placed individually in sealed plastic bags after collection to prevent escape of any associated ectoparasites until they could be processed. Age, sex, and reproductive status were determined for each animal. Blood samples were taken by cardiac puncture after the sternal and thoracic regions had been soaked externally with 70% ethanol. Blood was allowed to clot, transferred to the laboratory on wet ice, and spun down by centrifugation. The resultant clot and serum from each specimen were stored at 80 C until they could be tested by polymerase chain reaction (PCR). Additionally, multiple 2-mm ear-punch biopsies were obtained from each pinna after the ears had been surface-sterilized sequentially with 10% povidone-iodine (Betadine solution, The Purdue Frederick Company, Norwalk, CT) and 70% ethanol. Last, the entire body of each animal was examined for 20 min, and all ectoparasites found were put into 1.5-ml Eppendorf tubes containing 95% ethanol. Subadult ticks were identiþed to species by using published taxonomic keys and descriptions (Furman and Loomis 1984). To determine whether the prevalence of bacterial infection in attached I. pacificus ticks removed from each squirrel was related to the degree of tick engorgement as well as to the infection status of the host, an engorgement index (EI) was calculated for each

3 390 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 larval and nymphal tick tested. Our use of this index follows that of Falco et al. (1996), who expressed it as the ratio between body length and scutum width. An ocular micrometer was used to measure the length of the body dorsally from the base of the basis capitulum to the tip of the abdomen, and the width of the scutum at its widest point. Larval and nymphal ticks having an EI of 2.5 had swelled slightly and those with an index of 3.0Ð3.6 had engorged moderately. None of the ticks removed from squirrels had fed to repletion. For comparative purposes, wild-caught, host-seeking larvae (n 5) and nymphs (n 5) preserved in 95% ethanol were measured to determine the indices for unfed subadults. Likewise, Þve nearly replete larvae and nymphs removed from Þeld-derived western fence lizards, Sceloporus occidentalis, were measured after they, too, had been preserved in 95% ethanol. Borrelial Isolation Attempts. In efforts to isolate B. burgdorferi s.l., one to two drops of cardiac blood from all 10 squirrels, and two ear-punch biopsies from each of eight squirrels, were put into Eppendorf tubes containing 1.5 ml of BSK-H medium (Sigma, St. Louis, MO) with 25 l of rifampicin per milliliter. Cultures were incubated at 34 C and examined for presence of spirochetes at 400 magniþcation weekly for 1 mo by dark-þeld microscopy. Positive cultures were stored in glycerol stocks ( 25% glycerol by volume) at 80 C. DNA Extraction and Pathogen Detection in Isolates, Skin Biopsies, Blood, and Ticks. Blood clots and serum specimens from all 10 squirrels, two ear-punch biopsies per squirrel, and 120 immature ixodid ticks of two species (I. pacificus, 113; Dermacentor occidentalis, 7) removed from infested animals were tested individually for presence of B. burgdorferi s.l. and A. phagocytophilum. DNA was extracted from ticks following previously published methods (Lane et al. 2004). DNA was extracted from ear-punch biopsies by using the Qia DNeasy kit (QIAGEN, Valencia, CA) with a few modiþcations. Two ear-punch biopsies per squirrel that had been stored in 95% ethanol were placed in a 1.5-ml tube and allowed to air dry before adding ATL buffer. DNA extracts were prepared from 50 l of serum and 50 l of clotted blood from each animal. To prepare borrelial isolates for identiþcation, the frozen stocks were thawed and then grown in 5 ml of BSK-H medium. Next, the cultures were spun down three times at 14,000 g for 10 min apiece, the supernatants were discarded, and the pellets were washed with sterile phosphate-buffered saline. After the third supernatant was removed, 100 l oflte buffer (10 mm Tris, ph 8.0; 0.1 mm EDTA; 0.1% Tergitol) was added, and the resultant suspension was vortexed and then boiled for 10 min. After each suspension was vortexed once more, it was frozen at 20 C until PCR was performed. Details of the PCR assays and sequencing followed those of Lane et al. (2004) with a few modiþcations. Ear-punch biopsies from wild-caught squirrels that had been infected with either B. burgdorferi (squirrel 1) or A. phagocytophilum (squirrel 5) were used as positive controls, and PCR water was used as a negative control, with each run. B. burgdorferi s.l. infection was assessed with a nested PCR format that speciþcally targets the 5S-23S rrna spacer region (Lane et al. 2004). A. phagocytophilum infection was evaluated by targeting the 16S rrna gene (Massung et al. 1998). The amplicons were sequenced on an ABI 3730 Sequencer (Applied Biosystems, Foster City, CA). The sequences were aligned using the software Sequencher 4 (Gene Code, Ann Arbor, MI). Statistical Analyses. The association between the EI and B. burgdorferi s.l. infection prevalence in I. pacificus subadults was evaluated with the Wilcoxon test (two-tailed). Borrelial infection prevalences were compared within life stages and among engorgement categories by using either the 2 test when numbers permitted or FisherÕs exact test. The homogeneity of borrelial infection prevalences between stages and across EI categories was tested with the MantelÐHaenszel test before performing individual FisherÕs exact tests within EI categories. Results Tick Burdens and EIs. Totals of 83 larvae and 51 nymphs of I. pacificus, and Þve larvae and two nymphs of D. occidentalis, were collected from the 10 squirrels. Thus, the mean numbers ( SD, range) of subadult ticks per squirrel were 8.3 (12.1, 0Ð37) for larval and 5.1 (4.2, 0Ð14) for nymphal I. pacificus and 0.5 (0.8, 0Ð2) for larval and 0.2 (0.4, 0Ð1) for nymphal D. occidentalis. The EIs for I. pacificus subadults removed from squirrels ranged from 1.4 to 3.3 for larvae and from 1.8 to 5.0 for nymphs. For unfed, host-seeking ticks, the unengorged indices averaged (range 1.6Ð 1.7) for the larvae and (range 1.8Ð2.1) for the nymphs. By comparison, the EIs averaged (range 3.7Ð4.2) and (range 5.1Ð5.5) for almost fully replete larvae and nymphs, respectively, that had been removed from wild-caught western fence lizards at one of the study sites (James II). Isolation of Spirochetes. Freshly removed earpunch biopsies (EPBs) from three of eight squirrels yielded spirochetal isolates in BSK-H medium, but efforts to cultivate spirochetes from the whole blood of all 10 squirrels were unsuccessful. Spirochetes were observed in positive cultures within 1Ð2 wk of Þeld collection (Table 1). PCR Results. EPBs from eight of 10 squirrels were positive for B. burgdorferi, one was positive for A. phagocytophilum, and none was coinfected (Table 1). EPBs from eight squirrels (numbers 1 and 4Ð10) were tested for borreliae by both PCR and culture. PCR was more sensitive than the culture method for detecting spirochetes, and the concordance between the two methods was 62.5%. SpeciÞcally, only three of six animals whose ear biopsies were PCR-positive also yielded isolates, and two (squirrels 5 and 6) were negative by both assays (Table 1). Sequencing analyses revealed that spirochetes present in the three

4 May 2005 LANE ET AL.: GRAY SQUIRRELS AND LYME DISEASE SPIROCHETES 391 Table 1. Detection of A. phagocytophilum and B. burgdorferi s.l. in western gray squirrels by PCR analyses of blood specimens, ear-punch biopsies, and naturally attached I. pacificus larvae Squirrel no. a Clot Serum EPB b I. pacificus larvae I. pacificus larvae Clot Serum EPB (no. positive/no. tested) (no. positive/no. tested) A. phagocytophilum B. burgdorferi s.l. 1 NA NA 2 (1/21) (4/21) 3 (0/9) (3/9) 4 (0/19) c (18/19) 5 (0/3) (1/3) 6 (0/2) (1/2) 7 (0/9) c (3/9) 8 (0/1) c (0/1) 9 NA NA 10 NA NA Data for nymphal ticks are excluded here because positive ticks could have acquired their infections while feeding previously in the larval stage on a different host. NA, not applicable. a Squirrels were collected at Þve sites designated S1 pasture (squirrel 1), James II (2, 3, and 9), Hunt Club (4 and 5), Tank (6 and 7), and Beasley (10). b Ear-punch biopsies had been preserved in 95% ethanol before testing. c Additional ear-punch biopsies (n 2) removed from these squirrels immediately post mortem yielded isolates of B. burgdorferi 7Ð10 d after they had been put into BSK-H medium. Similar isolation attempts were made from other squirrels, except 2 and 3. isolates and in DNA extracts from alcohol-preserved EPBs from the same animals were B. burgdorferi. None of the 10 serum specimens was positive for either B. burgdorferi s.l. or A. phagocytophilum, but one of the clots contained A. phagocytophilum (Table 1). Overall, 47% (30/64) of I. pacificus larvae removed from seven squirrels contained B. burgdorferi s.l. (Table 2) notwithstanding the fact that 61% (39/64) of them had fed little, if at all (i.e., had an EI of 1.4Ð1.8 compared with a mean index of 1:6 for an unfed larva). Assuming that infected larvae had acquired spirochetes from their hosts and not transovarially (see Discussion), testing attached ticks was somewhat more sensitive for detecting the presence of spirochetes than was the EPB method. Of seven squirrels (squirrels 2Ð8) that were infested with larvae, six animals yielded one or more infected larvae and the remaining animal (squirrel 8) was parasitized by a single uninfected larva (Table 1). The latter squirrel was infested with three nymphs, however, two of which contained B. burgdorferi s.l. (Table 2). In contrast, EPBs taken from only Þve of these seven squirrels were PCRpositive for B. burgdorferi s.l. (Tables 1 and 2). The prevalence of spirochetal infection in I. pacificus larvae removed from Þve of the eight PCR-positive squirrels (47%, n 59) was similar to that for the few larvae removed from the two EPB-negative animals (40%, n 5) (Table 1). Sequencing analyses of the 30 PCR-positive I. pacificus larvae revealed that the amplicons contained B. burgdorferi (n 25), B. bissettii (n 3), or uncharacterized B. burgdorferi s.l. (n 2). The two squirrels whose EPBs were negative by both culture and PCR each yielded a larva that was infected with either an uncharacterized B. burgdorferi s.l. (squirrel 5) or B. bissettii (squirrel 6). The three B. bissettii-positive amplicons originated from larvae collected from two widely spaced woodlands, James II (n 1) and Tank (n 2). The former site comprises a black oakðmadrone forest that abuts on chaparral and grassland, Table 2. Percentage of I. pacificus larvae and nymphs removed from wild-caught western gray squirrels that were PCR-positive for B. burgdorferi s.l. in relation to their engorgement status No. larvae positive/no. tested by EI b No. nymphs positive/no. tested by EI Squirrel no. Infection status a 1.4Ð Ð Ð Ð Ð Ð Ð /1 1/1 2 1/17 2/3 1/1 0/6 1/3 0/3 3 1/5 1/3 1/1 0/1 1/6 2/4 4 2/3 10/10 5/5 1/1 1/1 2/3 1/1 5 1/3 6 1/1 0/1 0/2 0/2 7 3/9 0/2 0/1 8 0/1 1/2 1/1 9 0/3 0/1 0/1 10 1/1 3/3 Totals (% positive) 9/39 (23) 13/17 (76) 6/6 (100) 2/2 (100) 1/3 (33) 6/26 (23) 4/11 (36) 4/9 (44) Of the 30 positive amplicons from larvae, 25 were identiþed as B. burgdorferi s.s., three as B. bissettii, and two as uncharacterized B. burgdorferi s.l. Of the 15 positive amplicons from nymphs, 13 were identiþed as B. burgdorferi s.s. and two could not be sequenced. a Determined by PCR analyses of ear-punch biopsies (see Table 1). b EI is the ratio of the length of the tick (i.e., from the posteromedial margin of the basis capitulum to the tip of the abdomen) to the greatest width of the scutum.

5 392 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 whereas the latter contains a large rock outcrop and is located adjacent to a natural pond. One of the uncharacterized B. burgdorferi s.l. also was detected in a tick from the James II site, and the other was from a site (Hunt Club) that borders chaparral. By comparison, the Þve I. pacificus larvae that were removed from the two squirrels (5 and 6) that had a PCR-positive EPB or clot for A. phagocytophilum tested negative for this rickettsia, whereas one (1.7%) of 59 larvae removed from Þve of the eight squirrels having PCR-negative biopsies or clots was infected (Table 1). The latter larva, which had fed moderately (EI of 3.2), was coinfected with B. burgdorferi, which was the only tick found to be infected dually. Fifteen (31%) of 49 I. pacificus nymphs were infected with B. burgdorferi s.l. (Table 2). All of the positive amplicons contained B. burgdorferi except for two that were not sequenced successfully. Two (4%) I. pacificus nymphs were infected solely with A. phagocytophilum. B. burgdorferi was detected in one of Þve larvae and in zero of two nymphs of D. occidentalis obtained from squirrels. The infected larva had been removed from a squirrel (10) that was EPB-positive for B. burgdorferi and parasitized by four I. pacificus nymphs that contained the same spirochete. Prevalence of Borreliae in Ticks by Engorgement Status and Life Stage. Consistent with the observed trend of increasing prevalence of infection among larvae that had imbibed more blood (Table 2), infected larvae had signiþcantly higher EIs than uninfected larvae (P ). Thus, 23% of the 39 I. pacificus larvae that had an EI of 1.8 were infected versus 84% of those (n 25) having an EI of 1.9 (Table 2). When the data for the larvae having EIs of 1.8 are broken down, the prevalence of infection among larvae that had EIs ranging from 1.4 to 1.6 (23.8%, n 21) versus from 1.7 to 1.8 (22.2%, n 18) were similar. Notably, an EI of 1.6 is at or below the average for an unfed, host-seeking I. pacificus larva. In contrast, the EIs among infected nymphs were not signiþcantly greater than they were among uninfected nymphs (P 0.28; Table 2). Although large, the difference in the overall infection prevalence for larvae (47%) versus nymphs (31%) was not statistically signiþcant (P 0.08; 2 test). A test of homogeneity across EI categories indicated a lack of consistent differences between stages (P 0.04; MantelÐHaenszel test). The individual comparisons of infection prevalence between stages within EI categories revealed signiþcantly higher infection prevalences in larvae having EIs of 1.9Ð2.4 (P ; FisherÕs exact test) and 2.5Ð3.0 (P ; FisherÕs exact test) in contrast to nymphs. Discussion Squirrels as Hosts of Borreliae. Several criteria have been used by medical entomologists to implicate a vertebrate species as a reservoir host of a vector-borne zoonotic agent (DeFoliart et al. 1987, Lane 1994, Kahl et al. 2002). To be considered a reservoir of a tickborne agent, a vertebrate minimally must be infected with the agent with some frequency in nature; it must be fed upon by at least two trophic stages (often the larva and nymph) of a vector tick; and it must retain its infectivity long enough so that it can serve as a source of infection for uninfected ticks that subsequently feed on it. The western gray squirrel satisþes these criteria: at least 80% of squirrels from Þve separate dense woodlands were infected with B. burgdorferi; squirrels had abundant contact with both the larvae and nymphs of a proven primary vector tick, I. pacificus; 47% of naturally acquired larval ticks that had attached to, and fed partially on, squirrels contained B. burgdorferi; and acquisition of spirochetes was associated positively with the degree of engorgement of attached larval ticks. Behavioral factors also contribute to the reservoir potential of S. griseus. Squirrels spend a considerable amount of time foraging on the ground, especially during the morning, and most of their young leave their arboreal nests about mid-april (Ingles 1947, Carraway and Verts 1994). I. pacificus nymphs are active during the morning and afternoon over a broad range of temperatures and relative humidities (Lane et al. 2004), and the weaning period of juvenile squirrels coincides with the time of year when host-seeking activities by I. pacificus larvae and nymphs in leaf litter areas are rising or peaking in northwestern California (Eisen et al. 2001, 2003b). Little is known about the longevity of S. griseus, and studies are needed to investigate the duration of B. burgdorferi infection in squirrels along with other aspects of their reservoir potential. In captivity, squirrels have been held for as long as 11 yr (Ross 1930). The western gray squirrel is the Þrst vertebrate restricted spatially to woodlands, a heterogeneous mixture of habitats in which humans can be at risk of exposure to B. burgdorferi-infected I. pacificus nymphs (Clover and Lane 1995; Tälleklint-Eisen and Lane 1999; Eisen et al. 2003b, 2004a; Lane et al. 2004), to be incriminated as a primary reservoir host of this spirochete in the far-western United States. S. griseus is composed of three subspecies (S. g. anthonyi, griseus, and nigripes) that collectively range from north central Washington southward to Baja California (Carraway and Verts 1994, Jameson and Peeters 2004). S. g. griseus, which was evaluated in the current study, is by far the most widespread subspecies, and its distribution closely parallels that of human Lyme disease cases throughout much of California. In Mendocino County, squirrels were observed at varying frequencies in oak woodlands and oakðdouglas Þr woodlands but not in redwoodðtan oak woodlands, during widespread surveys of I. pacificus nymphs and their small vertebrate hosts between May and July 2002Ð2003 (L.E., R.J.E., and R.S.L., unpublished data). All eight squirrels whose EPBs were PCR-positive, and isolates cultivated from three of them, contained B. burgdorferi as did 83 and 100% of the amplicons sequenced from PCR-positive I. pacificus larvae and nymphs, respectively. However, the two squirrels that were EPB-negative for spirochetes (5 and 6) were

6 May 2005 LANE ET AL.: GRAY SQUIRRELS AND LYME DISEASE SPIROCHETES 393 parasitized by I. pacificus larvae infected either with B. bissettii or B. burgdorferi s.l. Similarly, three other larvae removed from EPB-positive squirrels contained B. bissettii (3 and 7) or an uncharacterized B. burgdorferi s.l (2). All Þve I. pacificus larvae infected with either B. bissettii or uncharacterized B. burgdorferi s.l. spirochetes in the current study had EIs of 1.6 or 1.7, which are like those of unfed larvae, and neither spirochete was detected in the blood or ear tissues of their hosts (Table 1). Moreover, four larvae that had EIs of 1.5Ð1.8 were infected with B. burgdorferi. Although an as yet unidentiþed spirochete is passed occasionally via the eggs of infected I. pacificus females to their F 1 generation larvae (Lane and Burgdorfer 1987), the best evidence to date suggests that transovarial passage of spirochetes in natural populations of I. pacificus ticks is a rare phenomenon (Schoeler and Lane 1993). Nevertheless, our current Þndings underscore the possibility that at least some of the infected I. pacificus larvae removed from squirrels may have acquired spirochetes transovarially. That is, larvae taken from two EPB-negative and Þve EPB-positive squirrels were found to have comparable infection prevalences (40 versus 47%), 23% of larvae having EIs 1.8 contained spirochetes, and Þve of 30 larvae were infected with B. burgdorferi s.l. spirochetes that were not detected in the squirrels upon which they had attached. We therefore sought to determine whether unfed, host-seeking I. pacificus larvae inhabiting one of our study sites occasionally harbor spirochetes acquired transovarially. Accordingly, we collected several hundred hostseeking larvae by dragging leaf litter, branches and logs at the James II site in 2004, and tested 450 of them by PCR for B. burgdorferi s.l. as well as for A. phagocytophilum. To minimize the probability that the progeny of only a few females would be represented in our collections, we sampled a broad area encompassing 1.0Ð1.5 ha on three separate occasions (30 April and 5 and 10 May), and tested 150 larvae (i.e., in pools averaging Þve ticks) per sampling occasion. The James II site was selected because it typically has abundant populations of both squirrels (unpublished data) and I. pacificus subadults (Tälleklint-Eisen and Lane 1999, 2000; Eisen et al. 2002, 2004a; Lane et al. 2004), all three squirrels collected there in 2003 were infected with B. burgdorferi, and two of the Þve larvae that contained either B. bissettii or an unclassiþed B. burgdorferi s.l. in 2003 originated there. Borrelia and Anaplasma spp. were not detected in any of the pools. These results, albeit gathered 1 yr after the squirrels had been collected, reafþrm those of Schoeler and Lane (1993) and indicate that if transovarial passage of B. burgdorferi s.l. occurs in I. pacificus at the James II site, it indeed must occur infrequently. They also mirror earlier evidence suggesting that transovarial transmission of A. phagocytophilum does not occur in I. ricinus or I. scapularis (MacLeod and Gordon 1933, Ogden et al. 1998, Oteo et al. 2001, Levin et al. 2002). Two other explanations may be proffered to account for the high prevalence of borrelial infection among larval ticks having low EIs. One is that the western gray squirrel is such a potent host of B. burgdorferi that larvae attaching to it must only imbibe small quantities of blood (or other infective ßuids or tissues) to acquire spirochetes during the initial slowfeeding phase of engorgement. Another is that some larvae could acquire spirochetes while attached alongside infective nymphs by cofeeding transmission. Of these, the second postulate seems least likely because tight clusters of cofeeding larvae and nymphs were not found on any of the 10 squirrels. Rather, larvae and nymphs sparsely infested several bodily regions, including the ears, eyelids, chin, throat, abdomen, forelegs, and especially the anterior venter. The relation between the engorgement indexes of I. pacificus larvae and acquisition of spirochetes from naturally infected squirrels at various intervals postattachment warrants investigation. These kinds of data also are unavailable for the blacklegged tick, Ixodes scapularis Say, but 78% of the I. scapularis larvae that had attached to a B. burgdorferi-infected hamster for only 18 h acquired spirochetes and macroscopically seemed to be unfed (Piesman 1991). Morever, the EI for I. scapularis nymphs does not begin to increase measurably until ticks have been attached to a host for 24 h (Yeh et al. 1995, Falco et al. 1996). If both phenomena were discovered to occur in I. pacificus larvae, they might explain why 23% of ßat larvae were PCR-positive for spirochetes (Table 2). Globally, S. griseus is one of several squirrels in the genus Sciurus found to be hosts of B. burgdorferi s.l. or Ixodes spp. ticks that transmit such spirochetes. In the eastern United States, the eastern gray squirrel, Sciurus carolinensis, and the fox squirrel, Sciurus niger, are infested with larvae and nymphs of I. scapularis (Carey et al. 1980, Main et al. 1982, Magnarelli et al. 1984, Schulze et al. 1986, Godsey et al. 1987, Fish and Dowler 1989, Mannelli et al. 1993, Levin et al. 2002, LoGiudice et al. 2003), and antibodies against B. burgdorferi were detected in 50% (n 30) of S. carolinensis from Connecticut and 60% (n 5) from Wisconsin (Magnarelli et al. 1984, Godsey et al. 1987). In New York State, S. carolinensis and another sciurid, the red squirrel (Tamiasciurus hudsonicus) have been identi- Þed as important dilution hosts that reduce the force of transmission of B. burgdorferi relative to the primary reservoir, the white-footed mouse (Peromyscus leucopus) (LoGiudice et al. 2003). Both S. carolinensis and S. niger have been introduced into California and can be found in many urban areas and adjacent woodlands (Jameson and Peeters 2004). In Europe, the introduced gray squirrel, S. carolinensis, and the native red squirrel, Sciurus vulgaris, are important hosts of B. burgdorferi s.l., principally B. afzelii or B. burgdorferi, and of the tick Ixodes ricinus L. (Craine et al. 1995, 1997; Humair and Gern 1998). We detected B. burgdorferi s.l. spirochetes in 39.8% (45/113) of larvae and nymphs collectively, which is virtually identical to the prevalence of B. afzelii or B. burgdorferi infection (39.6%) in I. ricinus larvae (n 16) and nymphs (n 211) that had attached to Þve road-killed S. vulgaris in Switzerland (Humair and Gern 1998). In the latter study, however, the preva-

7 394 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 lence of infection was determined by isolation of spirochetes in BSKII medium versus PCR in the current study. Besides I. pacificus, the only other species of tick that has been recorded from S. griseus to date is D. occidentalis (Kohls 1937, Cross 1969, Furman and Loomis 1984; this study). The Þnding that one of Þve D. occidentalis larvae that had been attached to a B. burgdorferi-infected squirrel contained the same spirochete, although of interest, is not surprising. D. occidentalis rarely has been found infected with B. burgdorferi s.l. (reviewed by Lane 1996, Holden et al. 2003), but this tick is an inefþcient experimental vector of B. burgdorferi (Brown and Lane 1992, Lane et al. 1994). Any hematophagous arthropod feeding on a bacterium-infected host potentially may acquire, but not necessarily maintain or possess, the capacity to transmit such bacteria to a naõ ve host. Squirrels as a Host of A. phagocytophilum. This is the Þrst time that the western gray squirrel has been documented to contain A. phagocytophilum. Inexplicably, both squirrels that were PCR-positive for this bacterium tested negative for spirochetes, even though B. burgdorferi s.l. was detected in an I. pacificus larva removed from each of them. The role of S. griseus in the enzootic cycle of A. phagocytophilum in the Hopland area seems to be of much less importance than its involvement in the transmission of B. burgdorferi. A. phagocytophilum was detected in just two of 10 squirrels, one (2%) of 64 I. pacificus larvae, and two (4%) of 49 nymphs. The one positive larva was coinfected with B. burgdorferi, the Þrst such record of dual infection with these bacteria in an I. pacificus larva. Coinfection with both bacteria has been reported before in host-seeking I. pacificus nymphs and adults obtained from dense woodlands or adjoining habitats (Lane et al. 2001, 2004). Heretofore, several species of small-to-large mammals, as well as Dermacentor spp. and I. pacificus ticks, from various localities in California were reported to contain A. phagocytophilum (Holden et al. 2003, reviewed by Foley et al. 2004). Only the dusky-footed wood rat has been implicated as a primary reservoir despite the fact that the conclusive laboratory transmission studies have not been conducted yet (Nicholson et al. 1998, 1999; Castro et al. 2001). In Connecticut, the congener S. carolinensis satisþes the major criteria for a competent reservoir of A. phagocytophilum, i.e., squirrels are exposed frequently to the agent in nature, they are susceptible to infection, and they can serve as a source of infection for vector ticks that feed on them (Levin et al. 2002). In conclusion, since 2002, we have evaluated 50 species of birds and mammals as hosts of vector ticks and spirochetes in an all-out effort to identify the primary reservoir host(s) of B. burgdorferi s.l. in dense woodlands throughout Mendocino County (Eisen et al. 2004b, c; this study). PCR and sequencing analyses revealed that many vertebrate species are exposed to one or more characterized or uncharacterized members of this spirochetal complex, but to date only the western gray squirrel has fulþlled the major criteria that deþne a primary reservoir of the human pathogen B. burgdorferi. We therefore conclude that this squirrel is a keystone species in maintaining enzootic foci of B. burgdorferi in at least some subtypes of dense woodlands in northwestern California. Our Þndings with respect to A. phagocytophilum, although of less certain signiþcance, suggest that S. griseus could serve as a secondary host of this rickettsia. Knowledge gleaned during this study ultimately may prove useful for modifying preexisting, cost-effective control strategies (e.g., host-targeted methods) for reducing the density of infected vector ticks in rural or semirural residential settings where squirrels are plentiful and people are at elevated risk for contracting Lyme disease or other tick-borne diseases (Lane et al. 1992, 1998; Fritz et al. 1997). Acknowledgments We thank J. E. Kleinjan for technical assistance. This research was supported in part by grant AI22501 from the National Institutes of Health and by a generous gift from A. Henry to R.S.L. References Cited Brown, R. N., and R. S. Lane Lyme disease in California: a novel enzootic transmission cycle of Borrelia burgdorferi. Science (Wash. DC) 256: 1439Ð1442. Brown, R. N., and R. S. Lane Reservoir competence of four chaparral-dwelling rodents for Borrelia burgdorferi in California. Am. J. Trop. Med. Hyg. 54: 84Ð91. Burgdorfer, W., R. S. Lane, A. G. Barbour, R. A. Gresbrink, and J. R. Anderson The western black-legged tick, Ixodes pacificus: a vector of Borrelia burgdorferi. Am. J. Trop. Med. Hyg. 34: 925Ð930. Carey, A. B., W. L. Krinsky, and A. J. Main Ixodes dammini (Acari: Ixodidae) and associated ixodid ticks in south-central Connecticut, USA. J. Med. Entomol. 17: 89Ð99. Carraway, L. N., and B. J. Verts Sciurus griseus, pp. 1Ð7. In Mammalian species. No American Society of Mammalogists, Lawrence, KS. Castro, M. B., W. L. Nicholson, V. L. Kramer, and J. E. Childs Persistent infection in Neotoma fuscipes (Muridae: Sigmodontinae) with Ehrlichia phagocytophila sensu lato. Am. J. Trop. Med. Hyg. 65: 261Ð267. Clover, J. R., and R. S. Lane Evidence implicating nymphal Ixodes pacificus (Acari: Ixodidae) in the epidemiology of Lyme disease in California. Am. J. Trop. Med. Hyg. 53: 237Ð240. Craine, N. G., S. E. Randolph, and P. A. Nuttall Seasonal variation in the role of grey squirrels as hosts of Ixodes ricinus, the tick vector of the Lyme disease spirochaete, in a British woodland. Folia Parasitol. 42: 73Ð80. Craine, N. G., P. A. Nuttall, A. C. Marriott, and S. E. Randolph Role of grey squirrels and pheasants in the transmission of Borrelia burgdorferi sensu lato, the Lyme disease spirochaete, in the U.K. Folia Parasitol. 44: 155Ð 160. Cross, S. P Behavioral aspects of western gray squirrel ecology. Ph.D. dissertation, University of Arizona, Tucson. DeFoliart, G. R., P. R. Grimstad, and D. M. Watts Advances in mosquito-borne arbovirus/vector research. Annu. Rev. Entomol. 32: 479Ð505.

8 May 2005 LANE ET AL.: GRAY SQUIRRELS AND LYME DISEASE SPIROCHETES 395 Eisen, R. J., L. Eisen, and R. S. Lane Prevalence and abundance of Ixodes pacificus immatures (Acari: Ixodidae) infesting western fence lizards (Sceloporus occidentalis) in northern California: temporal trends and environmental correlates. J. Parasitol. 87: 1301Ð1307. Eisen, L., R. J. Eisen, and R. S. Lane Seasonal activity patterns of Ixodes pacificus nymphs in relation to climatic conditions. Med. Vet. Entomol. 26: 235Ð244. Eisen, L., M. C. Dolan, J. Piesman, and R. S. Lane. 2003a. Vector competence of Ixodes pacificus and I. spinipalpis (Acari: Ixodidae), and reservoir competence of the dusky-footed woodrat (Neotoma fuscipes) and the deer mouse (Peromyscus maniculatus), for Borrelia bissettii. J. Med. Entomol. 40: 311Ð320. Eisen, R. J., L. Eisen, M. B. Castro, and R. S. Lane. 2003b. Environmentally related variability in risk of exposure to Lyme disease spirochetes in northern California: effect of climatic conditions and habitat type. Environ. Entomol. 32: 1010Ð1018. Eisen, L., R. J. Eisen, C.-C. Chang, J. Mun, and R. S. Lane. 2004a. Acarologic risk of exposure to Borrelia burgdorferi spirochaetes: long-term evaluations in north-western California, with implications for Lyme borreliosis riskassessment models. Med. Vet. Entomol. 18: 38Ð49. Eisen, L., R. J. Eisen, and R. S. Lane. 2004b. The roles of birds, lizards, and rodents as hosts for the western blacklegged tick, Ixodes pacificus. J. Vector Ecol. 29: 295Ð308. Eisen, R. J., L. Eisen, and R. S. Lane. 2004c. Habitat-related variation in infestation of lizards and rodents with Ixodes ticks in dense woodlands in Mendocino County, California. Exp. Appl. Acarol. 33: 215Ð233. Falco, R. C., D. Fish, and J. Piesman Duration of tick bites in a Lyme disease-endemic area. Am. J. Epidemiol. 143: 187Ð192. Fish, D., and R. C. Dowler Host associations of ticks (Acari: Ixodidae) parasitizing medium-sized mammals in a Lyme disease endemic area of southern New York. J. Med. Entomol. 26: 200Ð209. Foley, J. E., P. Foley, R. N. Brown, R. S. Lane, J. S. Dumler, and J. E. Madigan Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States. J. Vector Ecol. 29: 41Ð50. Fritz, C. L., A. M. Kjemtrup, P. A. Conrad, G. R. Flores, G. L. Campbell, M. E. Schriefer, D. Gallo, and D. J. Vugia Seroepidemiology of emerging tickborne infectious diseases in a northern California community. J. Infect. Dis. 175: 1432Ð1439. Furman, D. P., and E. C. Loomis The ticks of California (Acari: Ixodida). Bull. Calif. Insect Surv. 25: 1Ð239. Godsey, Jr., M. S., T. E. Amundson, E. C. Burgess, W. Schell, J. P. Davis, R. Kaslow, and R. Edelman Lyme disease ecology in Wisconsin: distribution and host preferences of Ixodes dammini, and prevalence of antibody to Borrelia burgdorferi in small mammals. Am. J. Trop. Med. Hyg. 37: 180Ð187. Gordus, A. G., and J. H. Theis Isolation of Borrelia burgdorferi from the blood of a bushy-tailed wood rat in California. J. Wildl. Dis. 29: 478Ð480. Holden, K., J. T. Boothby, S. Anand, and R. F. Massung Detection of Borrelia burgdorferi, Ehrlichia chaffeensis, and Anaplasma phagocytophilum in ticks (Acari: Ixodidae) from a coastal region of California. J. Med. Entomol. 40: 534Ð539. Humair, P.-F., and L. Gern Relationship between Borrelia burgdorferi sensu lato species, red squirrels (Sciurus vulgaris) and Ixodes ricinus in enzootic areas in Switzerland. Acta Trop. 69: 213Ð227. Ingles, L. G Ecology and life history of the California gray squirrel. Calif. Fish Game 33: 139Ð158. Jameson, E. W., Jr., and H. J. Peeters Mammals of California. University of California Press, Berkeley, CA. Kahl, O., L. Gern, L. Eisen, and R. S. Lane Ecological research on Borrelia burgdorferi sensu lato: terminology and some methodological pitfalls, pp. 29Ð46. In J. S. Gray, O. Kahl, R. S. Lane, and G. Stanek [eds.], Lyme borreliosis: biology, epidemiology and control. CABI Publishing, Wallingford, United Kingdom. Kohls, G. M Hosts of the immature stages of the PaciÞc Coast tick Dermacentor occidentalis Neum. (Ixodidae). Public Health Rep. 52: 490Ð496. Lane, R. S Infection of deer mice and piñon mice (Peromyscus spp.) with spirochetes at a focus of Lyme borreliosis in northern California, U.S.A. Bull. Soc. Vector Ecol. 15: 25Ð32. Lane, R. S Competence of ticks as vectors of microbial agents with an emphasis on Borrelia burgdorferi, pp. 45Ð67. In D. E. Sonenshine and T. N. Mather [eds.], Ecological dynamics of tick-borne zoonoses. Oxford University Press, New York. Lane, R. S Risk of human exposure to vector ticks (Acari: Ixodidae) in a heavily used recreational area in northern California. Am. J. Trop. Med. Hyg. 55: 165Ð173. Lane, R. S., and R. N. Brown Wood rats and kangaroo rats: potential reservoirs of the Lyme disease spirochete in California. J. Med. Entomol. 28: 299Ð302. Lane, R. S., and W. Burgdorfer Potential role of native and exotic deer and their associated ticks (Acari: Ixodidae) in the ecology of Lyme disease in California, USA. Zentralbl. Bakteriol., Mikrobiol. Hyg. Ser. A. 263: 55Ð64. Lane, R. S., and W. Burgdorfer Transovarial and transstadial passage of Borrelia burgdorferi in the western black-legged tick, Ixodes pacificus (Acari: Ixodidae). Am. J. Trop. Med. Hyg. 37: 188Ð192. Lane, R. S., and W. Burgdorfer Spirochetes in mammals and ticks (Acari: Ixodidae) from a focus of Lyme borreliosis in California. J. Wildl. Dis. 24: 1Ð9. Lane, R. S., and P. E. Lavoie Lyme borreliosis in California: acarological, clinical, and epidemiological studies. Ann. N.Y. Acad. Sci. 539: 192Ð203. Lane, R. S., and J. E. Loye Lyme disease in California: interrelationship of ixodid ticks (Acari), rodents, and Borrelia burgdorferi. J. Med. Entomol. 28: 719Ð725. Lane, R. S., S. A. Manweiler, H. A. Stubbs, E. T. Lennette, J. E. Madigan, and P. E. Lavoie Risk factors for Lyme disease in a small rural community in northern California. Am. J. Epidemiol. 136: 1358Ð1368. Lane, R. S., R. N. Brown, J. Piesman, and C. A. Peavey Vector competence of Ixodes pacificus and Dermacentor occidentalis (Acari: Ixodidae) for various isolates of Lyme disease spirochetes. J. Med. Entomol. 31: 417Ð424. Lane, R. S., L. E. Casher, C. A. Peavey, and J. Piesman ModiÞed bait tube controls disease-carrying ticks and ßeas. Calif. Agric. 52(2): 43Ð47. Lane, R. S., C. A. Peavey, K. A. Padgett, and M. Hendson Life history of Ixodes (Ixodes) jellisoni (Acari: Ixodidae) and its vector competence for Borrelia burgdorferi sensu lato. J. Med. Entomol. 36: 329Ð340. Lane, R. S., J. E. Foley, L. Eisen, E. T. Lennette, and M. A. Peot Acarologic risk of exposure to emerging tickborne bacterial pathogens in a semirural community in northern California. Vector Borne Zoonotic Dis. 1: 197Ð 210. Lane, R. S., D. B. Steinlein, and J. Mun Human behaviors elevating exposure to Ixodes pacificus (Acari: Ix-

9 396 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 odidae) nymphs and their associated bacterial zoonotic agents in a hardwood forest. J. Med. Entomol. 41: 239Ð248. Levin, M. L., W. L. Nicholson, R. F. Massung, J. W. Sumner, and D. Fish Comparison of the reservoir competence of medium-sized mammals and Peromyscus leucopus for Anaplasma phagocytophilum in Connecticut. Vector Borne Zoonotic Dis. 2: 125Ð136. LoGiudice, K., R. S. Ostfeld, K. A. Schmidt, and F. Keesing The ecology of infectious disease: effects of host diversity and community composition on Lyme disease risk. Natl. Acad. Sci. USA 100: 567Ð571. MacLeod, J., and W. S. Gordon Studies in tick-borne fever of sheep. I. Transmission by the tick, Ixodes ricinus, with a description of the disease produced. Parasitology 25: 273Ð283. Magnarelli, L. A., J. F. Anderson, W. Burgdorfer, and W. A. Chappell Parasitism by Ixodes dammini (Acari: Ixodidae) and antibodies to spirochetes in mammals at Lyme disease foci in Connecticut, USA. J. Med. Entomol. 21: 52Ð57. Main, A. J., A. B. Carey, M. G. Carey, and R. H. Goodwin Immature Ixodes dammini (Acari: Ixodidae) on small animals in Connecticut, USA. J. Med. Entomol. 19: 655Ð664. Mannelli, A., U. Kitron, C. J. Jones, and T. L. Slajchert Ixodes dammini (Acari: Ixodidae) infestation on mediumsized mammals and blue jays in northwestern Illinois. J. Med. Entomol. 30: 950Ð952. Massung, R. F., K. Slater, J. H. Owens, W. L. Nicholson, T. N. Mather, V. B. Solberg, and J. G. Olson Nested PCR assay for detection of granulocytic ehrlichiae. J. Clin. Microbiol. 36: 1090Ð1095. Nicholson, W. L., S. Muir, J. W. Sumner, and J. E. Childs Serologic evidence of infection with Ehrlichia spp. in wild rodents (Muridae: Sigmodontinae) in the United States. J. Clin. Microbiol. 36: 695Ð700. Nicholson, W. L., M. B. Castro, V. L. Kramer, J. W. Sumner, and J. E. Childs Dusky-footed wood rats (Neotoma fuscipes) as reservoirs of granulocytic ehrlichiae (Rickettsiales: Ehrlichieae) in northern California. J. Clin. Microbiol. 37: 3323Ð3327. Ogden, N. H., K. Bown, B. K. Horrocks, Z. Woldehiwet, and M. Bennett Granulocytic Ehrlichia infection in ixodid ticks and mammals in woodlands and uplands of the U.K. Med. Vet. Entomol. 12: 423Ð429. Oteo, J. A., H. Gil, M. Barral, A. Pérez, S. Jimenez, J. R. Blanco, V. Martinez de Artola, A. García-Pérez, and R. A. Juste Presence of granulocytic ehrlichia in ticks and serological evidence of human infection in La Rioja, Spain. Epidemiol. Infect. 127: 353Ð358. Peavey, C. A., and R. S. Lane Transmission of Borrelia burgdorferi by Ixodes pacificus nymphs and reservoir competence of deer mice (Peromyscus maniculatus) infected by tick-bite. J. Parasitol. 81: 175Ð178. Peavey, C. A., R. S. Lane, and J. E. Kleinjan Role of small mammals in the ecology of Borrelia burgdorferi in a peri-urban park in north coastal California. Exp. Appl. Acarol. 21: 569Ð584. Piesman, J Experimental acquisition of the Lyme disease spirochete, Borrelia burgdorferi, by larval Ixodes dammini (Acari: Ixodidae) during partial blood meals. J. Med. Entomol. 28: 259Ð262. Ross, R. C California Sciuridae in captivity. J. Mammal. 11: 76Ð78. Schoeler, G. B., and R. S. Lane EfÞciency of transovarial transmission of the Lyme disease spirochete, Borrelia burgdorferi, in the western blacklegged tick, Ixodes pacificus (Acari: Ixodidae). J. Med. Entomol. 30: 80Ð86. Schulze, T. L., G. S. Bowen, M. F. Lakat, W. E. Parkin, and J. K. Shisler Seasonal abundance and hosts of Ixodes dammini (Acari: Ixodidae) and other ixodid ticks from an endemic Lyme disease focus in New Jersey, USA. J. Med. Entomol. 23: 105Ð109. Tälleklint-Eisen, L., and R. S. Lane Variation in the density of questing Ixodes pacificus (Acari: Ixodidae) nymphs infected with Borrelia burgdorferi at different spatial scales in California. J. Parasitol. 85: 824Ð831. Tälleklint-Eisen, L., and R. S. Lane Spatial and temporal variation in the density of Ixodes pacificus (Acari: Ixodidae) nymphs. Environ. Entomol. 29: 272Ð280. Vredevoe, L. K., J. R. Stevens, and B. S. Schneider Detection and characterization of Borrelia bissettii in rodents from the central California coast. J. Med. Entomol. 41: 736Ð745. Wright, S. A., M. A. Thompson, M. J. Miller, K. M. Knerl, S. L. Elms, J. C. Karpowicz, J. F. Young, and V. L. Kramer Ecology of Borrelia burgdorferi in ticks (Acari: Ixodidae), rodents, and birds in the Sierra Nevada foothills, Placer County, California. J. Med. Entomol. 37: 909Ð918. Yeh, M.-T., J. M. Bak, R. Hu, M. C. Nicholson, C. Kelly, and T. N. Mather Determining the duration of Ixodes scapularis (Acari: Ixodidae) attachment to tick-bite victims. J. Med. Entomol. 32: 853Ð858. Received 16 July 2004; accepted 16 December 2004.