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Tick Burdens on Peromyscus leucopus Rafinesque and Infection of Ticks by Borrelia spp. in Virginia
Cheryl L. Tanner, Frank K. Ammer, Ronald E. Barry, and Ellen Y. Stromdahl

Southeastern Naturalist, Volume 9, Issue 3 (2010): 529–546

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2010 SOUTHEASTERN NATURALIST 9(3):529–546 Tick Burdens on Peromyscus leucopus Rafinesque and Infection of Ticks by Borrelia spp. in Virginia Cheryl L. Tanner1,*, Frank K. Ammer2, Ronald E. Barry3, and Ellen Y. Stromdahl4 Abstract - The emergence of Southern Tick-Associated Rash Illness (STARI) questions the validity of reported Lyme disease (LD) cases throughout the southern United States. Acute symptoms are identical, and an efficient method of diagnosis is currently unavailable, with the cause(s) in question. The etiologic agent of LD, Borrelia burgdorferi, is transmitted by Ixodes scapularis (Black-legged Tick), while Amblyomma americanum (Lone Star Tick) is known to carry B. lonestari, the bacterium associated with cases of STARI. Peromyscus leucopus (White-footed Mouse) is a very efficient reservoir for these bacteria and tolerant of their vectors. We sampled small (<3 ha) and large (>3 ha) forest patches in Virginia for tick densities, burdens, and infestation rates by collecting ticks by drag sampling and from mice. We also tested ticks for infection by Borrelia. Although mouse density was greater in small patches, tick densities, burdens, and infestation rates did not differ with patch size. B. burgdorferi was not detected in any tick species, but 3.4% of nymphal and 5.9% of adult Lone Star Ticks removed from vegetation were infected with B. lonestari. Introduction The incidence of Lyme Disease (LD) has been on the rise since the Centers for Disease Control and Prevention (CDC) began monitoring in 1982 for this common vector-borne infectious disease (CDC 2004). Although cases have been reported in every state (CDC 2008), LD is endemic in the northeastern and north-central portions of the country, where approximately 95% of reported cases occur (CDC 2004, Dennis et al. 1998). The etiologic bacterial agent of LD, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, and Brenner, is maintained in an enzootic cycle in nature and is transmitted by Ixodes scapularis Say (Black-legged Tick) in the eastern United States to reptilian, avian, and mammalian hosts (Burgdorfer et al. 1982, Mather 1993, Ostfeld and Keesing 2000b, Steere et al. 1977). Peromyscus leucopus Rafinesque (White-footed Mouse) is considered the primary reservoir for B. burgdorferi in the northeastern United States (Clark et al. 2001; Donahue et al. 1987; Levine et al. 1985; Mather 1993; Ostfeld 2002; Ostfeld and Keesing 2000a, b; Spielman et al. 1985; Telford et al. 1990) because of its abundance, limited home range, and ability to acquire and maintain B. burgdorferi. (Oliver et al. 2003, Spielman et al. 1993). 1USDA Forest Service, 932 North Fork Cherry Road, Richwood, WV 26261. 2Department of Biology, Frostburg State University, Frostburg, MD 21532. 3Biology Department, Bates College, Lewiston, ME 04240. 4Entomological Sciences Program, US Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD 21010. *Corresponding author - 530 Southeastern Naturalist Vol. 9, No. 3 The Black-legged Tick, its major hosts, and B. burgdorferi thrive in deciduous forests, but controversy persists over the effect of forest patch size on the abundance of Black-legged Ticks and prevalence of B. burgdorferi. Allan et al. (2002) concluded that the risk of contracting LD is higher in forest patches of ≤2 ha compared to larger patches, but the results of Wilder and Meikle (2004) suggested no difference to LD risk between small and large forest patches. Patch area is only one of many factors that contribute to the local incidence of LD. Habitat and microclimate variables as well as density of Odocoileus virginianus Zimmerman (White-tailed Deer) contribute greatly to the distribution of the disease-causing bacteria, their vectors, and their hosts. Complicating the diagnosis of the disease in the southern United States is the emerging illness, Southern Tick-Associated Rash Illness (STARI), the symptoms of which are indistinguishable from those of LD, except for the lack of chronic manifestations in STARI. STARI is associated with the bite of Amblyomma americanum L. (Lone Star Tick) and, though still under investigation, may be caused by the bacterium B. lonestari Barbour, Maupin, Teltow, Carter, and Piesman. The Lone Star Tick has a variety of hosts (Bishopp and Trembley 1945, Kollars et al. 2000, Sonenshine 1979). It currently ranges from central Texas northward to Missouri and eastward in a broad swath across the southern United States continuing northward in a thin band up the coast to Maine (CDC 2005, Keirans and Lacombe 1998), including areas of sympatry with the Black-legged Tick. This overlap raises questions about the diagnoses of LD patients in the South, given similar acute symptoms, the lack of a reliable standard clinical test for either disease, and physician-related factors (James et al. 2001, Kirkland et al. 1997). Investigations into vectors for B. lonestari have almost exclusively focused on the Lone Star Tick because this is the predominant species found in areas of STARI cases. However, Taft et al. (2005) detected B. lonestari in a Black-legged Tick from New York and in another from Massachusetts, but they did not detect B. lonestari in Dermacentor variabilis Say (American Dog Tick). Due to borreliacidal activity in its saliva, the American Dog Tick is capable of acquiring but not maintaining B burgdorferi (Johns et al. 2000, Piesman and Happ 1997, Piesman and Sinsky 1988). Perhaps the same borreliacidal factor prevents the maintenance of B. lonestari. On the other hand, because the Black-legged Tick is able to maintain B. burgdorferi (Lane et al. 1991, Piesman and Happ 1997, Piesman and Sinsky 1988), we believe it may also maintain infection with B. lonestari, as evidenced by Taft et al. (2005). At the time of this study, only 4 cases of LD had been reported across 3 of the 4 Virginia counties in which the study area is located (VDH 2001), and laboratory studies have shown only 4% of Black-legged Ticks in the state to be infected with B. burgdorferi (VDH 1997), as compared to 20–100% in focal portions of the country. Factors that contribute to the lower prevalence of B. burgdorferi in I. scapularis in the South include differences in feeding behavior of the vector, host diversity, and reservoir competencies of the 2010 C.L. Tanner, F.K. Ammer, R.E. Barry, and E.Y. Stromdahl 531 hosts upon which the vector feeds (Lane et al. 1991; Oliver 1996; Ostfelt and Keesing 2000a, b). The average infection rate of B. lonestari in Lone Star Ticks throughout its range is approximately 2% (Armstrong et al. 2001; Bacon et al. 2003, 2005; Barbour et al. 1996; Clark 2004; Stromdahl et al. 2003; Varela et al. 2004), and PCR analyses of Lone Star Ticks in Fort A.P. Hill, Caroline County, VA, revealed infection rates of 4.3% (13/299) in adult ticks and 2.8% (6/211) in nymphs (Stromdahl et al. 2003). The main objectives of this study were to 1) examine the relationship of tick densities to forest patch size and season; 2) examine the relationship between forest patch size and Black-legged Tick, Lone Star Tick, and American Dog Tick burdens on small mammals, particularly the White-footed Mouse; 3) compare infection rates of B. burgdorferi and B. lonestari between small (<3 ha) and large (>3 ha) forest patches; and 4) investigate the presence of B. lonestari in the Lone Star Tick, Black-legged Tick, and American Dog Tick. We hypothesized that 1) tick burdens on White-footed Mice would be greater in small patches because of lower mammalian diversity and higher densities of mice in the absence of competitors and predators; 2) if Blacklegged Tick and Lone Star Tick burdens on White-footed Mice were greater in smaller patches, minimum infection rates (MIRs) of B. burgdorferi and B. lonestari would be greater in those ticks; and 3) because the Black-legged Tick and American Dog Tick have been shown to acquire B. burgdorferi, these species also would acquire the closely related B. lonestari. Materials and Methods Study area Fredericksburg and Spotsylvania County Memorial National Military Park (FRSP) is located in both the Appalachian Piedmont and Atlantic Coastal Plain physiographic provinces of Virginia (FRSP 2005, Linzey 1998), in Caroline, Orange, Spotsylvania, and Stafford counties. The majority of forests are mixed deciduous with canopy species comprised of Quercus spp. (oaks), Carya spp. (hickories), Acer spp. (maples), Liquidamber styraciflua L. (Sweet Gum), and Liriodendron tulipifera L. (Tulip Poplar) and understories of Vaccinium angustifolium Aiton (Late Lowbush Blueberry), Toxicodendron radicans L. (Poison Ivy), Kalmia latifolia L. (Mountain Laurel), Prunus spp. (cherries), and Cornus spp. (dogwoods) (Petrides 1986, Tanner 2007). Study sites Forest patches were chosen according to size and habitat type using aerial photos and subsequently ground-truthed. We attempted to locate forest patches similar in habitat, but avoidance of the interannual impact of masting events was not possible because oaks are abundant in FRSP (Barry et al. 2008, NPS 2006). However, we proceeded under the assumption that the close proximity of all sites is sufficient to assume negligible local variation. Three small (<3-ha) and three large (>3-ha) forest patches were chosen for 532 Southeastern Naturalist Vol. 9, No. 3 replication, with a patch being defined as a forest fragment surrounded on ≥ 3 sides by nonforested habitat and, if on only 3 sides, bordered on the 4th side by a hard edge such as a road or stream. The habitat variables of canopy cover, ground cover, leaf-litter depth, and coarse woody debris (CWD) volume were quantified and averaged seasonally (Tanner 2007). The microclimate variables of ambient temperature, relative humidity (RH), soil pH, and soil moisture were recorded daily (Tanner 2007). Pellet-group surveys adapted from Mayle et al. (2000) and Wemmer et al. (1996) were conducted seasonally to estimate White-tailed Deer usage at each site. Tick collection Ticks were collected from late April through October daily from small mammals and monthly by drag sampling. Small-mammal trapping is considered the most accurate sampling technique for estimating Black-legged Tick density in areas of low prevalence like Virginia (Lane et al. 1991, Solberg et al. 2003). All juvenile ticks were removed with forceps from each rodent and counted to determine tick burdens. All ticks were stored in 70% ethanol (Clark et al. 2001, Falco and Fish 1992, Ostfeld et al. 1996b) for later identification (Pratt 1961; Ward’s Natural Science n.d.) and lab determination of Borrelia spp. infection. Tick densities within patches were determined through drag sampling with checks every 20 m (Daniels et al. 2000, Falco and Fish 1992, Schulze and Jordan 2001). Ticks were counted, collected, and stored in 70% ethanol (Clark et al. 2001, Falco and Fish 1992, Ostfeld et al. 1996b) for later identification and lab determination of Borrelia spp. infection. Mammal sampling Sherman live traps (7.6 x 8.9 x 22.9 cm) were arrayed in a grid configuration at each site to sample the nymphal and larval stages of ticks on mice from late April through mid-October 2005. The number of traps used depended on the size of the patch being sampled. Small patches contained 49 traps at 10-m intervals in 7 x 7 grids for an effective trapping area of approximately 1.12 ha. The boundary strip method (half the mean maximum distance moved) was used to determine the effective area of the grid (Krebs 1999, Otis et al. 1978, Parmenter et al. 2003, Wilson and Anderson 1985). Trapping effort in large patches was 100 traps at 10-m intervals in 10 x 10 grids to account for increased area, yielding an effective trapping area of approximate 3.28 ha. In larger patches, 1 side of the grid was located along an edge of the forest patch, and the rest of the grid extended into the interior of the patch to incorporate both edge and interior habitats. Although small patches inherently contain more edge than large patches because of the increased edge-to-interior ratio, traps in small patches were arrayed in the same configuration for consistency. Traps were baited with a mixture of peanut butter, bird seed, and oats and were checked once per day. To create blocking effects, 1 small and 1 large grid were sampled simultaneously for 2 or 3 consecutive days (the 2010 C.L. Tanner, F.K. Ammer, R.E. Barry, and E.Y. Stromdahl 533 schedule was adjusted from 3 days in the beginning to 2 for the remainder of the study) approximately every 2 weeks. Captured mice were examined for larval and nymphal ticks, which usually attach to the pinnae (Ostfeld et al. 1993). Species, age, sex, reproductive status, and weight of each mouse were recorded, and each individual was marked using the toe-clipping method (Rudran 1996) for individual identification. Sampling and handling procedures conformed to Frostburg State University IACUC requirements and guidelines approved by the American Society of Mammalogists (Animal Care and Use Committee 1998). Prevalence of Borrelia spp. Ticks were refrigerated at 4 ºC in 70% ethanol until they were processed for isolation of their DNA. Most nymphs and larvae were pooled for extractions according to location, species, developmental stage, and feeding status. However, all adult ticks, Black-legged Tick nymphs, and any ticks that were lone captures of their species and life stage from a drag session or host were processed individually. Each individual and pool was dissected with a sterile disposable 18-gauge NoKor needle on a sterile glass surface. Extractions were performed using both an IsoQuick Nucleic Acid Extraction Kit (ORCA Research Inc., Bothell, Washington) and a Qiagen DNeasy Tissue Kit (Qiagen Inc., Valencia, CA) according to manufacturer protocols. DNA isolates were stored at 4 ºC until amplification by polymerase chain reaction (PCR). PCR reactions were carried out using puReTaq Ready-To-Go PCR Beads (GE Healthcare, Piscataway, NJ) following the manufacturer’s protocol. Each 25-μl reaction contained 1 puReTaq™ Ready-To-Go™ PCR Bead, purified water, 1 μM of each primer, and approximately 50 ng/μl and 0.682– 1.429 ng/μl of template DNA for tick and Borrelia spp., respectively. All reactions contained a sample of verified Borrelia spp. DNA and a sample of purified water as positive and negative controls, respectively. PCR products were electrophoresed on 2% agarose gels stained with ethidium bromide and visualized with an ultraviolet (UV) transilluminator. To verify extraction of DNA, an approximate 300-bp portion of the mitochondrial 16S rRNA tick gene was amplified using primers 16S+2 and 16S-1 (Black and Piesman 1994), with a band at approximately 300 bp being accepted as presence of tick DNA. Each PCR run contained a sample of purified water as a negative control (Stromdahl et al. 2001). After tick DNA was verified, DNA of Borrelia spp. was amplified by nested PCR using flagellin gene primers described by Barbour et al. (1996), with bands at approximately 240 bp from internal reactions being considered positive for Borrelia spp. infection (Stromdahl et al. 2003). PCR samples positive for Borrelia spp. were further analyzed by nested PCR using primers specific for each bacterium. Samples were amplified using the p66 gene primers a, a′, f, and f′ described by Rosa et al. (1991) specific for B. burgdorferi, with bands at approximately 240 bp being considered positive for B. burgdorferi infection (Rosa et al. 1991, Stromdahl et al. 2001). Samples were also amplified using nested flagellin gene primers modified from Barbour et al. (1996) by P.C. 534 Southeastern Naturalist Vol. 9, No. 3 Williamson (University of North Texas Health Science Center, Forth Worth, TX, unpubl. data) (Table 1) to target B. lonestari, with bands at approximately 240 bp considered positive for B. lonestari infection (Barbour et al. 1996, Stromdahl et al. 2003). All PCR reactions were prepared in a UV hood and run on a PTC 200 DNA Engine (MJ Research, Inc., Waltham, MA). Minimum infection rates (MIRs) were calculated by assuming only 1 tick per positive pool as infected. All samples positive for Borrelia spp. were purified using a Quantum Prep Gel Slice Kit (Bio-Rad Laboratories, Hercules, CA) and sent to SeqWright DNA Technology Services (Houston, TX) for sequencing. Sample sequences were then compared to known sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST) accessed through the National Center for Biotechnology Information (NCBI; Bethesda, MD) webpage (NCBI 2006). Statistical analyses Densities (D = N/A) of White-footed Mice were calculated using relative abundance (individuals per 100 trap nights) as N and effective area (A) of the grid (Krebs 1999, Otis et al. 1978, Parmenter et al. 2003, Wilson and Anderson 1985). Relative abundance was chosen to represent N to incorporate trapping effort because captures in 2 of 3 large sites were consistently very low compared to small sites due to persistent trap disturbance by Procyon lotor (L.) (Raccoon). One-way ANOVA was used to test for significant differences in mouse densities between patch sizes. Mean densities of ticks obtained from drag sampling were expressed per 1000 m2 and tested by repeated measures (rm) ANOVA for interaction and statistical significance between patch sizes and seasons. Because relatively few nymphs were found on mice (17 of 200 ticks removed), larvae and nymphs were combined into the category of “juveniles” for analysis of tick burdens. Hereafter the term “tick burden(s)” refers to the combination of juvenile life stages on mice. Mean tick burdens were calculated by dividing the total number of ticks found on mice by the total number of mice captured (i.e., number of ticks per mouse). Too few mouse captures in certain seasons at some sites prevented seasonal analysis of tick burdens. Therefore, tick burdens were combined into site means to examine the effect of only patch size by 1-way ANOVA. Only the 1st mouse capture per individual per 2- or 3-day session was included in tick burden analyses to allow reinfestation and an equal opportunity for infestation of all mice at each Table 1. Borrelia lonestari primer sequences modified from Barbour et. al. (1996) by P.C. Williamson. Primer Sequence Bl-Fla 522 F 5'-GGTACATATTCAGATGCAGACAGAGGG Bl-Fla 1182 R 5'-GCACTTGATTTGCTTGTGCAATCATAGCC Bl-Fla 667 F 5'-CTGAAGAGCTTGGAATGCAACCTGC Bl-Fla 907 R 5'-GAGCTAATCCCACCTTGAGCTGG 2010 C.L. Tanner, F.K. Ammer, R.E. Barry, and E.Y. Stromdahl 535 sampling event. Because sampling was repeated on the same experimental units, rmANOVA (Zar 1999) was used to test for significant difference in tick burdens between mouse 1) ages, 2) sexes, and 3) weights after these variables were categorized. Mean infestation burdens were calculated by dividing the total number of ticks found on infested mice by the number of mice that were infested (i.e., number of ticks per infested mouse). Infestation rates (percentages of mice infested) were calculated by dividing the number of infested mice by the total number of mice captured and multiplying by 100. Mean infestation burdens and infestation rates were analyzed for treatment effects of patch size by 1-way ANOVA. Simple linear regression (Zar 1999) was used to test for functional dependence of mean tick burdens on mouse densities and the relationship between tick densities and mouse densities. The relationship between tick burdens on mice and tick densities determined from drags could not be tested because of species- and life stage-specific behavior resulting in insufficient data. Certain species and life stages of ticks were found on either drags or rodents but never both. Infection rates of B. lonestari could not be compared with tick burdens because the bacterium was detected only in Lone Star Ticks, which were found only on drags and not on mice. Data in percent format (infestation rates) were arcsine square-root transformed before analyses. All data were tested for compliance with the assumptions of normality and homoscedasticity using Kolmogorov-Smirnov and Levene’s tests, respectively. Most normality and almost all equal variance assumptions were met, but ANOVA is powerful and robust, can tolerate minor violations of assumptions (Zar 1999), and therefore was used to analyze mouse densities, tick densities, tick burdens, infestation burdens, and infestation rates. All samples were random, with sites chosen based on their sizes, but samples within taken randomly. Samples that could not be considered independent because data were recorded repeatedly from the same 6 sites were analyzed with rmANOVA (Zar 1999). All ANOVA, regression, normality, and homoscedasticity tests were performed using MINITAB Release 14 statistical software (Minitab Inc., State College, Pennsylvania). The criterion for statistical significance for all tests was P < 0.05. Results Mammals captured and White-footed Mouse demography Seven species of small mammals were captured. P. leucopus comprised 74% of small mammals captured and 84% of total captures. Mean (± SE) density of white-footed mice was significantly greater in small forest patches (x̅small = 4.2 ± 2.0 mice/ha, x̅large = 1.0 ± 0.3 mice/ha; F = 8.01; d.f. = 1, 4; P < 0.05). Tick density Drags yielded no American Dog Tick larvae or nymphs or Black-legged Tick larvae or adults (Table 2). American Dog Tick juveniles quest at the 536 Southeastern Naturalist Vol. 9, No. 3 base of vegetation and are rarely caught on drags (Sonenshine 1993), and perhaps the timing of our sampling from late April through October did not include the period of peak abundance of Black-legged Tick adults (early and late fall). We found no single-factor effect of forest patch size (F = 0.00; d.f. = 1, 12; P > 0.05), season (F = 2.53; d.f. = 2, 12; P > 0.05), or patch size x season interaction (F = 0.02; d.f. = 2, 12; P > 0.05) on density of adult American Dog Ticks (Table 3). No single-factor effect of patch size (F = 0.11; d.f. = 1, 12; P > 0.05), season (F = 3.53; d.f. = 2, 12; P > 0.05), or patch size x season interaction (F = 0.07; d.f. = 2, 12; P > 0.05) on density of nymphal Black-legged Ticks was detected. No patch size x season interaction (F = 0.50; d.f. = 2, 12; P > 0.05), or single-factor effect by patch size (F = 0.71; d.f. = 1, 12; P > 0.05) or season (F = 2.11; d.f. = 2, 12; P > 0.05), on density of adult Lone Star Ticks was found. Density of nymphal Lone Star Ticks (Table 3) did not differ significantly by patch size (F = 0.64; d.f. = 1, 12; P > 0.05), season (F = 2.10; d.f. = 2, 12; P > 0.05), or interaction of patch size x season (F = 0.36; d.f. = 2, 12; P > 0.05). Because of a significant patch size x season interaction (F = 4.65; d.f. = 2, 12; P < 0.05), main effects of patch size and season on density of larval Lone Star Ticks could not be analyzed. However, regression analysis revealed no functional dependence of density of Lone Star Tick larvae on density of mice (F = 3.29; d.f. = 1, 4; P > 0.05). Similarly, there was no functional dependence of density of Lone Star Tick nymphs (F = 0.24; d.f. = 1, 4; P > 0.05), Lone Star Tick adults (F = 0.32; d.f. = 1, 4; P > 0.05), Black-legged Tick nymphs (F = 0.04; d.f. = 1, 4; P > 0.05), or American Dog Tick adults (F = 0.87; d.f. = 1, 4; P > 0.05) on density of mice. We found no single-factor effect of forest patch size (F = 0.18; d.f. = 1, 12; P > 0.05), season (F = 0.66; d.f. = 2, 12; P > 0.05), or Table 2. Sources and total numbers of ticks tested for, and minimum infection rates (MIRs) of ticks by, Borrelia spp. B. burgdorferi B. lonestari Source of ticks % of Sample MIR Sample MIR Tick species/life stage Drags Rodents* Total sample size (%) size (%) Amblyomma americanum Larvae 72 0 72 13.1 72 0 72 0.0 Nymphs 237 0 237 43.2 237 0 237 3.4 Adults 17 0 7 3.1 17 0 17 5.9 Dermacentor variabilis Larvae 0 129 129 23.5 129 0 129 0.0 Nymphs 0 15 15 2.7 15 0 15 0.0 Adults 7 0 7 1.3 7 0 7 0.0 Ixodes scapularis Larvae 0 49 49 8.9 49 0 49 0.0 Nymphs 20 2 22 4.0 22 0 22 0.0 Adults 0 0 0 0.0 0 0 0 0.0 Totals 353 195 548 100 *All rodents are Peromyscus leucopus except for 1 Mus musculus from which 2 D. variabilis larvae were removed. 2010 C.L. Tanner, F.K. Ammer, R.E. Barry, and E.Y. Stromdahl 537 patch size x season interaction (F = 0.02; d.f. = 2, 12; P > 0.05) on density of White-tailed Deer as reflected by usage indices. Tick burdens All but 2 ticks (of any species) were encountered on White-footed Mice; 2 larval American Dog Ticks were found on a single Mus musculus L. (House Mouse). No Lone Star Ticks of any life stage or adult ticks of any species were documented on mice (Table 2). Black-legged Tick (F = 3.27; d.f. = 1, 4; P > 0.05) and American Dog Tick (F = 1.20; d.f. = 1, 4; P > 0.05) burdens on newly captured mice did not differ between forest patch sizes (Table 4). Similarly, when recaptures were included in the model, Black-legged Tick (F = 1.23; d.f. = 1, 4; P > 0.05) and American Dog Tick (F = 1.57; d.f. = 1, 4; P > 0.05) burdens did not differ by patch size (Table 4). Mean tick burdens of infested mice (Table 4) did not differ with patch size for either the American Dog Tick (F = 0.00; d.f. = 1, 4; P > 0.05) or Black-legged Tick (F = 0.41; d.f. = 1, 4; P > 0.05). Infestation rates by the American Dog Tick (F = 2.78; d.f. = 1, 4; P > 0.05) and Black-legged Tick (F = 2.70; d.f. = 1, 4; P > 0.05) did not differ with patch Table 3. Mean (± SE) overall and seasonal tick densities (per 1000 m2) in small (<3 ha; S) and large (>3 ha; L) deciduous forest patches. Season Species Patch size Overall Spring Summer Fall Dermacentor variabilis adults S 1.1 (1.1) 3.2 (3.2) 0.0 (0.0) 0.0 (0.0) L 1.1 (0.7) 3.0 (2.0) 0.4 (0.4) 0.0 (0.0) Ixodes scapularis nymphs S 2.1 (2.1) 6.4 (6.4) 0.0 (0.0) 0.0 (0.0) L 2.8 (1.1) 6.7 (1.3) 1.9 (0.4) 0.0 (0.0) Amblyomma americanum adults S 1.3 (1.1) 3.2 (3.2) 0.8 (0.8) 0.0 (0.0) L 3.5 (2.4) 8.9 (6.8) 1.1 (0.6) 0.4 (0.4) A. americanum nymphs S 18.3 (10.8) 23.8 (19.2) 31.0 (27.5) 0.0 (0.0) L 32.0 (14.0) 57.8 (33.2) 37.0 (20.5) 1.1 (0.6) Table 4. Mean (± SE) overall tick burdens and infestation rates of newly captured mice by 2 species of ticks in small (<3 ha; S) and large (>3 ha; L) deciduous forest patches. Patch size Variable S L Burdens on mice (ticks/mouse)* Dermacentor variabilis 1.2 (0.4) 0.6 (0.3) Ixodes scapularis 0.1 (0.1) 0.3 (0.2) Burdens on infested mice (ticks/mice)* D. variabilis 2.5 (0.4) 2.5 (0.4) I. scapularis 1.6 (0.3) 2.3 (0.4) Infestation rates (%)* D. variabilis 41.9 15.1 I. scapularis 5.8 17.4 *Includes recaptures (see text). 538 Southeastern Naturalist Vol. 9, No. 3 size (Table 4). Neither Black-legged Tick (F = 2.40; d.f. = 1, 8; P > 0.05) nor American Dog Tick (F = 0.55; d.f. = 1, 8; P > 0.05) burdens differed signifi- cantly by mouse age. No significant difference by mouse sex was detected in Black-legged Tick (F = 0.01; d.f. = 1, 8; P > 0.05) or American Dog Tick (F = 0.29; d.f. = 1, 8; P > 0.05) burdens. Black-legged Tick (F = 0.84; d.f. = 2, 12; P > 0.05) and American Dog Tick (F = 0.61; d.f. = 2, 12; P > 0.05) burdens did not differ significantly by mouse weight. Neither juvenile Black-legged Tick (F = 1.66; d.f. = 1, 4; P > 0.05) nor American Dog Tick (F = 0.00; d.f. = 1, 4; P > 0.05) burdens were functionally dependent on mouse density. Borrelia spp. minimum infection rates All 188 tick individuals and pools yielded tick DNA, with further analysis confirming 9 samples positive for B. lonestari infection. No Lone Star Tick larvae, Black-legged Ticks, or American Dog Ticks were infected (Table 2). All ticks species were negative for B. burgdorferi infection; i.e., an MIR of 0% regardless of life stage. All positive samples for B. lonestari originated from Lone Star Ticks, with an MIR of 5.9% (1 of 17) for adults and 3.4% (8 of 237) for nymphs. MIRs could not be analyzed between forest patch sizes because of inadequate tick sample sizes within each site. All Borrelia-positive nucleotide sequences aligned with known B. lonestari sequences in the Genbank database for further confirmation of sample results. Most but not all samples contained some unidentified nucleotides on either side of the target loci. However, all target sequences aligned 100% with known sequences. Sample sequence lengths ranging from 177–206 nucleotides aligned perfectly (100% with no mismatches or gaps) with several known sequences originated from 13 states (including Virginia) throughout the range of the Lone Star Tick. Discussion Competitive and predatory pressures on White-footed Mice should be reduced in smaller forest patches, allowing one of the most competent reservoirs for B. burgdorferi to flourish (Nupp and Swihart 1996, 1998). While our research supports this assertion with greater mouse density in small forest patches, tick densities in small and large patches did not differ, and the percentage of mice infested with ticks also did not differ with patch size, perhaps due to sample size. Densities of Lone Star Tick larvae, nymphs, or adults, American Dog Tick adults, or Black-legged Tick nymphs removed from vegetation were not dependent on mouse density. We expected greater tick burdens on mice where there was a higher mouse density, but Blacklegged Tick and American Dog Tick burdens on mice showed no dependence on mouse density. Neither Black-legged Ticks nor American Dog Ticks demonstrated selection for mouse age, sex, or weight in our investigation (Tanner 2007). Collectively, these observations suggest that, in some locations, factors other than the density of competent reservoirs for B. burgdorferi may be as important as patch size in LD ecology. 2010 C.L. Tanner, F.K. Ammer, R.E. Barry, and E.Y. Stromdahl 539 Mast production is a major determinant of deer activity and abundance of White-footed Mice (McShea and Schwede 1993; Ostfeld 1997, 2002; Ostfeld et al. 1996a), but many other habitat and microclimate variables share in determining tick survival, densities, and burdens once eggs are laid (Lane et al. 1991, Ostfeld 1997, Ostfeld et al. 1996a). Leaf-litter depth, canopy cover, ambient temperature, relative humidity, soil moisture, and soil pH differed seasonally in our investigation (Tanner 2007) and may have determined tick activity and survival, but such variation between late April and October when sampling occurred had no apparent role in determination of tick densities in our study. Canopy cover, ground cover, and soil moisture differed between small and large patches and may have affected mouse density (Tanner 2007). However, differences in these habitat variables did not effect differences in tick densities in patches differing in size. Variables that affect deer movement, such as masting activity (Koenig and Knops 2002, McShea and Rappole 1992, McShea and Schwede 1993), have a greater effect on tick densities because their eggs will be laid where they detach from the deer. However, deer usage showed no difference with patch size in our study, which was expected with a lack of difference in tick densities and burdens. That tick densities and burdens were low compared to other studies (Allan et al. 2002, Armstrong et al. 2001, Clark et al. 2001, Kollars et al. 2000), regardless of patch size, suggests that conditions in FRSP are not optimal for tick promulgation and/or survival. Seasonal occurrence of the 3 tick species at this Virginia site (Table 2) was similar to that reported from other Mid-Atlantic sites (Schulze et al. 1986). Stromdahl et al. (2008) detected a low prevalence (4/1320 = 0.3% MIR) of B. lonestari in Lone Star Tick larvae. Failure to detect B. lonestari in larvae in this study was probably due to a comparatively small sample size and the low expectation of transovarial transmission. The MIRs of Lone Star Ticks by B. lonestari in this study (5.9% adult and 3.4% nymphal) are within the range (1–5%) reported by numerous researchers throughout the South (Armstrong et al. 2001; Bacon et al. 2003, 3005; Barbour et al. 1996; Clark 2004; Varela et al. 2004) and in Caroline County, VA (4.3% adult and 2.8% nymphal; Stromdahl et al. 2003), further supporting its distribution and incidence. B. lonestari nucleotide sequences from Lone Star Ticks matched 100% with those reported from 11 southern states—Arkansas, Florida, Georgia, Kansas, Kentucky, Maryland, Missouri, North Carolina, South Carolina, Tennessee, and Virginia— and New Jersey and New York. These findings do not support our hypothesis of higher bacterial prevalence accompanying higher mouse densities in smaller patches, but sample sizes were relatively small. Infection rates of ticks by B. lonestari may be relatively low in the South (Armstrong et al. 2001; Bacon et al. 2003, 2005; Barbour et al. 1996; Clark 2004; Varela et al. 2004), but infection rates by B. burgdorferi appear even lower. Approximately 95% of LD cases occur in the northeastern and northcentral portions of the US, but cases are scant and scattered in the South, where the vector is also established (CDC 2004, Dennis et al. 1998). In 540 Southeastern Naturalist Vol. 9, No. 3 the South, the Lone Star Tick is more abundant than the Black-legged Tick (Armstrong et al. 2001, Bishopp and Trembley 1945, Childs and Paddock 2003, James et al. 2001, Varela 2004). This finding is consistent with the results of our tick sampling in which Lone Star Ticks comprised almost 60% of all ticks collected (drags and burdens), and Black-legged Ticks only accounted for approximately 13%. Our sample size for Lone Star Ticks was much greater than that for Black-legged Ticks, which could have led to greater detection ability of B. lonestari compared to B. burgdorferi. The American Dog Tick was negative for both B. lonestari and B. burgdorferi, suggesting that the species is a poor vector for the former, in contrast to what we hypothesized. However, sample size was relatively low, with larvae represented disproportionately. Infection with B. burgdorferi activates an antimicrobial defense in American Dog Ticks that rapidly kills the bacterium (Johns et al. 2000), so that even if the tick acquires the pathogen it does not maintain it. Perhaps the same is true of its infection with B. lonestari. That American Dog Ticks in this study did not acquire B. lonestari does not mean that the ability is absent. Perhaps its efficiency of acquiring B. lonestari is similar to that of acquiring B. burgdorferi. Mukolwe et al. (1992), Piesman and Happ (1997), and Piesman and Sinsky (1988) have documented low acquisition levels of B. burgdorferi in American Dog Ticks, whereas we and Taft et al. (2005) documented none. Our sample sizes may have been too small to detect Borrelia spp. in American Dog Ticks. Contrary to our hypothesis, we found no Black-legged Ticks infected with B. lonestari. However, Taft et al. (2005) documented 2 Black-legged Ticks infected with B. lonestari in the northeastern portion of the country. Few tests have been conducted to determine other possible vectors of B. lonestari other than its principal vector, the Lone Star Tick. Coinfections with pathogens in different genera also have been documented for the Blacklegged Tick (Schauber et al. 1998, Stromdahl et al. 2008), so it is possible that this species can transmit both B. burgdorferi and B. lonestari. Further testing with larger sample sizes is necessary to determine other possible vectors for B. lonestari, and the investigation into the bacterium’s role in STARI is ongoing. The few potential hosts captured (Tanner 2007) that were not Whitefooted Mice may serve as “dilution” or “rescue” species during times of low densities of the principal host (Ostfeld and Keesing 2000a, b). Incidental small-mammal captures included the House Mouse, Blarina brevicauda Say (Northern Short-tailed Shrew), Tamias striatus L. (Eastern Chipmunk), Didelphis virginianus Kerr (Virginia Opossum), Sciurus carolinensis Gmelin (Gray Squirrel), and Rattus norvegicus Berkenhout (Norway Rat), all of which have demonstrated some level of reservoir competence (Craine et al. 1997, Gage et al. 1995, Gern et al. 1998, Godsey et al. 1987, Markowski et al. 1998, Mather 1993, Matuschka et al. 1997, McLean et al. 1993, Ostfeld and Keesing 2000b, Slajchert et al. 1997, Telford et al. 1990). Lizards seen in the study sites were Eumeces fasciatus L. (Five-lined Skink), E. laticeps Schneider (Broadhead 2010 C.L. Tanner, F.K. Ammer, R.E. Barry, and E.Y. Stromdahl 541 Skink), and Sceloporus undulatus Green (Northern Fence Lizard). Any of them may serve as “rescue” or “dilution” species (Ostfeld and Keesing 2000b), but the reservoir competencies of these species are unknown. Based on our research, we do not recommend active management for LD or STARI at FRSP. Because of the time, cost, and effort required, management procedures are feasible only in areas with high risk factors, including high tick burdens and densities, and high prevalence of the tickborne pathogen. The relatively low density of Black-legged Ticks within the Park, the low burdens of the species on reservoir mice, and the absence of B. burgdorferi in all tick species collected suggest only a remote probability of contracting LD in FRSP. Even though no Lone Star Ticks were found on reservoir mice, the density of this species was the greatest of the 3 studied, and B. lonestari was detected. American Dog Ticks yielded the lowest density but highest burdens, yet no bacteria. These tick densities and burdens, together with low Borrelia spp. prevalence, suggest a low risk of contracting STARI, if B. lonestari is shown to be the causative agent, and an even lower probability of contracting LD in the Fredericksburg region. Acknowledgments We thank Drs. Durland Shumway and Lance Revennaugh for statistical expertise and Drs. Wayne Yoder and Rich Robbins (the latter at the Defense Pest Management Information Analysis Center/Armed Forces Pest Management Board of the Walter Reed Army Medical Center, Washington, DC) for assistance with initial tick identification. Field housing, equipment, and funding were provided by the National Park Service (NPS) under Cooperative Agreement 1443DCA309701200 with Ronald Barry. NPS Natural Resource Manager Gregg Kneipp provided GIS maps and frequent general support. We thank Dr. William Seddon and the Frostburg State University Department of Biology for providing laboratory supplies. We also thank Mrs. Valerie Fritz for obtaining both field and laboratory supplies and Ms. Bebe Elrick for her continuous help and support. C.L. Tanner thanks her family for their unyielding support, encouragement, and confidence. Literature Cited Allan, B.F., F. Keesing, and R.S. Ostfeld. 2002. 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