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1 Department of Environmental Science, Policy and Management, 137 Mulford Hall, University of California, Berkeley, California 94720, USA
3 Corresponding author (email: blane{at}nature.berkeley.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADDENDUMLITERATURE CITED |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADDENDUMLITERATURE CITED |
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Despite their contemporary popularity as a game species, a review of the literature published since 1950 revealed that little has been reported about their associated microparasites (e.g., Mycoplasma spp., pox virus) and nothing about their macroparasites (e.g., ticks or lice), in the far-western United States (Jessup et al., 1983; Lutz and Crawford, 1987; Charlton, 2000). Most of what is known about the parasites of wild turkeys is based on research conducted in the eastern United States (Davidson and Wentworth, 1992). Furthermore, wild turkeys have not been examined heretofore as either hosts, or reservoir hosts, for the bacterial agent of Lyme disease, Borrelia burgdorferi sensu stricto (s.s.), in North America. Host, as used herein with respect to B. burgdorferi s.s., merely denotes a vertebrate species that may become infected naturally with this bacterium. In contrast, a reservoir is a proven natural host of a vector tick, serves as a source of infection for uninfected ticks that feed on it, and amplifies the number of infected ticks in a given area (Kahl et al., 2002).
In 2003, a study was initiated to determine the status, food habits, and other biologic parameters of Rio Grande wild turkeys (M. gallopavo intermedia) in three Sonoma County state parks: Annadel State Park (ASP), Jack London State Historic Park (JLSP), and Sugarloaf Ridge State Park (SRSP). Wild turkeys first appeared in these parks around 1992 and became sufficiently abundant by 2002 to arouse concern that this nonnative bird was disrupting park ecosystems by negatively impacting native plants or animals (Barrett, unpubl. data). As one component of the study, we evaluated the capacity of turkeys to serve as a host of ticks, lice, and borreliae belonging to the B. burgdorferi sensu lato (s.l.) complex, particularly the human pathogen B. burgdorferi s.s.
Lyme disease is endemic in Sonoma County, and adults of the primary regional tick vector, the western black-legged tick (Ixodes pacificus), have been found infected naturally there (Burgdorfer et al., 1985). The reported incidence of Lyme disease in Sonoma County was within the range of 1.0 to 5.0 cases per 100,000 person-years between 1990 and 2000 (Fritz and Vugia, 2001). Our specific objectives were to determine the seasonal distribution and abundance of ixodid ticks infesting wild turkeys in these parks, especially ASP, over a 1-yr period; to ascertain what species of lice also parasitize turkeys; to determine if host-seeking I. pacificus nymphs inhabiting leaf litter/fir-needle areas at ASP are infected with B. burgdorferi s.l.; and to discover by laboratory bioassays if turkeys could serve as natural hosts of B. burgdorferi s.s.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADDENDUMLITERATURE CITED |
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Turkeys were collected from ASP (n=97, 85.8% of total), SRSP (n=12, 10.6%), or JLSP (n=4, 3.5%) in east-central Sonoma County, California, USA (Fig. 1
). Because the borders of all three parks are located within a radius of ~4.l km, their populations of turkeys are treated here as a metapopulation. The principal study area, ASP (38°25'53''N, 122°36'49''W), is a 2,023-ha multipurpose, heavily used recreational area located on the eastern outskirts of the city of Santa Rosa (population, ~156,000). The topography is characterized by rolling hills with grassland, woodlands, meadows, intermittent streams, and a 10.5-ha lake. At least 14 vegetative types are present with Douglas fir (Pseudotsuga menziesii), California black oak (Quercus kelloggii), grassland, California bay (Umbellularia californica), coast live oak (Quercus agrifolia), and common manzanita (Arctostaphylos manzanita) comprising most of the ground cover (Barrett et al., 1988). The climate is Mediterranean with cool, moist winters and hot, dry summers. Rainfall occurs largely in winter and early spring, and averages about 762 mm per year.
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Turkey collections
Turkeys were collected monthly from 30 September 2003 to 9 September 2004 by shooting with a .22-caliber rifle in accordance with collecting permits issued by the California Department of Parks and Recreation, the California Department of Fish and Game, and in compliance with procedures approved by the Animal Care and Use Committee at the University of California, Berkeley. Permission was obtained to collect up to 12 turkeys per month.
Birds were processed in the field after a postmortem interval of approximately 0.25 hr to 3 hr. The age (juvenile or adult), gender, weight, and standard ornithologic measurements were recorded for each bird, and all birds were inspected cursorily for presence of ticks. Any ticks found were stored in 95% ethanol for later specific identification. Blood was withdrawn from the jugular vein, the heart, or an internal blood vessel with a 27 g 1/2 inch needle (Becton Dickinson, Franklin Lakes, New Jersey, USA) and injected into a 4- or 5-ml vacutainer tube containing EDTA (Becton Dickinson). Next, the head of each bird was cut off at the base of the neck and placed into a labeled plastic bag on wet ice. In the laboratory, the blood samples and heads were frozen until they could be tested by PCR (blood) or carefully inspected for ticks and lice (heads and necks).
After thawing, the turkey heads were examined for lice as well as ticks for 
10 min with a dissecting microscope at a magnification of 13x. The plastic bags in which the heads were frozen also were inspected for presence of ticks or lice. The specific location of attached ticks on the head or neck was noted, and all ectoparasites found were preserved in 95% ethanol for subsequent taxonomic determination. Ticks were identified to species with a dissecting microscope at magnifications up to 90x using Furman and Loomis (1984) and an unpublished key to immature ixodid ticks (Kleinjan and Lane, unpubl. data), and voucher specimens were deposited in the collection of R.S.L. Lice were slide mounted and identified with a compound microscope at magnifications ranging from 40x to 400x using several taxonomic works (Clay, 1938; Price and Graham, 1997; Price et al., 2003), and by comparison with reference specimens in the collections of the Natural History Museum, London, and the Smithsonian Institution, Washington, D.C. Voucher specimens have been deposited in the Natural History Museum (London).
Collection of questing Ixodes pacificus nymphs
On 8 May 2004, host-seeking I. pacificus nymphs were collected from fir-needle/leaf-litter areas beneath two turkey-roosting sites and two foraging areas at ASP. Two of us used standardized flannel tick-drags, 1 m by 1.25 m, to sample each site for 1 hr apiece. Sampling at sites 1–4 was begun at 11:30 AM, 2:00 PM, 3:12 PM, and 4:51 PM, respectively. The predominant tree species at all sites were Douglas fir and California bay with lesser elements of coast live oak and madrone. Besides leaf or fir-needle litter, the ground at each site contained a variable amount of branches and tree limbs. All ticks found were preserved in 95% ethanol and identified as described above. Fifty nymphs obtained from each site were assayed for borrelial infection by PCR.
DNA extraction and PCR
Blood specimens from turkeys were stored in 4- or 5-ml EDTA tubes at –20 C, and ticks were stored in 95% ethanol, prior to DNA extraction. DNA was extracted from 10 µl of turkey blood and the entire bodies of ticks using the DNeasy Tissue Kit (QiaGen, Chatsworth, Massachusetts, USA) according to the manufacturer’s instructions. Our total DNA extraction protocol requires 5–10 µl of bird blood to yield ~20 µg to 100 µg of DNA. DNA from blood samples and ticks was eluted in final volumes of 200 µl and 100 µl of AE buffer, respectively.
Presence of borreliae was determined by PCR using primer sets targeting the 5S–23S rRNA intergenic spacer region (Lane et al., 2004). PCR assays used 3 µl of each DNA extract as a template in a total reaction volume of 25 µl. All PCR mixtures contained 2.5 µl of 10x PCR buffer (Applied Biosystems, Foster City, California, USA), 2.5 µl of 8 mM dNTP, 1.5 µl of 25 mM MgCl2, 1 µl of 10 µM primers, and 0.2 µl of 5 unit/µl Taq polymerase (Applied Biosystems). Cycling conditions involved an initial 4 min denaturation at 94 C followed by amplification cycles, each consisting of a 40-sec denaturation at 94 C, a 40-sec annealing at 52–58 C, and a 1-min extension at 72 C. These cycles were followed by a 10-min extension at 72 C. Positive controls (CA4 strain) and negative controls (autoclaved distilled water) were included in each run.
Sequence alignment and phylogenetic analysis
Positive amplicons were characterized by sequence analysis of the 5S–23S rRNA intergenic spacer region. The amplicons were purified with the Qiaquick PCR Purification Kit (QiaGen, Valencia, California, USA). Each 10-µl cycle sequencing reaction contained 6.5 µl of PCR-grade water, 0.5 µl of Big Dye Terminator Ready Reaction Mix (Applied Biosystems), 0.5 µl of 3.2 µmol of the primer that was used to produce the PCR product, 1.5 µl of 5x Sequencing Dilution Buffer (Applied Biosystems), and 1 µl of purified PCR product. All cycle-sequencing products were purified with Sephadex Centri-Sep columns (Edge Biosystems, Gaithersburg, Maryland, USA) and run on an ABI 3100 (Applied Biosystems).
Complement sensitivity
To determine if turkeys could serve as reservoir hosts of Lyme disease spirochetes, the sensitivity of B. burgdorferi s.s. isolate CA4 to complement proteins present in domestic turkey serum was determined. To that end, four in vitro serum assays were performed in 96-well microtiter plates (Dynatech Laboratories & Co., Chantilly, Virginia, USA) as described previously (Kuo et al., 2000), that is, untreated (preimmune serum), heat treated (56 C for 30 min), EDTA treated (10 mM), and EGTA treated (10 mM) with MgCl2 (4 mM). Each well contained a final volume of 100 µl, 50% of which was either treated or untreated serum and the remainder BSK-II culture medium containing viable spirochetes. The survivability of spirochetes was determined with a Petroff-Hausser counting chamber (Hausser Scientific Co., Horsham, Pennsylvania, USA) at 1 and 2 hr postinoculation by examining the viability of 100 individual spirochetes in each of 10-µl aliquots. Spirochetes were considered dead if they were nonmotile or lysed. Spirochetes were inoculated into BSK-II culture medium as a positive control, and into preimmune serum from a western fence lizard (Sceloporus occidentalis), which previously had been demonstrated to be highly bacteriolytic for B. burgdorferi s.s. (Lane and Quistad, 1998; Kuo et al., 2000).
Blood specimens were drawn from four Nicholas large white domestic turkeys at the Nicholas Turkey Breeding Farms in Sonoma, California, USA. All birds were males 17 wk of age. Although their feed had been medicated with an antiprotozoal drug (amprolium hydrochloride) during the first 6 wk to 8 wk of life, none of them had been treated with antibiotics or exposed to vector ticks (Dr. Yan Ghazikhanian, pers. commun.) The blood samples were transported to the Berkeley campus on wet ice, refrigerated at 4 C for several hours, centrifuged and then the sera were removed and stored at –80 C prior to testing.
Statistical analyses
All prevalence data were evaluated in either 2 x 2, or 2 x 4, chi-square contingency tables. A probability value of 5% was set for rejection of the null hypothesis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADDENDUMLITERATURE CITED |
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In total, 113 turkeys were collected including 69 males and 44 females; of these, 25 were juveniles and 88 adults. Significantly fewer females (n=2) than males (n=21) (P=0.009), and more adults (n=22) than juveniles (n=1) (P=0.075), were collected in fall. Most adults were males (61/88), whereas the majority of juveniles were females (17/25, P=0.0007).
Fifty (44.2%) turkeys were infested by 361 ixodid ticks representing three species; these included 248 western blacklegged ticks (I. pacificus), 112 rabbit ticks (Haemaphysalis leporispalustris), and one American dog tick (Dermacentor variabilis) (Table 1). Ignoring season, the prevalence of all ticks combined was unrelated to the age (P=0.16) or sex of turkeys (P=0.86). Most ticks (290/361, 80.3%) were found on the head or neck during the 10-min laboratory inspections; the remainder were collected during the cursory field inspections. Attached ticks (n=101, 28.0% of total) were distributed almost equally on the head (53) and neck (48) regions. Among those affixed to the head, 83% were found on the crown (n=23, 43.4%), around the eye (n=11, 20.8%), and on the auricular (n=10, 18.9%). Low percentages were removed from the dewlap (5.7%), snood (5.7%), around the mandibles (3.8%), or in the suborbital region (1.9%).
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0.0001). Seven (14.0%) of the infested birds were co-infested with I. pacificus and H. leporispalustris. The proportion of the two most prevalent tick species differed significantly by life stage (P0.0001). Specifically, 86.3% of the I. pacificus were nymphs, whereas 82.1% of the H. leporispalustris were larvae (Table 1
). Two turkeys contributed about one half of all the I. pacificus or H. leporispalustris obtained from all birds. A juvenile female collected at ASP on 19 May 2004 was parasitized by 131 I. pacificus (1 larva, 130 nymphs) and seven H. leporispalustris nymphs. This bird was the lightest (2,950 g) of nine juvenile females (mean±SD=3,722±446; range, 2,950–4,200 g) taken at ASP in spring 2004. An adult female collected at ASP on 17 August 2004 was parasitized by 55 H. leporispalustris larvae. The weight of this bird (3,750 g) was slightly higher than the average weight (mean±SD=3,514±273; range, 3,250–4,000 g) of seven adult females collected at ASP in summer 2004.
Larvae and nymphs of I. pacificus were present on turkeys in winter and spring but were most prevalent in spring (Fig. 2A). The nymphs also occurred on birds in summer, but neither life stage was found on birds in fall. One I. pacificus female was removed from a turkey in spring. Larvae of H. leporispalustris infested turkeys in summer and fall, especially the former, whereas nymphs were found on low percentages of birds taken in spring, summer, and fall (Fig. 2B). The prevalence of I. pacificus and H. leporispalustris larvae or nymphs on turkeys (Table 2) did not differ significantly by the age or sex of the bird (P values range from 0.69 to 0.90). A single D. variabilis male was collected from a turkey in fall.
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Three species of lice (Insecta: Phthiraptera) were removed from the heads or necks of 20 (17.7%) of the turkeys (Table 3). Fourteen birds were infested by one species of louse, five birds by two species, and one bird by all three species. These included the cosmopolitan chicken body louse Menacanthus stramineus, which accounted for 12.5% of the 64 lice enumerated, the large turkey louse Chelopistes meleagridis (37.5% of total), and the slender turkey louse Oxylipeurus polytrapezius (50% of total). Forty-seven (73.4%) of the lice were collected in summer versus 4.7, 7.8, and 14.1% in fall, winter, and spring, respectively. Further, 14 of the louse-infested birds were parasitized by one (n=11 birds) or two (n=3 birds) species of ticks.
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Blood was drawn successfully from 90 (79.6%) of the turkeys and tested by PCR. One specimen (1%) was positive for B. burgdorferi s.l., and sequencing analysis revealed that the genospecies was B. burgdorferi s.s. This bird, an adult male, was collected at ASP on 21 October 2003. It was parasitized by a D. variabilis male, which proved to be PCR negative for borrelial DNA.
Sensitivity of B. burgdorferi s.s. to turkey-serum complement
Over 50% of spirochetes inoculated into domestic turkey sera were alive after l hr of incubation at room temperature, as compared with zero of those injected into lizard serum and ~86% introduced into BSK-II culture medium (Table 4). Similar results were observed after 2 hr of incubation (data not shown). Heat treatment (56 C, 30 min) abolished the moderate bactericidal activity of turkey serum and the complete bactericidal activity of lizard serum (Table 4). Adding EDTA to sera from turkeys or the western fence lizard similarly reduced spirochetal mortality. The addition of EGTA plus MgCl2 to turkey or lizard sera resulted in bactericidal activity like that observed for untreated sera.
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Drag-sampling leaf litter/fir-needle areas in four turkey-roosting or foraging areas for 8 hr at ASP yielded 430 I. pacificus nymphs for a mean of 53.8 ticks/hr (SD=23.1, range=23–91/hr). Additionally, two larvae and four males of I. pacificus, one Ixodes spinipalpis nymph, nine H. leporispalustris nymphs, and 13 nymphs, three males and one female of Dermacentor occidentalis were collected.
Prevalence of B. burgdorferi s.l. in questing I. pacificus nymphs
Borrelia burgdorferi s.l. DNA was detected in 15.0% (range, 8–28% per site) of the 200 nymphs tested (Table 5). Twenty-eight of the 30 positive amplicons were sequenced and, of these, 27 were determined to be the human pathogen B. burgdorferi s.s. and one was identified as an unclassified B. burgdorferi s.l.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADDENDUMLITERATURE CITED |
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The wild turkey is indigenous to the North American continent (Dickson, 1992). Following numerous translocations during the 20th century, this bird now is widespread throughout much of the continental United States (Stangel et al., 1992) including the three major geographic foci where humans contract Lyme disease, the Northeast, the Upper Midwest, and the Far West. Nonetheless, wild turkeys have not been evaluated heretofore as potential hosts of Lyme disease spirochetes, and there have been no previously published records of wild turkeys being naturally infested with either of the two primary Ixodes spp. tick vectors of B. burgdorferi s.s. in the United States, I. pacificus in the Far West and the black-legged tick Ixodes scapularis in the East. Here we report new records for three species of ixodid ticks parasitizing wild turkeys including I. pacificus, the detection of B. burgdorferi s.s. DNA in a low percentage of birds (1%, n=90), and the moderate sensitivity of B. burgdorferi s.s. to complement proteins present in sera from domestic turkeys. Moreover, three species of lice common on wild turkeys in the eastern United States are recorded for the first time from this host in California.
During preceding investigations of the role of birds in the ecology of B. burgdorferi s.l. in northern California, I. pacificus immatures (i.e., larvae, nymphs or both) were removed from three (13%) of 24 avian species in an oak-woodland (Manweiler et al., 1990), eight (24%) of 34 species in an oak/pine woodland (Wright et al., 2000), 13 (38%) of 34 species from either chaparral or woodland-grass habitats (Slowik and Lane, 2001), and 23 (51%) of 45 species in an isolated canyon containing mixed hardwoods (Wright et al., 2006). In two of those studies, when the avian sample size was 10 for birds infested by I. pacificus, the prevalence and the mean intensities (mean number of ticks per infested bird species) were relatively low and ranged from 7.1–10.0% and 1.0–3.0 ticks (Manweiler et al., 1990) and 4.2–30.8% and 1.0–2.0 ticks (Slowik and Lane, 2001), respectively. In another study, the prevalence (2.2–100%) and intensity data (1.0–7.8) were considerably higher (Wright et al., 2006). In the fourth study, these data were not provided, but the average number of ticks removed from infested birds representing five species ranged from 0.08 to 1.14 when the avian sample size exceeded 10 (Wright et al., 2000). The foregoing studies are not strictly comparable because sampling was not uniform with respect to the seasons when birds were sampled.
Among potential avian hosts in California, the prevalence (35.4%), and especially the mean intensity (6.2 ticks) of I. pacificus immatures on wild turkeys examined year-round during the present study were moderately high. In spring alone, 66.7% of the turkeys collected were infested by I. pacificus, and the mean number of ticks per bird was 9.0 (data not shown). To determine the actual significance of any vertebrate species as a host of a particular tick, the area-wide abundance of the host must be determined relative to that of other potential hosts, as well as the tick burdens present on each host species. Our abundance data for I. pacificus larvae and nymphs must be considered underestimates because the entire body of each bird was examined superficially in the field, whereas only the head/neck regions were inspected thoroughly in the laboratory with the aid of a dissecting microscope and proper lighting conditions. In future studies involving wild turkeys, the entire body of each bird should be inspected exhaustively to obtain a more reliable estimate of tick burdens.
Overall, birds seem to contribute less to the maintenance of I. pacificus larvae or nymphs in oak/Pacific-madrone woodlands, and adjacent grasslands or chaparral than do lizards and western gray squirrels (Sciurus griseus) (Eisen et al., 2004). Infestation of lizards (western fence lizards; alligator lizards [Elgaria spp.]) and western gray squirrels by I. pacificus immatures (means of 9–35 larvae and 5–6 nymphs per animal) was several times higher than it was for dusky-footed wood rats (Neotoma fuscipes), deer mice (Peromyscus spp.), and birds (means of 0.9–3.5 larvae and 0–0.3 nymphs). In dense woodlands, lizards alone hosted 84% of I. pacificus larvae and 91% of nymphs removed from all animals (lizards, birds, rodents) collected simultaneously (Eisen et al., 2004).
Elsewhere in the continental United States, wild turkeys have been recorded as hosts of four ixodid (Amblyomma americanum, Amblyomma cajennense, Haemaphysalis chordeilis, Rhipicephalus sanguineus) and two argasid ticks (Argas miniatus, Argas persicus) (Cooley and Kohls, 1944; Bishopp and Trembley, 1945; Kellogg et al., 1969; Jacobson and Hurst, 1979; Davidson and Wentworth, 1992; Mock et al., 2001). Two of these tick species (A. americanum, H. chordeilis) reportedly cause losses among wild turkeys (Bishopp and Trembley, 1945). Losses or incapacity directly attributable to high tick burdens were not observed during the present study. The most heavily tick-infested turkey (138 ticks) was the lightest juvenile female collected in spring, and it weighed 21% less than the average bird in its age/sex cohort taken at that time of year. However, the weight of the only other abundantly parasitized bird (55 ticks) was close to the average for its cohort/sex.
Although the other primary vector of B. burgdorferi s.s. in the United States, I. scapularis, has never been reported from wild turkeys, an experimental study in which hundreds of I. scapularis immatures were placed directly on five captive wild birds demonstrated that no larvae and few nymphs actually engorged on turkeys (Ostfeld and Lewis, 1999). The authors concluded that wild turkeys are unlikely to serve as natural hosts of I. scapularis larvae or nymphs, which is consistent with the absence of published collection records indicative of such a tick/host association.
Lice typically are the most commonly reported ectoparasites of wild turkeys. Eight species of chewing lice have been recorded from wild turkeys in the United States (Davidson and Wentworth, 1992). The three species we found (M. stramineus, C. meleagridis, O. polytrapezius) were known previously to infest wild turkeys in seven to 10 states from other geographic regions (Davidson and Wentworth, 1992). Our data indicate that lice were most prevalent on birds in summer, but both the prevalence and abundance data should be regarded circumspectly, if not as gross underestimates. We did not attempt to collect lice from turkeys in the field, and our search was confined solely to the head and neck regions in the laboratory. Moreover, M. stramineus feeds in part on feathers, C. meleagridis is a niche specialist restricted in distribution to feathers on the neck and breast of its host, and O. polytrapezius exploits turkey wing feathers (Johnson and Clayton, 2003)
Menacanthus stramineus parasitizes many species of domestic poultry worldwide, but the wild turkey generally is considered to be its native host because this louse has never been recorded from any other wild avian species (Price and Graham, 1997). It is the most prevalent and destructive species of louse infesting modern poultry in the United States (Axtell, 1999). Menacanthus stramineus also is considered to be a possible vector of various avian viral and bacterial disease agents (Derylo, 1970) because, unlike most chewing lice, its diet includes host blood as well as feathers, hair, and skin debris (Derylo and Gogacz, 1974).
Two species of Chelopistes occur in North America North of Mexico, and C. meleagridis is by far the most common (Price et al., 2003). This louse is a cosmopolitan parasite of domestic turkeys and is a common parasite of wild turkeys in the United States. It also has been recorded from the ocellated turkey (Meleagris ocellata) of southern Mexico and Central America (Clay, 1941). Oxylipeurus polytrapezius was the most prevalent louse among the three species collected. Although it is a common parasite of both domestic and wild turkeys, its life history and habits are poorly known. Its slender morphology enables it to exploit the wing feathers, where it can slot between barbules to escape the preening activities of its host (Johnson and Clayton, 2003).
Turkeys as hosts of B. burgdorferi s.s.
The fact that B. burgdorferi s.s. DNA was detected in blood from only 1% of wild turkeys collected year-round suggests that this bird is not an important host of this particular Lyme disease spirochete. The wild turkey is just the second avian species from the far western United States found to contain B. burgdorferi s.s. DNA in its blood. A specimen from a hermit thrush (Catharus guttatus) collected from a wooded canyon in the Sutter Buttes of northern California was PCR positive for this spirochete (Wright et al., 2006). The low prevalence of spirochetal infection detected in turkeys during the present study is noteworthy because most turkeys were collected at ASP where 15% of the I. pacificus nymphs assayed were infected with B. burgdorferi s.l. in spring, and 86% of all the I. pacificus removed from birds were nymphs. Thus, at least some of the turkeys had been, or were being, fed upon by spirochete-infected nymphs at the time of collection. We did not test the relatively few I. pacificus larvae (n=33) obtained from turkeys because no larval ticks were found attached to the one infected bird. Curiously, that bird was collected in fall when I. pacificus nymphs usually are inactive, and the only tick found on it, a D. variabilis male, was PCR negative for B. burgdorferi s.s.
The complement system plays an important role in defense against infection (Sim and Dodds, 1997). Among other functions, it promotes and regulates the lysis or phagocytosis of foreign cells including gram-negative bacteria (e.g., borreliae), and it interacts with the adaptive immune system. The level of complement activity in serum has been found to be an important intrinsic factor determining the reservoir competence of lizards, birds, and mammals for several genospecies of B. burgdorferi s.l. (Isogai et al., 1994; Kurtenbach et al., 1998; Kuo et al., 2000; Nelson et al., 2000). Our complement-protein assays demonstrated that untreated (preimmune) serum from domestic turkeys was moderately bacteriolytic for B. burgdorferi s.s. because 45% of spirochetes introduced into it were dead within 1 hr. In contrast, 100% of spirochetes injected into untreated lizard serum died within 1 hr, as expected (Lane and Quistad, 1998; Kuo et al., 2000). Taken together, the assays implicate components of the alternative complement pathway as a significant source of much of the observed bacteriolytic activity: heat or EDTA treatment of domestic-turkey serum largely arrested bacteriolysis, whereas a lack of treatment or the addition of EGTA/MgCl2 allowed turkey serum to retain its moderate borreliacidal effects.
In related studies, serum from northern bobwhite quail (Colinus virginianus) was nonlytic for the B-31 type strain of B. burgdorferi s.s. and only partially lytic for two strains of the related spirochete Borrelia bissettii (Ullmann et al., 2003). Like turkeys, serum from pheasant was moderately lytic for B. burgdorferi s.s., whereas it was either nonlytic or highly lytic for four other genospecies of B. burgdorferi s.l. (Kurtenbach et al., 1998). In vitro complement studies such as these, and reservoir-competence trials using xenodiagnostic larval ticks and wild-caught birds (e.g., Ginsberg et al., 2005), demonstrate that avian species differ characteristically in their capacity to host and disseminate various genospecies of B. burgdorferi s.l. group spirochetes.
We used sera from domestic turkeys rather than sera from wild turkeys in our complement assays because these birds represent a single species (M. gallopavo), and we did not have a source of pre-immune serum from captive wild birds that had been held in a tick-free environment. Nevertheless, the complement-test results, when considered alongside the low spirochetal infection prevalence detected in blood specimens of field-derived turkeys and the low infestation prevalence by I. pacificus larvae, suggest that the Rio Grande wild turkey is an inefficient host of B. burgdorferi s.s. in northern California woodlands. A competent reservoir of a tick-borne disease agent must not only be susceptible to the agent and capable of serving as a source of infection for un-infected ticks that feed on it, but it also must be fed upon by at least two life stages of a vector tick in order to amplify the infection in nature.
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ADDENDUM |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADDENDUMLITERATURE CITED |
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). All 33 larvae were PCR negative for Lyme disease-group spirochetes, which reconfirms our conclusion that the Rio grande wild turkey is an inefficient host of B. burgdorferi s.s.
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ACKNOWLEDGMENTS |
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. S.K. Wikel and P.C. Giclas are thanked for their helpful counsel concerning the complement assays and their interpretation. A portion of this research was supported by the Louisiana State University Museum of Natural Science. This research was funded in part by grants from the California Department of Parks and Recreation to R.H.B., the National Institutes of Health (AI22501) and a generous gift from A. Henry to R.S.L., and the National Science Foundation (DEB-0107891) to V.S.S. |
FOOTNOTES |
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LITERATURE CITED |
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Received for publication 15 December 2005.
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