Ticks on birds.
A total of 19,631 captures of birds, representing 105 species, occurred in 48,030 net hours for an overall netting success rate of 40.9 captures per 100 NH (see Tables S1 and S2 in the supplemental material). Net success was significantly higher in October and November (53.3 birds/100 NH) versus May through August (33.4 birds/100 NH), reflecting the influx of fall migrants. Recaptures comprised 19.5% of all capture events. Gray Catbird, Myrtle Warbler, American Goldfinch, and White-throated Sparrow were the most abundant species with a sample size of over 1,000 captures of each species (see Tables S1 and S2 in the supplemental material). Of all captures, 65.9, 30.6, and 3.5% of birds were hatch-year, after hatch-year, and of unknown age class, respectively; 30.7, 27.7, and 41.6% of the birds were male, female, and of undetermined sex, respectively.
A total of 2,074 captures (10.6% of all captures) were infested with ticks, representing 1,799 individual birds with ticks. A total of 12,301 ticks were removed from birds, with a mean infestation burden of 5.9 ticks per infested bird. Of all ticks, 86.4% were I. dentatus (10,363 larvae and 265 nymphs), 13.4% were Haemaphysalis leporispalustris (1,535 larvae and 118 nymphs), 0.1% were I. scapularis (7 larvae and 6 nymphs), and 0.06% were Dermacentor variabilis (6 larvae). PCR of the tick ITS-2 region resulted in I. scapularis sequences that matched with 100% homology to published I. scapularis sequences, whereas the ITS-2 regions of I. dentatus and H. leporispalustris were not previously deposited. The most commonly parasitized bird species, in which at least one-third of all individuals were infested, included Brown Thrasher, Carolina Wren, Eastern Towhee, White-throated Sparrow, Song Sparrow, Lincoln's Sparrow, Hermit Thrush, American Robin, and Yellow-breasted Chat (see Table S1 in the supplemental material). In total, 285 birds were noted to harbor ticks but were released without removing ticks. Of the remaining birds, 84.6, 23.5, 0.2, and 0.06% of parasitized birds harbored I. dentatus, H. leporispalustris, I. scapularis, and D. variabilis, comprising 7.8, 2.2, 0.02, and 0.005% of all birds, respectively. Coinfestation of birds with both I. dentatus and H. leporispalustris simultaneously was found for 8.2% of all parasitized birds; this rate is ~2.4 times lower than what would be expected by chance (χ2 = 1,051; df = 3; P < 0.001). All ticks were at immature stages, with the exception of a single adult female I. dentatus collected from a House Wren. The mean burdens of I. dentatus larvae and nymphs on infested birds were 7.0 and 1.7, respectively. The mean burdens of H. leporispalustris larvae and nymphs on infested birds were 4.2 and 1.2, respectively.
Across all sampling, only three individual birds harbored I. scapularis: a Swainson's Thrush, a Hermit Thrush, and a Connecticut Warbler. All three were hatch-year birds upon their first capture during in late August and September of 2004-2005; none were recaptured subsequently. The Hermit Thrush harbored 6 larval and 5 nymphal I. scapularis ticks, in addition to 2 H. leporispalustris larvae. The Connecticut Warbler harbored a single I. scapularis larva. The Swainson's Thrush harbored a single I. scapularis nymph, in addition to 10 and 3 H. leporispalustris larvae and nymphs, respectively.
Ticks on mammals.
At Pitsfield, no I. scapularis were found on 65 white-footed mice and 59 chipmunks trapped in May and June of 2005-2009. Concurrently, at Van Buren State Park, the positive control site for I. scapularis 90 km away, 69.3% of 179 mice and 100% of 23 chipmunks were infested with I. scapularis. D. variabilis was present on small mammals from both areas (Table ). Nineteen captures of eastern cottontails at Pitsfield represented 15 individual rabbits of which 71.4% were infested by ticks. The most common species were I. dentatus and H. leporispalustris; no I. scapularis ticks were found on rabbits (Table ). A total of 18 nontarget mammals, comprising seven additional species, were captured, and none harbored I. scapularis (Table ).
TABLE 1. Mammal infestation prevalence with various tick species, mammal infection prevalence with B. burgdorferi, and mammal-derived tick prevalence of infection with B. burgdorferi at the Pitsfield Banding Station and Van Buren State Park, May to October, 2004-2009 (more ...) B. burgdorferi in bird-associated ticks.
A total of 2,202 ticks/larval pools from 1,579 individual birds were assayed for infection with Borrelia species; these infested birds from which we tested ticks were a random subset of all infested birds. Of these, 78 (3.5%) ticks/larval pools were sequence confirmed positive for B. burgdorferi, and these infected ticks were removed from 73 different birds (4.6% of infested birds from which ticks were tested). An additional 18 samples produced IGS bands at ~900 bp in size (a finding indicative of either B. burgdorferi or B. andersonii) that were not successfully sequenced. Accordingly, the reported tick infection prevalence is a minimum. A minimum of 0.49% of all birds in our study carried B. burgdorferi-infected ticks (10.6% of birds carried ticks; 4.6% of infested birds carried infected ticks). B. burgdorferi infection prevalence in nymphs was significantly greater than that of larval pools (5.2 and 3.2%, respectively; P = 0.02). B. burgdorferi was detected in I. dentatus, H. leporispalustris, and I. scapularis (Table ). Of the small number of I. scapularis found in our study, 1 of the 5 nymphal I. scapularis ticks removed from the Hermit Thrush was infected with B. burgdorferi.
Prevalence of infection with B. burgdorferi in ticks removed from birds, 2004-2007, at the Pitsfield Banding Stationa
Aggregating all 4 years of the study, B. burgdorferi was present in all months sampled (i.e., May-November), with the highest monthly prevalences of 11 to 12% in May and July. From mid-June through mid-August—a period that largely excludes the spring and fall migrations in our area—we detected 19 B. burgdorferi-positive ticks/pools, comprising 24% of all positives. Of these mid-summer positive samples, 8 (42.1%) were from hatch-year birds, which is indicative of local pathogen maintenance.
Our data on production of infected larval ticks (i.e., natural xenodiagnosis) implicate 20 species of bird as reservoir competent for B. burgdorferi (see Table S1 in the supplemental material). As further evidence of there being local infectious hosts, nine individual birds of four host species were each associated with multiple infected ticks/tick pools that were removed during multiple capture events (i.e., American Robin, Song Sparrow, Swainson's Thrush, and White-throated Sparrow).
B. burgdorferi in mammal-associated ticks.
A total of 150 adults, nymphs, and larval pools removed from white-footed mice, chipmunks, eastern cottontails, and nontarget mammals at Pitsfield were assayed for infection with B. burgdorferi. The only host species associated with positive ticks was the eastern cottontail, in which a minimum of 4 of 122 (3.3%) I. dentatus adults or nymphs were sequence confirmed to be positive for B. burgdorferi, and an additional 4 were positive for either B. burgdorferi or B. andersonii (no sequence obtained; Table ). At Van Buren State Park, a total of 376 nymphs and larval pools removed from white-footed mice and chipmunks were assayed for infection with B. burgdorferi, 109 (29.0%) of which were positive (Table ).
B. burgdorferi in mammal ear biopsy specimens.
At Pitsfield, 1 of 61 (1.6%) and 2 of 55 (3.6%) white-footed mouse and chipmunk ears, respectively, tested positive for B. burgdorferi, whereas the equivalent prevalences at Van Buren were 30.4 and 34.8%, respectively (Table ). At Pitsfield, a minimum of 4 of 20 (20%) cottontail ear biopsy specimens were positive for B. burgdorferi, and an additional two rabbits were positive for either B. burgdorferi or B. andersonii (no sequence obtained). None of 14 ear biopsy specimens from the nontarget mammals tested positive.
B. burgdorferi genotypes.
B. burgdorferi IGS PCR products were successfully sequenced from 80 samples, including nine mammal-associated samples and 71 ticks/larval pools removed from birds. Of the 80 sequences, 10% were interpreted as mixed strain infections due to the presence of double-nucleotide peaks at polymorphic sites (six tick samples removed from birds, one chipmunk ear, and one tick removed from a rabbit). Among all IGS strains, we found no evidence for recombination using Sawyer's test. Within the 500-nucleotide IGS fragment that we analyzed, 58 sites were found to be polymorphic, including one indel block of 7 nucleotides (treated as a single polymorphism).
In all, 25 IGS strains were found among 72 samples, including strains from all three RST groups. Of all of the strains, 32% were “ubiquitous” (i.e., also found in association with classic, I. scapularis
-driven transmission cycles in the Northeast [10
] and/or the Midwest [S. A. Hamer, unpublished data]), and 68% were “unique” (i.e., novel IGS mutants thus far found only at Pitsfield in cryptic transmission; Fig. ). The only strains found more than once were ubiquitous strains; all unique strains were singletons, many of which were single- or double-nucleotide polymorphisms of ubiquitous strains. A minimum spanning network of Pitsfield strains shows, for example, that there were many unique strains very similar to the ubiquitous strain IGS 2D, including six different single nucleotide polymorphism mutants and three different double nucleotide polymorphism mutants (Fig. ). The strain evenness, a component of diversity, is illustrated by the relative sizes of the symbols in Fig. , in which the most abundant strains are depicted by the largest symbols (IGS 2D, Midwest B, and Midwest K). A rarefaction curve to assess strain richness suggests that true B. burgdorferi
strain richness at Pitsfield is vast, since the rate at which new strains were found per unit of individuals sequenced was not yet asymptotic. We detected 25 IGS strains within 72 samples derived from investigations of over 19,000 birds and a small number of mammals; from these data, the Chao-1 nonparametric estimator of true species richness is 246 ± 101 strains.
FIG. 1. Minimum spanning network of B. burgdorferi IGS haplotypes collected from Pitsfield Banding Station, 2004-2007. Dashed rectangles delineate the three main RST groups. Haplotypes are represented by solid ovals or rectangles; the solid rectangles represent (more ...)
The single B. burgdorferi-positive from an I. scapularis nymph removed from a bird was type “Midwest A” of RST group 2 (Fig. ), a ubiquitous strain with a wide Midwestern distribution. There was no difference in the proportion of unique strains in bird-associated ticks during the migratory (1 May to 15 June; 15 August to 31 November) versus nonmigratory (15 June to 15 August) seasons (23.6 and 30%, z-ratio, 043; P = 0.67). Of the seven B. burgdorferi sequences identified in mammal tissues or mammal-derived ticks, all but one were ubiquitous strains, with all three RST groups represented (Fig. ). The single infected white-footed mouse ear was infected with Midwest A, the chipmunk ear was infected with Midwest B, and the I. dentatus adults removed from rabbits were infected with IGS 2D (n = 2), IGS 1A (n = 1), and Midwest K (n = 1).