Extensive Distribution of the Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, in Multiple Tick Species Parasitizing Avian and Mammalian Hosts across Canada

Lyme disease, caused by the spirochetal bacterium, Borrelia burgdorferi sensu lato (Bbsl), is typically transmitted by hard-bodied ticks (Acari: Ixodidae). Whenever this tick-borne zoonosis is mentioned in medical clinics and emergency rooms, it sparks a firestorm of controversy. Denial often sets in, and healthcare practitioners dismiss the fact that this pathogenic spirochetosis is present in their area. For distribution of Bbsl across Canada, we conducted a 4-year, tick–host study (2013–2016), and collected ticks from avian and mammalian hosts from Atlantic Canada to the West Coast. Overall, 1265 ticks representing 27 tick species belonging to four genera were collected. Of the 18 tick species tested, 15 species (83%) were positive for Bbsl and, of these infected ticks, 6 species bite humans. Overall, 13 of 18 tick species tested are human-biting ticks. Our data suggest that a 6-tick, enzootic maintenance cycle of Bbsl is present in southwestern B.C., and five of these tick species bite humans. Biogeographically, the groundhog tick, Ixodes cookei, has extended its home range from central and eastern Canada to southwestern British Columbia (B.C.). We posit that the Fox Sparrow, Passerella iliaca, is a reservoir-competent host for Bbsl. The Bay-breasted Warbler, Setophaga castanea, and the Tennessee Warbler, Vermivora peregrina, are new host records for the blacklegged tick, Ixodes scapularis. We provide the first report of a Bbsl-positive Amblyomma longirostre larva parasitizing a bird; this bird parasitism suggests that a Willow Flycatcher is a competent reservoir of Bbsl. Our findings show that Bbsl is present in all provinces, and that multiple tick species are implicated in the enzootic maintenance cycle of this pathogen. Ultimately, Bbsl poses a serious public health contagion Canada-wide.


Spirochete Detection
After identification, ticks were sent to three different laboratories for Bbsl testing because the first two institutions had policy changes during this study, and halted tick testing. , bacteria from live ticks were cultured in Barbour-Stoenner-Kelly (BSK) medium, whereas dead ticks were processed directly for DNA extraction followed by PCR (polymerase chain reaction) amplification. The DNA detection protocol was previously described [28][29][30]. Even though Persing et al. employed both the flagellin (flaB) gene and the major outer surface protein A (OspA) gene [28], this lab (J.F.A.) only used the OspA gene. Bona fide negative and positive controls were utilized.
In the third phase, ticks were directly put in 94% ethyl alcohol, and sent promptly by courier to the lab (J.E.F.). The protocol used for detecting Bbsl is outlined in Barbour et al. [32].
Since the occurrence of B. miyamotoi, a relapsing fever group spirochete, is deemed to be rare (<1%), we did not screen ticks for this microorganism. The PCR primers that we used were designed and applied in the study to specifically detect known species in the B. burgdorferi sensu lato complex.
In this study, the infection rate is the number of ticks infected with Bbsl divided by the number of ticks tested.

Molecular Tick Identification of Ixodes cookei
In order to confirm the identification of I. cookei collected in southwestern British Columbia, we used molecular methodology, and barcoded a larval tick (17-5A85L) hatched from the eggs of a gravid female, which was collected at North Saanich, Vancouver Island. The molecular identification was conducted at the Centre of Biodiversity Genomics (CBG), University of Guelph with accession number BIO-18-060. The DNA extract is being held at −80 • C at the same location. The collection data and barcode sequence is stored in BOLD, and can be accessed in the BOLD dataset at: dx.doi.org./10.5883/DS-IGAK. Eight co-infestations (seven double, one triple) were identified ( Table 6). The triple co-infestation consisted of Ixodes angustus (male, female), Ixodes pacificus (western blacklegged tick) (nymph, Bbsl-positive), and Ixodes spinipalpis (nymph). When we looked at earlier tick-host studies [7,33], and compared their findings with our dataset, we discovered a number of range extensions for certain tick species, namely I. cookei, Ixodes gregsoni (a mustelid-feeding tick), I. spinipalpis, Ixodes rugosus, and Ixodes texanus (raccoon tick) (Figure 1). From a medical standpoint, we collected an I. spinipalpis nymph from a human; the nymph tested negative for Bbsl. Because I. spinipalpis has vector competence for Bbsl, this tick species has the potential to transmit Lyme disease spirochetes to people.
In southwestern Ontario, 27 larval and nymphal specimens of A. longirostre, which are native to the Neotropics, were collected from neotropical songbirds during northward spring migration.
In order to answer a long-standing question of how long I. cookei live, we determined the longevity of one generation of I. cookei: 4201 days (11.51 years). The breakdown for the developmental life stages (egg, larva, nymph, and adult) was 55 days, 614 days, 919 days, and 2613 days (female), respectively. This dataset provides rudimentary information on the sustainment of I. cookei, and represent the longest living individuals for each developmental life stage.
In this study, we report many novel host records for ticks on birds and mammals ( Table 6). The two focal study areas comprise: (a) Pacific region: Metchosin-Victoria-Vancouver-Maple Ridge in southwestern British Columbia (Tables 2 and 3) and (b) Eastern region: London-St. Thomas-Simcoe-Toronto in southern Ontario (Tables 4 and 5). In southwestern B.C., 22 I. cookei were collected from three different mammal species (American mink, Pacific raccoon, and striped skunk). In addition, I. pacificus, Ixodes rugosus, Ixodes soricis (shrew tick), I. spinipalpis, and I. texanus were collected from mammals on Vancouver Island (Figure 1). Excluding I. soricis and including the avian coastal tick, Ixodes auritulus, we put forward a 6-tick, enzootic maintenance transmission cycle of Lyme disease spirochetes in southeastern region of Vancouver Island.
In southwestern Ontario, 27 larval and nymphal specimens of A. longirostre, which are native to the Neotropics, were collected from neotropical songbirds during northward spring migration.
In order to answer a long-standing question of how long I. cookei live, we determined the longevity of one generation of I. cookei: 4201 days (11.51 years). The breakdown for the developmental life stages (egg, larva, nymph, and adult) was 55 days, 614 days, 919 days, and 2613 days (female), respectively. This dataset provides rudimentary information on the sustainment of I. cookei, and represent the longest living individuals for each developmental life stage.            We provide two novel tick-host records for blacklegged ticks, Ixodes scapularis (northern populations previously considered Ixodes dammini), on passerine birds. A fully engorged I. scapularis nymph was collected from a Bay-breasted Warbler, Setophaga castanea, on 15 May 2015 at Ste-Anne-de-Bellevue, Québec during spring migration. This replete nymph underwent ecdysis to a female in 72 days. Likewise, two I. scapularis larvae were collected from a hatch-year Tennessee Warbler, Vermivora peregrina, at Ruthven Park, Ontario on 1 September 2013 during fall migration.
In the Pacific region, the most common avian host parasitized by ticks was the Song Sparrow, whereas in the Eastern region, the Common Yellowthroat, a ground-foraging passerine, was most frequently parasitized by ixodid ectoparasites (Figure 2).
We report 34 I. angustus ticks (29 females, five males) parasitizing a red squirrel at East Sooke, British Columbia on 28 October 2013; this heavy tick infestation constitutes the first documentation of Bbsl-positive I. angustus on this sciurid species in Canada (Figure 3).
We collected 24 I. rugosus nymphs from a river otter at Esquimalt, B.C.; not only is this collection a new host record, it is the first account of this tick species on Vancouver Island. Additionally, we collected a single I. texanus female from a Pacific raccoon at Colwood, Vancouver Island; this collection is the southernmost record in British Columbia. We provide two novel tick-host records for blacklegged ticks, Ixodes scapularis (northern populations previously considered Ixodes dammini), on passerine birds. A fully engorged I. scapularis nymph was collected from a Bay-breasted Warbler, Setophaga castanea, on 15 May 2015 at Ste-Anne-de-Bellevue, Québec during spring migration. This replete nymph underwent ecdysis to a female in 72 days. Likewise, two I. scapularis larvae were collected from a hatch-year Tennessee Warbler, Vermivora peregrina, at Ruthven Park, Ontario on 1 September 2013 during fall migration.
In the Pacific region, the most common avian host parasitized by ticks was the Song Sparrow, whereas in the Eastern region, the Common Yellowthroat, a ground-foraging passerine, was most frequently parasitized by ixodid ectoparasites (Figure 2).
We report 34 I. angustus ticks (29 females, five males) parasitizing a red squirrel at East Sooke, British Columbia on 28 October 2013; this heavy tick infestation constitutes the first documentation of Bbsl-positive I. angustus on this sciurid species in Canada (Figure 3).
We collected 24 I. rugosus nymphs from a river otter at Esquimalt, B.C.; not only is this collection a new host record, it is the first account of this tick species on Vancouver Island. Additionally, we collected a single I. texanus female from a Pacific raccoon at Colwood, Vancouver Island; this collection is the southernmost record in British Columbia.

Molecular Tick Identification of Ixodes cookei
A gravid I. cookei female was collected from a striped skunk on 15 June 2017 at Saanich, Vancouver Island, B.C. This female laid eggs that hatched into larvae (BIO-18-060), and one of these larvae was barcoded at BIO (Biodiversity Institute of Ontario), University of Guelph, Ontario. The We provide two novel tick-host records for blacklegged ticks, Ixodes scapularis (northern populations previously considered Ixodes dammini), on passerine birds. A fully engorged I. scapularis nymph was collected from a Bay-breasted Warbler, Setophaga castanea, on 15 May 2015 at Ste-Anne-de-Bellevue, Québec during spring migration. This replete nymph underwent ecdysis to a female in 72 days. Likewise, two I. scapularis larvae were collected from a hatch-year Tennessee Warbler, Vermivora peregrina, at Ruthven Park, Ontario on 1 September 2013 during fall migration.
In the Pacific region, the most common avian host parasitized by ticks was the Song Sparrow, whereas in the Eastern region, the Common Yellowthroat, a ground-foraging passerine, was most frequently parasitized by ixodid ectoparasites (Figure 2).
We report 34 I. angustus ticks (29 females, five males) parasitizing a red squirrel at East Sooke, British Columbia on 28 October 2013; this heavy tick infestation constitutes the first documentation of Bbsl-positive I. angustus on this sciurid species in Canada (Figure 3).
We collected 24 I. rugosus nymphs from a river otter at Esquimalt, B.C.; not only is this collection a new host record, it is the first account of this tick species on Vancouver Island. Additionally, we collected a single I. texanus female from a Pacific raccoon at Colwood, Vancouver Island; this collection is the southernmost record in British Columbia.

Molecular Tick Identification of Ixodes cookei
A gravid I. cookei female was collected from a striped skunk on 15 June 2017 at Saanich, Vancouver Island, B.C. This female laid eggs that hatched into larvae (BIO-18-060), and one of these larvae was barcoded at BIO (Biodiversity Institute of Ontario), University of Guelph, Ontario. The

Molecular Tick Identification of Ixodes cookei
A gravid I. cookei female was collected from a striped skunk on 15 June 2017 at Saanich, Vancouver Island, B.C. This female laid eggs that hatched into larvae (BIO-18-060), and one of these larvae was barcoded at BIO (Biodiversity Institute of Ontario), University of Guelph, Ontario. The barcoding analysis confirmed the identification as I. cookei, and the nucleotide sequence was deposited in GenBank with accession number: MH338173.
Based on a sequence length of 620 bp, this larva had a 99.8% similarity match to three other I. cookei in BOLD. This molecular tick identification verifies the establishment of I. cookei in southwestern B.C.

Spirochete Detection
We sampled a wide cross section of ticks collected coast to coast, and tested 18 tick species for Bbsl (Table 1). Of the 18 tick species tested, 15 species (83%) were positive for Bbsl and, of these systemically infected ticks, 6 species bite humans. In the Western region, we collected 25 I. rugosus nymphs (one from a striped skunk, and 24 from a river otter), but none was positive for Lyme disease spirochetes. It is noteworthy that 11 (35%) of 31 I. angustus adults were positive for Bbsl. In the Pacific coastal region, we detected Bbsl in three tick species parasitizing birds ( Table 2) and, likewise, in seven tick species infesting mammals (Table 3).
In southwestern B.C., 5 (23%) of 22 I. cookei, which were collected from three different mammalian hosts (i.e., Pacific raccoon, American mink, and striped skunk) were positive for Bbsl (Table 3). Specifically, the biogeographic breakdown for Bbsl-positive I. cookei in southwestern B.C. was Mainland, 2 and Vancouver Is., 3. Our data provide newfound evidence of Lyme disease spirochetes in I. cookei in far-western Canada.
A single I. soricis tick was collected from a roadkill vagrant shrew on the southern shoreline of Vancouver Island, B.C.; it was not tested for Lyme disease spirochetes, but instead, kept as a voucher specimen.
Notably, 17 (77%) of 22 I. auritulus larvae collected from a Fox Sparrow, Passerella iliaca, were positive for Bbsl, which suggests that this ground-frequenting songbird is a reservoir-competent host.
In the eastern Canadian region, we detected Bbsl in two tick species parasitizing birds (Table 4) and, similarly, in four species infesting mammals (Table 5). Notably, 16 (59%) of 27 I. scapularis nymphs collected from ground-foraging songbirds during northward spring migration were infected with Lyme disease spirochetes. Additionally, we collected Bbsl-infected I. scapularis larvae from a northern short-tailed shrew, which reaffirms that this small mammal is a reservoir-competent host.
In the laboratory (J.F.A.), the majority of cultures became contaminated, and only one culture produced motile spirochetes. This culture was PCR positive for Bbsl, but it was not sent for DNA sequencing.
Of epidemiological significance, 1 of 27 A. longirostre ticks was positive for Bbsl; this is the first report of a Bbsl-positive A. longirostre parasitizing a bird in North America. In addition, we provide the first report of Bbsl in I. brunneus and I. texanus in Canada.

Discussion
We detected Bbsl in 15 of 18 tick species tested, and unveiled multiple host records nationwide ( Table 6). In certain regions of Canada, we encountered several tick species in a complex enzootic maintenance cycle of Bbsl. In one particular locality on the Pacific coast, we encountered several tick species parasitizing avian and mammalian hosts, and some of these host-seeking ticks bite humans. Surprisingly, we observed instances where ticks had shifted their host range, or were noted for the first time. Because we collaborated closely with biologists and wildlife rehabilitators in certain locations, we were able to collect a proportionally larger number of ticks in these areas. From the Atlantic Ocean to the Pacific Ocean, we detected the Lyme disease spirochete in a continuum of 15 different tick species.

Tick Vector Competency for B. burgdorferi Sensu Lato
In total, eight tick species in our study are known to be competent vectors of Lyme disease spirochetes. They include Ixodes affinis, I. angustus, Ixodes dentatus (rabbit-associated tick), Ixodes minor, Ixodes muris (mouse tick), I. pacificus, I. scapularis, and I. spinipalpis [38]. Because all motile life stages (larvae, nymphs, and adults) of I. auritulus had significant Bbsl infection prevalence, and this tick species is exclusively parasitizes birds, we further indicate that I. auritulus has vector competence for Bbsl (Table 2) [39].

Ticks as Bridge Vector for B. burgdorferi Sensu Lato
In this study, we uncovered eight tick co-infestations (seven double species, one triple species) on vertebrates. When a Bbsl-infected tick parasitizes its host, it can transmit spirochetes during its blood meal. If the host is spirochetemic, it can then act as a source of Lyme disease spirochetes to other cofeeding ticks, either the same tick species or a different tick species. When these engorging ticks have fed to repletion, they drop off and undergo ecdysis before they can transmit spirochetes to suitable vertebrate hosts, including humans. Depending on the developmental life stage of the tick, the molt normally takes five to eight weeks. Both I. pacificus and I. scapularis are noted as the primary bridge vectors of Lyme disease spirochetes to humans [40].

Dispersal of Vector-Borne Pathogens
During this study, four genera (i.e., Amblyomma, Dermacentor, Haemaphysalis, and Ixodes) of ticks were collected from wild birds and terrestrial mammals across Canada. With the exception of I. affinis, I. minor, and Amblyomma species, the remainder of tick species are established and survive successfully in Canada. Migratory songbirds import bird-feeding ticks into Canada annually during spring migration, and these songbird-transported ticks and associated avifauna may be infected with a wide range of vector-borne, zoonotic pathogens. These bird parasitisms include Bbsl [10][11][12][13][14][15]41], Anaplasma phagocytophilum [12,42], Babesia spp. [42], Bartonella spp. [43], and vector-borne viruses [44,45]. Neotropical passerines migrate across national and intercontinental borders, and become long-range vectors for any zoonotic pathogen that they harbor. Overall, dispersal of Bbsl-infected ticks along migration routes is an important mechanism in the establishment of new endemic foci of tick-borne diseases [46].
Since passerines and raptors are parasitized by bird-feeding ticks, they provide an interconnecting link for Bbsl back and forth across the Salish Sea. More epidemiologic details on avian and mammalian hosts in this region are provided in Table 6. In the Pacific Northwest, I. auritulus is the most frequently occurring tick species parasitizing wild birds, namely raptors [30] and passerines [39]. In addition, several mammals act as reservoirs in an ongoing enzootic maintenance cycle of Bbsl along B.C.'s Pacific coast (Table 3).
Along the Pacific B.C. coast, I. auritulus plays a vital role in maintaining the presence of Lyme disease spirochetes. In all, 49 (48%) of 102 I. auritulus (larvae, nymphs, and adults) in this study were infected with Bbsl. Similarly, Scott et al. detected Bbsl in 31% of I. auritulus [39]. Although I. auritulus only parasitizes avifauna, both birds and mammals eat these ixodid ectoparasites, and may become systematically infected by oral inoculation [56].

London-St. Thomas-Simcoe-Toronto Region
The most commonly occurring tick species in our study was the blacklegged tick (Tables 4 and 5). East of the Rocky Mountains, I. scapularis [57,58] is the most prevalent tick species on passerine migrants during northward spring migration (Table 4). Ecologically, the peak questing activity of nymphs coincides with peak spring migration of northbound passerine migrants.
Bbsl-positive I. scapularis larvae were collected from a northern short-tailed shrew, which reinforces that this insectivore is a reservoir host. This parasitism is consistent with other researchers who found that this shrew is a reservoir-competent host [59][60][61]. Because there is no transovarial transmission of Bbsl in I. scapularis females [62], we are confident that the source of infection was this spirochetemic host. This high-energy insectivore burrows through leaf litter and the humus layer, and has ample opportunity to become parasitized by Bbsl-infected I. scapularis immatures, especially in a Lyme disease endemic area.

Geographical Distribution of Ticks
We document new Canadian foci where certain tick species have been collected for the first time. Our novel tick findings may have been oversights by earlier tick studies [7,8] or unnoticed biogeographical shifts. Any ticks that are not transported by wild birds must have a terrestrial mode of transportation to occupy a new area.
In eastern Canada, we noticed a biogeographical shift in I. gregsoni. An archetype study reported this mustelid tick at Ignace, Ontario [33]. We report I. gregsoni 200 km further west at Jim Lake, Ontario ( Figure 1).

Mammal Parasitisms Reflect Established Ticks
This study provides many mammalian, tick-host associations across Canada. The highlights of these first-time records of mammal parasitisms are reported in Table 6. Since terrestrial mammals have a localized home range, they typically signify that a given tick species is established in the vicinity.
In the present study, 25 (93%) of the 27 tick species parasitize mammalian hosts, including humans. Not only do mammal parasitisms signify the presence of a tick species in a locality, Bbsl-positive ticks indicate that Lyme disease spirochetes are cycling enzootically in the area. These established populations may be hundreds of kilometres from where the zoonotic pathogens were acquired. In particular, one established population of blacklegged ticks on Corkscrew Island, which is located in northwestern Ontario, has a Bbsl infection prevalence of 73%; this insular location has the highest mean infection prevalence of Lyme disease spirochetes in I. scapularis adults ever reported in Canada [64].

Ixodes cookei Established on Vancouver Island
We provide the first report of I. cookei in far-western Canada and the West Coast of North America. We collected five I. cookei (one female, one nymph, three larvae) from a Pacific raccoon at North Saanich; this tick collection is the first record of I. cookei west of Manitoba (Table 6). Additionally, a gravid female was collected from a striped skunk on Vancouver Island, and it laid eggs that subsequently produced viable larvae. One of these larvae was barcoded for tick identification, and confirmed as I. cookei. The parasitism of a terrestrial mammal is the only viable mode of passage to Vancouver Island. Since I. cookei is not a bird-feeding tick [23], it must have been introduced via a terrestrial host that serves as a reservoir. Not only were Bbsl-positive I. cookei collected on Vancouver Island, they were collected from mammals in the lower Fraser Valley on the B.C. mainland. In the case of I. cookei on Vancouver Island, we hypothesize that a traveller from an indigenous area in central or eastern Canada took a terrestrial animal, such as a dog, to Vancouver Island, B.C., and the mammalian host was infested with a gravid I. cookei female and a male. During the trip westward, the female mated with the male and, upon arrival, the replete female dropped off into the leaf litter in a woodland habitat and, subsequently, laid eggs that hatched into viable larvae. Thus, a new I. cookei population was initiated on Vancouver Island. Since I. cookei had not previously been discovered in southwestern British Columbia, we contend that this previously undetected population became established recently, and is clearly not attributed to climate change [66].

Composite 6-Tick Enzootic Cycle of Bbsl on Vancouver Island
Whenever there are two or more tick species feeding concurrently on a host, they can transmit Bbsl, via the reservoir host, from one cofeeding tick species to another cofeeding tick species. Alternatively, one tick species can infect a reservoir-competent host and, after the blood meal, another tick species can subsequently acquire Bbsl from this spirochetemic host. Regardless of the sequel of feeding, co-infestations provide a direct way to maintain an enzootic transmission cycle of Lyme disease spirochetes. Using six vertebrate hosts and six tick species (i.e., I. angustus, I. auritulus, I. cookei, I. pacificus, I. spinipalpis, and I. texanus) from this study, we show a complete enzootic circuit of Bbsl from one tick species to another (Figure 4). With the exception of I. auritulus, these tick species bite people. In essence, this continual interconnecting link fulfills a multifaceted enzootic maintenance cycle of Bbsl. eggs that subsequently produced viable larvae. One of these larvae was barcoded for tick identification, and confirmed as I. cookei. The parasitism of a terrestrial mammal is the only viable mode of passage to Vancouver Island. Since I. cookei is not a bird-feeding tick [23], it must have been introduced via a terrestrial host that serves as a reservoir. Not only were Bbsl-positive I. cookei collected on Vancouver Island, they were collected from mammals in the lower Fraser Valley on the B.C. mainland. In the case of I. cookei on Vancouver Island, we hypothesize that a traveller from an indigenous area in central or eastern Canada took a terrestrial animal, such as a dog, to Vancouver Island, B.C., and the mammalian host was infested with a gravid I. cookei female and a male. During the trip westward, the female mated with the male and, upon arrival, the replete female dropped off into the leaf litter in a woodland habitat and, subsequently, laid eggs that hatched into viable larvae. Thus, a new I. cookei population was initiated on Vancouver Island. Since I. cookei had not previously been discovered in southwestern British Columbia, we contend that this previously undetected population became established recently, and is clearly not attributed to climate change [66].

Composite 6-Tick Enzootic Cycle of Bbsl on Vancouver Island
Whenever there are two or more tick species feeding concurrently on a host, they can transmit Bbsl, via the reservoir host, from one cofeeding tick species to another cofeeding tick species. Alternatively, one tick species can infect a reservoir-competent host and, after the blood meal, another tick species can subsequently acquire Bbsl from this spirochetemic host. Regardless of the sequel of feeding, co-infestations provide a direct way to maintain an enzootic transmission cycle of Lyme disease spirochetes. Using six vertebrate hosts and six tick species (i.e., I. angustus, I. auritulus, I. cookei, I. pacificus, I. spinipalpis, and I. texanus) from this study, we show a complete enzootic circuit of Bbsl from one tick species to another (Figure 4). With the exception of I. auritulus, these tick species bite people. In essence, this continual interconnecting link fulfills a multifaceted enzootic maintenance cycle of Bbsl.

Amblyomma Ticks Transported to Canada
Amblyomma ticks are transported into Canada by neotropical songbirds from the Neotropics. In order for these ixodid ectoparasites to sustain the transcontinental flight, they must remain attached for sustained periods of time. In the case of A. americanum, larvae typically feed 3 to 6 days, whereas nymphs commonly feed 4 to 7 days (J.D.S., unpublished data). Because many neotropical songbirds are marathon flyers, they are capable of transporting neotropical ticks long distances. During the flight, bird-feeding ticks gradually take a blood meal, and are fully engorged when they arrive in Canada. Based on these circumstantial factors, certain neotropical songbirds (e.g., flycatchers, thrushes, and warblers) transport slow-feeding Amblyomma ticks thousands of kilometres to Canada.
With respect to Amblyomma ticks, we collected A. americanum, A. dissimile, A. longirostre, A. maculatum, and A. rotundatum from neotropical and southern temperate songbirds in central and eastern Canada during northward spring migration. Additionally, Scott et al. previously reported an A. americanum (nymph) on a neotropical songbird (Swainson's Thrush) in northwestern Alberta during spring migration [13]. Based on winter hardiness studies in southwestern Ontario, which is the southernmost region in Canada, we determined that A. americanum larvae do not overwinter (J.D.S., unpublished data). After migration, songbird-transported A. americanum larvae and nymphs will undergo ecdysis in late spring, and subsequently bite vertebrates, including people. However, the larvae will neither survive subzero Canadian winters, nor sustain an A. americanum population. For example, a neotropical songbird can import an A. americanum nymph into Canada in May during northward spring migration. Over the next 35 to 60 days, this replete tick will molt to either a male or a female, and starts host-seeking. A female will typically bite dogs, outdoor cats, and humans in August. Additionally, songbirds can introduce ticks infected with pathogens from southern climes that cause invasive zoonoses in domestic and wildlife mammals in Canada. Based on the geographic origin of Amblyomma spp., it is highly unlikely that these invasive ticks from tropical or semi-tropical regions will colonize in Canada.

Long-Distance Transport of Neotropical Ticks to Canada
Notably, a Bbsl-positive A. longirostre larva was collected from a Willow Flycatcher at Toronto, Ontario; this bird parasitism provides the first report of Lyme disease spirochetes in this neotropical tick species in North America. The immature stages preferentially parasitize passerine birds, while adults are found on rodents. As adults, A. longirostre have the potential to transmit pathogens to other large mammals, including humans.
Overall, we collected 27 A. longirostre larvae and nymphs from neotropical songbirds during spring migration. A combination of ecological factors facilitate the fast, long-distance movement of Amblyomma ticks from Brazil to Canada. From a zoogeographical perspective, A. longirostre ticks have a home range in north-central South America; Brazil is a significant part of this indigenous area [26]. As well, many neotropical passerines, which migrate to the northern boreal forest, have their wintering grounds in Brazil. Certain songbirds, such as the Willow Flycatcher fly long distances during migratory flight. In fact, they will fly up to 8000 km to Canada during northward spring migration [67]. During fall migration, a Willow Flycatcher flew 2217 km for 48 h nonstop from Harrison, Illinois to Minatitlan, Veracruz, Mexico with a flight pace of 45 km/h, or 1109 km/day [68]. At this flight pace, a Willow Flycatcher could fly from Brazil to Canada, a distance of 5240 km, in 4.7 days. With adequate food reserves and sustained southerly tailwinds, a Willow Flycatcher has the potential to transport Amblyomma ticks from Brazil to Canada.

Avian Host Records for I. scapularis
We document the first host records of I. scapularis parasitizing a Tennesse Warbler and a Bay-breasted Warbler. Both of these neotropical songbirds have their breeding range primarily in the northern boreal forest. With respect to the Bay-breasted Warbler, a fully engorged I. scapularis nymph was collected from this ground-foraging songbird at Ste-Anne-de-Bellevue, Québec during north-bound spring migration; this parasitism is a new tick-host record.
During fall migration, two I. scapularis larvae were collected from a hatch-year Tennessee Warbler. This bird parasitism indicates that this fall migrant was parasitized by the two larvae at a more northern location. Since one larva was Bbsl-positive, the spirochetal infection could have been transmitted transovarially via the mother bird to her eggs, and passed onward to her offspring and, thus, to this fledgling. When the fledgling was parasitized by two I. scapularis larvae, one larva became infected. Since the Bbsl-infected larva had not had a previous blood meal, the mother bird could be a competent reservoir. Although it is possible that maternal-neonatal transmission of Lyme disease spirochetes occurred between the mother bird and its offspring, it is more likely that the fledgling was bitten by a Bbsl-infected tick while foraging for food after leaving the nest, and became spirochetemic. In essence, immature stages of I. scapularis parasitize migratory songbirds during bidirectional migrations. Nymphs typically parasitize passerine migrants in the late spring, whereas I. scapularis larvae commonly parasitize songbirds in late summer and early fall.

Novel Bird Parasitisms
Ticks normally bite wild birds on the head and neck [11]; however, we document a tick parasitizing its avian host in the buccal cavity. Specifically, a fully engorged I. scapularis nymph was detached from the base of the mouth of an American Kestrel nestling [37]. This 3-week-old nestling was collected at Mirabel, Québec shortly after leaving the nest. This bird parasitism reveals the first collection of I. scapularis from an American Kestrel in Canada.
Migratory songbirds, which are heavily infested with ticks, have the potential to initiate new foci of ticks hundreds of kilometers from their original source [42,55,69]. Even though spring passerine migrants transport ticks to northern latitudes, these engorged ticks may not molt to the next live stage. Each tick species has its own photoperiod requirements to activate and undergo ecdysis [19]. In the case of I. scapularis, this tick species requires at least 14 hours of daylight for larvae and nymphs to molt to the next developmental life stage [66]. In actuality, the expansion and establishment of I. scapularis in northern areas is limited by photoperiod.
In this study, we detected Lyme disease spirochetes in 17 (77%) of 22 I. auritulus larvae feeding on a Fox Sparrow. The presence of Bbsl in a replete I. auritulus larva does not automatically confirm that a bird is a reservoir-competent host. However, since I. auritulus larvae attached to Fox Sparrows have been consistently positive for Bbsl, it is highly likely that the Fox Sparrow is a competent reservoir of Lyme disease spirochetes. More specifically, our results are congruent with other studies that show Bbsl-positive I. auritulus larvae collected from Fox Sparrows [11,35]. In order to confirm reservoir competency in birds, Richter et al. conducted a xenodiagnostic study using spirochete-free I. scapularis larvae to show that certain passerines, such as the American Robin, are reservoir-competent hosts of Bbsl [70]. Since transovarial transmission of Bbsl is not apparent in I. auritulus females, we postulate that Fox Sparrows are reservoir-competent hosts.
Ixodes auritulus harbors a diversity of Bbsl genomospecies. For example, Scott et al. documented B. burgdorferi sensu stricto, plus three other genotypes, in bird-feeding I. auritulus ticks collected in southwestern B.C. [13]. In the same province, Scott et al. identified Borrelia lanei (formerly Borrelia genomospecies 2), which is another member of the Bbsl complex, in I. spinipalpis adults collected from an eastern cottontail along the southern fringe of Vancouver Island (Table 6) [34]. Additionally, Scott & Foley discovered Borrelia americana, a member of the Bbsl complex, in an I. auritulus tick collected from a ground-foraging songbird in British Columbia (Table 6) [35]. Moreover, Banerjee et al. isolated Borrelia bissettiae (formerly B. bissettii; culture number 1340) from I. angustus larvae in southwestern B.C. [71]. Since B. bissettiae is cycling enzootically in this coastal area, it is likely that this borrelial species is dispersed by wild birds and attached ticks.

Epidemiological Significance of Ticks on Songbirds
It is noteworthy that three tick species (i.e., I. affinis, I. dentatus, and I. minor) were transported into central and eastern Canada during northward spring migration [14,59,60]; two of these extralimital ticks (I. affinis and I. minor) are known to be enzootic vectors of Bbsl. Although I. affinis and I. minor seldom bite humans, it appears that they are more important enzootic vectors of Bbsl in the southeastern U.S.A. than I. scapularis [36]. Of epidemiologic significance, Bbsl-infected, songbird-transported I. scapularis ticks have been documented as far west and as far north as Peace River, Alberta [72]. In the same study, Bbsl-infected I. scapularis nymphs were also reported in Atlantic Canada in Cape Breton Island, Nova Scotia, and the province of Newfoundland and Labrador. East of the Rocky Mountains, Bbsl-positive I. scapularis have been collected from songbirds in all provinces (Alberta, Saskatchewan, Manitoba, Ontario, Quebec, New Brunswick, Prince Edward Island, Nova Scotia, and Labrador and Newfoundland) [9][10][11][12][13][14][15]37,59,72]. These bird parasitisms underpin the fact that people do not have to visit an endemic area to contract Lyme disease.
We collected bird-rabbit ticks, Haemaphysalis leporispalustris, from songbirds and lagomorphs in several areas Canada-wide, and some of these ticks were positive for Bbsl. Our findings are consistent with Banerjee et al. who cultured Lyme disease spirochetes from H. leporispalustris collected in northwestern Alberta [73]. It is worth mentioning that H. leporispalustris larvae and nymphs are the predominant tick species that parasitize fall passerine migrants in central and eastern Canada. Of note, H. leporispalustris, a bird-and lagomorph-feeding ectoparasite, has transcontinental distribution in Canada [8]. Songbirds widely disperse H. leporispalustris immatures, and lagomorphs are present in each province to act as terrestrial hosts. Lagomorphs are competent reservoirs of Bbsl, especially for the genomospecies B. andersonii [74,75]. In Table 6, we have highlighted some of the significant H. leporispalustris parasitisms that were positive for Bbsl. Although a rare occurrence, H. leporispalustris is known to bite humans [76].

Conclusions
Our findings show that Bbsl has wide distribution across Canada, and multiple tick species are involved in the enzootic maintenance cycle of this highly adaptive spirochete. Of 18 species tested, 15 were Bbsl-positive by PCR. Our data provide many tick-host firsts for indigenous and extralimital ticks parasitizing wild birds and terrestrial mammals coast to coast. The groundbreaking discovery of I. cookei on Vancouver Island suggests that this tick species has undergone a major geographic shift in Canada. In addition, wild birds transport bird-feeding ticks to new foci during long-distance migrations. Of special note, a Bbsl-positive A. longirostre was transported from the Neotropics by a Willow Flycatcher. In all, 16 of the 27 tick species in this study are bird-feeding ticks, and the majority are known to harbor and transmit tick-borne pathogens. Notably, six tick species were positive for Bbsl within the southeastern region of Vancouver Island, and suggest that these ectoparasites are involved in a 6-tick, enzootic maintenance cycle of Bbsl. Not only do wild birds widely disperse Lyme disease vector ticks countrywide, terrestrial mammals maintain Bbsl in numerous localized Lyme disease foci. Notably, 6 of 15 tick species that were infected with Bbsl bite humans. Ultimately, healthcare professionals must be cognizant that multiple tick species and their vertebrate hosts perpetuate Lyme disease spirochetes and associated tick-borne pathogens. These tick-borne zoonoses have spawned a major public health crisis throughout Canada.
Author Contributions: J.D.S. was responsible for study design and coordinating this tick-host project. J.F.A., B.C.B., K.L.C. and J.E.F. conducted molecular testing of ticks and analysis of PCR amplicons. L.A.D. confirmed the identification of ticks. All authors read and approved the final manuscript.
Funding: Funding was provided in part by the Mary Alice Holmes Memorial Foundation.