Monitoring of Nesting Songbirds Detects Established Population of Blacklegged Ticks and Associated Lyme Disease Endemic Area in Canada

This study provides a novel method of documenting established populations of bird-feeding ticks. Single populations of the blacklegged tick, Ixodes scapularis, and the rabbit tick, Haemaphysalis leporispalustris, were revealed in southwestern Québec, Canada. Blacklegged tick nymphs and, similarly, larval and nymphal rabbit ticks were tested for the Lyme disease bacterium, Borrelia burgdorferi sensu lato (Bbsl), using PCR and the flagellin (flaB) gene, and 14 (42%) of 33 of blacklegged tick nymphs tested were positive. In contrast, larval and nymphal H. leporsipalustris ticks were negative for Bbsl. The occurrence of Bbsl in I. scapularis nymphs brings to light the presence of a Lyme disease endemic area at this songbird nesting locality. Because our findings denote that this area is a Lyme disease endemic area, and I. scapularis is a human-biting tick, local residents and outdoor workers must take preventive measures to avoid tick bites. Furthermore, local healthcare practitioners must include Lyme disease in their differential diagnosis.

When the tick salivary glands are infected with Lyme disease spirochetes, Ixodes ticks can transmit Bbsl to the host in less than a day [25], especially during nymphal blood meals. Based on North American studies, Bbsl is normally transmitted in 24-48 h; however, when the salivary glands are infected with Lyme disease spirochetes, transmission to a host can occur in less than 16 h [26,27]. Using European Bbsl strains and Ixodes ricinus ticks, Bbsl has been found disseminated in mice within 12 h [26].
In order for a location to meet the criteria for an established population of I. scapularis, an area must have at least six individual ticks or at least two of the three motile life stages identified within a single collection period [28]. Normally the collection period is one year; however, in the present study, it was two months.
The aim of this study was: (1) to determine whether tick-infested songbirds monitored during the nesting period are a means of pinpointing unrecognized populations of ticks and (2) to ascertain if these bird-feeding ticks are infected with tick-borne, zoonotic pathogens.

Tick Collection
Ticks were collected from songbirds during the period 8 June to 2 August 2019 at Montée Biggar (45.088226 • N, 74.215678 • W) in southwestern Québec, Canada ( Figure 1). Live ticks were collected from passerines using hardened stainless steel, #5, superfine-tipped forceps (BioQuip Products, Rancho Dominguez, CA). Live ticks were put into a transparent 8.5 mL, 15.7 mm × 75 mm, round-bottomed polypropylene tube (Sarstedt, Montréal, Québec, CA); each tube contained ticks from a single host. In order to keep live ticks from escaping, a piece of tulle netting (3-cm diameter) was placed over the mouth of the tube, and a push cap, which had a 7-mm hole, was promptly inserted into tube opening. This technique prevented live ticks from escaping and, at the same time, provided ventilation for live ticks. For mailing, polypropylene tubes were put in a double-zipper plastic bag with slightly moistened paper towel to sustain high humidity. All ticks were sent directly to the lab (J.D.S.) for identification. Live, partially and fully engorged larval and nymphal ticks were held to molt (metamorphose to the next developmental life stage) to confirm the species identification and, in certain cases, reveal transstadial passage of tick-borne, zoonotic pathogens. Taxonomic keys and scientific articles were employed to confirm ixodid tick identifications, namely I. scapularis larvae [29,30] and nymphs [30,31] and, likewise, Haemaphysalis leporispalustris (rabbit tick) larvae [29] and nymphs [32].
Engorged ticks were exposed to a long-day photoperiod of 16:8 (L:D) h. Background and processing information (i.e., host species, leg band number, geographic location, date collected, collector's name, developmental life stage, degree of engorgement) were logged for ticks from each avian host. After identification, ticks were stored in 2-mL microtubes (Sarstedt, Montréal, Québec, Canada) containing 95% ethyl alcohol to preserve them and stabilize DNA.

Spirochete Detection
DNA was extracted from ixodid ticks preserved in 95% ethyl alcohol using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. Quantitative real-time PCR (qPCR) was used in a combined thermocycler/fluorometer (ABI Prism 7700; Applied Biosystems, Foster City, CA) to screen for B. burgdorferi s.l. DNA. A probe (6FAM-TTC-GGT-ACT-AAC-TTT-TAG-TTA-A) with a 3' end modified with minor groove binding protein and labelled with corresponding dye, forward (5'-GCT-GTA-AAC-GAT-GCA-CAC-TTG-GT-3') and reverse (5'-GGC-GGC-ACA-CTT-AAC-ACG-TTA-G-3') primers targeting a region of the 16S rRNA gene were used [33,34]. Water negative controls were included in each run, while positive controls consisted of DNA from cultured Bbsl. Ticks were considered positive if the cycle threshold was <40, and there was a characteristic amplification curve. To identify B. burgdorferi s.l. genospecies in qPCR-positive samples, conventional PCR using primers (5'-AAR-GAA-TTG-GCA-GTT-CAA-TC-3' and 5'-GCA-TTT-TCW-ATT-TTA-GCA-AGT-GAT-G-3') that target the flagellin (flaB) gene were performed as previously described [35]. Modifications were made to use GoTaq Green Master Mix (Promega, Madison, WI). Gel electrophoresis was performed on conventional PCR products using 1% agarose gel stained with Gelstar (Lonza, Rockland, ME). Amplicons of approximately 497 bases were excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, USA), and sequenced using an ABI 3730 sequencer (Davis Sequencing, Davis, CA). Results were examined for accuracy of base determination, and end-read errors were trimmed [36]. We compared sequences to those in the GenBank database using the Basic Local Alignment Search Tool (BLAST), National Center for Biotechnology Information (NCBI), Bethesda, MD [37]. The MUSCLE algorithm was used to perform sequence alignments for fla gene sequences from ticks and from reference Borrelia genospecies obtained from the NCBI GenBank database [38].

Tick Collection
In total, 68 ticks were collected from 23 songbirds representing five families within the Order Passeriformes (Table 1). These songbird-derived ticks include 64 blacklegged ticks (nymphs, n = 63; larva, n = 1) and four H. leporispalustris (nymphs, n = 3, larva, n = 1). It is noteworthy that all tick collections were made before southward migration started and, therefore, all ticks were locally acquired.

Spirochete Detection
All ticks testing positive for Bbsl belonged to the genospecies B. burgdorferi sensu stricto. Significantly, 14 (42%) of 33 I. scapularis nymphs tested were positive (Table 1). A single I. scapularis larva was collected, but not tested for Bbsl. All larval and nymphal H. leporispalustris were negative for Bbsl. Four I. scapularis nymphs were collected from an American Robin on 22 July 2019, and each was positive for Bbsl. Of epidemiological merit, five (100%) of five I. scapularis nymphs collected from American Robins were positive for Bbsl. As well, seven I. scapularis nymphs collected from an American Robin were pooled for Bbsl testing; the pool was positive for Bbsl. This pool was not included in the Bbsl prevalence calculations. A total of 23 ticks were not tested for Bbsl because they got hot in transit, and died and spoiled.
Bbsl DNA sequences from this study were submitted to GenBank, and the accession numbers are listed in Table 2.

Discussion
In this study, we exposed I. scapularis and H. leporispalustris populations by monitoring a wooded area in southwestern Québec during the nesting and fledgling period. This deciduous woodland turned out to be a breeding colony of I. scapularis infected with Lyme disease spirochetes. The presence of Bbsl in these bird-feeding I. scapularis nymphs indicates that this nesting area is endemic for Lyme disease. Most importantly, these bird parasitisms during the nesting and fledgling periods provide a unique means of identifying established populations of ticks.

Nesting Area Marks Established Population of Blacklegged Ticks
Firstly, a Common Yellowthroat parasitized by 12 I. scapularis nymphs signifies an established population in this nesting environs (Figure 2) [28]. Secondly, a Veery was parasitized by immature stages of I. scapularis (two nymphs, one larva), and these two developmental stages on a single host within a single year, indicate that this locale is an established population [28]. When engorged I. scapularis nymphs molt, they become adults in late June to mid-August. Consistent with other bird-tick-Bbsl studies reporting Bbsl infection prevalence which range from 31% to 59% in I. scapularis nymphs in the Great Lakes area [5,7,8,14], we obtained an infection prevalence of 42%. Since these Bbsl-infected nymphs will molt to adults, we extrapolate that the Bbsl infection prevalence for I. scapularis adults will be approximately 42% at this locale. Thirdly, when we combine all of the three motile life stages (larvae, nymphs, adults), there is an established population in this woodland locality. In three different ways, we meet the surveillance criteria for an established population of I. scapularis ticks [28]. Based on the Bbsl infection prevalence, we show that this area is clearly endemic for Lyme disease. The determination of whether blacklegged ticks form an established population in an area is often determined by active surveillance. However, in this study, we have shown that ticks collected from songbirds during the nestling and fledgling period are, indeed, an established population. The June-July period is the time of year when songbirds stay in close proximity to the nest. Ecologically, the present bird-tick-pathogen study was conducted during the nesting and fledgling period, which coincides with the peak questing period of nymphal blacklegged ticks [39]. As a result, ground-foraging songbirds are frequently parasitized by host-seeking I. scapularis nymphs.

Short-Distance Flights Signify Tick Populations
During the nesting and fledgling period, songbirds forage contiguous to the nest, and maintain short-distance trips back and forth for food. Short runs ensure that the eggs in the nest stay warm and protected from predators. Additionally, during the fledgling period, ground-foraging songbirds make hasty flights near the nest, and promptly return to feed their young. As the ground-frequenting songbirds forage through the leaf litter or grass refuge searching for arthropods, they may become parasitized by host-seeking (questing) ticks. In the present study, bird banders, who were conducting demographic monitoring and collecting data related to songbird activity, had the opportunity to collected bird-feeding ticks. Notably, in late July, songbirds are in moult (shed and replace feathers), and only have localized flight activity. Based on the criteria for an established population [28], we were able to ascertain that there were solo populations of I. scapularis and H. leporispalustris ticks in the nesting and fledgling area at Montée Biggar, Québec. We were also able to determine that this nesting area is endemic for Lyme disease.

Songbirds as Reservoir Hosts
Certain bird species are reservoir hosts of Bbsl. An American Robin (female, second year) was parasitized with four I. scapularis nymphs, and each nymph was infected with Bbsl ( Figure 3). Anderson et al. isolated Lyme disease spirochetes from the liver of a Veery [43] and, also, from I. scapularis larvae feeding on this avian host. These acarologists also isolated Bbsl from larval I. scapularis feeding on Rose-breasted Grosbeaks and Common Yellowthroats suggesting that these songbirds are also reservoir-competent hosts. Other researchers provide substantive evidence that the Veery, Song Sparrow, Common Yellowthroat are competent reservoirs of Bbsl [17,44,45]. Using xenodiagnostic methods, Richter et al. placed unfed spirochete-free I. scapularis larvae on Bbsl-inoculated American Robins [46] and, when replete, let these fully engorged larvae molt to nymphs. These unfed nymphs were then placed on spirochete-free American Robins and, subsequently, became infected with Lyme disease spirochetes. These findings reveal that American Robins are competent reservoirs. Similarly, Anderson et al. found that American Robins are reservoir hosts of Bbsl [18]. In the present study, five (100%) of five I. scapularis nymphs parasitizing American Robins were infected with Bbsl. In addition, six (32%) of 19 I. scapularis nymphs collected from Veeries were infected with Bbsl. Since we did not take any blood samples from host birds, we do not know whether any songbirds were spirochetemic for Bbsl.
Of enzootic significance, a partially engorged I. scapularis nymph molted to an unfed female, and this female was positive for Bbsl; this molt demonstrates transstadial passage of Bbsl. This enzootic finding show that Bbsl can move to the next developmental life stage at this site.

H. leporispalustris Vector Competency
In this study, we did not identify Bbsl in H. leporispalustris ticks. However, Banerjee et al. detected Lyme disease spirochetes in H. leporispalustris ticks collected from a snowshoe hare, Lepus americanus, in northern Alberta [47]. As well, Scott et al. discovered Borrelia lanei-like spirochetes and a Babesia divergens-like piroplasm concurrently in a H. leporispalustris (female) collected from an eastern cottontail, Sylvilagus floridanus, in southern Manitoba [48]. Scott & Durden provide the first record of a Bbsl-positive H. leporispalustris (nymph) collected from an avian host (Swainson's Thrush) in Canada [14]. Previously, Scott et al. found Bbsl in a H. leporispalustris larva parasitizing a passerine (Canada Warbler) in Québec suggesting that this tick species is a reservoir-competent host [8]. During southbound, fall migration in Canada, larval and nymphal H. leporispalustris frequently parasitize passerines, and are widely dispersed in southern regions.

Ticks Co-infest Songbirds
One Veery was co-infested with H. leporispalustris (one nymph and one larva), and I. scapularis (two nymphs). Not only is there a breeding colony of I. scapularis present in this Laurentian River basin, an established population of H. leporispalustris is also there. Since these ixodid ticks were collected during the nesting and fledgling period, this bird parasitism denotes a cohabitation of two tick species in this sylvan locale, and signifies that these two tick species are sympatric. The Veery has trans-border and trans-equatorial migration during its northward spring flight, and has a breeding range in southern Canada, including southwestern Québec and northern United States; the wintering range is in central and southeastern Brazil (Figure 4). During the breeding, nesting, and fledgling period, Veeries have localized activity in juxtaposition to the stationary nest. When the young have fledged the nest, these passerines replenish their fat reserves, and prepare for the southbound trek to wintering ranges in southern latitudes during August and September. In late July, they typically moult in preparation for the southbound marathon flight.

Definition of Lyme Disease Endemic Area
We put forth the following definition for a Lyme disease endemic area based on comprehensive, tick and Lyme disease research across Canada [8], and similarly, conducting field studies and monitoring multiple established tick populations [4,8,12,14,19,22,[48][49][50][51][52][53][54][55][56][57][58]: A Lyme disease endemic area is defined as an established population of vector ticks infected with the Lyme disease bacterium, Borrelia burgdorferi sensu lato, at a geographic locality in a single collection period. The present bird-tick-pathogen study at Montée Biggar, Québec meets this definition for a Lyme disease endemic area.

Conclusions
By monitoring nesting songbirds, we demonstrate a novel method of pinpointing tick populations. Nesting songbirds have short-distance flight, and reflect bird-feeding ticks acquired locally. By testing songbird-derived ticks during the nesting and fledgling period, we obtained an infection prevalence of 42% for I. scapularis nymphs, and discovered that this locality is a Lyme disease endemic area. This high level of Bbsl infectivity, reveals that outdoor people working in this newfound, Lyme disease endemic area during snow-free days encounter a public health risk. Any individuals frequenting this area should take extra precautions to avoid tick bites, and do full-body tick checks at the end of the day. Because Borrelia burgdorferi sensu stricto is pathogenic to humans, anyone who is bitten by a tick or has Lyme disease symptoms should seek medical attention. Since chronic Lyme disease is a pernicious, debilitating infection, healthcare practitioners must take special steps to screen symptomatic patients for this incapacitating spirochetosis.
Author Contributions: J.D.S. was responsible for study design, coordinating this tick-host-microbe project, and writing the manuscript. J.E.F., E.L.P. and M.S.S. conducted molecular testing of ticks and analysis on PCR amplicons. All authors have read and agreed to the published version of the manuscript.