Presence of Babesia odocoilei and Borrelia burgdorferi Sensu Stricto in a Tick and Dual Parasitism of Amblyomma inornatum and Ixodes scapularis on a Bird in Canada

Wild birds transport ticks into Canada that harbor a diversity of zoonotic pathogens. However, medical practitioners often question how these zoonotic pathogens are present in their locality. In this study, we provide the first report of an Amblyomma inornatum tick cofeeding with a blacklegged tick, Ixodes scapularis, which parasitized a Veery, Catharus fuscescens—a neotropical songbird. Using the flagellin (flaB) gene of the Lyme disease bacterium, Borrelia burgdorferi sensu lato, and the 18S rRNA gene of the Babesia piroplasm, a malaria-like microorganism, we detected Borrelia burgdorferi sensu stricto and Babesia odocoilei, respectively, in an I. scapularis nymph. After the molt, these ticks can bite humans. Furthermore, this is the first documentation of B. odocoilei in a tick parasitizing a bird. Our findings substantiate the fact that migratory songbirds transport neotropical ticks long distances, and import them into Canada during northward spring migration. Health care practitioners need to be aware that migratory songbirds transport pathogen-laden ticks into Canada annually, and pose an unforeseen health risk to Canadians.


Introduction
Ticks have medical and veterinary importance because they are vectors of multiple zoonotic microorganisms [1]. Hard-bodied ticks (Acari: Ixodidae) often harbor a wide array of bacterial, piroplasmic, and viral microorganisms, and transmit them to vertebrate hosts [1][2][3]. Wild birds provide long-distance dispersal of ixodid ticks, whereas terrestrial vertebrates facilitate short-distance movement. In the temperate zone of North America, Lyme disease and human babesiosis are the most frequent tick-borne diseases [4].
Lyme disease is a zoonotic disease caused by members of the Borrelia burgdorferi sensu lato (Bbsl) complex, and is generally transmitted to humans by blood-feeding ticks [5]. Worldwide, there are at least 23 genospecies in this complex, and B. burgdorferi sensu stricto is one of the genospecies that is pathogenic to humans. Not only do Ixodes species transmit Bbsl, certain Amblyomma and Rhipicephalus ticks have been reported to transmit Bbsl in east-central South America [6]. Morphologically, Bbsl is pleomorphic; it has diverse forms (i.e., spirochetes, blebs, The aim of this study was to determine whether passerines are continuing to transport extralimital ticks to Canada and to ascertain whether they are harboring any zoonotic pathogens that have not been identified previously.

Tick Collection
Ticks were collected from a neotropical songbird by bird banders using fine-pointed, stainless steel forceps. Live ticks were put in a transparent, round-bottom, 8.5 mL polypropylene tube (15.7 mm × 75 mm, round based) (Sarstedt, Montréal, Québec, Canada). The mouth of the tube was covered with tulle netting (3-cm diameter) to allow ventilation for ticks. A polyethylene push cap with a 7-mm hole was placed into the mouth of the tube to secure the tulle netting, and prevent ticks escaping. Each tube, which contained the ticks from one host, was placed in a double-zipped plastic bag with a slightly moistened paper towel to maintain high humidity. All ticks were sent to the lab for identification (J.D.S.). The Amblyomma nymph was tentatively identified using a taxonomic key [27] and, following the nymph-adult molt, Amblyomma taxonomic keys for adults indigenous in the Western Hemisphere were used [28,37]. Similarly, for Ixodes nymphs, a nymphal taxonomic key was used [38]. Ixodes species were exposed to a long-day photoperiod of 16:8 h (Light:Dark), while Amblyomma ticks from the Neotropics were held at a photoperiod of 12:12 h (L:D). Complete records (i.e., geographic location, tick collection date, tick species, developmental life stage, and host species) were logged for each tick collection. To preserve ticks, they were stored in 2-mL microtubes containing 95% ethyl alcohol. The Amblyomma sp. female, 18-5A70B, was compared to specimens in the U.S. National Tick Collection.

DNA Sequence Analysis
PCR products from the Babesia 18S rRNA and the Borrelia flaB positive samples were purified using the Wizard®SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). DNA templates were sequenced [41] using both the forward and reverse primers used in the nested PCRs. Investigator-derived sequences were compared with those obtained by searching the GenBank database (National Center for Biotechnology Information) using the Basic Local Alignment Search Tool (BLAST) [42], and aligned using Clustal X [43].
Nucleotide sequence accession numbers: The DNA nucleotide sequences for the Babesia 18S and Bbsl flaB gene fragments obtained from ticks in this study were deposited in the GenBank database with accession numbers MK628544 and MK620851, respectively.

Molecular Tick Identification
The methodology used for the molecular identification of the Amblyomma sp. female, 18-5A70B, was carried out at the Centre of Biodiversity Genomics (CBG), University of Guelph. The algorithm was previously described [44].

Identification of Amblyomma Tick
An Amblomma sp. nymph was collected from a Veery, Catharus fuscescens, on 16 May, 2018, at the Ruthven Park National Historic Site Banding Station, Haldimand Bird Observatory, Cayuga, Ontario: 42.97 N, 79.87 W. Using a photoperiod of 12:12 h (L:D), this fully engorged Amblyomma nymph molted to a female in 37 d. The newly formed female was allowed to become fully sclerotized before it was morphologically identified. The author (J.D.S.) provided preliminary identification, and the co-author (L.A.D.) provided verification of the identification. Taxonomic keys for adult Amblyomma species in the Neotropics were also employed to confirm the identification of this tick species [28,37]. This female specimen was also compared with confirmed A. inornatum females in the U.S. National Tick Collection, matched favorably with them, and the species was confirmed as A. inornatum.

Identification of Tick-Borne Pathogens
Based on DNA sequence analysis, B. burgdorferi sensu stricto for Bbsl, and B. odocoilei were detected in the I. scapularis nymph.

Molecular Tick Assessment
Molecular analysis of the Amblyomma specimen (18-5A70B) was undertaken, but we were unable to amplify a valid barcode sequence for this tick specimen.

Discussion
We provide the first report of A. inornatum (nymph) and an I. scapularis (nymph) cofeeding on a bird in Canada. Even though these two tick species have individually been documented on passerines, this is the first-ever account of coinfestation on a bird anywhere. This discovery is congruent with previous bird-tick studies which show that migratory songbirds transport Amblyomma ticks from neotropical areas into Canada. Specifically, the I. scapularis nymph was coinfected with Bbsl and B. odocoilei. Ultimately, these tick-borne, zoonotic pathogens may be a serious health risk to humans.

Identification of Amblyomma inornatum
The coinfestation of I. scapularis and A. inornatum on a bird in Canada is a first. Even though Ogden et al. reported A. inornatum on wild birds in Canada, they did not provide details, such as background information, a morphological description, or confirmation of identification [17]. Since there are at least 57 Amblyomma spp. In the neotropical region, including the Caribbean sub-region, identifying larvae and nymphs morphologically makes for unreliable identifications [45]. Taxonomic keys are not available to delineate all of these immature species. When these larval and nymphal Amblyomma ticks are collected from spring migratory songbirds in Canada, there is a strong possibility for misidentification because they are outside their established geographical distribution. Moreover, the account of an A. inornatum male on a bird in the Ogden et al. study is questionable because adults are not known to parasite passerines.
Within a tick-conducive microenvironment, replete nymphs (i.e., I. scapularis, A. inornatum) will molt to adults, and start questing for suitable vertebrate hosts, including humans. A photograph of an I. scapularis female parasitizing a human shows that ticks often take a blood meal in concealed locations on the body (Figure 1). This female was attached on the nape of the neck where it had been feeding for 4 days. If this tick was infected with tick-borne pathogens, it could have easily transmitted tick-borne pathogens to the boy. Exudates formed around the tick when it was disturbed by hair brushing. If this female had become fully engorged, and was not found and removed, it could have caused tick paralysis (if it was genetically predisposed to synthesize paralysis biotoxins).
In order to confirm the identification of A. inornatum in the present study, we allowed the fully engorged nymph to molt to an adult (female). After the nymph-adult molt, the Amblyomma female was keyed to A. inornatum using two Amblyomma keys for adults [28,37]. Morphologically, the distinguishing characteristics include: 1) Coxa I with an external spur that is much longer than the internal spur and 2) scutum inornate. The specimen matched favorably with confirmed A. inornatum females in the U.S. National Tick Collection. We provide confirmatory evidence of A. inornatum in Canada.

Flight Path of Veery
Veeries start their northward migratory flight from their wintering grounds in central and southeastern South America. From there, they head northward to breeding grounds in north-central and northeastern U.S.A. These night-time fliers may use transoceanic pathways or employ overland routes through Central America [46]. Veeries can fly up to 285 km/night [47]. At this flight pace, a Veery could fly from the Mexico-U.S. border to Ruthven Park, Ontario, a distance of 2600 km, in 9 d. The Veery has a low percentage of body fat and, thus, must stop periodically along the flight path to replenish its energy reserves [47]. This timeline indicates that the A. inornatum nymph is a slow feeder during migratory flight. Moreover, a fully engorged tick normally drops from its host when the peripheral sensory organs deduce a suitable microhabitat [1]. Since A. inornatum is native to Central America, Mexico, and southern Texas, the Veery must have followed the Mississippi Flyway northward and proceeded through the Ohio River valley to Ruthven Park, Ontario.
In their indigenous distribution, A. inornatum nymphs have peak questing activity from February through May [30]. This host-seeking activity period corresponds with the northbound migration of Veeries en route through Central America. Likewise, the host-seeking activity period of I. scapularis nymphal ticks in the upper Midwest overlaps peak migration of Veeries [48]. Since the I. scapularis nymph was partially engorged, it most likely parasitized the Veery in the Ohio River valley. Both the fully engorged A. inornatum and the partially engorged I. scapularis were collected from the host bird at Ruthven Park in mid-May during peak spring migration, which coincides with the peak questing period of nymphs for both A. inornatum and I. scapularis.

Sequence of Zoonotic Infection During Flight of Veery
The presence of Bbsl and B. odocoilei in the I. scapularis nymph, but not in the A. inornatum nymph, indicates that the Veery was likely not infected with these two pathogens. Based on the flight pace of the Veery and the extent of engorgement of the I. scapularis nymph, it is most likely that the host bird was parasitized in the Ohio valley two days before arrival and banding at Ruthven Park. The I. scapularis nymph possibly acquired Bbsl and B. odocoilei while taking a blood meal as a larva. More studies are needed to determine what vertebrate hosts simultaneously harbor both Bbsl and B. odocoilei. Even though A. inornatum is a non-indigenous tick in Canada, nymphs have ample time to molt during late spring and early summer to either males or females. Subsequently, both A. inornatum and I. scapularis females have adequate time to molt and transmit these tick-borne zoonotic microbes to humans.
When a pathogen-laden tick is cofeeding on a songbird with other ticks, it can transmit pathogens to these attached ticks. As soon as the host bird becomes parasitemic, it can systematically transmit pathogens to engorging ticks. For instance, Babesia sporozoites are stored in the tick salivary glands, and transmitted once the tick starts to take a blood meal from its host [12]. Endogenous transmission of sporozoites promptly occurs during the initial stage of engorgement. Not only can an infected bird be a reservoir, it can be an enzootic bridge for tick-borne pathogens. Whenever songbird-transported ticks are infected with one or more of these pathogens, they can subsequently induce acute illness in unsuspecting patients.

Tick-Host-Pathogen Dynamics
Cervids play an important role in the enzootic transmission cycle of B. odocoilei. The isolation of B. odocoilei from the blood of white-tailed deer, Odocoileus virginianus, was first achieved in Texas, and reveals that this cervid is a reservoir host [49,50]. Biogeographically, this piroplasm overlaps with the distribution of I. scapularis [50]. White-tailed deer are hosts of all three motile stages (larvae, nymphs, adults) of I. scapularis, and support the reproduction of this hematophagous ectoparasite. Ecologically, both white-tailed deer and I. scapularis perpetuate the enzootic transmission cycle of B. odocoilei. Transovarial and transstadial transmission of B. odocoilei occur in I. scapularis and, upon tick feeding, Babesia sporozoites are promptly transmitted, and entry ensues and multiplication takes place within the erythrocytes of cervid hosts [50]. Whenever larval or nymphal I. scapularis parasitize a white-tail deer, and become infected with B. odocoilei, they molt to the next life stage and can subsequently transmit sporozoites to vertebrate hosts, including humans. Paradoxically, Bbsl and Babesia odocoilei have different enzootic transmission pathways with cervid hosts. White-tailed deer are refractory to Bbsl, whereas cervids are reservoir-competent hosts of B. odocoilei [49,50].
With regard to avian hosts, Anderson and Magnarelli isolated Bbsl from the blood of passerine birds (i.e., Gray Catbird, Common Yellowthroat and American Robin) [51]. Pointedly, Anderson et al. isolated Bbsl from the liver of a Veery and, likewise, from I. scapularis larvae collected from Veeries [52]. In addition, McLean et al. cultured Bbsl from the blood of a Song Sparrow, Melospiza melodia, in the upper Midwest [53]. In the southeastern U.S.A., Durden et al. documented Bbsl in skin biopsies removed from passerine birds, and revealed that songbirds act as disseminators of Lyme disease spirochetes [54]. Richter et al. used spirochete-free xenodiagnostic I. scapularis larvae to show that the American Robin, Turdus migratorius, is a reservoir-competent host of Bbsl [55]. Since there is essentially no transovarial transmission of Bbsl in I. scapularis females [56], the I. scapularis nymph must have acquired Lyme disease spirochetes directly from the host Veery or indirectly when the nymph was feeding as a larva on a Bbsl-infected, reservoir-competent host. Pathologically, I. scapularis is known to carry and transmit at least 10 different tick-borne, zoonotic pathogens [1]. Upon repletion, some individual, fully engorged I. scapularis females can cause tick paralysis if they are not removed promptly.
In Europe, Babesia microti has been detected in Ixodes ricinus larvae feeding on European Robins, Erithacus rubecula [57]. In addition, Babesia divergens has been detected in bird-feeding I. ricinus, which is a competent vector of several tick-borne pathogens. In North America, Hersh et al. revealed that certain passerines (i.e., Wood Thrushes, Veeries, Gray Catbirds, American Robins) are reservoir competent hosts for B. microti [58]. They also documented a triple infection (i.e., B. microti, Bbsl, and Anaplasma phagocytophilum) in an I. scapularis nymph collected from a Veery. Based on our findings, and those of other researchers, Veeries play a significant role in the enzootic transmission dynamics of Bbsl and certain Babesia species.
Although there is uncertainty at this point in time, B. odocoilei may possibly be pathogenic to humans. This piroplasm is in the same sister group, namely the Babesia sensu stricto clade (i.e., EU1, a European genotype; Babesia divergens; Babesia divergens-like species) that is pathogenic to humans [59,60]. The EU1 strains are most closely related to B. odocoilei, but they differ significantly over the entire 18S rRNA gene from B. odocoilei. When Babesia serology is conducted, B. odocoilei may be cross-reacting with more familiar Babesia spp. (i.e., Babesia microti, Babesia duncani). More studies are needed to culture B. odocoilei from patients and to juxtapose them with Babesia serology.

Impact of Babesia and Borrelia burgdorferi Infections on Humans
Globally, there are over 100 recognized species of Babesia and, in North America, they infect a wide range of avian and mammalian hosts, including humans [61]. These apicomplexan piroplasms are pleomorphic, and have four diverse forms (i.e., gametes, sporozoites, merozoites, and trophozoites) [12]. When Babesia-infected ticks feed on vertebrates, sporozoites are transmitted from the tick's salivary glands without delay, and invade the hosts' red blood cells. Not only is this intraerythocytic parasite transmitted by ticks, it can be transmitted by blood transfusion [62] and transplacental transmission [63,64]. Common symptoms include fatigue, chills, sweats, headache, muscle aches, listlessness, dullness, nausea, and sleep disturbance. Cases of babesiosis can present with a high parasite burden, severe pathology, and fatal outcomes. Human babesiosis infections may recrudesce and persist and, ultimately, the resulting parasitemia can cause a fulminating, life-threatening zoonosis. Due to non-specific symptoms and possible coinfection with other tick-borne pathogens, human babesiosis is often misdiagnosed and underreported [65]. Although there are Babesia case reports in Canada, only one nationwide study of B. duncani has been published in the scientific literature [66].
Based on the MyLymeData, an online patient registry for chronic Lyme disease, 44% of the respondents had Babesia as the most common coinfection [4]. This study also revealed that psychiatric disorders (52%) were the most common misdiagnosis [4]. Concurrent Lyme disease and human babesiosis is typically more grave than either zoonosis by itself [35,90]. The co-occurrence of Lyme disease and human babesiosis typically makes clinical symptoms more severe, intense, and long-lasting [90,91]. Bbsl weakens the immune system, and makes these co-occurring infections more difficult to treat.

Conclusions
We provide the first report of B. odocoilei in a tick parasitizing a bird. The presence of B. odocoilei and Bbsl in a bird-feeding I. scapularis nymph indicates that this vector tick may subsequently transmit a dual infection to a suitable host, including a human. The cofeeding of A. inornatum and I. scapularis ticks on a Veery is significant because it is the first-ever account of these two tick species simultaneously parasitizing a bird. This bird parasitism also reveals that neotropical songbirds can transport ticks long distances and introduce foreign pathogenic microbes from the Neotropics, Mexico, and the United States, and widely disperse them across Canada during northward spring migration. Since migratory songbirds transport multiple ticks and associated zoonotic pathogens from southern latitudes into Canada, health care practitioners must watch for, and fervently treat these tick-borne zoonoses with due diligence.
Author Contributions: J.D.S. was responsible for study design and coordinating this tick-host project. K.L.C. 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.