Detection of Babesia odocoilei in Ixodes scapularis Ticks Collected in Southern Ontario, Canada

Tick-borne zoonotic diseases have an economic and societal impact on the well-being of people worldwide. In the present study, a high frequency of Babesia odocoilei, a red blood cell parasite, was observed in the Huronia area of Ontario, Canada. Notably, 71% (15/21) blacklegged ticks, Ixodes scapularis, collected from canine and feline hosts were infected with B. odocoilei. Consistent with U.S. studies, 12.5% (4/32) of questing I. scapularis adults collected by flagging in various parts of southwestern Ontario were positive for B. odocoilei. Our data show that all B. odocoilei strains in the present study have consistent genetic identity, and match type strains in the GenBank database. The high incidence of B. odocoilei in the Huronia area indicates that this babesial infection is established, and is cycling enzootically in the natural environment. Our data confirm that B. odocoilei has wide distribution in southern Ontario.

The primary aim was to determine the distribution of B. odocoilei in questing and animal-derived I. scapularis ticks in southern Ontario.

Tick Collection
Questing ticks were collected by flagging low-lying vegetation in the southern part of southwestern Ontario within the Carolinian forest region from 24 to 26 April 2019. At a more northerly location in the Huronia area, veterinarians and technicians collected attached, engorged ticks from domestic dogs and domestic cats from 2 to 27 May 2019. These attached ticks were removed using superfine-tipped stainless steel forceps, and ticks from each host were stored in a tightly sealed microtube containing 94% ethanol. Each microtube was labelled with a tick identification number. A white, vinyl-backed flannel cloth attached to a telescoping, aluminum pole was employed to collect questing ticks. Blood-fed and questing ticks were identified to species by using microscopy and taxonomic keys [34,42].

DNA Extraction, PCR, and Sequencing
To extract DNA from unfed ticks, an ammonium hydroxide protocol (unfed ticks) [35], or the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocol for animal tissue was used. The resulting DNA was stored at −20 • C until PCR was performed. Amplification of the 18S rRNA gene of Babesia was performed as previously described using the BJ1 (5 -GTC-TTG-TAA-TTG-GAA-TGA-TGG-3 ) and BN2 (5 -TAG-TTT-ATG-GTT-AGG-ACT-ACG-3 ) primers [43]. Amplicons were visualized by UV transillumination on a 1% agarose gel containing GelStar nucleic acid stain (Lonza, Rockland, ME, USA), and those that were 400−500 nucleotides in length, were excised from the gel, and prepared for DNA sequencing to confirm Babesia presence and species using the QIA amp DNA Kit (Qiagen, Valencia, CA, USA). DNA sequencing was performed at the University of California Davis Sequencing facility using the Big Dye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and PCR primers.

Phylogenetic Analysis
End-reading errors were removed from sequences and, when possible, ambiguous nucleotides were manually corrected. Sequences were compared to those published in GenBank using the BLAST database search program (https://blast.ncbi.nlm.nih (3 November 2020). The phylogenetic tree was constructed based on select published sequences of Babesia species (B. odocoilei, B. bovis, B. conradae, B. divergens, B. duncani, B. canis canis, B. gibsoni, B. microti, and B. vulpes) downloaded from GenBank. Babesia bovis was used as the outgroup species. All sequences were trimmed to the same length (445 nucleotides, including those absent in some species), and were aligned using the MUSCLE algorithm [44]. Phylogeny was resolved using the maximum likelihood method in MEGA 10.0.5 [45]. This general time reversible model facilitated gamma distribution and invariant sites (number of discrete categories equals five) was determined by jModeltest 2.1.10 [46]. Consensus was achieved by bootstrapping based on 1000 pseudoreplicate datasets generated from the original sequence alignments.

Tick Collection
Between 24 April and 22 May 2019, a grand total of 53 I. scapularis adults (males, n = 13; females, n = 40) were collected from 14 locations in southern Ontario). These collections were conducted within two different forested areas (Great Lakes-St. Lawrence and Carolinian). Flagging was done in the Carolinian forest region, whereas canine-and feline-derived ticks were obtained from three veterinary clinics located in the Huronia area ( Figure 1). Collections of I. scapularis comprised of 32 ticks (males, n = 13; females, n = 19) collected by flagging, and 21 females collected from 21 companion animals (domestic dogs, n =17; domestic cats, n = 4). outgroup species. All sequences were trimmed to the same length (445 nucleotides, including those absent in some species), and were aligned using the MUSCLE algorithm [44]. Phylogeny was resolved using the maximum likelihood method in MEGA 10.0.5 [45]. This general time reversible model facilitated gamma distribution and invariant sites (number of discrete categories equals five) was determined by jModeltest 2.1.10 [46]. Consensus was achieved by bootstrapping based on 1000 pseudoreplicate datasets generated from the original sequence alignments.

Tick collection
Between 24 April and 22 May 2019, a grand total of 53 I. scapularis adults (males, n = 13; females, n = 40) were collected from 14 locations in southern Ontario). These collections were conducted within two different forested areas (Great Lakes-St. Lawrence and Carolinian). Flagging was done in the Carolinian forest region, whereas canine-and felinederived ticks were obtained from three veterinary clinics located in the Huronia area (Figure 1). Collections of I. scapularis comprised of 32 ticks (males, n = 13; females, n = 19) collected by flagging, and 21 females collected from 21 companion animals (domestic dogs, n =17; domestic cats, n = 4).

Babesia Detection
Of the 53 ticks, 35.8% (n = 19) tested positive for Babesia DNA (Tables 1 and 2). The majority of positive ticks were from dogs (68.4%, n = 13), plus two from cats (10.5%), and four by flagging (21.1%; Table 2). All positive samples were confirmed to be B. odocoilei based on 99.72-100% similarity to sequences published in GenBank (Table 1). With the exception of three male ticks collected at flagging sites (Dundas, Turkey Point, Wainfleet bog), all Babesia-positive ticks were adult females (Table 1).   Between one and eight ticks (mean = 3.79) were collected from each location, and Babesia-positive ticks were collected from ten of the 14 sampled locations. The number of positive I. scapularis ticks collected at each positive location in both forested area was low (1)(2)(3)(4)(5), but prevalence ranged between 12.5-100% (mean = 65.06%; Table 1). Only ten of the 19 B. odocoilei sequences were of ample quality and length to perform phylogenetic analysis (CN19-89-CN19-99). These sequences were 100% similar to one another and to three reference strains of B. odocoilei downloaded from GenBank (Table 2, Figure 2). All sequences were trimmed to 445 nucleotides in length (including those absent to some species) and aligned using the MUSCLE algorithm. Phylogeny was resolved using a gamma distribution with invariant sites, and consensus was achieved by bootstrapping based on 1000 pseudoreplicate datasets generated from the original sequence alignments. Alphanumeric values in brackets denote published sequences. Babesia bovis is the outgroup species. The scale bar signifies the percentage of genetic variation along tree branches. Sequences from ticks are available in GenBank (accession numbers: MW182504MW182513).

Discussion
Here we report the presence of Babesia in I. scapularis ticks from ten of 14 sampled locations in Ontario, Canada, collected either by flagging low-lying vegetation or from domestic cats and dogs (Table 1). Babesia DNA was detected in 19 (35.8%) of 53 collected ticks. Our results are in concordance with previous studies conducted in the U.S.A. where 1115% of the questing blacklegged tick adults collected from established populations were positive for B. odocoilei [47,48]. Although the gender of I. scapularis adults may seem disproportionate (Table 1), the ratio of males to females are balanced in nature genetically reflecting a 50:50 ratio. Based on deer tracks along trails, we observed that an I. scapularis-O. virginianus interface was present in each of the woodland and ecotone locations flagged. The high incidence of B. odocoilei in I. scapularis ticks in the Huronia area suggests that an epizootic, babesial infection is present in the local white-tailed deer population [2325]. To our knowledge, this is the first time that B. odocoilei has been detected in ticks collected from dogs and cats in Canada. Researchers in Indiana reported one B. odocoileipositive I. scapularis in 15 adults collected from a domestic dog [49]. Globally, certain other Babesia spp. (B. canis sensu stricto, B. gibsoni, B. microti, B. vogeli) can infect either cats or All sequences were trimmed to 445 nucleotides in length (including those absent to some species) and aligned using the MUSCLE algorithm. Phylogeny was resolved using a gamma distribution with invariant sites, and consensus was achieved by bootstrapping based on 1000 pseudoreplicate datasets generated from the original sequence alignments. Alphanumeric values in brackets denote published sequences. Babesia bovis is the outgroup species. The scale bar signifies the percentage of genetic variation along tree branches. Sequences from ticks are available in GenBank (accession numbers: MW182504−MW182513).

Discussion
Here we report the presence of Babesia in I. scapularis ticks from ten of 14 sampled locations in Ontario, Canada, collected either by flagging low-lying vegetation or from domestic cats and dogs (Table 1). Babesia DNA was detected in 19 (35.8%) of 53 collected ticks. Our results are in concordance with previous studies conducted in the U.S.A. where 11−15% of the questing blacklegged tick adults collected from established populations were positive for B. odocoilei [47,48]. Although the gender of I. scapularis adults may seem disproportionate (Table 1), the ratio of males to females are balanced in nature genetically reflecting a 50:50 ratio. Based on deer tracks along trails, we observed that an I. scapularis-O. virginianus interface was present in each of the woodland and ecotone locations flagged. The high incidence of B. odocoilei in I. scapularis ticks in the Huronia area suggests that an epizootic, babesial infection is present in the local white-tailed deer population [23][24][25]. To our knowledge, this is the first time that B. odocoilei has been detected in ticks collected from dogs and cats in Canada. Researchers in Indiana reported one B. odocoilei-positive I. scapularis in 15 adults collected from a domestic dog [49]. Globally, certain other Babesia spp. (B. canis sensu stricto, B. gibsoni, B. microti, B. vogeli) can infect either cats or dogs [50]. In addition, B. conradae is associated with canine-feeding ticks and host dogs [51,52]. Babesia DNA, determined to be 97.8% similar to B. odocoilei, has been detected in ticks collected from dogs in Japan [53], whilst B. vogeli and B. microti have been detected in ticks associated with pet cats [54][55][56].
Babesia odocoilei can either be maintained in I. scapularis ticks by transstadial passage and/or transovarial transmission. All B. odocoilei-positive ticks in the present study were adults, and it is likely that they became infected during a previous developmental life stage while feeding on an infected reservoir host. As we did not draw blood from domestic cats and domestic dogs, we do not know if these companion animals play a role as reservoir hosts of B. odocoilei. This aspect warrants further investigation.
During the past half century, several tick-pathogen studies in North America have demonstrated that B. odocoilei has a wide distribution. In the U.S.A., the pathogen has been reported in ticks and vertebrate hosts as far north and east as Maine [47,49,57], as far south as Texas [58], and as far west as California [32]. The presence of B. odocoilei in California is notable because it has been detected in desert bighorn sheep, a non-cervid vertebrate host, which is outside the normal distribution of I. scapularis ticks [32]. Of biogeographical significance, B. odocoilei has been detected in the western blacklegged tick, Ixodes pacificus, and, thus, could be a vector infecting bovids in California. North of the Canada−U.S. border, B. odocoilei has been reported in Ontario [35,[39][40][41], Quebec [35], and Saskatchewan [26]. Additionally, other epidemiological studies have reported B. odocoilei causing fatal outcomes in bovid species in captivity [32,33].
Ecologically, the number of I. scapularis adults in the spring is lower than the full complement of adults in the fall because the number of questing females goes down as they parasitize hosts, and remain in quiescence until spring to lay eggs. By spring, the number of adults is predictably lower. As I. scapularis females parasitize suitable hosts, the number of questing females decreases.
Of epidemiological significance, B. microti has also been detected in the Huronia area in the Ixodes cookei (groundhog tick) [39]. As I. cookei does not parasitize songbirds, B. microti is most likely established in the area. Therefore, people may become infected with this babesial piroplasm.
Songbirds play an important role in the wide dispersal of B. odocoilei-positive I. scapularis [39][40][41]. Therefore, people do not have to visit an endemic area to contract babesiosis. The symbiosis of blacklegged ticks and white-tailed deer in a sylvatic habitat provides the strategic components for a B. odocoilei endemic area. White-tailed deer are hosts of I. scapularis, especially males and female adults, and promote the propagation of this tick species. Currently, there is no risk map in Ontario for Babesia, so there is no way to see where these areas are located. Holding fast to the dogma that one must visit an endemic area might hamper the assessment and whereabouts of these areas by epidemiologists and healthcare providers. Any of the B. odocoilei-positive sites in southern Ontario may have been initiated by songbirds infested with B. odocoilei-infected I. scapularis larvae and nymphs. Migratory songbirds can transport bird-feeding ticks from as far south as equatorial South America [59][60][61][62][63][64][65][66][67], and potentially start a new foci of I. scapularis that is hundreds of kilometres from its original source [68][69][70][71]. These established populations of I. scapularis may unknowingly be infected with B. odocoilei.
Even though the pathogenicity in cats, dogs, and humans has not been clarified for B. odocoilei, this piroplasm is in the same clade/group as Babesia divergens and Babesia venatorum (Figure 2), both of which are pathogenic to humans [1,4]. In essence, healthcare providers must be vigilant to look for human babesosis in symptomatic patients, especially when bitten by blacklegged ticks.

Conclusions
We provide the first documentation of B. odocoilei in I. scapularis ticks collected from domestic dogs and cats in Canada. Of 21 ticks collected from domestic cats and domestic dogs, 71% were confirm positive for B. odocoilei. Babesia odocoilei-positive I. scapularis Pathogens 2021, 10, 327 7 of 9 ticks collected by flagging low-level vegetation exhibited widespread distribution in Ontario. Since white-tailed deer and songbirds transport B. odocoilei across the Ontario landscape, vertebrate hosts do not need to visit an endemic area to become infected with this babesial piroplasm.
Author Contributions: J.D.S. provided sourcing, collection, identification of ticks, writing of manuscript, and coordination of tick data. E.L.P. conducted analysis of dataset, writing manuscript, and completing phylogenetic analysis. M.S.S. tested ticks and compiled data spreadsheets. J.E.F. supervised lab procedures. All authors have read and agreed to the published version of the manuscript.
Funding: Funding for this study was facilitated in part by the Mary Alice Holmes Memorial Foundation and, also, by a philanthropic donor Diane Kindree. J.E.F. and E.L.P. acknowledge funding from the Pacific Southwest Regional Center of Excellence for Vector-Borne Diseases allocated by the U.S. Centers for Disease Control and Prevention (Cooperative Agreement 1U01CK000516).
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.