Emerging Tick-Borne Pathogens in Central Canada: Recent Detections of Babesia odocoilei and Rickettsia rickettsii

Abstract

Background:

The spread of emerging tick-borne pathogens has steadily increased in Canada with the widespread establishment of tick vectors and vertebrate hosts. At present, Borrelia burgdorferi, the bacterium causing Lyme disease, is the most common tick-borne pathogen in Canada and primarily transmitted by Ixodes scapularis. A low prevalence of other emerging tick-borne pathogens, such as Anaplasma phagocytophilum, Babesia species, Borrelia miyamotoi, and Francisella tularensis have also been detected through surveillance efforts in Canada. Although Rickettsia rickettsii has been historically detected in Haemaphysalis leporispalustris in Canada, the current prevalence and geographic extent of this pathogen is unknown.

Material and Methods:

In this study, we assessed the presence and prevalence of several emerging tick-borne pathogens in ticks and hosts collected through tick dragging and small mammal trapping in Central Canada.

Results:

Nested PCR testing detected three pathogen species in ticks, with Babesia odocoilei and B. burgdorferi in I. scapularis in addition to R. rickettsii in H. leporispalustris. Three pathogen species were detected in small mammals by nested PCR including B. odocoilei in Blarina brevicauda, Babesia microti in Peromyscus leucopus, and a Hepatozoon species in P. leucopus and Peromyscus maniculatus. B. burgdorferi and Babesia species were the pathogens most often detected in our samples, suggesting they are widely distributed across Central Canada. We also detected B. odocoilei and R. rickettsii beyond their known geographic distribution.

Conclusions:

Our results provide evidence that emerging tick-borne pathogens may be present outside defined risk areas identified by current surveillance efforts in Canada. As a result, emerging tick-borne pathogens introduced by the dispersal of infected ticks by migratory birds or maintained by hosts and vectors through cryptic transmission cycles may go undetected. More comprehensive testing including all tick life stages and additional tick-borne pathogens will help detect the spread and potential risk of emerging or re-emerging tick-borne pathogens for human and wildlife populations throughout Canada.

Introduction

Emerging tick-borne pathogens have increased in prevalence in Canada with climate warming, habitat fragmentation, and changes in the abundances and distributions of tick and host populations (Bouchard et al, 2019; Leo et al, 2016; Ogden and Lindsay, 2016). Pathogen transmission cycles are sustained by contact between ticks and their hosts (Radolf et al, 2012). Generalist tick species feed on a wide variety of hosts, including Ixodes scapularis feeding on mammals, birds, and humans and Haemaphysalis leporispalustris feeding on lagomorphs and birds (Keirans et al, 1996; Kollars and Oliver, 2003; Lindquist and Wu, 2016). Although some host specificity may occur depending on local host availability (McCoy et al, 2013), specialist tick species feed on select hosts such as Ixodes banksi and beavers (Lindquist and Wu, 2016).

Birds and small mammals are competent reservoir hosts that successfully feed large numbers of ticks and have greater probabilities of infecting feeding ticks with pathogens (Bouchard et al, 2011; LoGiudice et al, 2003; Ogden et al, 2008; Scott et al, 2019; Zinck and Lloyd, 2022). As a result, emerging tick-borne pathogens may be detected in areas beyond currently defined risk areas in Canada owing to the dispersal of infected ticks by migratory birds and the distributional shifts of host populations (Fiset et al, 2015; Garcia-Elfring et al, 2017; Roy-Dufresne et al, 2013; Simon et al, 2014).

The predominant tick-borne pathogen found in sentinel surveillance efforts in Canada is Borrelia burgdorferi, the bacterium causing Lyme disease (Guillot et al, 2020). Lyme disease prevalence has grown rapidly to become the most common tick-borne disease in North America (Gasmi et al, 2017; Nelder et al, 2018). The first case of another Borrelia species in Canada, Borrelia miyamotoi, was reported in 2013 (Bouchard et al, 2019). These spirochetes are primarily transmitted by I. scapularis and Ixodes pacificus ticks as well as rodents, especially Peromyscus mice (Kulkarni et al, 2015).

Other emerging tick-borne pathogens targeted by current surveillance efforts in Canada are expected to increase in the future, as Ixodidae transmit 40% of documented emerging vector-borne zoonotic diseases globally (Swei et al, 2020). Many pathogens including Anaplasma phagocytophilum and Babesia microti are primarily transmitted by Ixodes ticks and small mammals in Canada (Bouchard et al, 2019; Kulkarni et al, 2015). More recently, Babesia odocoilei has been found in cervids and I. scapularis in Canada (Mathieu et al, 2018; Milnes et al, 2019; Pattullo et al, 2013). In contrast, Francisella tularensis, the bacterium causing tularemia, is transmitted by Dermacentor, Amblyomma, and Haemaphysalis ticks. Tularemia can also spread through contact with infected mammals, infective aerosols, or arthropod bites (Gabriele-Rivet et al, 2016; Petersen et al, 2009).

Rickettsia rickettsii, the bacterium causing Rocky Mountain spotted fever, is vectored by Dermacentor, Haemaphysalis, and Rhipicephalus ticks. In Canada, the historical extent of R. rickettsii has relied on surveys and human cases owing to its rare occurrence (Bouchard et al, 2019; Humphreys and Campbell, 1947; Kulkarni et al, 2015). This bacterium has been found previously in H. leporispalustris ticks and dogs in Ontario, Nova Scotia, and Western Canada (Gary et al, 2006; Leighton et al, 2001; Wood and Artsob, 2012). Although H. leporispalustris does not typically bite humans, this tick species is important for the maintenance of R. rickettsii strains of variable virulence in ecosystems (Freitas et al, 2009; Parker et al, 1951). The current prevalence and geographic extent of R. rickettsii strains in H. leporispalustris ticks in Canada are unknown.

Only two of these emerging tick-borne pathogens are listed as nationally notifiable diseases in Canada: Lyme disease and tularemia (Bouchard et al, 2019). However, current sentinel surveillance efforts in Canada primarily target A. phagocytophilum, Borrelia species, and Babesia species (Guillot et al, 2020; Wilson et al, 2022). As certain emerging tick-borne pathogens are not reportable to public health agencies, it is challenging to assess their degree of establishment and spread across Canada. In this study, we assess the presence and prevalence of several emerging tick-borne pathogens along the northward edge of their range in Ontario and Quebec, Canada. We report recent detections of B. odocoilei and R. rickettsii in southeastern Quebec outside of their known geographic extent based on ongoing surveillance efforts. Our results highlight the need for comprehensive pathogen testing to detect the presence and monitor the spread of emerging tick-borne pathogens throughout Canada.

Materials and Methods

Sixteen forested sites of varying risk for Lyme disease were sampled in July and August 2019 in Central Canada [Institut national de santé publique du Québec, 2018; Ontario Agency for Health Protection and Promotion (Public Health Ontario), 2018]. At each site, tick dragging and small mammal trapping were conducted across three 40 meters by 70 meters grids. Within each grid, a 1 m² cotton flannel was dragged along four transects over low-lying vegetation and checked every 10 meters. Questing ticks were removed and placed into microvials with 95% ethanol and were later identified to the species using standard taxonomic keys (Egizi et al, 2019; Lindquist and Wu, 2016).

Small mammal trapping was conducted over 3 consecutive nights using 84 Sherman live traps (H.B. Sherman Traps, Inc.). Targeted species included mice (Peromyscus leucopus and Peromyscus maniculatus), shrews (Blarina brevicauda and Sorex cinereus), voles (Microtus pennsylvanicus and Myodes gapperi), and jumping mice (Napaeozapus insignis and Zapus hudsonius). Traps were baited with peanut butter and oatmeal, a water source (apple), and nesting material (cotton ball) in the late afternoon and checked the following morning. Nontargeted species and juveniles of targeted species were immediately released. Target species were killed in the field with isoflurane inhalation overdose followed by cervical dislocation. Owing to serious injuries, one red squirrel (Tamiasciurus hudsonicus) and two hairy-tailed moles (Parascalops breweri) were killed.

The liver of each specimen was dissected and placed into microvials with 95% ethanol. Feeding ticks were removed from the host and placed into microvials with 95% ethanol. All tick and small mammal specimens were accessioned in the collections of the Redpath Museum at McGill University (Montreal, Canada). All procedures were approved by McGill University (AUP No. 2019-8086), the Quebec Ministère des Forêts, de la Faune et des Parcs (SEG permit No. 2019-06-04-008-00-S-F), and the Ontario Ministry of Natural Resources and Forestry (WSCA No. 1093495).

Questing larval ticks were pooled together for testing by grid, with 2–10 larvae per pool. Feeding larval ticks found on the same individual host were pooled together, ranging from 1 to 10 larvae per pool. Questing and feeding nymphs and adults were tested individually. All DNA extractions and PCR were performed by Geneticks, Inc. As in Wills et al (2018), cleaned ticks were cut and homogenized using a microtube pestle in AquaGenomic solution. Samples were incubated in a heat block at 60°C for 45 min, vortexed briefly, and centrifuged for 4 min at 13,300 rpm. The supernatant was transferred to a microvial with 50 μL isopropanol, inverted, and centrifuged as before. After decanting the supernatant, the remaining DNA pellet was rinsed with 50 μL of 70% ethanol and left to air dry for 15 min at room temperature.

This pellet was resuspended with 50 μL of 1 mM Tris pH 8.0 and incubated in a heat block at 60°C for 1 h. DNA from mammal livers was extracted using the Thermo Scientific GeneJET Genomic Purification Kit (Thermo Fisher Scientific). The following modifications were made to the Mammalian Tissue Genomic DNA Purification protocol (Protocol A, 2016): 10 mg of liver was used, the extra centrifugation step 9 was included to remove residual solution, and no additional elution buffer was required after sitting for 5 min before centrifugation. DNA was used directly for PCR and then stored at −20°C.

Nested PCRs to identify Peromyscus specimens to species level were run using species-specific COIII primers following Tessier et al (2004). An initial denaturation time of 5 min was used. PCR products were run on 3% agarose gel, stained with Eco-Stain (Bio Basic, Markham, Canada), and visualized using a blue light transilluminator.

Nested PCRs were performed to target several pathogens in our tick and mammal specimens. The primers and conditions used are described in detail in Supplementary Table S1. All specimens were tested for A. phagocytophilum, Babesia species, and Borrelia species. B. burgdorferi and B. miyamotoi were tested once using the 5S–23S intergenic space (IGS) region. If a band was visible (i.e., positive PCR), two more replicates were conducted to identify false positives. An additional test using the flaB gene was performed to confirm the presence of B. burgdorferi (Supplementary Fig. S1). B. microti and B. odocoilei were targeted once with the 18S ribosomal RNA (rRNA) gene using the mic 494 and odo563 inner primers, respectively (Supplementary Fig. S2). If a band was visible, mic494 and/or odo563 primers were then tested twice more to distinguish false positives. An additional primer set targeting one Babesia species was used to confirm positive testing. The p44 gene was targeted to test for A. phagocytophilum.

Targeted testing of F. tularensis (fdx gene) and R. rickettsii (RRi6 hypothetical protein gene) was conducted for H. leporispalustris specimens, as these pathogens are typically found in this tick species, but not in Ixodes species. Amplified DNA was visualized on a 1.8% agarose gel stained with Eco-Stain (Bio Basic) using a blue light transilluminator. If no band was visualized, the sample was considered negative for that pathogen.

Amplified products were purified before sequencing using a cotton cushion following Sun et al (2012). The fragments were spun for 7 min at 5000 rpm, which were then reamplified using the corresponding inner primers to concentrate the amplicons for sequencing. Sanger DNA sequencing was performed at Bio Basic DNA Sequencing with the forward inner PCR primers.

All sequences were assessed for ambiguous base calls, end-reading errors, and quality scores using the 4Peaks software. Sequences with quality scores <20 were not included in our analyses. A MEGABLAST search using the nucleotide BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi#_blank) in GenBank was used to determine the pathogen species of a sequence.

Results

A total of 644 ticks were collected, with 506 larvae, 136 nymphs, and 2 adults (Table 1). For I. scapularis, we found 382 questing ticks including 255 larvae (29 pools), 126 nymphs, and 1 adult male in addition to 65 feeding ticks comprising 57 larvae (17 pools) and 8 nymphs. For H. leporispalustris, we only found 195 questing ticks at 4 sites in Quebec, which consisted of 194 larvae (22 pools) and 1 nymph. One Ixodes auritulus nymph feeding on one M. gapperi at Rose Hill and one Ixodes marxi adult female feeding on one T. hudsonicus at Saint-Majorique were found.

Table 1.

Total Number and Number of Infected Pools of Ticks at Our Study Sites in Ontario and Quebec, Canada

Site	Latitude	Longitude	Haemaphysalis leporispalustris		Ixodes auritulus	Ixodes marxi	Ixodes scapularis			Host of feeding ticks	Pathogen species found in ticks
Site	Latitude	Longitude	Larval pools	Nymph	Nymph	Adult	Larval pools	Nymph	Adult	Host of feeding ticks	Pathogen species found in ticks
(1) 3 Ridges Farm	42.70	−81.03	0	0	0	0	0	0	0	None	None
(2) New New Age Farm	42.73	−80.84	0	0	0	0	Q: 12 (0/12) F: 7 (0/7)	Q: 15 (2/15) F: 2 (0/2)	0	Four Peromyscus leucopus,^a two Napaeozapus insignis	Borrelia burgdorferi, Babesia odocoilei
(3) North Tract	44.08	−79.31	0	0	0	0	0	F: 2 (0/2)	0	Two N. insignis	None
(4) Brown Hill Tract	44.21	−79.36	0	0	0	0	0	0	0	None	None
(5) Upjohn Nature Reserve	45.08	−79.36	0	0	0	0	0	0	0	None	None
(6) Dyer Memorial Nature Reserve	45.40	−79.15	0	0	0	0	0	0	0	None	None
(7) Rose Hill Nature Reserve	45.16	−77.22	0	0	F: 1 (0/1)	0	0	0	0	One Myodes gapperi	None
(8) Kirkview Farm	45.42	−74.67	0	0	0	0	0	Q: 4 (0/4) F: 1 (1/1)	0	One P. leucopus	B. burgdorferi
(9) Saint-Polycarpe	45.33	−74.39	0	0	0	0	F: 1 (0/1)	Q: 1 (0/1)	0	One P. leucopus	None
(10) Saint-Valentin	45.18	−73.35	0	0	0	0	Q: 4 (0/4) F: 4 (0/4)	Q: 67 (19/67) F: 1 (0/1)	0	Three P. leucopus, one M. gapperi	B. burgdorferi, B. odocoilei
(11) Henryville	45.12	−73.21	0	0	0	0	Q: 10 (0/10) F: 3 (1/3)	Q: 26 (4/26) F: 2 (0/2)	Q: 1 (0/1)	Two P. leucopus,^a one Peromyscus maniculatus, one M. gapperi, one human (not tested)	B. burgdorferi, B. odocoilei
(12) Lefebvre	45.74	−72.41	Q: 1 (0/1)	0	0	0	0	0	0	None	None
(13) Parc Sanctuaire de Saint-Majorique	45.94	−72.53	0	0	0	F: 1 (0/1)	Q: 2 (0/2) F: 2 (1/2)	Q: 12 (2/12)	0	One Blarina brevicauda, one P. maniculatus, one Tamiasciurus hudsonicus (not tested)	B. burgdorferi, B. odocoilei
(14) Serpentine-de-Coleraine Ecological Reserve	45.98	−71.37	Q: 19 (4/19)	Q: 1 (0/1)	0	0	Q: 1 (0/1)	Q: 1 (0/1)	0	None	Rickettsia rickettsii
(15) Frontenac National Park	45.82	−71.20	Q: 1 (1/1)	0	0	0	0	0	0	None	R. rickettsii
(16) Saint-Sylvestre	46.37	−71.12	Q: 1 (0/1)	0	0	0	0	0	0	None	None

Larvae were tested together in pools, which were separated by grid if questing and by host if feeding. Nymphs and adults were tested individually. Questing pools are indicated by a “Q” and ticks found feeding on small mammals are indicated by an “F.” Numbers in parentheses represent the number of infected and total pools of ticks. These two infected small mammal individuals harbored different pathogen species than the feeding larvae, with one P. leucopus with a Hepatozoon spp. from New New Age Farm and one P. leucopus with Babesia microti from Henryville.

Infected small mammal host.

For questing I. scapularis, 29 larval pools and an adult male tested negative for all pathogens. However, 27 of 126 questing nymphs (21.42%) tested positive for a pathogen (Fig. 1 and Table 1). Five nymphs were infected with B. odocoilei (3.97%), including one nymph at New New Age Farm, Henryville, and Saint-Valentin, and two nymphs at Saint-Majorique. Similarly, 22 nymphs were infected with B. burgdorferi (17.46%), with 1, 3, and 18 nymphs at New New Age Farm, Henryville, and Saint-Valentin, respectively. For H. leporispalustris, 5 of 22 questing larval pools were infected with R. rickettsii (22.72%), with 4 pools at Coleraine and 1 pool at Frontenac. One uninfected H. leporispalustris nymph was also found at Coleraine.

FIG. 1.

Pathogens detected in ticks at our study sites in Ontario and Quebec, Canada. Circle size is representative of the abundance of local ticks, where larger circles denote greater tick abundances. No ticks were detected at sites with orange crosses. Circle coloration demonstrates the proportional results of pathogen testing for tick pools, which consisted of larval pools, individual nymphs, and individual adults. The proportion of the circle in light blue represents the tick pools that were negative for pathogen testing. Proportions of tick pools that were positive and harboring Babesia odocoilei (turquoise), Borrelia burgdorferi (light purple), or Rickettsia rickettsii (dark purple) are noted on the map. Study sites include (1) 3 Ridges Farm, (2) New New Age Farm, (3) North Tract—York Regional Forest, (4) Brown Hill Tract—York Regional Forest, (5) Upjohn Nature Reserve, (6) Dyer Memorial Nature Reserve, (7) Rose Hill Nature Reserve, (8) Kirkview Farm, (9) Saint-Polycarpe, (10) Saint-Valentin, (11) Henryville, (12) Lefebvre, (13) Parc du Sanctuaire Saint-Majorique, (14) Serpentine-de-Coleraine Ecological Reserve, (15) Frontenac National Park, (16) Saint-Sylvestre. The land cover map was extracted from provincial rasters of the 2019 annual crop inventories by Agriculture and Agri-Food Canada—Agroclimate (2019).

Feeding ticks were found on 22 small mammals (Table 1). For feeding I. scapularis ticks, 2 of 17 larval pools (11.76%) and 1 of 8 nymphs (12.50%) were infected (Fig. 1 and Table 1). One feeding larva from Henryville was infected with B. odocoilei, whereas its P. leucopus host was infected with B. microti. A larva feeding on an uninfected P. maniculatus from Saint-Majorique and a nymph feeding on an uninfected P. leucopus at Kirkview Farm were infected with B. burgdorferi. The feeding I. auritulus and I. marxi tested negative for all pathogens.

We did not detect similar infection rates in small mammals (Fig. 2 and Table 2). Only 4 of 105 small mammal hosts tested positive for a pathogen (3.8%). One B. brevicauda from Saint-Sylvestre tested positive for B. odocoilei and one P. leucopus from Henryville tested positive for B. microti. Two Peromyscus individuals tested positive for a Hepatozoon species, with one P. leucopus from New New Age Farm and one P. maniculatus from Henryville. We found a feeding larva on both infected P. leucopus, with one uninfected larva from New New Age Farm and one larva infected with B. odocoilei from Henryville.

FIG. 2.

Pathogens detected in small mammals in Ontario and Quebec, Canada. Circle size is representative of the abundance of collected small mammals, where larger circles denote greater abundances of collected small mammal. Circle coloration demonstrates the proportional results of pathogen testing for individual small mammals. The proportion of the circle in light blue represents the collected small mammals that were negative for pathogen testing. Individual small mammals that were positive and harboring Babesia odocoilei (turquoise), Babesia microti (pink), and a Hepatozoon species (teal) are noted on the map. Study sites include (1) 3 Ridges Farm, (2) New New Age Farm, (3) North Tract—York Regional Forest, (4) Brown Hill Tract—York Regional Forest, (5) Upjohn Nature Reserve, (6) Dyer Memorial Nature Reserve, (7) Rose Hill Nature Reserve, (8) Kirkview Farm, (9) Saint-Polycarpe, (10) Saint-Valentin, (11) Henryville, (12) Lefebvre, (13) Parc du Sanctuaire Saint-Majorique, (14) Serpentine-de-Coleraine Ecological Reserve, (15) Frontenac National Park, (16) Saint-Sylvestre. The land cover map was extracted from provincial rasters of the 2019 annual crop inventories by Agriculture and Agri-Food Canada—Agroclimate (2019).

Table 2.

Total Number and Number of Infected Small Mammal Individuals at Our Study Sites in Ontario and Quebec, Canada

Site	Latitude	Longitude	Peromyscus leucopus	Peromyscus maniculatus	Blarina brevicauda	Sorex cinereus	Microtus pennsylvanicus	Myodes gapperi	Parascalops breweri	Napaeozapus insignis	Pathogen species found in small mammals
(1) 3 Ridges Farm	42.70	−81.03	1 (0/1)	0	0	0	0	0	1 (0/1)	0	None
(2) New New Age Farm	42.73	−80.84	6 (1/6)	0	0	0	0	0	1 (0/1)	11 (0/11)	Hepatozoon spp. (P. leucopus)
(3) North Tract	44.08	−79.31	1 (0/1)	0	0	0	1 (0/1)	0	0	4 (0/4)	None
(4) Brown Hill Tract	44.21	−79.36	5 (0/5)	0	0	0	0	0	0	0	None
(5) Upjohn Nature Reserve	45.08	−79.36	2 (0/2)	0	0	0	0	0	0	0	None
(6) Dyer Memorial Nature Reserve	45.40	−79.15	0	1 (0/1)	0	0	0	0	0	1 (0/1)	None
(7) Rose Hill Nature Reserve	45.16	−77.22	0	3 (0/3)	0	0	0	2 (0/2)	0	2 (0/2)	None
(8) Kirkview Farm	45.42	−74.67	5 (0/5)	0	0	0	0	0	0	0	None
(9) Saint-Polycarpe	45.33	−74.39	8 (0/8)	0	5 (0/5)	0	0	0	0	0	None
(10) Saint-Valentin	45.18	−73.35	3 (0/3)	0	0	0	0	5 (0/5)	0	1 (0/1)	None
(11) Henryville	45.12	−73.21	2 (1/2)	3 (1/3)	0	0	0	1 (0/1)	0	0	Hepatozoon spp. (P. maniculatus) Babesia microti (P. leucopus)
(12) Lefebvre	45.74	−72.41	0	0	3 (0/3)	0	0	2 (0/2)	0	0	None
(13) Parc du Sanctuaire Saint-Majorique	45.94	−72.53	0	1 (0/1)	1 (0/1)	0	0	4 (0/4)	0	4 (0/4)	None
(14) Serpentine-de-Coleraine Ecological Reserve	45.98	−71.37	0	3 (0/3)	0	0	0	1 (0/1)	0	0	None
(15) Frontenac National Park	45.82	−71.20	0	0	0	1 (0/1)	0	0	0	0	None
(16) Saint-Sylvestre	46.37	−71.12	0	0	8 (1/8)	0	0	1 (0/1)	0	1 (0/1)	Babesia odocoilei (B. brevicauda)

Numbers in parentheses represent the number of infected and total small mammal individuals. Only four small mammal specimens were infected across our sites. Two Peromyscus individuals were infected with a Hepatozoon spp., with one P. leucopus from New New Age Farm and one P. maniculatus from Henryville. One P. leucopus individual was also infected with B. microti from Henryville. One B. brevicauda individual from Saint-Sylvestre was infected with Babesia odocoilei.

Discussion

We detected B. odocoilei and R. rickettsii in ticks and small mammals beyond their distributional limits based on current surveillance efforts in southeastern Quebec. No recent detections have been reported for R. rickettsii in Haemaphysalis or Dermacentor ticks in Canada (Dergousoff et al, 2009; Teng et al, 2011; Wood and Artsob, 2012; Wood et al, 2016; Yunik et al, 2015). Historically, R. rickettsii has been identified in H. leporispalustris ticks in Alberta, British Columbia, Ontario, and Nova Scotia, but has not been detected in Quebec (Humphreys and Campbell, 1947; Wood and Artsob, 2012). In Canada, B. odocoilei was first detected through cervid infections (Mathieu et al, 2018; Pattullo et al, 2013). More recently, our study as well as others have detected B. odocoilei in I. scapularis ticks in Central Canada (Milnes et al, 2019; Robinson et al, 2022; Scott and Pesapane, 2021; Scott et al, 2021; Scott et al, 2020). We also report for the first time B. odocoilei being detected in a shrew in Canada.

These pathogens use two modes of transmission in ticks including transovarial transmission from female to larvae and transstadial transmission between immature and adult stages—the latter relying on a bloodmeal taken from a competent host (Freitas et al, 2009; Moore et al, 2018; Roth et al, 2017; Zembsch et al, 2021). The dispersal of ticks infected with Babesia species and R. rickettsii by migratory songbirds has allowed the spread of these pathogens in Canada (Scott et al, 2021; Scott et al, 2020; Scott et al, 2019). Local competent hosts can maintain these pathogens in the environment, but feeding ticks may not become infected owing to low levels of pathogen circulation and variable strain virulence (Freitas et al, 2009; McDade and Newhouse, 1986).

Transovarial transmission can help spread these pathogens without requiring an infected host, although not all offspring may become infected owing to partial transovarial transmission, leading to low larval infection rates (Freitas et al, 2009; Zembsch et al, 2021). Therefore, it is unknown how quickly these emerging or re-emerging pathogens will spread in Canada owing to biotic barriers affecting pathogen transmission.

We also detected a Hepatozoon species in two white-footed mice. This pathogen is vectored by many arthropods including mosquitoes, ticks, mites, fleas, and flies, which are its definitive hosts (Smith, 1996). In Canada, competent intermediate hosts of this parasite include snakes, frogs, and small mammals (Boulianne et al, 2007; Léveillé et al, 2021; Léveillé et al, 2020). This parasite is transmitted to a mouse or another intermediate host through the accidental or intentional ingestion of an arthropod with infective oocytes. Arthropods consist of a large portion of the diet of Peromyscus mice, thereby providing greater opportunities for parasite transmission (Wolff et al, 1985). The parasite will then develop in its intermediate host, where it will be ready to infect future feeding arthropods (Smith, 1996).

The most prevalent pathogen among our samples was B. burgdorferi. Current surveillance efforts in Canada detect a high incidence of B. burgdorferi in ticks and vertebrate hosts, with an increase in positive I. scapularis ticks from 5.9% to 23% in the past decade in Central Canada (Gasmi et al, 2017; Gasmi et al, 2016; Guillot et al, 2020; Kulkarni et al, 2019; Milnes et al, 2019; Nelder et al, 2014; Slatculescu et al, 2020). Lower prevalence of A. phagocytophilum, B. miyamotoi, and B. microti have also been found in I. scapularis ticks through surveillance efforts in Canada (Guillot et al, 2020; O'Brien et al, 2016; Wilson et al, 2022). We only found B. microti in one P. leucopus individual in an endemic region of Quebec.

Surprisingly, no small mammals tested positive for B. burgdorferi. We tested liver tissues, which have been used for B. burgdorferi detection in wild rodents (Zinck and Lloyd, 2022). Higher B. burgdorferi detection rates might have been found if tail, tongue, ear samples (Zawada et al, 2020), or lung tissues (André et al, 2017) were used. A study analyzing liver microbiomes in P. leucopus found a low number of Borrelia sequences with next-generation sequencing, which were undetectable by classic endpoint PCR (André et al, 2017). However, we used longer B. burgdorferi fragments here (∼340 or 605 bp) than in André et al (2017) (142 bp). Therefore, B. burgdorferi may not be detected in some samples because of low concentrations or longer amplicons.

The primary tick-borne pathogens of interest for sentinel surveillance efforts in Canada are A. phagocytophilum, Borrelia species, and Babesia species (Guillot et al, 2020). Typically, these pathogens are tested in nymphs and adults. Larvae are rarely tested for pathogens, as transovarial transmission would be required. However, tick-borne pathogens are expected to increase in the coming decades, as tick and host populations continue to expand into new areas in Canada. Therefore, future surveillance efforts in Canada should focus on two key aspects for more comprehensive emerging tick-borne pathogen testing. First, pathogen testing should be performed on all tick life stages to detect possible transovarial transmission. Second, testing of nontargeted pathogens such as Rickettsia species can help detect pathogens that may be introduced by the dispersal of infected ticks by migratory birds or maintained through cryptic transmission cycles (Hamer et al, 2011). As a result, surveillance efforts would better detect the spread and potential risk of emerging or re-emerging tick-borne pathogens for human and wildlife populations throughout Canada.

Conclusions

Our study identified two emerging tick-borne pathogens, B. odocoilei and R. rickettsii, outside of their known range in Quebec. We also detected cases of B. burgdorferi, B. microti, and Hepatozoon spp. in tick and small mammal specimens in Central Canada. We demonstrate that all tick life stages should be tested comprehensively for pathogens, especially with the increased presence and spread of emerging or re-emerging tick-borne pathogens in Canada.

Footnotes

Acknowledgments

The authors express their appreciation to the landowners and stakeholders who allowed access to their property for field sampling. The authors gratefully thank all their assistants for their invaluable help in the field: Daniella Cross, Connia Ren, Dania Shaban, Ellen Bidulka, Jihane Benbahtane, Christina Provost, and Sébastien Cyr. The authors also thank Robbin Lindsay and Antonia Dibernardo for sharing the protocols used by the National Microbiology Lab for DNA extraction and PCR testing of tick samples.

Authors' Contributions

K.E.C., J.T.K., and V.M. conceived the study. K.E.C. led the methodology, data collection, formal analysis, visualizations, and wrote the article. J.T.K. and V.M. jointly supervised the research, helped design analyses, and contributed to writing.

Author Disclosure Statement

The authors declare no conflict of interest.

Funding Information

This study was funded by Hydro-Québec and Natural Sciences and Engineering Research Council fellowships to K.E.C. J.T.K. is supported by funds from a Natural Sciences and Engineering Research Council Discovery Grant—RGPIN-2017-147544, Natural Sciences and Engineering Research Council Discovery Accelerator Supplement, and the University Research Chair in Macroecology and Conservation at the University of Ottawa. V.M. is supported by a Natural Sciences and Engineering Research Council Discovery Grant—RGPIN-2017-03839.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Table S1

References

Agriculture and Agri-Food Canada—Agroclimate, Geomatics and Earth Observation Division, Science and Technology Branch. Annual Space-Based Crop Inventory for Canada, 2019 (Raster). 2019. Available from: https://open.canada.ca/data/en/dataset/ba2645d5-4458-414d-b196-6303ac06c1c9 [Last accessed: October 5, 2021].

André

, Mouton

, Millien

, et al. Liver microbiome of Peromyscus leucopus, a key reservoir host species for emerging infectious diseases in North America. Infect Genet Evol, 2017; 52:10–18; doi: 10.1016/j.meegid.2017.04.011

Bouchard

, Beauchamp

, Nguon

, et al. Associations between Ixodes scapularis ticks and small mammal hosts in a newly endemic zone in southeastern Canada: Implications for Borrelia burgdorferi transmission. Ticks Tick-Borne Dis, 2011; 2(4):183–190; doi: 10.1016/j.ttbdis.2011.03.005

Bouchard

, Dibernardo

, Koffi

, et al. Increased risk of tick-borne diseases with climate and environmental changes. Can Commun Dis Rep, 2019; 45(4):83–89; doi: 10.1474/5/ccdr.v45i04a02

Boulianne

, Evans

, Smith

. Phylogenetic analysis of Hepatozoon species (Apicomplexa: Adeleorina) infecting frogs of Nova Scotia, Canada, determined by ITS-1 sequences. J Parasitol, 2007; 93(6):1435–1441; doi: 10.1645/GE-1041.1

Dergousoff

, Gajadhar

, Chilton

. Prevalence of Rickettsia species in Canadian populations of Dermacentor andersoni and D. variabilis . Appl Environ Microbiol, 2009; 75(6):1786–1789; doi: 10.1128/AEM.02554-08

Egizi

, Robbins

, Beati

, et al. A pictorial key to differentiate the recently detected exotic Haemaphysalis longicornis Neumann, 1901 (Acari, Ixodidae) from native congeners in North America. Zookeys, 2019; 818:117–128; doi: 10.3897/zookeys.818.30448

Fiset

, Tessier

, Millien

, et al. Phylogeographic structure of the white-footed mouse and the deer mouse, two Lyme disease reservoir hosts in Québec. PLoS One, 2015; 10(12):e0144112; doi: 10.1371/journal.pone.0144112

Freitas

LHT

, Faccini

JLH

, Labruna

. Experimental infection of the rabbit tick, Haemaphysalis leporispalustris, with the bacterium Rickettsia rickettsii, and comparative biology of infected and uninfected tick lineages. Exp Appl Acarol, 2009; 47(4):321–345; doi: 10.1007/s10493-008-9220-4

10.

Gabriele-Rivet

, Ogden

, Massé A, et al. Eco-epizootiologic study of Francisella tularensis, the agent of tularemia, in Quebec wildlife. J Wildl Dis, 2016; 52(2):217; doi: 10.7589/2015-04-096

11.

Garcia-Elfring

, Barrett

, Combs

. et al. Admixture on the northern front: Population genomics of range expansion in the white-footed mouse (Peromyscus leucopus) and secondary contact with the deer mouse (Peromyscus maniculatus). Heredity, 2017; 119:447–458; doi: 10.1038/hdy.2017.57

12.

Gary

, Webb

, Hegarty

, et al. The low seroprevalence of tick-transmitted agents of disease in dogs from southern Ontario and Quebec. Can Vet J, 2006; 47(12):1194–1200.

13.

Gasmi

, Ogden

, Lindsay

, et al. Surveillance for Lyme disease in Canada: 2009–2015. Can Commun Dis Rep, 2017; 43(10):194–199; doi: 10.1474/5/ccdr.v43i10a01

14.

Gasmi

, Ogden

, Leighton

, et al. Analysis of the human population bitten by Ixodes scapularis ticks in Quebec, Canada: Increasing risk of Lyme disease. Ticks Tick-Borne Dis, 2016; 7(6):1075–1081; doi: 10.1016/j.ttbdis.2016.09.006

15.

Guillot

, Badcock

, Clow

, et al. Sentinel surveillance of Lyme disease risk in Canada, 2019: Results from the first year of the Canadian Lyme Sentinel Network (CaLSeN). Can Commun Dis Rep, 2020; 46(10):354–361; doi: 10.1475/4/ccdr.v46i10a08

16.

Hamer

, Hickling

, Sidge

, et al. Diverse Borrelia burgdorferi strains in a bird-tick cryptic cycle. Appl Environ Microbiol, 2011; 77(6):1999–2007; doi: 10.1128/AEM.02479-10

17.

Humphreys

, Campbell

. Plague, Rocky Mountain spotted fever, and tularaemia surveys in Canada. Can J Public Health, 1947; 38(3):124–130.

18.

Keirans

, Hutcheson

, Durden

, et al. Ixodes (Ixodes) scapularis (Acari: Ixodidae): Redescription of all active stages, distribution, hosts, geographical variation, and medical and veterinary importance. J Med Entomol, 1996; 33(3):297–318; doi: 10.1093/jmedent/33.3.297

19.

Kollars

, Oliver

. Host associations and seasonal occurrence of Haemaphysalis leporispalustris, Ixodes brunneus, I. cookei, I. dentatus, and I. texanus (Acari: Ixodidae) in southeastern Missouri. J Med Entomol, 2003; 40(1):103–107; doi: 10.1603/0022-2585-40.1.103

20.

Kulkarni

, Berrang-Ford

, Buck

, et al. Major emerging vector-borne zoonotic diseases of public health importance in Canada. Emerg Microbes Infect, 2015; 4(6):e33; doi: 10.1038/emi.2015.33

21.

Kulkarni

, Narula

, Slatculescu

, et al. Lyme disease emergence after invasion of the blacklegged tick, Ixodes scapularis, Ontario, Canada, 2010–2016. Emerg Infect Dis, 2019; 25(2):328–332; doi: 10.3201/eid2502.180771

22.

Leighton

, Artsob

, Chu

, et al. A serological survey of rural dogs and cats on the southwestern Canadian prairie for zoonotic pathogens. Can J Public Health Rev Can Santé Publique, 2001; 92(1):67–71; doi: 10.1007/BF03404848

23.

Leo

, Gonzalez

, Millien

. Multi-taxa integrated landscape genetics for zoonotic infectious diseases: Deciphering variables influencing disease emergence. Genome, 2016; 59(5):349–361; doi: 10.1139/gen-2016-0039

24.

Léveillé

, El Skhawy

, Barta

. Multilocus sequencing of Hepatozoon cf. griseisciuri infections in Ontario eastern gray squirrels (Sciurus carolinensis) uncovers two genotypically distinct sympatric parasite species. Parasitol Res, 2020; 119(2):713–724; doi: 10.1007/s00436-019-06583-5

25.

Léveillé

, Zeldenrust

, Barta

. Multilocus genotyping of sympatric Hepatozoon species infecting the blood of Ontario ranid frogs reinforces species differentiation and identifies an unnamed Hepatozoon species. J Parasitol, 2021; 107(2):246–261; doi: 10.1645/20-18

26.

Lindquist

, Wu

. A Handbook to the Ticks (Ixodida: Ixodidae, Argasidae) of Canada. Biological Survey of Canada: Ottawa; 2016.

27.

LoGiudice

, Ostfeld

, Schmidt

, et al. The ecology of infectious disease: Effects of host diversity and community composition on Lyme disease risk. Proc Natl Acad Sci U S A, 2003; 100(2):567–571; doi: 10.1073/pnas.0233733100

28.

Mathieu

, Pastor

, Berkvens

, et al. Babesia odocoilei as a cause of mortality in captive cervids in Canada. Can Vet J, 2018; 59(1):52–58.

29.

McCoy

, Léger

, Dietrich

. Host specialization in ticks and transmission of tick-borne diseases: A review. Front Cell Infect Microbiol, 2013; 3:57; doi: 10.3389/fcimb.2013.00057

30.

McDade

, Newhouse

. Natural history of Rickettsia rickettsii . Annu Rev Microbiol, 1986; 40(1):287–309; doi: 10.1146/annurev.mi.40.100186.001443

31.

Milnes

, Thornton

, Léveillé AN, et al. Babesia odocoilei and zoonotic pathogens identified from Ixodes scapularis ticks in southern Ontario, Canada. Ticks Tick-Borne Dis, 2019; 10(3):670–676; doi: 10.1016/j.ttbdis.2019.02.016

32.

Moore

, Pulscher

, Caddell

, et al. Evidence for transovarial transmission of tick-borne rickettsiae circulating in Northern Mongolia. PLoS Negl Trop Dis, 2018; 12(8):e0006696; doi: 10.1371/journal.pntd.0006696

33.

National Institute of Public Health of Quebec [in French]. Carte de risqué d'acquisition de la maladie de Lyme selon les municipalités du Québec. Gouvernement du Québec: Québec; 2018.

34.

Nelder

, Russell

, Lindsay

, et al. Population-based passive tick surveillance and detection of expanding foci of blacklegged ticks Ixodes scapularis. and the Lyme disease agent, Borrelia burgdorferi, in Ontario, Canada. PLoS One 2014;9(8):e105358; doi: 10.1371/journal.pone.0105358

35.

Nelder

, Wijayasri

, Russell

, et al. The continued rise of Lyme disease in Ontario, Canada: 2018. Can Commun Dis Rep, 2018; 44(10):231–236; doi: 10.1474/5/ccdr.v44i10a01

36.

O'Brien

, Delage

, Scalia

, et al. Seroprevalence of Babesia microti infection in Canadian blood donors. Transfusion, 2016; 56(1):237–243; doi: 10.1111/trf.13339

37.

Ogden

, Lindsay

, Hanincova

, et al. Role of migratory birds in introduction and range expansion of Ixodes scapularis ticks and of Borrelia burgdorferi and Anaplasma phagocytophilum in Canada. Appl Environ Microbiol, 2008; 74(6):1780–1790; doi: 10.1128/AEM.01982-07

38.

Ogden

, Lindsay

. Effects of climate and climate change on vectors and vector-borne diseases: Ticks are different. Trends Parasitol, 2016; 32(8):646–656; doi: 10.1016/j.pt.2016.04.015

39.

Ontario Agency for Health Protection and Promotion (Public Health Ontario). Ontario Lyme Disease Map 2018: Estimated Risk Areas. Queen's Printer for Ontario: Toronto, ON; 2018.

40.

Parker

, Pickens

, Lackman

, et al. Isolation and characterization of Rocky Mountain spotted fever rickettsiae from the rabbit tick Haemaphysalis leporispalustris Packard. Public Health Rep (1896), 1951; 66(15):455; doi: 10.2307/4587691

41.

Pattullo

, Wobeser

, Lockerbie

, et al. Babesia odocoilei infection in a Saskatchewan elk (Cervus elaphus canadensis) herd. J Vet Diagn Invest, 2013; 25(4):535–540; doi: 10.1177/1040638713491746

42.

Petersen

, Mead

, Schriefer

. Francisella tularensis: An arthropod-borne pathogen. Vet Res, 2009; 40(2):07; doi: 10.1051/vetres:2008045

43.

Radolf

, Caimano

, Stevenson

, et al. Of ticks, mice and men: Understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol, 2012; 10(2):87–99; doi: 10.1038/nrmicro2714

44.

Robinson

, Jardine

, Koffi

, et al. Range expansion of Ixodes scapularis and Borrelia burgdorferi in Ontario, Canada, from 2017 to 2019. Vector Borne Zoonotic Dis, 2022; 22(7):361–369; doi: 10.1089/vbz.2022.0015

45.

Roth

, Lane

, Foley

. A molecular survey for Francisella tularensis and Rickettsia spp. in Haemaphysalis leporispalustris (Acari: Ixodidae) in Northern California. J Med Entomol, 2017; 54(2):492–495; doi: 10.1093/jme/tjw202

46.

Roy-Dufresne

, Logan

, Simon

, et al. Poleward expansion of the white-footed mouse (Peromyscus leucopus) under climate change: Implications for the spread of Lyme disease. PLoS One, 2013; 8(11):e80724; doi: 10.1371/journal.pone.0080724

47.

Scott

, Clark

, Coble

, et al. Detection and trasstadial passage of Babesia species and Borrelia burgdorferi sensu lato in ticks collected from avian and mammalian hosts in Canada. Healthcare, 2019; 7(4):155; doi: 10.3390/healthcare7040155

48.

Scott

, Pascoe

, Sajid

, et al. Detection of Babesia odocoilei in Ixodes scapularis ticks collected from songbirds in Ontario and Quebec, Canada. Pathogens, 2020;9(10):781; doi: 10.3390/pathogens9100781

49.

Scott

, Pascoe

, Sajid

, et al. Detection of Babesia odocoilei in Ixodes scapularis ticks collected in southern Ontario, Canada. Pathogens, 2021; 10(3):327; doi: 10.3390/pathogens10030327

50.

Scott

, Pesapane

. Detection of Anaplasma phagocytophilum, Babesia odocoilei, Babesia sp., Borrelia burgdorferi sensu lato, and Hepatozoon canis in Ixodes scapularis ticks collected in eastern Canada. Pathogens, 2021; 10(10):1265; doi: 10.3390/pathogens10101265

51.

Simon

, Marrotte

, Desrosiers

, et al. Climate change and habitat fragmentation drive the occurrence of Borrelia burgdorferi, the agent of Lyme disease, at the northeastern limit of its distribution. Evol Appl, 2014; 7(7):750–764; doi: 10.1111/eva.12165

52.

Slatculescu

, Clow

, McKay

, et al. Species distribution models for the eastern blacklegged tick, Ixodes scapularis, and the Lyme disease pathogen, Borrelia burgdorferi, in Ontario, Canada. PLoS One, 2020; 15(9):e0238126; doi: 10.1371/journal.pone.0238126

53.

Smith

. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol, 1996; 82(4):565–585; doi: 10.2307/3283781

54.

Sun

, Sriramajayam

, Luo

, et al. A quick, cost-free method of purification of DNA fragments from agarose gel. J Cancer, 2012; 3:93–95; doi: 10.7150/jca.4163

55.

Swei

, Couper

, Coffey

, et al. Patterns, drivers, and challenges of vector-borne disease emergence. Vector Borne Zoonotic Dis, 2020; 20(3):159–170; doi: 10.1089/vbz.2018.2432

56.

Teng

, Lindsay

, Bartlett

, et al. Prevalence of tick-borne pathogens in the South Okanagan, British Columbia: Active surveillance in ticks (Dermacentor andersoni) and deer mice (Peromyscus maniculatus). Br Columbia Med J, 2011; 53:122–127.

57.

Tessier

, Noël

, Lapointe

F-J

. A new method to discriminate the deer mouse (Peromyscus maniculatus) from the white-footed mouse (Peromyscus leucopus) using species-specific primers in multiplex PCR. Can J Zool, 2004; 82(11):1832–1835; doi: 10.1139/z04-173

58.

Wills

MKB

, Kirby

, Lloyd

. Detecting the Lyme disease spirochete, Borrelia burgdorferi, in ticks using nested PCR. J Vis Exp, 2018;(132):e56471; doi:10.3791/56471

59.

Wilson

, Gasmi

, Bourgeois

A-C

, et al. Surveillance for Ixodes scapularis and Ixodes pacificus ticks and their associated pathogens in Canada, 2019. CCDR, 2022; 48(5):208–218; doi: 10.1474/5/ccdr.v48i05a04

60.

Wolff

, Dueser

, Berry

. Food habits of sympatric Peromyscus leucopus and Peromyscus maniculatus . J Mammal, 1985; 66(4):795–798; doi: 10.2307/1380812

61.

Wood

, Artsob

. Spotted fever group rickettsiae: A brief review and a Canadian perspective. Zoonoses Public Health, 2012; 59:65–79; doi: 10.1111/j.1863-2378.2012.01472.x

62.

Wood

, Dillon

, Patel

, et al. Prevalence of Rickettsia species in Dermacentor variabilis ticks from Ontario, Canada. Ticks Tick-Borne Dis, 2016; 7(5):1044–1046; doi: 10.1016/j.ttbdis.2016.06.001

63.

Yunik

MEM

, Galloway

, Lindsay

. Assessment of prevalence and distribution of spotted fever group rickettsiae in Manitoba, Canada, in the American dog tick, Dermacentor variabilis (Acari: Ixodidae). Vector Borne Zoonotic Dis, 2015; 15(2):103–108; doi: 10.1089/vbz.2014.1692

64.

Zawada

, von Fricken

, Weppelmann

, et al. Optimization of tissue sampling for Borrelia burgdorferi in white-footed mice (Peromyscus leucopus). PLoS One, 2020; 15(1):e0226798; doi: 10.1371/journal.pone.0226798

65.

Zembsch

, Bron

, Paskewitz

. Evidence for vertical transmission of Babesia odocoilei (Piroplasmida: Babesiidae) in Ixodes scapularis (Acari: Ixodidae). J Med Entomol, 2021; 58(6):2484–2487; doi: 10.1093/jme/tjab074

66.

Zinck

, Lloyd

. Borrelia burgdorferi and Borrelia miyamotoi in Atlantic Canadian wildlife. PLoS One, 2022; 17(1):e0262229; doi: 10.1371/journal.pone.0262229

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