The coccidian parasite Toxoplasma gondii is found worldwide infecting warm-blooded vertebrates. Felids are the definitive hosts; other species act as intermediate hosts. Squirrels (Sciuridae) generally have high population densities in cities and forage and cache food on the ground, where they may come into contact with T. gondii oocysts or be preyed upon by cats and other carnivores. This environment might make squirrels important intermediate hosts of T. gondii in cities, and infection rates could indicate environmental levels of oocysts in soil. We investigated whether urban squirrels would be more exposed to T. gondii infection than rural squirrels with samples collected from American red squirrels (Tamiasciurus hudsonicus), eastern grey squirrels (Sciurus carolinensis), northern flying squirrels (Glaucomys sabrinus), and least chipmunks (Tamias minimus) in and around Winnipeg, Manitoba, Canada. We tested 230 tissue samples from 46 squirrels for T. gondii DNA by quantitative PCR and 13 serum samples from grey squirrels for T. gondii antibodies by competitive ELISA. We found no evidence of infection in any squirrel, indicating that squirrels are probably not important intermediate hosts of T. gondii in cities and that consumption of oocysts in the soil in general may not be an important contributor to transmission in colder environments.

The coccidian parasite Toxoplasma gondii is found worldwide; Felidae are the only known definitive hosts for the parasite, and all mammal and bird species, including humans, may act as intermediate hosts (Hill et al. 2005). Squirrels (Sciuridae) are common inhabitants of cities and generally have higher population densities in urban compared with rural areas (Parker and Nilon 2008). Squirrels forage for food and cache on the ground in lawns, where they are likely to come into contact with T. gondii oocysts shed in cat feces. Like other prey species (Afonso et al. 2007), squirrels carrying T. gondii tissue cysts may act as a source of infection for cats and other predators. Consequently, squirrels are potentially important species for maintaining the T. gondii life cycle in cities. Squirrel infection rates with T. gondii also might serve as a proxy for soil contamination with oocysts, even if squirrels are not important intermediate host species of T. gondii. Knowledge of the role of squirrels in T. gondii infection dynamics in cities is limited. Our aim was to determine the prevalence of T. gondii infection in squirrel populations in and around Winnipeg, Manitoba, Canada. We hypothesized that because of the high population densities of both squirrels and cats in cities (Parker and Nilon 2008; Sims et al. 2008), urban squirrels might have a higher prevalence of T. gondii infection than rural squirrels.

We tested our hypothesis by molecular and serologic methods (Galeh et al. 2020). We collected frozen squirrel carcasses from trappers, wildlife rehabilitation centers, and pest control companies from 2017 to 2019. The organs were usable because the carcasses were frozen soon after death, and PCR can detect T. gondii DNA even in samples that are hindered by autolysis (Hurtado et al. 2001). Because T. gondii is a cyst-forming parasite, detection probability can differ between tissues (Elmore et al. 2016). Consequently, for each individual, we tested samples from multiple (two to six) organs to maximize the probability of detecting the parasite (Supplementary Material Table S1).

We tested 230 tissue samples from 46 squirrels belonging to four species for T. gondii DNA by quantitative PCR (Table 1; see Supplementary Material Table S2 for primers and probe sequences). Twenty-six carcasses were from urban locations within Winnipeg, and 20 were from rural locations approximately 30–250 km from Winnipeg (Fig. 1). There were 25 American red squirrels (Tamiasciurus hudsonicus), 16 eastern grey squirrels (Sciurus carolinensis), four northern flying squirrels (Glaucomys sabrinus), and one least chipmunk (Tamias minimus).

Table 1

The number of individuals, samples, and PCR tests carried out on squirrels collected in 2017–2019 from urban and rural sampling sites to survey Toxoplasma gondii prevalence in squirrel populations in and around the city of Winnipeg, Manitoba, Canada.

The number of individuals, samples, and PCR tests carried out on squirrels collected in 2017–2019 from urban and rural sampling sites to survey Toxoplasma gondii prevalence in squirrel populations in and around the city of Winnipeg, Manitoba, Canada.
The number of individuals, samples, and PCR tests carried out on squirrels collected in 2017–2019 from urban and rural sampling sites to survey Toxoplasma gondii prevalence in squirrel populations in and around the city of Winnipeg, Manitoba, Canada.
Figure 1

Map showing the urban and rural sampling locations in and around the city of Winnipeg, Manitoba, Canada, where a survey of Toxoplasma gondii prevalence in squirrel (Sciuridae) populations was conducted. The map on the left shows the locations and species of the 46 squirrel carcasses (varied shapes) used to collect tissue samples for PCR detection of T. gondii, and the urban and rural study sites (black triangles) where 13 samples of blood sera were collected from grey squirrels (Sciurus carolinensis) for testing of T. gondii antibodies. The map on the right is a close-up of the urban squirrel carcass locations (varied shapes) within the Winnipeg city perimeter.

Figure 1

Map showing the urban and rural sampling locations in and around the city of Winnipeg, Manitoba, Canada, where a survey of Toxoplasma gondii prevalence in squirrel (Sciuridae) populations was conducted. The map on the left shows the locations and species of the 46 squirrel carcasses (varied shapes) used to collect tissue samples for PCR detection of T. gondii, and the urban and rural study sites (black triangles) where 13 samples of blood sera were collected from grey squirrels (Sciurus carolinensis) for testing of T. gondii antibodies. The map on the right is a close-up of the urban squirrel carcass locations (varied shapes) within the Winnipeg city perimeter.

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For nucleic acid extraction, we processed tissues in two steps. First, 0.6 ml of buffer ATL (1063369, Qiagen, Hilden, Germany) was added to 100 mg of frozen tissue or 0.1 mL of sample (if liquid, e.g., thawed brain) in a 2-mL screw-cap tube and homogenized by Mini-Beadbeater-16 (BioSpec, Bartlesville, Oklahoma, USA) for 3 min. The homogenate was quickly spun (10 s at 5,000 × G), 70 µL of proteinase K was added, and the sample incubated for 2 h at 56 C. Buffer AL (600 µL; 1014594, Qiagen) was added into the proteinase K–treated homogenate, and the sample was incubated at 70 C for 10 min in a dry block. The samples were then centrifuged for 3 min at 10,000 × G. In the second step, 100 µL of ATL–proteinase K–AL homogenate was used for DNA extraction with 5× MagMAX Viral Isolation kit (AMB1836-5, Applied Biosystems, Vilnius, Lithuania).

Primers and probe for real-time PCR detection of T. gondii were designed according to the protocol of De Craeye et al. (2011; Supplementary Material Table S2). Cellular ribosomal RNA gene (18s) was targeted as an internal control of all sample extraction. The 18s primer and probe sequences were designed and modified from the study of De Craeye et al. (2011; Supplementary Material Table S2). Real-time PCR was conducted with TaqMan Fast Advanced Master Mix (4444557, Applied Biosystems) in a 7500 Real-Time PCR thermocycler (Applied Biosystems). The thermocycling program included an initial denaturation at 95 C for 2 min, followed by 40 cycles at 95 C for 5 s and 60 C for 33 s. The results were analyzed by 7500 Sequence Detection Software version 1.3.1 (Applied Biosystems, Foster City, California, USA). Two positive controls (nucleic acid) were used to confirm the efficiency of the real-time PCR.

We conducted live trapping of grey squirrels in one urban and one rural site between 5 June and 1 August 2019 (Fig. 1). The urban site in the city of Winnipeg, Manitoba, Canada, consisted of an approximately 10-ha park located on the University of Manitoba campus and a suburban neighborhood next to the park. The study site is bordered by the Red River and two major highways with high amounts of car traffic. Winnipeg is the largest city in the province of Manitoba with a population of 778,489 and a total land area of 46.433 km2 (Statistics Canada 2016). Winnipeg lies 239 m above sea level and has high seasonal climatic variation, with temperature varying from the extremes of around –31 C to –42 C between January and March to around 30 C to 35 C between June to September (Environment Canada 2020). The rural site was an approximately 34-ha forest patch next to an active honey farm near the twin cities Morden and Winkler in southern Manitoba (49°24′01.1″ N, 98°00′29.2″ W), bordered by agricultural land.

We used live traps (Tomahawk Live Trap Company, Tomahawk, Wisconsin, USA) to capture the squirrels at the study sites. Trapping was part of an ongoing effort to tag most of the individual squirrels at the urban and rural sites with passive integrated transponder (PIT) tags. Around 20–40 traps in total baited with peanut butter were set at the urban site, with around 80–100 traps at the rural site each trapping day. The number of traps was different for the two sites to ensure approximately the same number of squirrels was captured at both sites. After capture, squirrels were handled in a canvas capture bag, and we recorded the weight (g), body and tail length (cm), skull width (cm), age (adult or juvenile), reproductive status, and sex for each individual. We collected a minimum of 500 µL of blood from the femoral vein of each grey squirrel and stored the sample on ice until processing. Each squirrel was PIT tagged between the shoulder blades with PIT tags for further identification. We then released the squirrels at the place of capture. Our protocol (no. f16-003) was approved by the University of Manitoba animal care and use committee following Canadian Council on Animal Care guidelines. All blood samples were processed within 12 h of collection. We centrifuged the blood samples at 1,165 × g for 15 min and froze the serum at –20 C until used for testing.

We tested the serum from 13 blood samples from grey squirrels for antibodies (immunoglobulin G) against T. gondii with a commercially available indirect ELISA (Multi-species ID Screen kit, IDvet, Grabels, France) following the manufacturer's instructions. We read the optical density values at 450 nm in a spectrophotometer and calculated results with these values and kit controls expressed as sample-to-positive ratio percentage (S/P%). We considered samples with S/P% ≤40% negative; samples with S/P% between 40% and 50% inconclusive; and samples with S/P% >50% positive, following the kit's protocol. Because of the varied sources of squirrel carcasses, we were unable to obtain serologic samples from the dead squirrels tested by PCR. Meat juice serology is another good method for detecting T. gondii infection (Plaza et al. 2020). We did not use this method because most carcasses had little or no meat juice.

We did not detect T. gondii DNA on any of the 230 tissue samples (liver, heart, brain, lung, spleen, or kidney) obtained from the 46 squirrels. We also did not detect antibodies against T. gondii in the 13 blood sera samples from grey squirrels (Supplementary Material Table S3).

Molecular and serologic methods used together, as in our study, can be a more reliable way of estimating infection rate (Galeh et al. 2020). The distribution of T. gondii tissue cysts can be uneven and differ between tissues (Elmore et al. 2016), which may result in detection difficulties with PCR-based methods (Opsteegh et al. 2010). The sensitivity and specificity of antibody detection methods can also vary (Gilbert et al. 2013). The ELISA that we used, although not validated for squirrels, has been successfully used to detect T. gondii antibodies in wildlife (Roqueplo et al. 2011; Sharma et al. 2019), has high specificity and sensitivity compared with serologic tests such as the modified agglutination test (Sharma et al. 2019), and does not cross-react with other apicomplexan parasites—a factor known to limit the specificity of serologic assays (Hirota et al. 2010).

The prevalence of T. gondii in sciurids in cities is not well known. An earlier study conducted in Guelph, Ontario, Canada, found no evidence of infection from grey squirrels (n=16) or chipmunks (n=6; Tizard et al. 1978). A shortcoming of that study, as well as our own, is the small sample size. However, when interpreted together, these studies may indicate the potential for generally low T. gondii infection rates in squirrels in cities. In areas of natural habitat in the US and Europe, T. gondii has been found in the eastern grey squirrel (e.g., Smith and Frenkel 1995), western grey squirrel (Sciurus griseus, Soave and Lennette 1959), and Eurasian red squirrel (Sciurus vulgaris, e.g., Jokelainen and Nylund 2012). However, T. gondii prevalence in squirrels has usually been low (2/11 positive individuals, Smith and Frenkel 1995; 3/19, Jokelainen and Nylund 2012).

The prevalence of T. gondii in Manitoba, Canada, is poorly understood and no previous survey of T. gondii prevalence in squirrels exists from the province. Serologic testing in 1981 found that 19/72 cats and one polar bear (Ursus maritimus) tested positive; however, the results from 28 other species were all negative (Sekla et al. 1981). Moreover, the Canadian prairies generally seem to harbor a low prevalence of T. gondii in cats (3.4%), domestic sheep, and cattle (0%, Nation and Allen 1976), as well as in pigs (1% in Manitoba, 2.5% in Saskatchewan, Canada, Smith 1991). The reasons for this low prevalence are poorly understood, but the extreme climatic variations in Manitoba throughout the year (sustained fluctuation from 35 C in the summer to up to –42 C in the winter) might decrease the viability or infectivity of oocysts and tissue cysts, thereby lowering the overall T. gondii prevalence in the area (Dubey et al. 1970; Frenkel et al. 1975). However, T. gondii antibodies were found at relatively high rates in skunks (Mephitis mephitis; 28%) and raccoons (Procyon lotor; 27.5%) in Manitoba between 2002 and 2006 (Hwang et al. 2007). Given the presence of T. gondii in these carnivores in the province, our results may help us understand the ecology of T. gondii transmission. If infected squirrels are proxies for oocysts in the soil, our results suggest that encountering oocysts in the environment may be a less important mode of transmission in the Winnipeg area than predation or carrion consumption. Future work is needed to confirm this possibility and to improve our understanding of the infection dynamics of T. gondii in cities.

We thank Neil Pople, Andre Hamel, Amanda Salo, and virology lab members of the Manitoba Veterinary Diagnostic Services Laboratory for their helpful guidance and data collection. We also thank Mitchell Green, Kyle Lefort, and Paul O'Brien for their help with postmortem examinations and Constance Finney and Emily Jenkins for their excellent suggestions regarding the methods. We give special thanks to Vera and Phil Froese for giving their permission to work on their land. This study was supported by a Discovery Grant to C.J.G. from the Natural Sciences and Engineering Research Council of Canada (NSERC). R.P.K. and C.S. were additionally supported by the University of Manitoba Graduate Fellowships and a University of Manitoba Graduate Enhancement of Tricouncil funding grant to C.J.G.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-21-00171.

© Wildlife Disease Association 2023

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Supplementary data