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International Journal for Parasitology: Parasites and Wildlife logoLink to International Journal for Parasitology: Parasites and Wildlife
. 2025 Apr 3;27:101069. doi: 10.1016/j.ijppaw.2025.101069

Detection of Toxoplasma gondii (Types I, II, III and 12) and Sarcocystis spp. in the brains of river otter (Lontra canadensis) from Alberta, Canada

Kyle M Shanebeck a,⁎, Adrián Hernández-Ortiz b, Emily J Jenkins b, Philippe J Thomas c, Brent R Dixon d, Harriet Merks d, Clement Lagrue a,e
PMCID: PMC12019200  PMID: 40276325

Abstract

Toxoplasma gondii and Sarcocystis spp. are globally distributed coccidian parasites infecting endothermic vertebrates. Toxoplasma gondii is zoonotic, with widespread global prevalence in humans, domestic animals, and wildlife. Sarcocystis is a related and diverse genus, with species that use a range of definitive and intermediate hosts. In intermediate hosts, these tissue dwelling coccidians can be asymptomatic or cause disease through neural, hepatic, and transplacental infections. Semiaquatic mammals such as the North American river otter (Lontra canadensis) are at high risk of exposure to T. gondii and Sarcocystis spp. due to terrestrial runoff into freshwater environments. Their high trophic position and dual habitat use make them excellent sentinel species to monitor the presence of food and waterborne pathogens in ecosystems. Brain tissue was sampled from 89 river otters in Alberta, Canada. DNA of T. gondii was detected in 34 % of otters using magnetic capture sequence-specific DNA extraction and qPCR. Genotypes of T. gondii were identified using nested PCR and sequencing of the GRA6 and SAG2 genes, and included the most common clonal lineages in North America, Types I, II, and III, as well as Type-12 (X/A), which is highly pathogenic in sea otters. DNA of Sarcocystis spp. was detected in brain lysates of 30 % of otters via conventional PCR with primers targeting ITS1 and 18S ribosomal regions, and sequencing revealed S. lutrae and a species most closely related to, but distinct from, S. kitikmeotensis. This study suggests that river otters are exposed trophically to T. gondii shed by felids, and at least 2 species of Sarcocystis shed by unknown definitive hosts. Highly pathogenic S. neurona was not detected in this population, likely reflecting the absence of possum definitive hosts in northern Canada. The potential effects of T. gondii and Sarcocystis spp. on behaviour, health, and reproduction of river otters warrant further investigation.

Keywords: Toxoplasma gondii, Sarcocystis spp., Lontra canadensis, Wildlife disease, Zoonosis

Graphical abstract

Image 1

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Highlights

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    Toxoplasma gondii and Sarcocystis spp. detected in brains of river otters from Alberta, Canada.

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    Prevalence 34 % for T. gondii, 30 % for Sarcocystis spp.

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    Genotypes included Type-II and 12, which are potentially highly pathogenic.

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    High prevalence may indicate significant environmental contamination of oocysts.

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    May also indicate public health risks, especially for indigenous communities.

1. Introduction

Toxoplasma gondii and Sarcocystis spp. are protozoan parasites capable of infecting a wide range of endothermic vertebrates and are potential threats to wildlife and human health worldwide (Dubey and Hamir, 2000; Dubey, 2014; Fayer et al., 2015). The life cycle of T. gondii is complex, with felids the only definitive host and many vertebrates acting as intermediate hosts (Dubey, 2014). There are three infective stages, sporozoites, tachyzoites, and bradyzoites, which are infective in both intermediate and definitive hosts (Dubey, 2014; Attias et al., 2020). Asexual proliferation of tachyzoites occurs in various tissues, eventually forming cysts containing bradyzoites, often in muscle or neural tissue. Sexual reproduction occurs in the intestines of felids, with oocysts released in feces that sporulate in the environment (Hill et al., 2005; Dubey, 2014; Attias et al., 2020). Due to trophic transmission and a broad intermediate host range, T. gondii transmits through carnivory in food webs (Bachand et al., 2019). This makes generalist carnivores like mustelids excellent sentinel species to monitor the presence of T. gondii in ecosystems. Oocyst transmission through water is also common as they survive well in aquatic environments and are carried via run-off, making aquatic carnivores further suited as sentinels (Yan et al., 2016).

Sarcocystis species have an obligate two host life cycle which involves carnivore definitive hosts, with over a hundred species with variably well understood life cycles and host specificity (Dubey et al., 2016). Similar to T. gondii, they tend to be definitive host specific, but will use many vertebrates as intermediate host. Sporozoites are the infective stage for intermediate hosts, exposed through consumption of sporocysts shed by carnivore definitive hosts into the environment (Dubey et al., 2016). Asexual proliferation occurs in various tissues depending on species; sarcocysts containing bradyzoites form in tissue, and are the infective stage in definitive hosts that prey or scavenge the intermediate host (Dubey et al., 2016). After sexual reproduction in the intestine of the definitive host, sporulation occurs in the intestine (vs environment for T. gondii) and sporocysts are passed with feces. There is no evidence that Sarcocystis species can be passed from intermediate to intermediate host, as observed for T. gondii (Dubey et al., 2016).

Among the diversity of hosts susceptible to T. gondii and Sarcocystis spp. infections, mustelids, especially otters, have emerged as important sentinel species, providing insights into the transmission dynamics and genetic diversity of these parasites in terrestrial and aquatic ecosystems (Dubey and Hamir, 2000; Chadwick et al., 2013; Barros et al., 2018; Prakas et al., 2018; Sharma et al., 2019; Cotey et al., 2022; Miller et al., 2023). Infections by both T. gondii and Sarcocystis spp. have been reported in multiple otter species and are an important health risk for threatened otter populations (Gjerde and Josefsen, 2015; Cotey et al., 2022; Miller et al., 2023). Various clinical manifestations of toxoplasmosis (neurological, ocular, abortion, and congenital) are described in humans and variably reported in wildlife (Hill et al., 2005). Severity of clinical manifestations is often tied to specific clonal lineages (Dubey et al., 2011). Toxoplasmosis is an emergent disease of concern for multiple at-risk species such as sea otters in California, USA, and cetaceans and birds in Aotearoa New Zealand (Roberts et al., 2020a; Miller et al., 2023). In intermediate hosts, infection with Sarcocystis spp. may be subclinical to mild, though serious infections have been identified in both domestic animals and wildlife. Examples of this include S. cruzi and S. suihominis which can cause abortions in livestock, S. caninum which can cause neurological symptoms in dogs, and S. canis which causes severe and even fatal liver disease in bears (Dubey and Hamir, 2000; Fayer et al., 2015; Franco et al., 2018).

River otters (Lontra canadensis) are semi-aquatic mammals that occur across a broad swath of North America (Larivière and Walton, 1998). Once reduced by the fur trade, river otters have returned to a large portion of their historical range, though are still absent from the southern parts of Alberta, as well as parts of the central and southwest United States (Larivière and Walton, 1998; CITES, 2019; Roberts et al., 2020b). Important constituents of riparian ecosystems, river otters are meso-carnivores with top-down influence on fish and invertebrate species (Roemer et al., 2009). River otters feed on a broad range of riparian organisms including fish, frogs, turtles, waterfowl, crustaceans and other invertebrates, though fish make up the majority of their diet (Searing, 1979; Larivière and Walton, 1998). Being opportunistic predators, their diets are broad and tend to be determined by habitat, availability of food, and vulnerability of prey, and change by region and season (Searing, 1979). For example, in lake-dominated regions of Alberta, otters may increase the frequency of waterfowl and their eggs in diets likely due to easy availability of molting and nesting birds (Gilbert and Nancekivell, 1982).

Investigating the geographic range and prevalence of T. gondii and Sarcocystis spp. is essential to accurately predict disease risk for wildlife, domestic animals and humans. Studying the relationships between T. gondii genotypes and their wildlife hosts can provide valuable insights into the ecology and epidemiology of this parasite within aquatic ecosystems. Furthermore, understanding the genetic diversity of T. gondii in wildlife is crucial for assessing potential sources of zoonotic infection and devising targeted interventions to mitigate risk, especially for groups at greater risk such as First Nation communities (Elmore et al., 2012). Here we report detections of both T. gondii and Sarcocystis spp. in the brains of North American river otter (Lontra canadensis) from Alberta, Canada, including phylogenetic and genotypic analysis.

2. Materials and methods

Carcasses of 89 river otters were obtained from licensed fur trappers in Alberta, Canada, in association with the Alberta Trappers Association during the 2020-21 and 2021-22 trapping seasons. Carcasses were kept frozen at −20 °C until dissection. Brains were removed by opening the skull with a transverse cut through the frontal bone behind the orbital sockets with a hacksaw, followed by two lateral cuts from the zygomatic processes of the frontal bone to the foramen magnum. The separated sections of frontal and parietal bone were then carefully removed. The brain was extracted with dissecting scissors to cut away nerves and pull the dura mater from the skull, and then stored in sterile Whirl-Pak® bags (VWR, Mississauga, ON, Canada) and frozen at −20 °C. Tools were washed and bleached between each skull dissection.

2.1. Molecular detection of Toxoplasma gondii

Brains were tested for DNA of Toxoplasma gondii via magnetic capture sequence-specific DNA extraction and qPCR using primers for the 529 bp repeat element unique to T. gondii, following established protocols (Opsteegh et al., 2010). Real-time PCR amplification was performed in a Bio-Rad CFX 96 DNA thermal cycler (BioRad, Hercules, California, USA) using the primers Tox-9F (5′-AGGAGAGATA TCAGGACTGTAG-3′) and Tox-11R (5′-GCGTCGTCTC GTCTAGATCG-3′) (Opsteegh et al., 2010).

Each run was done in duplicate with positive, negative and ‘no template’ controls added. Heart tissue from an experimentally infected reindeer was used as a positive reference control (Bouchard et al., 2017). Additionally, one beef sample was used as a negative control, and two beef samples spiked with 2.5 × 105 and 2.5 × 106 tachyzoites/mL were used as positive controls. As a standard curve for quantification of positive samples we used a dilution series of T. gondii plasmid DNA. The reaction was considered positive if the Ct-value was less than or equal to 35, or negative if the Ct-value was equal to zero or above 35 (Bachand et al., 2019). These standards were fitted in a generalized linear model in R statistical software 4.4.2 (R Core Team, 2024), and the slope and y-intercept were used to predict the Log10(concentration) of tachyzoite-equivalents in the river otter samples, using the Ct-values from five standardized samples of beef muscle spiked with known concentrations (Bachand et al., 2019).

2.2. Genotyping of Toxoplasma gondii

Partial genotyping of T. gondii was done on a subset of 24 positive samples, with primers targeting the dense granule antigen (GRA6) and surface antigen 2 (SAG2) genes (Su et al., 2006). The outer PCR primers, GRA6 FO and GRA6 RO (Zakimi et al., 2006), and nested PCR primers, GRA6 F1 and GRA6 R1 (Opsteegh et al., 2010) were used. PCR master mix and cycling conditions as per Merks et al. (2025).

The SAG2 gene was amplified using a nested PCR approach (Howe et al., 1997) that separately amplified the 5′ and 3′ ends of the locus. The 5'end of the SAG2 locus was amplified using the outer PCR primers, SAG2-F4 and R4, and nested PCR primers, SAG2-F and SAG2-R2. The PCR was performed in a total volume of 50 μL containing 1 × GoTaq® G2 HotStart Green Master Mix, 500 nM of each primer SAG2-F4/SAG-R4, UltraPure™ DNase/RNase-free distilled water, and 5 μL template DNA. Two microliters of the outer PCR amplicon were used as the DNA template for the nested PCR. Reagent concentrations were the same as in the primary PCR reaction, but the primers used were SAG2-F/SAG2-R2. The 3′ end of the SAG2 locus was similarly analyzed with the primers SAG2-F3 and SAG2-R3 for the initial amplification, and SAG2-F2 and SAG2-R for the nested PCR. Amplification protocol for all reactions was modified from Howe et al. (1997) to 95 °C for 4 min followed by 35 cycles of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s, with a final extension step at 72 °C for 10 min and hold at 10 °C.

A positive conventional PCR result was based on the visualization of bands of the correct fragment length (bp) following agarose gel electrophoresis of the nested PCR products. Amplicons of the correct fragment length (bp), were purified using either the QIAquick PCR purification kit or QIAquick Gel Extraction kit (Qiagen, Mississauga, ON, Canada) following manufacturer's instructions.

The purified PCR products were subjected to bi-directional, cycle sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) as recommended by the manufacturer. Amplified sequence products were purified using a Wizard MagneSil green (Promega, Madison, WI, USA) sequencing reaction clean-up system, and capillary electrophoresis was performed on an ABI 3500 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). DNA sequences were assembled, edited and aligned using SeqScape v3 software (Applied Biosystems, Waltham, MA, USA). Resulting consensus sequences were aligned with representative GenBank sequence data from T. gondii GRA6 or SAG2 genes. Nucleotide BLAST analysis (Altschul et al., 1990) was performed on the assembled bi-directional fasta data using megablast with the standard nucleotide collection database.

In silico restriction fragment length polymorphism (RFLP) analysis was performed. Briefly, RFLP enzymes were virtually applied to each of the resulting sequences of the GRA6, 5′SAG2, and 3′SAG2 genes to identify SNPs that would produce distinctive cleavage patterns associated with T. gondii genotypes. Sequences were also compared against available sequences in the ToxoDB database (Harb and Roos, 2020) and GenBank (Clark et al., 2016). Sequences from the GRA6 gene were also aligned by MUSCLE as implemented in MEGA11 software (Kumar et al., 2018) with sequences as suggested by Khan et al. (2011). They were then used to create a phylogenetic tree with the Tamura-3 parameter method (Tamura, 1992), with evolutionary divergences estimated by calculating pairwise distance and bootstrapped at 500 replicates to infer likely genotypes. Typing was focused on phylogenetic analysis of the GRA6 region as it has been shown to be a good gene marker for genotyping T. gondii (Opsteegh et al., 2010; Abedian et al., 2024), and because in silico analysis of GRA6 and SAG2 cannot discriminate between Type-II and Type-12 (Su et al., 2006; Shapiro et al., 2019)

2.3. Molecular detection and species identification of Sarcocystis spp

A second DNA extraction was performed from 200 μL of lysates, using the DNeasy® Blood and Tissue Kit (QIAGEN Group, Germany) following manufacturer instructions. DNA was quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and extractions were stored at −20 °C until assayed using a nested PCR with pan-apicomplexan primers (External Forward DF 5′-TACCGATTGAGTGTTCCGGTG-3’; Internal Forward diF 5′-CGTAACAAGGTTTCCGTAGG-3’; External Reverse DR 5′-GCAATTCACATTGCGTTTCGC-3’; Internal Reverse diR 5′-TTCATCGTTGCGCGAGCCAAG-3′) targeting the Internal Transcribed Spacer 1 (ITS-1) region as per Michaels et al. (2016). Reactions for both external and internal primers involved initial denaturation of 94 °C for 3 min, 35 cycles of 95 °C for 40s, 59.5 °C for 40 s, 72 °C for 90 s, and a final extension of 72 °C for 4 min. Bands at a position of ∼450 bp corresponded to T. gondii and were ignored when testing for Sarcocystis spp. PCR products from bands observed at positions of ∼550 bp and ∼1000 bp were purified using QIAquick® PCR Purification Kit (QIAGEN Group, Germany) and sent for Sanger sequencing to the National Research Council in Saskatoon (Saskatchewan, Canada). Sequences were compared against those available in GenBank (https://blast.ncbi.nlm.nih.gov).

Additionally, the 18S rRNA gene of Sarcocystis spp. was targeted using the primers SarcoForward (5′-CGCAAATTACCCAATCCTGA-3) and SarcoReverse (ATTTCTCATAAGGTGCAGGAG-3′) (Moré et al., 2011) by a conventional PCR as per Hernández-Ortiz et al. (2023). Reactions involved initial denaturation of 95 °C for 4 min, 40 cycles of 94 °C for 40 s, 59 °C for 30 s and 72 °C for 1 min, and a final extension of 72 °C for 6 min.

ITS1 and 18S sequences of Sarcocystis spp. were analyzed for phylogenetic relatedness to published sequences, aligned by MUSCLE as implemented in MEGA11 software (Kumar et al., 2018), and the resulting alignment used to create phylogenetic trees with the Tamura-3 parameter method (Tamura, 1992). Evolutionary divergences were estimated by calculating pairwise distance and bootstrapped at 500 replicates.

2.4. Statistical analysis

Differences in presence/absence of Toxoplasma gondii and Sarcocystis spp. were analyzed with a binomial Generalized Linear Model (GLM) with the logit link function, using R statistical software 4.4.2 (R Core Team, 2024). The model included sex (female, male), age class (juvenile, sub-adult, adult, aged adult), region (location of registered trapline where animals were caught), and month of capture. Model significance was checked by Chi square test (“ANOVA” function), and goodness of fit was determined by deviance reported in the model summary.

3. Results

Of the 89 river otters tested, DNA of T. gondii was detected in 30 (34 %). Infection was biased towards males, with 41 % positive (n = 46) compared to 26 % in females (n = 43) (Table 1). Age was positively correlated to infection, with 25 % (n = 16) of juveniles (under 1 year of age) and 26 % (n = 31) of sub-adults (1 year of age) infected, compared to prime age adults which had a 45 % (n = 33) prevalence (Table 1). However, neither age nor sex were significant predictors of T. gondii infection in the GLM model. There was an average estimate of 48,639 (SD = 820,818, n = 30) and range of 586 to 4,061,167 tachyzoite-equivalents per 100 g of brain tissue. Four animals had over 1 million tachyzoite-equivalents per 100 g of tissue.

Table 1.

Prevalence of Toxoplasma gondii and Sarcocystis spp. in river otters by age class and sex. Number of infected individuals followed by percent of total sample size presented in parentheses. Age in years for each age class provided in parentheses, including juveniles which were individuals less than 1 year of age. Sample size (n) provided for each group. Numbers of individuals co-infected with both T. gondii and Sarcocystis spp. are also included.

Age class and Sex n Toxoplasma gondii positive (%) Sarcocystis spp. positive (%) Co-infections
Juveniles (<1) 16 4 (25) 1 (6) 0 (0)
Sub-adults (1) 31 8 (26) 10 (32) 3 (10)
Adults (2–5) 33 15 (45) 13 (39) 2 (6)
Aged Adults (6+) 9 3 (33) 3 (33) 1 (11)
Males 46 19 (41) 14 (30) 4 (8)
Females 43 11 (26) 13 (30) 2 (5)
Total 89 30 (35) 27 (30) 6 (7)
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Toxoplasma gondii was identified in all sampled regions of the province (Fig. 1), except in an area of central Alberta west of Athabasca where all 5 otters tested were negative. Prevalence was low in a cluster north-west of Slave Lake where only one otter was positive (n = 8). We collected the most samples from the lower Athabasca River basin sub-watershed in the east of the province (Fig. 1), where prevalence was 33 % (n = 63). Neither location (df = 12, p = 0.10) nor month of capture (df = 4, p = 0.14) were significant predictors of detection of T. gondii in our model.

Fig. 1.

Fig. 1

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Distribution of Toxoplasma gondii in river otters in Alberta. Results of magnetic capture real time qPCR for the detection of T. gondii in river otter brains from Alberta. Dots show all sampled otter brains by result of test, whether negative or positive for T. gondii infection, and scaled by the log-transformed number of tachyzoite-equivalents per 100 g of tissue as a measure of parasite intensity in the sample. Overlapping points are spread by jitter (h = 0.2, w = 0.2). Circled area includes all the samples from the Lower Athabasca River basin watershed, which was the largest sampling group from a single watershed.

Of the 89 river otters tested, DNA of Sarcocystis spp. was detected in 27 (30 %). Prevalence was identical between males (30 %, n = 46), and females (30 %, n = 43) and lower in juveniles (6 %, n = 16) compared to adults (Table 1). Sarcocystis spp. was also detected across the province except in two groups of animals collected east of Fort McMurray (n = 4) and in the center-north of the province (n = 3). In the Lower Athabasca River basin watershed, where most animals came from, prevalence was 30 % (n = 63). Again, no factors were significant in the model, although age was nearly significant (df = 3, p = 0.07).

3.1. Putative genotypes and phylogenetic analysis

Genotyping assigned on the basis of the GRA6 and SAG2 genes of T. gondii showed a mixture of clonal lineages, including the three most common in North America (Type I, II, and III) as well as Type-12 (also known as X or A) and potentially atypical infections (Khan et al., 2011) (Fig. 2; Table S1). Of the 24 T. gondii positives investigated, 14 sequences of the GRA6 region were obtained (accession nos. PQ625477 to PQ625491), and genotypes according to phylogenetic analysis of GRA6 included Type-I (3/14), Type-II (2/14), Type-12 (6/14), Type-III (3/14), and Type-III atypical (1/14) (Fig. 2).

Fig. 2.

Fig. 2

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Toxoplasma gondii genotype by GRA6 gene. Sequences of the GRA6 gene from river otter in Alberta (by individual ID number, denoted by prefix “RIOT” and bolded), compared against those available in GenBank (accession numbers) from North America, including their listed strain type. Groupings of genotype are provided according to the outline provided by Khan et al. (2011), with the tree rooted by sequences from South America. The evolutionary history was inferred using Maximum Likelihood and Tamura 3-parameter model (Tamura, 1992), bootstrapped at 500 replicates. The tree with the highest log likelihood (−1178.65) is shown. The percentage of trees in which the associated taxa clustered together is shown below the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura 3 parameter model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021).

For the 27 ITS1 sequences from Sarcocystis spp. (accession no. PQ605182, PV253212 to PV253232), one had 99.6 % identify with S. lutrae (accession no. PQ605181), and clustered strongly with other examples of S. lutrae (Fig. S1). Sequences from 26 animals matched best with S. kitikmeotensis, but generally below 90 % identity. In the phylogenetic tree, our samples clustered together separately from the single example of S. kitikmeotensis in 99 % of bootstrap replicates (Fig. S1). Only 3 sequences from the 18S region were obtained (accession nos. PV247947 to PV247949), which matched best to an unnamed species of Sarcocystis spp. (96.43 % identity) collected from a two-toed sloth in China, but clustered separately from the sloth sequence in the phylogenetic tree (Fig. S2). Unfortunately, there is only one partial 18S sequence available for S. kitikmeotensis, which did not overlap with our sequences for comparison.

4. Discussion

4.1. Toxoplasma gondii infection

This is the first study reporting detection of DNA of Toxoplasma gondii in river otters from Alberta, and one of only a few studies reporting infection in Lontra canadensis. We observed a prevalence of 34 % (n = 89), which is higher than previous studies based on detection of DNA of T. gondii in tissues; 24 % (n = 132) from brain tissue in North Carolina (Sanders et al., 2020), 28 % (n = 124) from muscle tissue in Michigan (Cotey et al., 2022), and as is often the case, somewhat lower than serological studies from Washington and Alaska, 38 % (n = 40) (Gaydos et al., 2007), and North Carolina, 45 % (n = 103) (Tocidlowski et al., 1997). Seroprevalence for T. gondii (which detects long lived antibodies in blood) tends to be higher than prevalence based on detection of DNA in tissues because cysts may be unevenly distributed in tissues, the amount of DNA may fall below detection limits, or animals may clear tissue infection while retaining antibodies (Bachand et al., 2019). Serological sensitivities may vary between field and laboratory methods and by species (Huertas-López et al., 2024).

Reports of prevalence vary among terrestrial carnivores in North America. By molecular identification in heart and brain tissue, 24 % (n = 68) of wolverines (Gulo gulo) from the Yukon were positive (Sharma et al., 2019a). By seroprevalence, 62 % (n = 127) of wolverines from the Northwest Territories (Sharma et al., 2019b), 41 % (n = 39) of red foxes (Vulpes vulpes) from the Nunavik Region of Quebec (Bachand et al., 2018), and 100 % (n = 38) of fishers (Martes pennanti) from Pennsylvania (Larkin et al., 2011) were positive for T. gondii. In Alaska, seroprevalence has been reported to be 9–18 % in wolves (Canis lupus), 15–43 % in black bears (Ursus americanus), and 18–25 % in grizzly bears (Ursus arctos) (Elmore et al., 2012). Seroprevalence in sea otters (Enhydra lutris) varies widely; as low as 0 % (n = 65) and 36 % (n = 80) in live-caught animals from Alaska and California (Miller et al., 2002), and as high as 93 % (n = 70) in opportunistically collected animals in Washington (Verma et al., 2018). This variability may be affected by sampling bias as opportunistically collected carcasses may be biased toward diseased individuals, compared to cross-sectional studies with random sampling (Shanebeck et al., 2022). Differences between molecular identification and seroprevalence, methodological biases, and patchy coverage between regions all limit our understanding of the geographic range and prevalence of T. gondii in wild populations.

In the current study, adult river otters had higher T. gondii prevalence than juveniles, and males were twice as likely to be infected as females, a pattern documented previously (Cotey et al., 2022). This may be due to the propensity of males to roam further than females, consume more terrestrial prey, or higher quantities of prey in general, which could increase their exposure to the parasite. Females tend to range less, especially when caring for young (Larivière and Walton, 1998; Blundell et al., 2000). However, Sanders et al. (2020) saw the opposite trend in L. canadensis in North Carolina, with prevalence among females 34 % (n = 76) compared to 19 % (n = 144) in males, and Tocidlowski et al. (1997) saw no difference in seroprevalence, also in North Carolina. Sex was not a significant predictor of infection in our study, which may be due to the opportunistic nature of sampling, which included multiple regions of the province, sometimes in small sample sizes.

Fecal contamination from wild felids is the ultimate source of infection by T. gondii. The Canada lynx (Lynx canadensis) is a confirmed host in Western Canada (Bouchard et al., 2023), and lynx are found throughout Alberta (Campbell and Strobeck, 2006). Mountain lions are also definitive hosts for T. gondii. However, they are mostly found in the Rocky Mountains and its foothills in Alberta (Knopff et al., 2013) and have limited overlap with our sample regions. River otters have a broad diet that varies by habitat, whether lake or river/stream. In Alberta, their diet is predominantly fish, making up as much as 91.1 % in stream and 78.9 % in lake habitats (Gilbert and Nancekivell, 1982). River otters also consume mammals, predominantly muskrat (Ondatra zibethicus) in lake habitats and snowshoe hare (Lepus americanus) in stream habitats (Gilbert and Nancekivell, 1982). Both muskrat and snowshoe hare are intermediate hosts of T. gondii and may be a source of infection (Ganoe et al., 2020; Bouchard et al., 2023). Fish may mechanically transport oocysts of T. gondii through filtering and concentrating oocysts in their gills, but natural infection has not been demonstrated (Moratal et al., 2020). DNA of T. gondii was recently detected in brain and heart of Arctic char (Salvelinus alpinus) in Canada, but this requires follow-up investigation (Merks et al., 2024).

Connections between agricultural land and freshwater contamination with oocysts of T. gondii include runoff from changes in soil structure and landscape, or from a higher likelihood of feral cats on farms (Yan et al., 2016; Cotey et al., 2022). However, most of our infected animals came from the lower Athabasca sub-watershed, where agriculture is limited. Toxoplasma gondii has also been detected in migratory geese in Canada (Bachand et al., 2019), and migratory wildlife may also be a source of infection. Diverse contamination origins could explain our observation of multiple clonal lineages, potentially acquired from more temperate regions, rather than highly diverse, “atypical” lineages in regions with high endemic transmission through the sexual cycle in felids. More research is needed to understand the effects of T. gondii infection in river otters and the prevalence of infection in wildlife hosts in Canada.

4.2. Sarcocystis spp. infection

DNA of Sarcocystis spp. was detected in 30 % of river otters, with one isolate identified as S. lutrae and the other 26 most closely related to, but likely a distinct species from, S. kitikmeotensis, previously described in another mustelid (wolverine) in northern Canada. Information about Sarcocystis spp. in L. canadensis in North America is limited to two reports of prevalence. Our study found similar tissue prevalence to a report from Michigan (29 %, n = 147) using DNA sequencing of muscle tissue, only identified as Sarcocystis spp. (Scimeca et al., 2020). Seroprevalence was higher in a study from Washington and Alaska where 72 % (n = 40) of coastal marine foraging river otters were exposed to S. neurona (Gaydos et al., 2007). Prevalence in river otters in our study was lower than in some other mustelid species, such as the 80 % prevalence in wolverine (Gulo gulo) based on muscle tissue from Canada (Dubey et al., 2010) and 83 % (n = 38) seroprevalence in fisher (Martes pennanti) from Pennsylvania (Larkin et al., 2011). This may reflect the broader diet and higher trophic status of these larger and more aggressive predators and scavengers. Finally, it is important to note that river otters in the current study are serving as intermediate hosts for Sarcocystis spp., and further work is needed to determine the definitive hosts. The definitive host for S. lutrae in Europe is thought to be a bird of prey (Gjerde and Schulze, 2014), and for S. kitikmeotensis, the possibility of wolverines serving as both definitive and intermediate hosts has been proposed, with transmission through cannibalism (Dubey et al., 2010).

4.3. Implications for river otter health

While T. gondii can encyst in various tissues, location in the brain is associated with clinical toxoplasmosis and likely represents more serious cases of infection (Hill et al., 2005; Sanders et al., 2020). Similarly, Sarcocystis species tends to be more pathogenic in neural tissue (Miller et al., 2010; Dubey et al., 2016). Infection with T. gondii in sea otters can be associated with meningitis and myocarditis (Miller et al., 2004), and toxoplasmosis the cause of death in a high proportion of cases, especially related to Type-12 (Miller et al., 2004; Shapiro et al., 2019). In sea otters, both Type-II and Type-12 have been associated with meningoencephalitis and death (Miller et al., 2011). Type-II may also be pathogenic in the Eurasian otter (Lutra lutra) (Viscardi et al., 2022). We detected both types in river otters in the current study. The clinical and pathological significance of these findings are unknown, as samples were not suitable for histopathology. Analysis of genotypes was based on only a few markers, which limits definitive conclusions, although GRA6 is generally considered a suitable marker (Abedian et al., 2024). Future work to fully analyze genotypes present in river otter is essential, especially considering the potentially new genotype identified in RIOT 21–35 (Fig. 2).

Our results indicate that cerebral infections of T. gondii may represent a health threat for river otters in Alberta, especially since 43 % (6/14) of genotyped infections were Type-12. Toxoplasma gondii, especially Type-12, is known to cause fatal encephalitis and stillbirths in their closest relative, the sea otter (Miller et al., 2004; Shapiro et al., 2019), as well as another close relative, the North American mink (Neogale vison) (Pridham and Belcher, 1958). Clinical toxoplasmosis can also be related to concurrent infection with morbilliviruses like canine distemper, which occurs in most species of mustelids, including L. canadensis and N. vison (Kimber and Kollias, 2000), and is widespread in Canadian wildlife (Philippa et al., 2004; Nelson et al., 2012). These viruses can have an immunosuppressive effect in the host, leading to more severe manifestations caused by T. gondii (Dubey et al., 2021). Almost all sequences of Sarcocystis from otters in the current study were of an unknown species, which to the best of our knowledge has not been previously documented. If this species frequently replicates in neural tissue like S. neurona, it may be of significant concern as well. Future research into the effects of T. gondii and Sarcocystis spp. in river otters in Western Canada is essential, including histopathology for evidence of inflammatory reactions and recovery of sarcocysts for further morphological and molecular characterization.

4.4. Implications for human communities

Toxoplasmosis is a global issue, affecting people of all backgrounds, and may account for a significant amount of unexplained disease burden worldwide (Flegr et al., 2014). Detection of T. gondii DNA in river otters does not pose a direct threat to human health as river otters are generally trapped for fur and not consumed by people. As well, to the best of our knowledge, there are no zoonotic species of Sarcocystis present in wildlife in Canada. However, as a sentinel species, the relatively high prevalence and diversity of T. gondii in river otters, detected across Alberta, is indicative of significant environmental contamination. This is a public health risk, especially for First Nation communities that subsist on wild sources of food and have poor access to clean water (Elmore et al., 2012). Studies have shown high seroprevalence in Alaskan Native communities and First Nation woman in Nunavik, Quebec (Elmore et al., 2012), however we could find no information from Alberta. Future research is needed to investigate the potential threat of T. gondii infections for Albertans, especially First Nation communities.

CRediT authorship contribution statement

Kyle M. Shanebeck: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Adrián Hernández-Ortiz: Methodology, Formal analysis. Emily J. Jenkins: Writing – review & editing, Supervision, Resources. Philippe J. Thomas: Resources, Project administration, Funding acquisition. Brent R. Dixon: Writing – review & editing, Project administration. Harriet Merks: Methodology, Investigation, Formal analysis. Clement Lagrue: Writing – review & editing, Supervision, Conceptualization.

Data availability

Sequences have been deposited in GenBank with the accession numbers PQ625477 to PQ625491 for T gondii GRA6 gene, PQ605181 for Sarcocystis lutrae ITS1, PQ605182 for Sarcocystis spp. ITS 1, PV247947 to PV247949 for Sarcocystis spp. 18S. Further sequences are available upon request.

Declaration of competing interest

None.

Acknowledgements

This research was funded in part by Environment and Climate Change Canada, and the Canada-Alberta Oil Sands Monitoring program (OSM). While this work was funded under the Oil Sands Monitoring Program and is a contribution to the Program, it does not necessarily reflect the position of the Program. Work at the Zoonotic Parasite Research Unit was also funded by an NSERC Discovery Grant held by Emily Jenkins.

We would like to thank and acknowledge the Indigenous communities and Métis Nations, whose traditional lands provided the river otters used for our study. Many thanks to the Alberta Trappers Association for logistical support in the collection of the animals, and the individual trappers who submitted carcasses for this study. Thank you to our undergraduate assistant Sydney Storvold who was an invaluable help during brain collection.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijppaw.2025.101069.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (136.8KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.docx (136.8KB, docx)

Data Availability Statement

Sequences have been deposited in GenBank with the accession numbers PQ625477 to PQ625491 for T gondii GRA6 gene, PQ605181 for Sarcocystis lutrae ITS1, PQ605182 for Sarcocystis spp. ITS 1, PV247947 to PV247949 for Sarcocystis spp. 18S. Further sequences are available upon request.

Articles from International Journal for Parasitology: Parasites and Wildlife are provided here courtesy of Elsevier

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