Toxoplasma gondii is a ubiquitous apicomplexan protozoal parasite of significant importance to human and animal health. Wild and domestic felids are the only definitive hosts, and asymptomatic cats can shed millions of environmentally resistant oocysts in their feces (Dubey and Frenkel, 1972; Fritz et al., 2012). Under optimal conditions, sporulated oocysts can remain viable for months (Dumètre and Dardé, 2003), and are infectious to all warm-blooded vertebrates (Tenter et al., 2000). Toxoplasma gondii infection can occur via consumption of food or water contaminated with sporulated oocysts, ingestion of tissue cysts in intermediate hosts, and transplacental transmission (Tenter et al., 2000).

In intermediate hosts, T. gondii tachyzoites spread systemically, often causing subclinical infection in healthy animals and humans. However, fatal neurological disease can occur, particularly in immunosuppressed individuals. Miscarriage, abortion, and congenital toxoplasmosis are also possible. For individuals surviving initial infection, T. gondii tachyzoites respond to the host immune response by converting to bradyzoite-filled tissue cysts in the central nervous system, muscles, and other tissues. If immunity wanes, quiescent tissue cysts can re-activate, again causing systemic disease (Dellacasa-Lindberg et al., 2007; DaSilva et al., 2010; Miller et al., 2018). These diverse transmission strategies and broad host range, coupled with substantial risk to human and animal health, make T. gondii one of the world’s most globally important pathogens.

Despite being restricted to terrestrial definitive hosts, T. gondii can cause morbidity and mortality in diverse marine mammal intermediate hosts, including phocids, otariids, cetaceans, sirenians, and sea otters (Miller et al., 2018). Southern sea otters (Enhydra lutris nereis) are especially impacted, with T. gondii infecting 62% of sea otters and serving as the primary or contributing cause of death for 8% of otters examined from 1998 through 2012 (Miller et al., 2020). Contributing factors include: 1) Proximity of sea otter habitat to coastal cities, where land-based runoff driven by rainfall can carry freshwater contaminated by oocyst-laden feline feces to nearshore waters, and 2) High consumption of marine bivalves and snails capable of concentrating oocysts from contaminated water (Miller et al., 2008b; Johnson et al., 2009; Krusor et al., 2015).

Although southern sea otters commonly have chronic sublethal T. gondii infections, fatal toxoplasmosis also occurs (Shapiro et al., 2016; Miller et al., 2020). Sea otters that have died due to toxoplasmosis usually have no pathognomonic gross lesions other than non-specific lymphadenosplenomegaly; histopathology is required to confirm infection and disease (Miller et al., 2018). Protozoal tissue cysts and occasional tachyzoites are most common in the central nervous system (CNS), accompanied by non-suppurative meningoencephalitis (Miller et al., 2018). For most T. gondii-infected sea otters, few parasites are observed microscopically outside of the CNS and occasionally the heart, suggestive of chronic infection (Miller et al., 2018). Death is usually attributed to chronic-active or recrudescent, T. gondii-mediated meningoencephalitis and/or myocarditis (Miller et al., 2020).

The four cases described herein exhibited a strikingly different lesion pattern, characterized by grossly apparent, diffuse, severe steatitis affecting all subcutaneous and internal adipose stores. Histopathology revealed severe inflammation and numerous tissue cysts and tachyzoites in most tissues, apart from the central nervous system, where parasite numbers and inflammation were comparatively mild. Multilocus sequence typing revealed that all otters with severe protozoal steatitis were infected with the same atypical T. gondii strain, sharing 100% identity across all examined loci. This previously described genotype (COUG or TgCgCa1), was isolated from two mountain lions (Puma concolor) during investigation of a large community outbreak of waterborne toxoplasmosis in humans in Canada (Aramini et al., 1999; Dubey et al., 2008; Dubey et al., 2020).

This is the first report of T. gondii-associated fatal steatitis and systemic toxoplasmosis in sea otters, and the first report of the COUG T. gondii genotype infecting any aquatic animal. The cases described here highlight detection of a previously unreported and virulent strain of T. gondii in threatened southern sea otters and underscores the need for detailed studies of T. gondii transmission at the land-sea interface.

This study describes an unusual and severe presentation of toxoplasmosis and steatitis in sea otters infected with a rare T. gondii strain not previously reported in any aquatic animal. Observed lesion and case presentation patterns differed substantially from prior descriptions of toxoplasmosis in sea otters, where cases of acute to subacute, severe, disseminated toxoplasmosis were reported mainly from fetuses and neonates (Miller et al., 2008a; Shapiro et al., 2016). In contrast with the cases described herein, sea otters with fatal toxoplasmosis usually have no gross lesions other than non-specific lymphadenosplenomegaly, and histopathology is required to confirm infection (Miller et al., 2018). Protozoa are typically numerous in the CNS, accompanied by non-suppurative meningoencephalitis, but are relatively rare in other tissues on conventional histopathology (Miller et al., 2018). The most common extra-CNS lesion reported from T. gondii-infected sea otters is protozoal myocarditis, and death is usually attributed to chronic, active meningoencephalitis or myocarditis (Miller et al., 2020). Although microscopic foci of T. gondii-associated steatitis, myositis, pancreatitis, and adrenalitis are occasionally observed, this finding is consistently mild in comparison to the brain and cardiac pathology.

The four cases described herein exhibit a strikingly different lesion pattern, characterized by grossly apparent, diffuse, severe steatitis of all subcutaneous and internal adipose stores.

Affected adipose was yellow-mottled and firm with a nodular appearance. Some cases also exhibited grossly visible pancreatic, pulmonary, adrenal, and cardiac pathology. Histopathology revealed severe inflammation with numerous intralesional protozoa in most tissues, apart from the CNS, where parasite numbers and inflammation were comparatively mild. Severe toxoplasmosis and protozoal steatitis was identified as the primary cause of death in all cases and inflamed tissues were strongly immunopositive, confirming the association of systemic inflammation with T. gondii infection.

These unusual cases also differed from prior reports of fatal toxoplasmosis in sea otters in other ways: In previous studies (Kreuder et al., 2003: Miller et al., 2020), sea otter deaths due to toxoplasmosis were relatively sporadic, with little variation by season or month. In contrast, all otters with fatal toxoplasmosis with steatitis stranded in February and March, which corresponds with the peak rainy season in California when land-sea runoff following rainfall events is highest. Wet season protozoal disease outbreaks have also been reported for S. neurona (Miller et al., 2010; Shapiro et al., 2012), and seasonal land-sea transport of infectious oocysts or sporocysts is considered the most likely route of sea otter infection by both parasites (Miller et al., 2002a; Miller et al., 2008b; Miller et al., 2010).

Affected otters were strongly seropositive for T. gondii IgG (≥1:10,240) and multilocus sequence typing at 13 loci revealed that all animals with this unusual lesion pattern were infected with the same atypical COUG strain of T. gondii (TgCgCa1). Despite decades of studies on the molecular epidemiology of T. gondii in coastal California, including genetic characterization of >140 T. gondii isolates obtained from southern sea otters (Miller et al., 2004; Sundar et al., 2008; VanWormer et al., 2014; Shapiro et al., 2015; Shapiro et al., 2016; Shapiro et al., 2019), this is the first report of the COUG strain in sea otters, or any aquatic species. To ensure that prior protozoal steatitis cases had not been missed, archival necropsy data (>1,000 sea otters from 1997 through 2022) from the California Department of Fish and Wildlife was reviewed for additional cases of diffuse, severe T. gondii-associated steatitis. None were found other than the current four cases, the earliest of which occurred in 2020.

Importantly, the COUG T. gondii strain appears to be capable of causing rapid mortality in ostensibly healthy, prime-aged adult sea otters. Three of the four cases were adult females, while one was an immature male. Possible spatial clustering of cases was also noted within a 26.5 km coastal region in San Luis Obispo County. However, given our small sample size, effects of sex, age, and stranding location cannot be statistically evaluated.

The COUG strain (ToxoDB PCR-RFLP genotype #66) is considered a rare, atypical North American strain circulating in wildlife. Phylogenetically, this strain clusters within the same major clade (D) as the archetypal Type II and atypical Type X strain, but within a distinctly characterized haplotype (11) that it shares with another atypical strain derived from a jaguar (GUY-2004-JAG1) (Lorenzi et al., 2016). The COUG strain was originally isolated via mouse bioassay from fecal samples from two mountain lions collected following epidemiological studies of a large waterborne toxoplasmosis outbreak in humans in British Columbia, Canada that occurred in 1995 (Dubey et al., 2008). One sample was found in the environment, while the second was collected per rectum from a mountain lion that was euthanized in the vicinity of the drinking water reservoir implicated in the outbreak; no information was reported regarding T. gondii-associated pathology in the euthanized mountain lion (Dubey et al., 2008). Fecal shedding of oocysts by mountain lions or other wild felids was postulated to have served as the source of the toxoplasmosis outbreak (Bowie et al., 1997; Aramini et al., 1999). However, because no information was published on genotype(s) of T. gondii in infected humans from the outbreak, there is insufficient evidence linking the COUG strain in sympatric felids to the human waterborne toxoplasmosis event. To our knowledge, the only other animal reported to be infected with a COUG genotype is a feral pig (Sus scrofa) from the Sierra Nevada Mountains in eastern California (Dubey et al., 2020).

Although this unique parasite strain could have been present before 2020, no prior molecular studies of T. gondii infection in sea otters, domestic cats, mountain lions, bobcats, canids, and other animals along the California coast identified any animals infected with the COUG T. gondii strain (Miller et al., 2008b; VanWormer et al., 2013; VanWormer et al., 2014; Shapiro et al., 2019). This knowledge, plus detection of a novel T. gondii-associated lesion pattern with high virulence that coincides temporally with detection of COUG T. gondii infection in stranded sea otters suggests that introduction of this highly pathogenic strain into coastal California wildlife occurred recently.

Geographical movement of T. gondii strains may occur via infected migratory birds, migration of terrestrial wildlife, or anthropogenic translocation of wild animals, livestock, and pets, including domestic cats (Galal et al., 2022). Though isolated previously from wild felids, domestic cats have been shown to contribute substantially more oocysts in coastal California watersheds when compared to bobcats and mountain lions (VanWormer et al., 2013), and cats could be exposed to this wildlife-adapted strain by feeding on wild prey.

It is also unclear at present whether multiple point sources of the COUG T. gondii strain may be spreading into the marine environment along the California coast. Multiple sources of exposure could explain the two geographically separate regions where the four cases described in this study were found. Another possibility is that the immature male was initially infected in the same region as the females but stranded elsewhere due to migration post-infection. Previous studies of tagged sea otters revealed that males may travel hundreds of km (Tinker et al., 2017), with immature males frequently dispersing beyond study areas for extended periods (J. Tomoleoni, pers. comm), suggesting that the immature male could have stranded in a different county than the original location of T. gondii infection. Interestingly, the three adult females stranded near Morro Bay, a confirmed, high-risk area for T. gondii Type X infection and mortality in sea otters (Shapiro et al., 2019). Numerous factors appear to contribute to these high-risk zones, including local geography, precipitation, climate, felid density, and other factors (VanWormer et al., 2013; VanWormer et al., 2014; Burgess et al., 2018; Miller et al., 2020). Given the apparent high virulence and unknown source of this atypical strain, as well as the potential health risks for threatened sea otters, other animals and humans, additional research is needed to clarify the host and geographical distribution of the COUG strain in North America.

This is the first report of severe steatitis associated with toxoplasmosis in sea otters, but similar lesions were reported in Hawaiian monk seals (Neomonachus schauinslandi) with fatal toxoplasmosis (Barbieri et al., 2016). Although the monk seal steatitis lesions appear similar grossly and microscopically, the T. gondii genotype(s) associated with monk seal infection were not reported. Protozoal steatitis was also reported in a red kangaroo and aborted goats with fatal toxoplasmosis (Chen and Alley, 1987; Dubey and Hartley, 1992).

Although T. gondii has been traditionally classified into three archetypal genotypes (Type I, II, and III), more advanced genotyping approaches and global characterization of parasite strains have contributed to much broader recognition of non-archetypal or “atypical” genotypes. Associations between infection by specific T. gondii genotypes and more severe disease have been reported in animals and people (Grigg et al., 2001; Khan et al., 2009; Melo et al., 2013). The relationship between strain genotype and virulence is well established in mouse models, with Type I isolates and most South American strains being highly virulent, whereas Type II and III strains are less virulent (Khan et al., 2009; Melo et al., 2013).

The atypical Type X strains (ToxoDB#5) have been classified within haplotype 12 and are predominantly found in North American wildlife (Dubey et al., 2011; Jiang et al., 2018). Southern sea otters are most frequently infected by Type X or X variant strains, which are more pathogenic in sea otters than Type II strains (Miller et al., 2004; Shapiro et al., 2019). In a study of 135 necropsied southern sea otters, 79% of T. gondii isolates were Type X or Type X variants, while the remaining 21% were Type II or Type II/X recombinants. Additionally, all enrolled otters with T. gondii identified as the primary cause of death were infected with Type X or Type X variants (Shapiro et al., 2019). In contrast, a study of mixed marine mammal species in the Pacific Northwest, including five northern sea otters (E. l. kenyoni) revealed no significant associations between T. gondii genotype and disease outcome (Gibson et al., 2011).

Although no assessment of associated pathology or disease outcome was included in descriptions of the COUG T. gondii strain from mountain lions and a wild pig (Dubey et al., 2008; Dubey et al., 2020), the COUG genotype has been shown to be highly virulent in mice and significantly more virulent than Type II strains (Khan et al., 2009). However, it was unclear if this enhanced virulence was due to a high parasite burden or a strong, parasite-associated inflammatory response. Substantial genetic differences between COUG and Type II T. gondii strains have also been reported (Minot et al., 2012).

Another study revealed that the COUG strain is unusual among T. gondii isolates in eliciting a Type I interferon response in both murine macrophages and human fibroblasts (Melo et al., 2013). Type I interferons (IFN), including IFN-α and -β, are immunomodulatory cytokines that serve as potent coordinators of antimicrobial responses. In addition to their well-recognized role in viral immunity, IFNs can also play important roles during parasitic infection by controlling parasite growth through activation of intracellular killing mechanisms or enhancing disease severity. According to Melo et al. (2013), recent reports that Type I IFNs suppress Type II IFN-triggered human anti-mycobacterial responses (Teles et al., 2013) could suggest that the Type I IFN response induced by COUG and other select atypical T. gondii strains could be associated with enhanced pathogenicity. In addition, Type I IFNs can affect host cellular lipid metabolism by inhibiting de novo cholesterol and fatty-acid synthesis and upregulating the uptake of exogenous lipids (Wu et al., 2016). Although Type I IFNs can induce metabolic changes that are critical for immune function (Wu et al., 2016), these changes could also provide selective advantages for highly lipophilic intracellular protozoa.

The striking tropism of the COUG strain of T. gondii for sea otter adipose tissue is unusual and noteworthy. Not only were parasites numerous throughout systemic adipose stores in association with severe inflammation, but on histopathology numerous parasites were concentrated along the periphery of lipid droplets within the cytoplasm of infected adipocytes ( Figure 7C ). Although T. gondii is known to scavenge host lipids to facilitate parasite replication and survival (Nolan et al., 2017), and host-parasite competition for lipid stores likely contributes to disease pathogenesis (Coppens, 2006), the degree of parasitism within adipose tissue in these cases was extreme when compared with hundreds of previous histologically and molecularly confirmed cases of toxoplasmosis in sea otters.

In addition to the grossly apparent steatitis and pancreatitis reported herein, another fascinating and potentially strain-associated difference for COUG T. gondii-infected sea otters is significant protozoal infection of smooth muscle cells within the uterine myometrium ( Figures 8B–D ) and duodenal tunica muscularis ( Table 2 ). Despite screening sea otter tissues for 24 years, one author (MM) has never observed this previously. Although T. gondii can infect virtually any nucleated cell type in warm-blooded animals and humans, infection of skeletal and cardiac muscle is most commonly reported, and skeletal muscle appears to be a preferred tissue for persistent T. gondii infection (Swierzy et al., 2014). The parasite load and inflammation in the uterine myometrium was particularly severe and could have been associated with fetal infection and abortion, although this was impossible to confirm at gross necropsy. Interestingly, uterine and gastric mural infection was also reported in Hawaiian Monk seals with fatal toxoplasmosis and protozoal steatitis (Barbieri et al., 2016), although the infecting genotype in the described Monk Seal cases have not been reported to date. Toxoplasma gondii-associated smooth muscle necrosis has also been reported in highly susceptible Australian marsupials (Canfield et al., 1990), and HIV-positive humans with protozoal pneumonia and gastritis (Nash et al., 1994; Alpert et al., 1996).

Although co-infection with S. neurona was identified via parasite isolation, immunohistochemistry, or PCR for two cases ( Supplementary Table 1 ), findings from extensive histopathology and immunohistochemistry confirm T. gondii as the primary cause of death in all four cases. All otters for which pericardial fluid was available for serology had high positive titers for T. gondii and low positive titers for S. neurona. Sarcocystis neurona was isolated in culture from the brain of Case 2; however, no S. neurona organisms were seen associated with inflammation in the brain histologically or immunohistochemically. While Case 4 exhibited sparse S. neurona immunopositive staining in heart, adipose and lung, DNA extracted from the brain, subcutaneous fat, omentum, lung, and spleen for this sea otter only yielded amplification at the ITS1 locus for T. gondii. As the parasite load of T. gondii was far greater and was closely associated with pathology in multiple tissues from all examined otters, S. neurona appeared to be incidental or of minimal clinical significance. However, it cannot be ruled out that S. neurona co-infection may have contributed to the severity of observed lesions.

This study had several limitations including a small sample size, varying postmortem duration, and freeze-thaw conditions for examined animals, which can decrease tissue quality and resolution. Additionally, steatitis altered the gross appearance of adipose stores and likely impacted standardized assessment of nutritional condition.

Our findings reveal a novel and concerning lesion pattern for southern sea otters with systemic toxoplasmosis that appears to be associated with an atypical T. gondii strain described in an aquatic animal for the first time. The unique combination of host, pathogen, and environmental factors contributing to this newly recognized manifestation of systemic toxoplasmosis in sea otters is unknown. Future directions should include expanded research on this seemingly highly pathogenic T. gondii strain, such as genomic studies to identify unique virulence factors, or transcriptomic investigations into host lipid metabolism and tropism. Additional studies are needed to evaluate the presence of this strain in a larger sample of coastal terrestrial animals, sea otters, and other aquatic wildlife, including screening animals with subclinical T. gondii infections. Investigating habitat or climate change factors that may have facilitated the spread of this rare atypical strain to coastal California will shed light on how anthropogenic activities can alter T. gondii ecology and epidemiology. The potential for immunosuppression following exposure to occult viruses, toxins, anthropogenic chemicals, and other causes also warrants investigation. Because T. gondii oocysts can be concentrated in invertebrates and transmitted to susceptible hosts via ingestion (Arkush et al., 2003), there is potential for this virulent pathogen to pose a public health risk for people consuming seafood, or ingesting water contaminated with this parasite. Sea otters have served as important sentinels for recognition of the COUG strain circulating in the coastal California terrestrial and marine environment, highlighting the need for ongoing research into spread and possible emergence of diverse T. gondii strains from coastal terrestrial vertebrates to marine ecosystems.

The data presented in the study are deposited in GenBank, accession numbers: OQ249665 (3’SAG2), OQ249666 (5’SAG2), OQ249667 (altSAG2), OQ249668 (Apico), OQ249669 (B1), OQ249670 (BTUB), OQ249671 (C22-8), OQ249672 (C29-2), OQ249673 (GRA6), OQ249674 (L358), OQ249675 (PKI), OQ249676 (SAG1), OQ249677 (SAG3).

In accordance with Section 109(h) of U.S. Marine Mammal Protection Act (MMPA), U.S. Fish and Wildlife Service (Service) regulations implementing the MMPA at 50 CFR 18.22(a), and in accordance with Service regulations implementing the US Endangered Species Act at 50 CFR 17.21(c)(3), samples used to complete this work were collected by an official or employees of the California Department of Fish and Wildlife (CDFW) in the course of their duties as an official or employee of CDFW.

MM: Completed gross necropsies for some enrolled otters; read out all case slides and compiled summary reports; envisioned, coordinated, and oversaw project completion; took gross photos and all photomicrographs; led completion of all summary image plates; substantial contribution to manuscript design, preparation, revision, editing and literature review; designed and contributed Tables 1 , 2 ; Supplementary Figure 2 , and Figures 1 through 8 . CN: Leadership role in manuscript writing, editing, and polishing of case discussion and literature review; helped coordinate and oversee project completion; assisted with gross necropsy for one enrolled otter and transported samples to UCD for diagnostic workup; provided substantial input regarding project design and completion, and manuscript design, preparation, revision, editing and literature review; final formatting and polishing of manuscript to meet publication guidelines. DS: Extracted DNA and performed PCR for all T. gondii genotyping assays for enrolled otters; established and maintained in vitro cultures for one otter and verified culture ID via PCR; assisted with sequence trimming and alignment; assisted with design and construction of Table 2 ; reviewed all histopathology slides and immunohistochemistry; designed and contributed Table 3 and Supplementary Figure 2 ; assistance with literature review, and manuscript writing and editing, especially sections of the discussion related to genotyping and the COUG T. gondii strain. FB: Assisted with gross necropsies, data, and sample collections; coordinated specimen subsampling and shipments, and immunohistochemistry shipments for diagnostic workup; conducted database queries of existing southern sea otter steatitis data; data organization; assisted with manuscript edits and submission. KG: Assisted with gross necropsies, data, and sample collection; coordinated archiving of samples and data to assist completion of summary reports; completed histopathology trimming of 2/4 enrolled otters; assisted with manuscript edits and table completion. AR: Assisted with histopathology trimming of 2/4 enrolled otters; assisted with manuscript edits and completed reformatting and optimization of all image montages for the manuscript. CY: Provided critical field recognition of cases enrolled in the study; assisted with gross necropsies, data, and sample collection; assisted with manuscript edits. Completed gross necropsy on 1 case. MH: Provided critical field recognition of cases enrolled in the study; assisted with gross necropsy for 1 case, data, and sample collection; assisted with manuscript edits. Created Supplementary Figure 1 . AP: Completed all serological testing for T. gondii and S. neurona for three cases and provided expertise to facilitate parasite isolation efforts; assisted with manuscript edits. KS: Supervised and funded parasite tissue isolation and protozoal parasite molecular detection and characterization at UC Davis; analyzed sequence data and interpreted molecular results; substantial assistance with literature review and suggestion of applicable manuscripts on genotyping details; substantial assistance with manuscript writing and editing, especially sections of the discussion related to genotyping and the COUG T. gondii strain. All authors contributed to the article and approved the submitted version.