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All issues Volume 32 (2025) Parasite, 32 (2025) 48 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 48
Number of page(s) 11
DOI https://doi.org/10.1051/parasite/2025045
Published online 01 August 2025

© G. Mao et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Toxoplasma gondii is an intracellular protozoan parasite that infects nearly all warm-blooded animals, including marine mammals [7, 10, 22]. Several studies have demonstrated that T. gondii is present in marine mammal species, including pinnipeds, cetaceans, sea otters, and polar bears [4, 7, 15, 22, 23, 35]. Infection typically occurs through ingestion of T. gondii sporulated oocysts from felines, tissue cysts from the definite host or intermediate host, or vertical transmission through the placenta [7]. Oocysts are the primary source of infection for T. gondii, either through direct ingestion or the ingestion of mechanical carriers such as bivalve mollusks, sardines, and anchovies, all of which can carry and concentrate oocysts [1, 18, 19, 21]. Marine mammals are considered sentinels of T. gondii oocyst pollution in marine environments [3, 5, 15, 20, 35]. Furthermore, vertical transplacental transmission of T. gondii has been described in chronically infected sea otters and Australian fur seals [14, 29]. However, the T. gondii infection status in fur seals is unknown, and the contribution of T. gondii infection to fetal loss is unknown. Therefore, the aim of this study was to explore whether T. gondii was present in the placenta, aborted fetuses, and other available tissues from fur seals.

Materials and methods

Ethics statement

All experiments were approved by the Institutional Animal Use Protocol Committee of Henan Agricultural University, China. All animals were handled in accordance with the Animal Ethics Procedures and Guidelines of China.

Sample collection

A total of 10 northern fur seals (Callorhinus ursinus) from zoos in China were observed from 2012 to 2024. Fur seal case #2 was imported from the United States (Alaska) in 2010, and cases #1 and #3–10 were from domestic breeding in China. All of them were fed frozen, thawed, commercially sourced fish imported from other countries. Samples were collected from these fur seals, including ten fresh placentas, two fresh stillbirths, two fresh pups, one fresh male, and four female fur seals with multiple organs fixed in formalin (Table 1). The samples were sent to the Veterinary Pathology Laboratory of Henan Agricultural University for pathology diagnosis, etiological diagnosis, and the detection of T. gondii. In addition, placental exudates were centrifuged and collected.

Table 1

Background and T. gondii infection in fur seals (Callorhinus ursinus) from China (2012–2024).

Detection of T. gondii antibodies in fur seals

The placental and heart exudates from fur seals were tested for T. gondii antibodies using a modified agglutination assay (MAT) [8]. First, we tested whether the sample was infected with T. gondii by two titers (1:25 and 1:200) on the sample received date. Then, retrospective study was performed on T. gondii positive samples in 2024 (double dilution from 1:16 to endpoint dilution). The MAT antigen was obtained from the University of Tennessee Research Foundation (Knoxville, TN, USA). Positive (VEG-infected mice) and negative (T. gondii-free mice) control serum samples were used in each 96-well U plate. The MAT cut-off titer was 1:25 [7, 20].

Pathology analysis

Fur seal tissue samples were fixed in formalin. Paraffin sections were prepared using conventional techniques, hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) [36]. The primary antibody used was a rabbit anti-T. gondii polyclonal antibody. Anti-rabbit IgG conjugated with Horseradish Peroxidase (HRP)/3,3'-Diaminobenzidine (DAB) (IHC detection kit, ab64261, Abcam, Cambridge, United Kingdom) was used as the secondary antibody. Placental tissue sections of sheep infected with T. gondii VEG strain (provided by Dr. Dubey) were used as positive controls for IHC staining. Toxoplasma gondii-free mice brains were used as negative controls. The primary antibody was diluted to 1:3,000 and incubated overnight at 4 °C. The secondary antibody was treated at 37 °C for 15 min. The signal was amplified with streptavidin-peroxidase and then visualized with DAB under a microscope. Finally, it was counterstained with hematoxylin, dehydrated, and mounted in neutral balsam.

DNA extraction from fur seal tissues and detection of T. gondii nucleic acid by PCR

Genomic DNA was extracted from the placenta, heart, liver, spleen, lungs, kidneys, and other tissue samples of the fur seals using a commercial kit (Tiangen Biotech, DP304, Beijing, China). Toxoplasma gondii-specific primers TOX5 and TOX8 were used to detect T. gondii nucleic acids [28, 37]. The PCR reaction system was 25 μL, which contained 12.5 μL of 2× PCR Mix (GDSBio, Guangzhou, China), 2 μL of primers (with a primer concentration of 0.2 μM), 2 μL of target DNA, and 8.5 μL of ddH2O. The PCR reaction involved initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min, extension at 72 °C for 1 min, for a total of 35 cycles, and finally extension at 72 °C for 10 min. The PCR product was approximately 450 bp, which indicated the presence of T. gondii pathogen nucleic acid in tissues. During DNA extraction and PCR amplification, a negative control (commercial negative samples from a T. gondii nucleic acid detection kit, Product ID: 25T, Shanghai Yan Qi Biological Technology Company, Shanghai, China) and a positive control (brain tissues from Me49 strain-infected mice) were included for each experimental batch.

Isolation of viable T. gondii from tissues of fur seals using mouse bioassay

Two methods were used to process fur seal tissue for mouse bioassay. One was taking approximately 50 g of fur seal tissue and digesting it with hog pepsin, and the other was taking approximately 5 g of fur seal tissue and grinding it thoroughly [7]. Tissue fluid was inoculated subcutaneously into Swiss mice (n = 2−5) and/or interferon gamma knockout mice (IFN-γ−/−, n = 1). The mice were observed and recorded (appetite, activity level) every day. Toxoplasma gondii parasites were detected in the brain and lungs of the dead mice. The blood of the surviving mice was collected at 30 days and 60 days post-inoculation (DPI), and their T. gondii infection status was determined via MAT (titer: 1:25, 1:200). If T. gondii tachyzoites, cysts, or antibodies were observed, the brain, heart, spleen, and other tissues were ground and inoculated into a new group of mice to preserve and isolate the viable strain.

Cell culture, genotyping, and morphological observation of T. gondii from fur seals

The tissue homogenates (brain, heart, lungs, spleen, and membranous lymph nodes) of T. gondii-positive mice were inoculated into Vero cells, and the fluid was changed twice per week. DNA was extracted and collected from T. gondii tachyzoites using commercial DNA kits, and the genotypes of T. gondii were determined using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) with 10 genetic markers [32]. Toxoplasma gondii virulence factors were identified by genotyping ROP5 and ROP18 polymorphisms [31]. Toxoplasma gondii reference DNA (n = 8) was included in the batches.

When numerous parasitophorous vacuoles were observed, a glutaral-paraformaldehyde mixture was added to the cell culture flask for pre-fixation, after which the cells were scraped off with a cell scraper and centrifuged, with the supernatant being discarded. The glutaral-paraformaldehyde mixture was then added to the precipitate for fixation, and the samples were sent to the Henan University of Chinese Medicine for transmission electron microscope (TEM) section production.

Evaluation of the virulence of T. gondii isolated from fur seals using Swiss mice

Toxoplasma gondii tachyzoites were collected from the cell culture, counted, and diluted 10-fold to 104, 103, 102, 101, 100, and <1 per mL. Following this, the diluted tachyzoites were inoculated intraperitoneally into five Swiss mice in each group, and the mice were checked daily [26]. At 30 and 48 DPI, the blood of mice was collected and tested for T. gondii IgG antibodies using MAT with titers between 1:25 and 1:200. This study could not be conducted over a longer period because of the COVID-19 epidemic in China, during which the laboratory was closed (48 DPI: 9 November 2022). The mice were euthanized at 48 DPI, and T. gondii cysts in the brain were counted [9]. In brief, the whole mouse brain was homogenized with 1 mL of saline (0.85% NaCl) and tissue cysts were counted microscopically in 50 μL homogenate, and the count was multiplied by 20 to obtain the number of tissue cysts per brain. Two replicates were performed for each mouse brain. Tissue samples from mice infected with T. gondii were fixed in formalin. Virulence was assessed based on the percentage of dead mice among the T. gondii-infected mice [26].

Statistical analysis

Data were expressed as the mean ± SEM using the GraphPad Prism 8.0, followed by one-way analysis of variance to test whether there were significant differences, with p < 0.05 considered statistically significant.

Results

The cause of death and T. gondii infection in fur seals

The background information, clinical symptoms, pathological changes, and T. gondii infection status of the fur seals (n = 10) are summarized in Table 1. The fur seal placenta was found zonary and belt-shaped (Fig. 1). Large quantities of hematoidin crystals (yellow rhombic plates, bilirubin) were observed in the intervillous space of the chorioallantoic membrane (Figure S1).

thumbnail Figure 1

Morphology of the placenta and samples from fur seal #2. A: Stillborn and placental tissue in 2017. B: Placenta in 2022. C: Zonary and belt-shaped placenta, dam, and its pup. D: Magnified C: surface of the chorioallantoic membrane.

Three fur seals (cases #2, #3, and #4) had no clinical abnormality and were alive. In the seven fur seals (cases #1, #5, #6, #7, #8, #9, and #10) that underwent detailed pathological autopsy and histopathological examination, T. gondii infection was not considered the primary cause of death, but it may have contributed to the death of one fur seal (case #7). Among them, one male (case #1) had died of hydropenia (kept out of water cages for 6 days), two pups (case #9 and the pup of case #4, both 2 months old) died of diarrhea, and four adult females died of the following: Mycobacterium tuberculosis infection (case #7), vitamin A deficiency (case #6), pleuropneumonia (case #10), and multiple tumors (case #5). Based on detailed T. gondii parasite examinations using PCR, MAT, and IHC data, five cases (cases #2, #3, #4, #7, and #8) were exposed to this parasite from the ten cases. However, three cases (cases #2, #7, and #8) resulted in stillbirths, while the other cases (cases #3 and #4) had viable fetuses.

Fur seal case #1, which was male, was placed into a cage for six days after it fought with the other fur seals, where it died of hydropenia. Microscopically, necrotizing myocarditis, hepatic steatosis, and gastroenteritis catarrhalis were observed.

Fur seal case #2 had a history of abnormal reproduction every other year from 2012 to 2015, and a follow-up study was performed. A total of five placentas (2016, 2017, 2018, 2022, and 2023) from case #2 were collected. In 2017, fur seal case #2 gave birth to a stillborn fetus in the third trimester of pregnancy. However, there was clear grey necrosis at the end of the fin of the stillborn fetus (Fig. 1). Microscopically, many blood vessel calcifications were found to have occurred in the placenta, and it was speculated that the blood vessels were blocked, resulting in anemia at the end of the fetal limb.

The mother of fur seal case #3 (born 2012) was fur seal case #2. Fur seal case #3 had its firstborn in 2016, and then birthed a second pup in 2019; it had no history of abortion.

Fur seal case #4 gave birth to a baby that died at 66 days of age in 2023. Pathology examination revealed suppurative bronchial pneumonia, hepatic and renal insufficiency, hemorrhagic necrotizing splenitis, and necrotizing enteritis.

Fur seal case #5 showed pulmonary sarcoma, liver insufficiency, thrombosis in the liver, spleen, kidney, and intestines, as well as uterine leiomyosarcoma, necrotizing adrenal inflammation, necrotizing thyroiditis, and necrotizing myocarditis. It had an abortion in 2022.

Arteriosclerosis in multiple organs (the uterus, spleen, kidney, stomach, and intestine) was observed in fur seal case #6 based on pathology. Furthermore, histiocytoma in the kidney, uterine fibroids, and lesions in the mucosal epithelium of the pulmonary airway system (vitamin A deficiency) were observed in case #6.

Fur seal case #7 showed multi-organ infectious granuloma (heart, lung, liver, intestine, spleen, uterus, stomach, and lymph nodes) and tested positive for acid-fast staining, which indicated Mycobacterium tuberculosis infection, confirmed by PCR and sequencing. It had a history of abortion, and the stillborn fetus was seropositive for T. gondii.

Fur seal case #8 showed placental focal necrosis, fetal cardiac insufficiency, and acute liver injury. Additionally, T. gondii was detected by MAT, PCR, IHC in the placental tissue of case #8, and T. gondii nucleic acid was detected in the heart, liver, and lungs of its stillborn fetus by PCR.

The pup of fur seal case #9 showed acute suppurative pneumonia, hypoproteinemia, acute enteritis, and multiple organ hypoplasia.

Fur seal case #10 exhibited fibrinous pleuropneumonia.

Detection of antibodies and pathogen T. gondii in fur seals

In this study, ten zoo-housed fur seals were tracked and monitored for their reproduction and T. gondii infection status. Among them, six of nine (67%, 95%CI: 35.09%–88.27%) females (case #2, #4, #5, #7, #8, and #9) had a history of poor reproduction. Based on their serology or etiology, five fur seals (case #2, #3, #4, #7, and #8; 50%, 95%CI: 23.66%–76.34%, 5/10) showed evidence of exposure to T. gondii.

Ten placental exudates, two heart exudates, and three fetal exudates from six adult fur seals (cases #1, #2, #3, #4, #7, and #8) were tested, and seven samples (50%, 95%CI: 26.80%–73.20%, 7/14) showed the presence of T. gondii antibodies using MAT; these samples belonged to four fur seals (cases #2, #3, #7, and #8). The exudates from four placentas of fur seal case #2 tested positive for anti-T. gondii antibodies, with titers of 1:128 (2015), 1:64 (2017), 1:256 (2018), and 1:2048 (2022). However, this fur seal produced three healthy fetuses and only one stillborn fetus in 2017. In 2023, the anti-T. gondii antibody titer in the placental exudate of fur seal case #2 was below 1:25. In 2012, fur seal case #2 gave birth to a healthy female fur seal, named fur seal case #3. The placental exudate of case #3 tested positive for T. gondii antibodies with titers of 1:256 (2019) and 1:64 (2025). However, it delivered four healthy fetuses in 2016, 2019, 2024, and 2025.

Eight placentas, multiple organs from seven fur seals, and two fetal tissues were examined for T. gondii antigens using IHC, and T. gondii antibody positive reactions were observed in five samples (four placentas and a lung) (29%, 95%CI: 12.99%–53.43%, 5/17); these samples belonged to four fur seals (cases #2, #4, #7, and #8) (Fig. 2). Toxoplasma gondii-like bradyzoites were mainly distributed in the vascular endothelial cells of placental trophoblasts.

thumbnail Figure 2

Morphology of T. gondii in the tissues of the fur seals. A–D: T. gondii antibody positive reaction (trigon) in the placentas of fur seal #2. A and C show IHC staining, while B (P#3445) and D (P#3559) show HE staining of serial sections A and C, respectively. E, F: T. gondii antibody positive reaction (trigon) in placentas of fur seal #4. E shows IHC staining, and F (P#3568) shows HE staining of the E serial section. G: T. gondii antibody positive reaction in the lung of fur seal #7 (P#3814) shown by IHC staining (trigon). H: T. gondii antibody positive reaction in the placenta of fur seal #8 (P#3841) shown by IHC staining (trigon). Bar = 50 μm.

Among the ten placentas, multiple organs from two fur seals, and two fetal tissue samples, 21% T. gondii nucleic acid (95%CI: 6.84%–48.32%, 3/14) was detected by PCR in the placental tissue of fur seal case #2 in 2022, as well as in the placenta and stillborn fetal heart, liver, and lung of case #8 (Table 1).

Isolation, genotyping, and transmission electron microscopy of T. gondii

Eight placental tissue homogenates (2016–2024) from four fur seals (cases #2, #3, #4, and #8) were inoculated into Swiss mice or IFN-γ–/– mice (Table 1). Toxoplasma gondii antibodies (MAT titer was ≥1:200) were detected in Swiss mouse M#43 in the Tox#12-9 group (inoculated placenta obtained in 2022 from case #2) at 30 DPI, and many cysts (n = 1,180) were found in the brain on 54 DPI. The mouse brain T. gondii cysts were successfully propagated in cell culture (19 DPI) and were named TgFurSealCHn1. Abundant electron-dense granules (3.74 ± 0.55, n = 62) and amylopectin granules (2.63 ± 0.33, n = 62) were observed in T. gondii tachyzoites through transmission electron microscopy (Fig. 3).

thumbnail Figure 3

Morphology of tachyzoites of TgFurSealCHn1 in cell culture under transmission electron microscopy. A: A group of TgFurSealCHn1 tachyzoites within the parasitophorous vacuolar membrane. B: Magnified image of A. Am: amylopectin granule, Co: conoid, Dg: electron-dense granule, Lb: lipid, Mn: microneme, Nu: nuclei of daughter tachyzoites, Pm: parasitophorous vacuolar membrane, Pv: parasitophorous vacuole, Rh: rhoptry. Bar = 2 μm.

The genotype of TgFurSealCHn1 was ToxoDB #5, which was determined using PCR-RFLP with 10 markers. In addition, the allele types of ROP18 and ROP5 genes were 2/2 (Fig. 4, Table 2).

thumbnail Figure 4

Genotyping of T. gondii TgFurSealCHn1 strain isolated from fur seals. 1: GT1, 2: PTG, 3: CTG, 4: TgCgCal, 5: MAS, 6: TgCatBr5, 7: TgCatBr64, 8: TgToucan (TgRsCr1), 9: TgFurSealCHn1, and M: Marker.

Table 2

Genotypes of T. gondii isolates from fur seal according to PCR-RFLP of 10 markers and virulence proteins.

Other fur seal placentas (cases #3, #4, and #8) failed to isolate viable T. gondii.

Evaluation of the virulence of the TgFurSealCHn1 strain in Swiss mice

After inoculation with tachyzoites from different gradients of TgFurSealCHn1, the survival times of Swiss mice infected with T. gondii were recorded (Table 3). Ten tachyzoites infected 100% of the mice (MAT titer, ≥1:200). Under 48 days observation period, the survival rates of mice were 20%, 80%, 100%, and 100% for the 104, 103, 102, and 10 tachyzoites, respectively. Cumulative mouse mortality was 7% after infection of 10 – 103 tachyzoites. The number of cysts in the mouse brains was examined, and there was no significant difference among the different tachyzoite gradient groups at 48 DPI (p > 0.05).

Table 3

Evaluation of the virulence of T. gondii TgFurSealCHn1 strain in Swiss mice (M ± SE).

Discussion

In the present study, T. gondii was found to be highly infectious in captive fur seals (50%), consistent with reported infection patterns in other marine animals [7, 20]. Reported exposure rates are 25.9% for sea otters (IFA cut-off 1:320) [2], 86.3% for dolphins (MAT cut-off 1:25) [27], and 32.8% for sea lions (IFA cut-off 1:40) [3]. Notably, T. gondii was detected in 7.5% of third trimester abortions in Australian fur seals [14]. Here, the infection rate of T. gondii in fur seals was higher than that of food animals of local origin (p < 0.05) [6].

Direct evidence of T. gondii infection in fur seal was observed by isolation of viable strain and was named TgFurSealCHn1. A viable strain of T. gondii was successfully isolated from fur seal case #2 placenta (2022) using a mouse biological method. Case #2 already had anti-T. gondii antibody in 2016 (1:128), and the placental exudate showed a T. gondii titer of 1:2,048 in 2022; T. gondii-like parasites and nucleic acids were also detected in placental tissue in 2022 by IHC and PCR. However, the puppy showed no abnormalities and survival. These results indicate that endogenous T. gondii vertical transmission during pregnancy or exogenous oocysts reinfection occurred in 2022. The former phenomenon has been reported in sheep [7, 16]; however, there are only a few reports on marine mammals. Furthermore, the serological reaction from the placental exudate of case #2 was inconsistent from 2016 to 2023. MAT can be used to detect IgG antibodies, with a continued increase in antibody levels, indicating the activation and proliferation of T. gondii, and decreased antibody levels indicating that the parasites may have been eliminated [8]. This phenomenon of fluctuating T. gondii antibody levels was also observed by Martins et al. in fur seals by MAT [20]. Interestingly, case #2 was serologically negative in placental exudate in 2023, yet HE and IHC staining still detected T. gondii-like parasites in the placenta. Cattle have shown transient antibody responses to T. gondii infection because they developed an effective immune response that may facilitate T. gondii elimination [11, 12]. Unlike other animals, which exhibit a sustained antibody response, the fur seal could have eliminated T. gondii and shown serologically negative results. Whether fur seals have a unique immune response to T. gondii infection (like that in cattle) needs to be further explored. Fur seals case #2 and case #4 had T. gondii-like parasites in the placenta in 2023; however, the serological reaction was negative. This phenomenon was associated with lower antibody titers in placental tissue exudate than in serum, or it could indicate that case #2 and case #4 were at the stage of acute T. gondii infection. Serum samples from these animals should be collected for further T. gondii confirmation, if possible. The T. gondii antibody in the placental exudate of fur seal case #3 was lower than 1:25 in 2016, but increased to 1:256 in 2019. This means that case #3 was postnatally exposed to T. gondii and indicates that cat T. gondii oocysts may contaminate the food or environment of the zoo. Oocysts can survive even at −20 °C for 28 days, indicating that standard freezing conditions are insufficient to eliminate T. gondii infectivity in frozen fish products [13].

Genotyping of TgFurSealCHn1 suggested potential T. gondii transmission via imported frozen fish or fur seal. The TgFurSealCHn1 was identified as ToxoDB PCR-RFLP genotype #5. ToxoDB #5 (along with ToxoDB #4 and #5, collectively forming haplotype 12) was the dominant type in wildlife from North America, including marine mammals (e.g., sea otters), but rarely in animals from the rest of the world and domestic animals [7, 10]. Studies have shown that different genotypes of T. gondii have important relationships with geographic regions [7]. The epidemic genotype of T. gondii is ToxoDB #9 in China, and only one strain of the ToxoDB#5 genotype has been reported in a caracal from China, which may have been spread from North America by marine animals, feral birds, or sea trade routes [24]. The transmission process of the T. gondii ToxoDB #5 strain in China is unknown. Fur seals in zoos from China were not checked for T. gondii infection status when imported from Alaska. Frozen sea fish are the main food for these animals. According to the distribution of ToxoDB #5, case #2, from which TgFurSealCHn1 was isolated, was a plausible carrier of the pathogen upon import or infection from oocysts in frozen fish imported from North American.

T. gondii ROP18 and ROP5 allele types were associated with virulence in mice. The allele types of virulence genes ROP18 and ROP5 (2/2) was predicted to be non-lethal in mice [31], which is consistent with the virulence evaluation of TgFurSealCHn1 in Swiss mice (survival rate 77%, 17/22, 48DPI) (Table 3). The mouse has been used as the main animal model for determining the virulence of T. gondii strains. Epidemiologic data suggest a potential association between T. gondii virulence in mice and disease manifestations in humans, and other susceptible animals [7, 26]. However, the severity of the toxoplasmosis following natural infection varies in intermediate hosts, and it is particularly challenging in captive or wild animals, in which detailed etiology and pathology surveys for T. gondii infection are rare [7, 35]. ToxoDB #5 T. gondii isolates (n = 117) from sea otters have been verified to be avirulent through mice assays, although most sea otters died of toxoplasmosis in these studies [30, 33, 34].

Vertical transmission of T. gondii in fur seals was proved in cases #2, #8, and #7 by serology, IHC, PCR, and the isolation of viable parasites from placenta. Vertical transmission of T. gondii causes fetal loss in several marine species, including dolphins and seals [17, 25]. Toxoplasma gondii transplacental transmission in sea otters was proved in a chronically infected dam by serology, IHC, and the isolation of viable parasites from the fetal brain [29, 30]. However, evidence of vertical transmission by isolating viable parasites from other marine mammals has not been reported. Here, T. gondii transplacental transmission in chronically infected fur seal case #2 was verified by serology (1:2018), IHC (placenta), PCR (placenta), and isolation of viable parasites from the placenta. Both the dam and fetus survived. In addition, the stillborn fetus and placenta from fur seal case #8 were proven to be infected with T. gondii by serology (dam 1:64, fetal <1:25), IHC (placenta positive, fetus negative), and PCR (placenta positive, fetal heart, liver, and lung positive), and the dam survived. Fur seal case #7 miscarried, and the aborted fetus was found to be positive for T. gondii by serology, and T. gondii tachyzoites were found in the lungs of fur seal case #7 by IHC, indicating possible vertical transmission of T. gondii.

Marine mammals are susceptible to T. gondii and are good sentinels for detecting marine pollution. Most T. gondii infections in people and animals are subclinical. However, sea otters are the most susceptible mammals in the United States [7, 10]. Toxoplasma gondii infections in sea otters can directly cause mortality due to meningoencephalitis [10, 34]. However, T. gondii infections were not lethal for fur seals (n = 5) in this study, although they were associated with abortion (3/5). Fur seals do not prey on the intermediate hosts of T. gondii, but they could be infected by the ingestion of oocysts from land sources or by ingesting mechanical transmitters from cold-blooded marine animals. In this survey, the high T. gondii infection rate in fur seals indicates that more attention should be paid to imported animals, frozen fish food, and oocyst-contaminated environments. The present study had several limitations. The first was the limited number of serum and tissue samples. The second was that the impact of T. gondii on marine mammals and the human care marine environment is not well understood. In the future, exploring the infection status of marine mammals and evaluating contamination by T. gondii oocysts will be of great significance for the health of marine animals and humans.

Conclusions

In the present study, T. gondii infection in chronically infected fur seal case #2 was verified by serology (1:2018), IHC (placenta), PCR (placenta), and isolation of viable parasites from the placenta. Genotyping analysis of this isolate indicates a probable North American origin and suggests vertical transmission of T. gondii in fur seals. Future follow-up studies on fur seal #2 and its offspring, including fur seal #3 may provide stronger support for these conclusions.

Acknowledgments

We thank Hongjie Ren, Liulu Yang, Shilin Xin, Niuping Zhu, Nan Jiang, Yaoyao Lu, and Yiheng Ma (Henan Agricultural University, China) for performing sample collection and animal management, and Chunlei Su (University of Tennessee, USA) for carefully checking the TgFurSealCHn1 genotypes and virulence factors.

Funding

This study was funded by Henan Province’s International Scientific and Technological Cooperation Projects (242102521041).

Conflicts of interest

The authors have no conflicts of interest to declare.

Supplementary material

thumbnail Figure S1:

Morphology of fur seal placenta and hematoidin crystal distribution as shown by microscopy. A: Large quantity of hematoidin crystals (trigon) were observed in the chorioallantoic membrane (fetal facial placenta). B: Magnified image of A (white square); hematoidin crystals are yellow rhombic plates (trigons).

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Cite this article as: Mao G, Guo B, Xie S & Yang Y. 2025. Isolation of Toxoplasma gondii from the placenta of northern fur seals (Callorhinus ursinus) and potential transplacental transmission of the parasite. Parasite 32, 48. https://doi.org/10.1051/parasite/2025045.

All Tables

Table 1

Background and T. gondii infection in fur seals (Callorhinus ursinus) from China (2012–2024).

In the text
Table 2

Genotypes of T. gondii isolates from fur seal according to PCR-RFLP of 10 markers and virulence proteins.

In the text
Table 3

Evaluation of the virulence of T. gondii TgFurSealCHn1 strain in Swiss mice (M ± SE).

In the text

All Figures

thumbnail Figure 1

Morphology of the placenta and samples from fur seal #2. A: Stillborn and placental tissue in 2017. B: Placenta in 2022. C: Zonary and belt-shaped placenta, dam, and its pup. D: Magnified C: surface of the chorioallantoic membrane.
In the text
thumbnail Figure 2

Morphology of T. gondii in the tissues of the fur seals. A–D: T. gondii antibody positive reaction (trigon) in the placentas of fur seal #2. A and C show IHC staining, while B (P#3445) and D (P#3559) show HE staining of serial sections A and C, respectively. E, F: T. gondii antibody positive reaction (trigon) in placentas of fur seal #4. E shows IHC staining, and F (P#3568) shows HE staining of the E serial section. G: T. gondii antibody positive reaction in the lung of fur seal #7 (P#3814) shown by IHC staining (trigon). H: T. gondii antibody positive reaction in the placenta of fur seal #8 (P#3841) shown by IHC staining (trigon). Bar = 50 μm.
In the text
thumbnail Figure 3

Morphology of tachyzoites of TgFurSealCHn1 in cell culture under transmission electron microscopy. A: A group of TgFurSealCHn1 tachyzoites within the parasitophorous vacuolar membrane. B: Magnified image of A. Am: amylopectin granule, Co: conoid, Dg: electron-dense granule, Lb: lipid, Mn: microneme, Nu: nuclei of daughter tachyzoites, Pm: parasitophorous vacuolar membrane, Pv: parasitophorous vacuole, Rh: rhoptry. Bar = 2 μm.
In the text
thumbnail Figure 4

Genotyping of T. gondii TgFurSealCHn1 strain isolated from fur seals. 1: GT1, 2: PTG, 3: CTG, 4: TgCgCal, 5: MAS, 6: TgCatBr5, 7: TgCatBr64, 8: TgToucan (TgRsCr1), 9: TgFurSealCHn1, and M: Marker.
In the text
thumbnail Figure S1:

Morphology of fur seal placenta and hematoidin crystal distribution as shown by microscopy. A: Large quantity of hematoidin crystals (trigon) were observed in the chorioallantoic membrane (fetal facial placenta). B: Magnified image of A (white square); hematoidin crystals are yellow rhombic plates (trigons).
In the text

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