Toxoplasma gondii in cetaceans of Brazil: a histopathological and immunohistochemical survey

Toxoplasmosis is a parasitic disease caused by the protozoan Toxoplasma gondii. In cetaceans, T. gondii infection is a significant cause of morbidity and mortality. Despite the worldwide range and broad cetacean host record of T. gondii infection, there is limited information on toxoplasmosis in cetaceans from the Southern hemisphere. We investigated the occurrence of T. gondii by histopathology and immunohistochemistry in tissue samples of 185 animals comprising 20 different cetacean species from Brazil. Three out of 185 (1.6%) animals presented T. gondii-associated lesions: a captive killer whale Orcinus orca, a free-ranging common bottlenose dolphin Tursiops truncatus and a free-ranging Guiana dolphin Sotalia guianensis. The main lesions observed in these animals were necrotizing hepatitis, adrenalitis and lymphadenitis associated with protozoal cysts or extracellular tachyzoites presenting immunolabeling with anti-T. gondii antibodies. This study widens the spectrum of species and the geographic range of this agent in Brazil, and provides the first reports of T. gondii infection in a captive killer whale and in a free-ranging common bottlenose dolphin in South America.


Introduction
Toxoplasma gondii is a zoonotic intracellular coccidian protozoan of the phylum Apicomplexa (DUBEY, 2008).This protozoan is one of the most common parasites of warm-blooded animals (DUBEY et al., 2004), and was described for the first time in Brazil andin Tunisia, in 1908 (NICOLLE &MANCEAUX, 2009;SPLENDORE, 2009).The first record of toxoplasmosis in cetaceans was in a Guiana dolphin Sotalia guianensis from Rio de Janeiro state, Brazil, in the 1970's (BANDOLI & OLIVEIRA, 1977).Since then, the exposure and infection by this protozoan has been detected through serological, pathological and molecular techniques in a wide variety of captive and free-ranging cetaceans worldwide (INSKEEP et al., 1990;OMATA et al., 2006;MAZZARIOL et al., 2012;GONZALES-VIERA et al., 2013;IQBAL et al., 2018).Most of these studies have covered individual cases or small groups of cetaceans (DI GUARDO et al., 2010;GONZALES-VIERA et al., 2013).Toxoplasmosis is a significant cause of morbidity and may lead to stranding and death, and is considered one of the most important emerging diseases in cetaceans worldwide ( VAN BRESSEM et al., 2009;DI GUARDO et al., 2010;GONZALES-VIERA et al., 2013;BIGAL et al., 2018).
The epidemiology of T. gondii transmission has been established in several terrestrial animals (e.g., cats, livestock), and to a lesser extent, aquatic species (e.g., shellfish), including transplacental transmission in susceptible pregnant animals (TENTER et al., 2000;CATÃO-DIAS et al., 2013;JARDINE & DUBEY, 2002;MILLER et al., 2008).In terrestrial animals, infection also occurs through ingestion of tissue cysts or contaminated food and/or water containing infecting sporulated oocysts released in feline feces.However, the transmission of T. gondii in cetaceans has not been fully elucidated.One major source of transmission in cetaceans could be the polluted marine environments in proximity with the coast, e.g., run-off of cat feces or soil contaminated with T. gondii oocysts from rivers, polluted effluents and ballast water from ships ( VAN BRESSEM et al., 2009).In utero transmission of T. gondii has been described in Risso's dolphin Grampus griseus and Indo-Pacific bottlenose dolphin Tursiops aduncus (JARDINE & DUBEY, 2002;RESENDES et al., 2002).For offshore marine ecosystems, T. gondii transmission patterns are still unclear.
The occurrence of T. gondii in marine mammals remains a poorly understood phenomenon, particularly in Brazil, where very limited information exists.Exposure to T. gondii was reported in Amazon river dolphins Inia geoffrensis (SANTOS et al., 2011), but to this date, only one cetacean species is known to be infected by T. gondii in Brazilian waters: the Guiana dolphin (BANDOLI & OLIVEIRA, 1977;GONZALES-VIERA et al., 2013).We hypothesized that T. gondii infection could be present in a wider number of cetacean species in Brazil than previous studies have suggested.Thus, the aim of this study was to evaluate retrospectively the occurrence of T. gondii infection in captive and free-ranging cetaceans employing histopathological and immunohistochemical (IHC) analyses on tissue samples obtained from a large marine mammal tissue bank in Brazil.

Materials and Methods
We evaluated formalin-fixed paraffin-embedded tissue samples stored at the Marine Mammal Tissue Bank of the Laboratory of Wildlife Comparative Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil.The samples included in the study came from partial or complete standard necropsies conducted on odontocetes and mysticetes between 1988 and 2014, provided by partner institutions attending marine mammal strandings, rehabilitation and incidental captures along the Brazilian coast.Tissues from 185 individuals of 20 different cetacean species (Table 1) were evaluated.Among the evaluated samples, three were from originally wild animals kept in captivity in two different amusement parks of the São Paulo state (Brazil): a killer whale Orcinus orca from Iceland kept in captivity in the 1980's, an Amazon river dolphin from the Amazon Basin, and a common bottlenose dolphin Tursiops truncatus of unknown origin.The remaining animals were all free-ranging species that stranded along the Brazilian coast, mostly from the southern region (43.9%;80/182), followed by the southeastern (30.2%; 55/182), and northeastern (25.8%; 47/182) regions (Figure 1).The examined tissues were: liver, kidney, lung, spleen, skeletal muscle, lymph node, adrenal gland, brain, uterus, intestines, heart and eye.
Formalin-fixed paraffin-embedded tissue sections from the aforementioned organs were sectioned at 5 µm thickness and stained with H&E.Additionally, special histochemical techniques -periodic acid-Schiff (PAS), Giemsa and Grocott's methenamine silver stains -were used in selected tissue sections to improve visualization of protozoan structures compatible with T. gondii (LUNA, 1992;POPPER et al., 1960).
v. 28, n. 3, july.-sept. 2019 397/402 397 Toxoplasma gondii in cetaceans of Brazil alcohols.Toxoplasma gondii antigen was retrieved by heating tissue sections in citrate buffer (pH=7.0)solution for seven minutes at 90 °C.The sections were blocked with 1% normal rabbit serum in PBS for 30 minutes followed by overnight incubation with a polyclonal goat anti-T.gondii primary antibody (1 in 400 dilution; VMRD Inc; Pullman).This antibody does not cross-react with Neospora caninum (CORBELLINI et al., 2002).The sections were washed in PBS and incubated for 30 minutes with biotinylated polyclonal anti-goat secondary antibody (1 in 600 dilution; Dako) as previously described (DI GUARDO et al., 2010).Amplification of the immunologic reaction was based on an avidin-biotin-peroxidase complex method (Elite ABC kit, Vector laboratories), following manufacturer's instructions.Labeling was 'visualized' with 3-amino-9-ethyl-carbazole (Sigma) and/or diaminobenzidine (DAB D-5637; Sigma), and sections were counterstained with Mayer's haematoxylin.Tissue sections in which the primary antibodies were replaced by phosphate buffered saline or nonimmune homologous serum served as negative controls.Sections of the adrenal gland of a Guiana dolphin infected by T. gondii (GONZALES-VIERA et al., 2013) were used as positive control.Morbillivirus immunohistochemistry was also performed on selected tissue sections of T. gondii positive cases to rule out potential coinfection, following a previously published protocol (SALIKI et al., 2002;GROCH et al., 2014), using a commercial monoclonal antibody against the nucleoprotein of Canine distemper virus (VMRD Inc.), known to cross-react with Cetacean morbillivirus.Positive tissue samples of an infected Guiana dolphin were selected as positive control (GROCH et al., 2014).

Results
Three of 185 (1.6%) animals were IHC-positive for T. gondii-: a captive killer whale, a free-ranging common bottlenose dolphin, and a free-ranging Guiana dolphin.These animals presented a variety of toxoplasmosis-compatible lesions, with positively labeled protozoal cysts and free tachyzoites.The occurrence of T. gondii in free-ranging species was 1.1% (2/182), whereas the occurrence in captive animals was 33.3% (1/3).A summary of T. gondii-associated lesions in these animals follows.Additional information regarding the microscopic findings of positive animals is available in Table 2.All three T. gondii positive animals were IHC-negative morbillivirus.No PCR techniques were employed in these cases because frozen tissue samples were not available.
Case No 1 (MM#452) was a 5-6 years-old juvenile (2,500 kg) male killer whale captured in Iceland in 1983, and one year later brought into an oceanarium located in the city of São Paulo, São Paulo state, Brazil.The animal remained in this facility until its

Discussion
Despite the widespread geographical range of T. gondii infection, in Brazil, the current knowledge regarding cetacean toxoplasmosis is limited to few species and studies (BANDOLI & OLIVEIRA, 1977;SANTOS et al., 2011;GONZALES-VIERA et al., 2013).Based on microscopical and immunohistochemical examinations, we provide the first description of T. gondii in a common bottlenose dolphin of South America, and in a captive killer whale.Furthermore, the identification of toxoplasmosis in a Guiana dolphin corroborates with previous observations regarding this species and geographic area, the Paranaguá Bay.On the other hand, in spite of the high prevalence of T. gondii antibodies reported in free-ranging Amazon river dolphins (SANTOS et al., 2011), our sampled captive Amazon river dolphin was T. gondii-negative.
In this study, we evaluated cetacean samples from geographic areas that correspond to a large portion of the Brazilian coastline, most of them coastal species, mainly franciscanas (Pontoporia blainvillei) (55.0%) and Guiana dolphins (14.5%).Coastal cetaceans, considered suitable sentinels of the marine environmental ecosystem and important indicators of marine pollution, are usually more exposed to anthropogenic activities (WELLS et al., 2004;BOSSART, 2011;MOURA et al., 2014).From these, we found histological and immunohistochemical evidences of toxoplasmosis in one Guiana dolphin.This is the third report of toxoplasmosis in this species (BANDOLI & OLIVEIRA, 1977;GONZALES-VIERA et al., 2013).This animal presented severe adrenalitis, similar to what was reported by Gonzales-Viera et al. (2013) in another Guiana dolphin from the same area (Paranaguá Bay) that died in the same year (1998).Given the marked pattern of site fidelity presented by Guiana dolphins (MOURA et al., 2014), this result suggests a common source of exposure to T. gondii and a possible negative impact of toxoplasmosis on this population.Although the low occurrence of T. gondii infection found in the present study does not allow us to infer further on this protozoan's geographic distribution, the location of the Guiana dolphin case and previous reports suggest that infection may occur at higher rates in coastal areas of Brazil.Coastal areas usually receive freshwater run-offs and terrestrial biologic pollutants, contributing to the presence of T. gondii (SHAPIRO et al., 2015).These results also suggest areas in which future research efforts should be concentrated.
Concerning killer whales, positive serology for T. gondii was reported in a killer whale calf that stranded alive in Japan, in 1988(MURATA et al., 2004), and T. gondii infection was identified by molecular techniques in a stranded killer whale from the Northeastern Pacific, in California (GIBSON et al., 2011).However, these reports lacked detailed pathological examinations.
In the present study, we provide the first histopathological and immunohistochemical evidences of toxoplasmosis in this species.Based on the severity and extent of characteristic multiorganic necroinflammatory foci, we believe that T. gondii played a major role in the death of the evaluated killer whale.In this case, potential environmental sources of T. gondii infectious forms may include water and/or food contamination in one or more of the following scenarios: (1) the facility's water system (e.g., used to clean fish and the enclosure, and fill the tank), especially considering that sporulated oocysts of T. gondii are viable in saltwater for at least two years (AUBERT & VILLENA, 2009); (2) contaminated food (e.g., fish); and (3) contaminated enclosure (e.g., contaminated fomites, personnel and/or presence of roaming cats).These findings highlight the importance of proper hygiene and husbandry in captive cetaceans, including properly treated water and reliable food sources free of T. gondii sporulated oocysts.Although unlikely, considering the physiopathology of toxoplasmosis in highly susceptible or 'less adapted' hosts (CATÃO-DIAS et al., 2013) -which could be the case for the killer whale -one should also consider the possibility of infection prior to the specimen's capture in the wild (e.g., transplacental or transmammary) or initial period in captivity (1984)(1985)(1986)(1987)(1988).In that case, the infection would have been quiescent until being activated by the stress of captivity and/or other predisposing factors (e.g., concomitant diseases) (LINDSAY et al., 2003;AUBERT & VILLENA, 2009;MAZZARIOL et al., 2012).
Toxoplasma gondii infections in common bottlenose dolphins have been observed worldwide; in the Mediterranean Sea and the Atlantic Ocean (CRUICKSHANK et al., 1990;INSKEEP et al., 1990;DI GUARDO et al., 1995;DUBEY et al., 2008;PRETTI et al., 2010;PROFETA et al., 2015;BIGAL et al., 2018), and also in captive specimens (DUBEY et al., 2009).Furthermore, it has also been detected in a related species from the Pacific Ocean -the Indo-Pacific bottlenose dolphin (JARDINE & DUBEY, 2002).However, this is the first report of T. gondii in an Atlantic bottlenose dolphin from South America (Figure 2), widening the geographic range of T. gondii occurrence for this species.
Because cetacean species' susceptibility to T. gondii infection is unknown, non-exposure and natural resistance to this agent should be considered in negative cases (DI GUARDO & MAZZARIOL, 2013).Omata et al. (2006) proposed that killer whales from Japan might be resistant to T. gondii; however, the evidence provided seemed slight because none of these animals presented antibodies against the agent.
Finally, this is the first report of a large-scale T. gondii survey based on histopathological and immunohistochemical examinations in cetaceans of Brazil.This study widens the spectrum of T. gondii-susceptible species and geographic range of this agent in Brazil and presents the first report of T. gondii-infection in a captive killer whale, and in a free-ranging Atlantic bottlenose dolphin from South America.Furthermore, it corroborates with previous observations of toxoplasmosis in Guiana dolphin and the geographic area of Paranaguá Bay.Further studies are warranted to characterize the circulating strains and clarify the distribution of this protozoan, its transmission route(s), unsolved pathogenetic mechanisms, cetacean host-specific susceptibilities and potential implications to the conservation of cetacean species of Brazil and mechanisms involved in human exposure.

Figure 1 .
Figure 1.Geographic distribution along the coastline of Brazil of Toxoplasma gondii-positive (green triangle) and -negative (red circle) animals, according to immunohistochemistry.

Table 1 .
Cetacean species evaluated in Brazil for Toxoplasma gondii by immunohistochemistry.

Table 2 .
Microscopic lesions in Toxoplasma gondii-positive animals in Brazil..43m total body length, that stranded alive and died shortly after stranding, on October 29 th , 2001, in Lagos Region, Rio de Janeiro state, Brazil (22°56'S, 42°19'W).Samples of lungs, liver, kidneys, and large intestine were collected upon necropsy.No gross findings were reported.The main microscopic findings observed were moderate to severe, multifocal random to coalescing necrotizing hepatitis with protozoan cysts compatible with T. gondii and moderate to marked, multifocal, acute fibrinosuppurative bronchopneumonia.Case No 3 (MM#67) was a 1.86 m-long adult, female Guiana dolphin that stranded dead on March 3 th , 1998, in Paranaguá Bay, Paraná state, Brazil (25°31'S, 48°30'W).No necropsy records were available.Samples of eye and adrenal glands were evaluated.Microscopically, the main finding was marked, multifocal, acute necrotizing adrenalitis with numerous protozoal cysts and extracellular tachyzoites compatible with T. gondii, and further highlighted with PAS and Giemsa stains.
NA = not available.