Nonhuman Primates and Wild Felines as Environmental Bioindicators of Toxoplasma gondii and Leishmania spp. from a Brazilian Zoo

Introduction

Toxoplasmosis and leishmaniasis are globally distributed zoonoses, involving multiple vertebrate hosts and impacting humans, animals, and environmental health (Villar-Echarte et al., 2021; Leite et al., 2008).

Toxoplasma gondii is a cosmopolitan protozoan parasite with a complex epidemiological cycle involving a wide range of warm-blooded vertebrates. Felids act as definitive hosts, being responsible for the sexual reproduction of the parasite and the shedding of environmentally resistant oocysts, which play a central role in environmental contamination. Intermediate hosts, including mammals and birds such as primates, become infected primarily through the ingestion of sporulated oocysts from contaminated soil, water, or food, or by consuming tissue cysts present in infected prey, leading to chronic infections characterized by the formation of tissue cysts, particularly in skeletal muscles (Carneiro et al., 2014; Ornellas et al., 2020). In zoological settings, infection may occur through the consumption of raw or inadequately processed meat, contaminated food or water, or exposure to feline feces. Clinical manifestations range from fever, lymphadenopathy, hepatosplenomegaly, diarrhea, and neurological signs to asymptomatic infections; however, in primates with arboreal habits, the disease is often acute and fatal and may progress to death without overt clinical signs (Catão-Dias et al., 2013; Epiphanio et al., 2003). The epidemiology of T. gondii is strongly influenced by ecological, environmental, and anthropogenic factors, including climate conditions, habitat fragmentation, feeding habits, the presence of domestic and wild felids, and human-mediated environmental changes. The parasite’s wide host range, high environmental persistence of oocysts, and frequent occurrence of subclinical infections facilitate its silent circulation among wild, domestic, and human populations, reinforcing its relevance as a zoonotic agent within the One Health framework (Hotea and Dărăbuș, 2023).

Leishmaniasis, caused by protozoa of the genus Leishmania (Trypanosomatidae), comprises mainly visceral and American tegumentary forms and is maintained through a complex epidemiological cycle involving multiple vertebrate hosts and phlebotomine sandfly vectors, primarily of the genera Lutzomyia in the New World and Phlebotomus in the Old World. Transmission occurs through the bite of infected sandflies, which inoculate promastigotes, while vectors acquire amastigotes during blood meals from mononuclear phagocytic cells. The transmission dynamics are strongly influenced by environmental, ecological, and socio-anthropogenic factors, including climate, vegetation, land use, urban expansion, and the presence of reservoir hosts. Wild mammals such as rodents, marsupials, primates, and carnivores play a key role in maintaining the sylvatic cycle, whereas domestic dogs are considered the main reservoirs in urban and periurban environments, particularly for visceral leishmaniasis. Disease progression is generally similar in domestic and wild animals; however, wild primates often exhibit mild or absent clinical signs, with necropsy findings including weight loss, hepatosplenomegaly, lymphadenopathy, and hemorrhages. Environmental changes that increase habitat fragmentation and contact among vectors, wildlife, domestic animals, and humans favor the emergence and reemergence of leishmaniasis, reinforcing its relevance as a neglected zoonotic disease within the One Health framework (Leite et al., 2008; Sapatera et al., 2022; Souza et al., 2014; Azami-Conesa et al., 2021; Kyari, 2024).

Species considered natural bioindicators are organisms whose presence, absence, abundance, physiological condition, or biological responses sensitively and measurably reflect environmental status, ecosystem quality, or the occurrence of ecological changes, such as pollution, habitat degradation, or pathogen circulation (Bahranipour et al., 2024).

Nonhuman primates (NHPs), due to their high susceptibility to diseases affecting humans as a result of close evolutionary proximity, are considered sentinels for several zoonotic diseases in epidemiological surveillance (Morato et al., 2025). Wild felines, in turn, may act as natural bioindicators of environmental health owing to their high trophic position, broad exposure to different ecological compartments, and ability to reflect pathogen circulation and environmental changes, particularly in areas at the interface between wild, domestic, and human environments (Macdonald and Loveridge, 2010; Hatam-Nahavandi et al., 2021).

Studies in zoo animals are essential within the One Health perspective, since both diseases have zoonotic potential and reservoirs in urban centers. They also reflect hygiene practices, structural planning, food management, and reservoir control. This study aimed to evaluate antibodies against T. gondii and Leishmania spp. in NHPs and wild felines from the Municipal Zoo in western Paraná, Brazil.

Materials and Methods

Results

Of the 19 primate samples, nine (47.37%) samples were reactive for anti-T. gondii antibodies, with one sample (5.26%) for a titration of 256, five samples (26.31%) for a titration of 1024, two samples (10.52%) for a titration of 4096, and one sample (5.26%) for a titration of 16,384 (Table 1).

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Table 1.

Research on anti-Toxoplasma gondii and anti-Leishmania Antibodies Using the Indirect Immunofluorescence Technique in Nonhuman Primates and Wild Felines Residing in the Municipal Zoo in a City in the Western Region of the State of Paraná, Brazil, 2023

Nonhuman primates
Samples	Toxoplasma gondii	Leishmania infantum	Leishmania amazonensis	Leishmania braziliensis
01 P	4096	—	—	—
02 P	1024	—	—	—
03 P	—	—	—	—
04 P	256	20	40	—
05 P	4096	—	—	—
06 P	—	—	—	—
07 P	—	—	—	—
08 P	1024	20	40	—
09 P	1024	—	—	—
10 P	1024	20	—	—
11 P	—	40	—	—
12 P	16,384	20	—	—
14 P	1024	—	—	—
15 P	—	—	—	—
17 P	—	—	—	—
18 P	—	—	20	—
19 P	—	—	—	—
Wild feline samples
01 PC	—	—	—	—
02 PC	—	—	—	—
03 PC	—	—	—	—
04 PC	—	—	—	—
01 PO	—	—	—	—
1 LP	—	—	40	80
3 LP	—	—	—	—

Source: Prepared by the authors. P, non-human primate; PC, Puma concolor; PO, Panthera onca; LP, Leopardus pardalis.

Related to the research of anti-Leishmania antibodies in NHPs, when only titers above 40 were considered, three samples (15.79%) were reactive: one sample (5.26%) was reactive for L. infantum, two samples (10.52%) were reactive for L. amazonensis, and no samples were reactive for L. braziliensis.

Of the seven samples from wild felines, none were reactive to the anti-T. gondii antibody test. However, there was one (14.28%) sample that was reactive for the anti-Leishmania amazonensis antibody with a titer of 40 and L. braziliensis with a titer of 80.

Discussion

Zoos serve as valuable sources for epidemiological studies, representing “artificial ecosystems” susceptible to pathogens from wild animals, synanthropic species, and humans (Ferreira et al., 2022). While the role of captive animals in the transmission of toxoplasmosis and leishmaniasis remains unclear, primates and wild felines are recognized as key reservoirs (Dahroug et al., 2010; Ornellas et al., 2020).

Regarding toxoplasmosis in free-living primates, a study in three regions of Bahia, Brazil, using the MAT technique, found antibodies in 2 out of 64 NHPs (3.13%) (Ferreira et al., 2022). In Italy, IIF detected 50% of felines and 95.4% of primates positive for T. gondii in zoo and circus animals (Marková et al., 2019). In Paraná, Brazil, seroprevalence in living primates was 30.2% for Cebus spp. and 17.6% for Alouatta caraya, with MAT titers of 16 (15/16) and 64 (1/16) (Garcia et al., 2005).

This study adopted a cut-off of 1:40 for primate positivity, consistent with previous studies in primates and similar to those in felines (Bouer et al., 2010; Marková et al., 2019).

Variations in T. gondii prevalence may result from exposure to infected animals such as birds and rodents, direct or indirect contact with feces from stray domestic cats, or contaminated food and water (Santos et al., 2021; Leite et al., 2008). Higher seropositivity is often linked to herbivorous diets, suggesting that forage, fruits, or water provided to these animals may be contaminated with T. gondii oocysts (Casagrande et al., 2013; Leite et al., 2008).

New World primates (NWPs), particularly the genera Leontopithecus and Callithrix, are highly susceptible to T. gondii, whereas Old World primates (e.g., Pan, Pongo, and Chlorocebus) show greater resistance (Dubey et al., 2021). NWPs exhibit three response patterns: (I) Callitrichinae (Saguinus, Leontopithecus, and Callithrix) experience severe disease with high mortality; (II) Cebidae (Saimiri and Aotus) and Atelidae (Alouatta, Ateles, and Lagothrix) show severe outbreaks with variable survival (15%–66%); and (III) Cebus spp. develop high, persistent IgG with mild symptoms and rare deaths. In this study, primates had high T. gondii titers but no clinical signs. Similarities between Cebus sp. and Sapajus sp. suggest comparable immune responses. These variations are likely influenced by ecological, behavioral, and strain-related factors (Martins et al., 2023; Catão-Dias et al., 2013).

High primate seropositivity may also result from caretaker-related contamination, such as irregular cleaning or exchange of shoes and clothing between enclosures (Santos et al., 2021). Although primates tested positive, it cannot be confirmed that exposure occurred within the zoo, as all animals were rescued or seized by environmental authorities (IAT, IBAMA, and CAFS) and may have been previously exposed. Before this study, newly arrived animals were not screened for diseases.

Related to leishmaniasis in primates, in the present study, 15.79% of samples were detected as positive for the anti-Leishmania antibodies, and this result contrasts a study carried out in the North Central region of Paraná, which although used the direct agglutination technique, also researched anti-Leishmania antibodies in capuchin monkeys and detected 53.33% (8/15) positive samples (Lopes et al., 2022). In the present study, the cutoff point for leishmaniasis positivity in NHPs was 1:40 in the IFI (Ferreira et al., 2022; Guiraldi et al., 2022; Santos et al., 2023).

NHPs can host Leishmania spp., allowing the protozoan to develop and reproduce within them; however, their role as reservoirs in the epidemiological cycle remains unclear. To function as a reservoir, several conditions must be met: The population size and lifespan must sustain a continuous food source for vectors; frequent contact between host and vector must occur to allow transmission; over 20% of the host population must be infected; the infection must be long-lasting and nonlethal to enable parasite survival during low vector periods; and parasites must be present in the skin and blood at levels sufficient for transmission to vectors (Lopes et al., 2022). None of the primates evaluated in the present study exhibited clinical signs of leishmaniasis at the time of sample collection.

The zoo’s microclimate, characterized by humidity, dense vegetation, organic matter, and low light, likely promotes frequent vector exposure, creating ideal conditions for phlebotomine larvae development and increasing vector populations (Sapatera et al., 2022; Lopes et al., 2022).

Future studies should examine T. gondii oocysts in enclosure soil and water to identify contamination sources and implement preventive measures.

In this study, no wild felines tested positive for toxoplasmosis, contrasting with the Rio de Janeiro Zoo, where ELISA detected 88.1% reactive samples (Villar-Echarte et al., 2021). The low prevalence of wild felids may be due to enclosure design, with large felids housed far from small felids and forest areas, limiting exposure to other animals. Predatory behavior and controlled food and water sources further reduce contact with sporulated oocysts (Bolais et al., 2022; Ferreira et al., 2022). Regarding the management of felids at the study site, the animals are fed previously frozen meat, as recommended to eliminate tissue cysts (Djurković-Djaković and Milenković, 2000), thereby reducing the potential risk of T. gondii contamination. Furthermore, the felines in this study were divided by sex, with males housed alone, with one or two animals per enclosure, according to IBAMA regulations. Since the enclosures are mostly made of cement and have a smaller area of land, this makes cleaning easier and reduces the chances of exposing the animals to possible sporulated/infectious oocysts (Kakakhel et al., 2021).

In the present study, one felid (14.28%) was positive for Leishmania spp. In domestic felines from Rio Grande do Norte, leishmaniasis testing via indirect immunofluorescence revealed 14 out of 91 positive samples, with titers from 1:40 to 1:320 (Bezerra et al., 2019). Studies in northern Italy showed even lower seropositivity (4.9%) with a cutoff of 1:80 (Spada et al., 2020). A study on 20 tigers in an Italian safari park evaluated L. infantum infection using immunofluorescence antibody testing and PCR, showing 45% and 25% positivity, respectively, with 5 animals positive in both tests (Cavalera et al., 2020). Variations in positive rates across studies may reflect differences in feline species tested and the cutoff points used (Braga et al., 2014; Coura et al., 2018; Bezerra et al., 2019).

These variations in prevalence may be linked to the distinct epidemiological characteristics and biomes of each region. The humid climate of North Central Paraná, combined with environments rich in organic matter and low light, favors the development of phlebotomine sandfly larvae, potentially increasing vector populations (Lopes et al., 2022; Chagas, 2017). In contrast, the western region of Paraná, where this study was conducted, features deep, dark red clayey soils that are friable, highly porous, and well-drained (Larach et al., 1984). This lower humidity further influences larval development.

In domestic cats, Leishmania infections may be asymptomatic or present nonspecific signs, mainly cutaneous (Santos et al., 2018). Clinical manifestations have not been reported in large felines positive for L. infantum (Cavalera et al., 2020) or L. chagasi (Dahroug et al., 2011). Low antibody titers in the present study may reflect either early-stage infections or past exposures, potentially explaining the absence of clinical signs and classifying the animals as asymptomatic (Lima et al., 2024; Ferreira et al., 2022). In this study, none of the wild felids exhibited clinical signs of Leishmania spp. infection.

The hypothesis of the presence of cross-reaction between the samples tested for toxoplasmosis and leishmaniasis is not raised in this work. There may be cross-reaction when the species in question is dogs; however, there is no evidence of this nature in felines and NHPs (Pennisi et al., 2015; Zanette et al., 2014).

The city where the zoo is located ranks 10th in Paraná for reported cases of American cutaneous leishmaniasis, with 285 cases recorded between 2001 and 2017. The disease is unevenly distributed across the state, with higher incidence in the North and West, particularly in areas near native and riparian forests, rivers, and basins, which favor high phlebotomine sandfly populations (PAHO, 2019). This may explain the circulation of the pathogen among wild primates and felines.

Leishmaniasis remains a major public health challenge in western Paraná, where this study was conducted. The presence of anti-Leishmania spp. antibodies in primates and wild felines suggests a potential risk of exposure for other zoo-resident species.

Paraná is a key region for the capture, transit, and export of wild animals, with 20,275 seizures between 1980 and 2002, mostly linked to illegal trade, particularly in the western areas of Cascavel and Toledo due to easy access to neighboring countries (Hernandez and Carvalho, 2006; Vidolin et al., 2004). The Cascavel Zoo receives rescued primates from trafficking or seizures, which may have been exposed to T. gondii and Leishmania spp. prior to arrival, while long-term resident large felines remain in more controlled conditions with lower exposure. Additionally, animals rescued from precarious situations may be more susceptible to infection due to stress, immunosuppression, and prior pathogen exposure, likely explaining the higher antibody prevalence in primates compared to felines.

Conclusions

This study evaluated the presence of antibodies against T. gondii and Leishmania spp. in NHPs and wild felines housed in the municipal zoo in western Paraná, Brazil, contributing to the understanding of the circulation of zoonotic protozoa in captive wildlife. The detection of seropositivity, particularly in NHPs, indicates environmental exposure to these pathogens, even in the absence of evident clinical signs, highlighting the silent and subclinical nature of these infections in wildlife populations.

From a One Health perspective, these findings reinforce the role of NHPs as sentinels of zoonotic pathogens and wild felines as bioindicators of environmental health. Studies conducted in zoological settings are essential for monitoring pathogen circulation at the interface between wildlife, domestic animals, humans, and the environment, providing valuable information for scientific research, conservation strategies, and the development of preventive and surveillance measures for zoonotic diseases. Furthermore, the study also underscores the scarcity of the regional literature, largely due to challenges in capturing and monitoring free-living animals.