Surveillance of infectious diseases in wildlife, particularly free-ranging NHPs, is crucial for identifying zoonoses and emerging or neglected diseases that pose risks to public health. Monitoring these diseases also enhances our understanding of ecological dynamics, assesses their impact on NHP populations, and aids in biodiversity conservation in natural and urban settings.

This study offers a groundbreaking overview of toxoplasmosis in free-ranging marmosets in Central Brazil. Our findings reveal a relevant incidence of fatal T. gondii infections (9.2%) in marmosets within the Brazilian Cerrado biome, rising to over 10% among free-ranging marmosets in the FD, with no apparent discernible influence from sex, age, or location. Variations in AFT prevalence across states may result from limited sample sizes in some regions (e.g., Mato Grosso State) or nonspecific local factors that influence diagnostic rates. Additionally, we estimated an overall prevalence of 50.7% of non-fatal T. gondii infections and a lethality rate of 20.3% for toxoplasmosis in free-ranging black-tufted marmosets in the FD, which are key findings that underscore the study’s significance.

While AFT is well-documented in captive neotropical NHPs in South America^20,21,22, reports of AFT in free-ranging neotropical NHPs remain limited to Brazil and may not accurately reflect the disease’s prevalence in wild populations^7,8,9,10. Most studies on AFT in wild NHPs estimated seroprevalence^{23,24,25,26,27}, but acute toxoplasmosis can cause death in susceptible animals before seroconversion, complicating its detection through serological methods such as the modified agglutination test (MAT)²⁸. This limitation underscores the challenges of determining AFT prevalence in free-ranging NHPs in Brazil, which remains largely unknown.

Similar to the high infection rate reported in this study, previous research detected T. gondii DNA in 17.9% of heart samples from 38 free-ranging common marmosets (Callithrix jacchus) with undetermined causes of death in Northeastern Brazil²⁹. AFT accounted for 25% of fatal infectious diseases and 4.6% of mortality causes in free-ranging NHPs in Rio Grande do Sul state⁸. Additionally, a retrospective analysis of 1,001 deaths among free-ranging callitrichids from the Brazilian Atlantic Forest reported a 1.6% prevalence of AFT in Rio de Janeiro⁹. Comparing toxoplasmosis prevalence across NHP populations reveals notable variations, likely influenced by differences in sample size, diagnostic methods, and ecological characteristics of regions such as the Atlantic Forest, Southern Brazil, Northeastern Semiarid, and Cerrado Biome in Central Brazil.

Similarly, the lethality of toxoplasmosis in Brazilian wild neotropical NHPs remains uncertain. Our study estimated a lethality rate of roughly 20% by analyzing both fatal and non-fatal cases of T. gondii infection in the overall studied population in FD. This finding underscores the disease’s significance as a cause of death in free-ranging NHPs. Despite potential biases, such as variations in the collection rate of dead animals and environmental and ecological variables, this study offers a novel perspective on toxoplasmosis in predominantly urbanized marmoset populations in Central Brazil.

In Central Brazil, AFT was diagnosed in both outbreaks and isolated cases, consistent with reports from captive neotropical NHPs and, more recently, free-ranging callitrichids in Rio de Janeiro^4,9,22,30. While most cases in our study involved single AFT diagnoses, it is possible that other family group members also died, but their carcasses were not found to be recovered for necropsy and sampling.

Our findings indicated a seasonal trend, with an increase in AFT cases during the dry season (May to September) and extending into the early rainy season (October to April) in Central Brazil. Seasonal variations in food availability and rainfall likely create environmental pressures influencing mortality patterns in primates³¹. Reduced food availability during the dry season³² may drive marmosets to forage more intensively, increasing their exposure to contaminated environments and food sources (e.g., domestic waste) with T. gondii oocysts, thereby elevating infection risk. However, the role of seasonality in AFT patterns among free-ranging NHPs remains poorly understood, warranting further research to clarify its impact on mortality trends and disease persistence in NHP populations.

The susceptibility of neotropical NHPs to toxoplasmosis is well-documented, and arboreal behavior is considered an evolutionary factor contributing to low resistance against soil-borne pathogens like T. gondii^5,6. Urban expansion and increased human activity in natural environments amplify interactions between urbanized marmosets and other threatening conditions such as electrocutions, leptospirosis, and Alphaherpesvirus infections, including T. gondii-contaminated environments and food sources^33,34,35,36. Urbanized free-ranging marmosets with AFT may also act as environmental sentinels, signaling geographical areas of potential risk for human infection¹⁰, and highlights the importance of diagnosing toxoplasmosis in urbanized free-ranging NHPs.

In this study, no clinical signs were observed in NHPs diagnosed with AFT, as the animals were found dead. Clinical reports of toxoplasmosis in free-ranging marmosets are rare, with nonspecific symptoms like depression, lethargy, tachypnea, and fever leading to death within 12 h⁷. Additional clinical signs have been described in captive neotropical NHPs, including abdominal distension, cough, and neurological symptoms such as tremors, incoordination, and paralysis^20,22,30,37.

The clinical course of toxoplasmosis varies with species susceptibility and treatment duration, with death reported in captive NHPs within 2 to 15 days after symptom onset^6,22,37,38. The acute and frequent fatal nature of toxoplasmosis in free-ranging callitrichids, combined with the rapid progression of the disease and lack of population monitoring, posed significant challenges in identifying clinical signs in affected animals in this study.

The primary gross findings in NHPs with AFT in Central Brazil included hepatomegaly with an accentuated lobular pattern, splenomegaly, pulmonary congestion, hemorrhage, and edema. Hepatomegaly with an accentuated lobular pattern was the main gross lesion in free-living marmosets with AFT. A free-living Brachyteles arachnoides with AFT showed similar gross findings and also hemoperitoneum, petechiae, ecchymoses in the lungs, kidneys, adrenal glands, hepatic lipidosis, and brain congestion⁷. Comparable lesions, especially involving the liver and lungs, have also been documented in captive neotropical NHPs across various countries^{21,22,30,38,39,40}.

Less commonly observed gross lesions, such as enteritis, ulcerative jejunitis^41,42, and mesenteric lymphadenopathy^39,42, were noted in captive NHPs but were absent in this study. Severe gross central nervous system (CNS) lesions are rare, with only one report of diffuse submeningeal hemorrhage in the cerebral cortex of a captive Aotus nigriceps with AFT³⁷. The lack of significant CNS macroscopic changes in AFT cases in our study underscores the low prevalence of such alterations, although the underlying pathogenesis remains unclear. Further research is warranted to characterize necropsy findings fully, as well as their clinical progression and fatal outcomes, particularly in free-ranging marmosets.

In all evaluated AFT cases, random hepatocellular necrosis with inflammatory infiltrate, predominantly histiocytic, was the primary microscopic hepatic lesion. Diffuse histiocytic splenitis was the second most frequent lesion. Necrotizing liver lesions and histiocytic spleen inflammation are hallmark findings in fatal T. gondii infections and have been consistently observed in both captive and free-living neotropical NHPs^{7,9,10,22,30,40}.

Severe cellular damage and host cell membrane rupture during parasite invasion and replication are closely associated with cell death and tissue necrosis⁴³, supporting the hepatic lesions detected in this study. Simultaneously, macrophages and antigen-presenting cells recognize T. gondii, triggering IL-12 and TNF-α production, which activates the Th1 (CD4 T cell) IFN-γ-dependent immune response^44,45,46,47. These immune mechanisms likely contributed to the observed microscopic lesions, with macrophages predominating in the liver and spleen of NHPs with AFT.

Pulmonary lesions, including lymphohistiocytic interstitial pneumonia and edema, were prominent in marmosets with AFT. In some cases, additional findings, such as type II pneumocyte hyperplasia and hyaline membrane formation, were indicative of diffuse alveolar damage (DAD)⁴⁸. Similar pulmonary lesions have been documented in 94% of free-ranging callitrichids with AFT in Rio de Janeiro State, as well as in individual cases involving B. arachnoides and C. penicillata^7,9,10. Captive NHPs worldwide have shown comparable pathological findings [39,42,40,30.22]. Type II pneumocyte hyperplasia and hyaline membrane formation associated with DAD⁴⁸ have also been described in severe AFT-associated pneumonia in captive neotropical NHPs^22,28,30.

Acute respiratory disease due to experimental T. gondii infection in cats begins with minimal pathological changes during tachyzoite invasion of respiratory epithelial and endothelial cells. However, significant pulmonary function impairment follows due to severe damage, including respiratory basement membrane denudation, fibrin production, and pneumocyte necrosis⁴⁹. Non-inflammatory lesions indicative of DAD may occur during the early stages of infection, but these changes are often visible only through electron microscopy, complicating the correlation between observed lesions and stages of pulmonary injury via light microscopy⁴⁹. It is plausible to hypothesize that the pulmonary lesions identified in free-ranging marmosets with AFT may share pathogenic mechanisms similar to those observed in experimentally infected cats.

Cardiac involvement was less frequent in this study, with myocarditis and cardiomyocyte necrosis being the most significant lesions observed. Cardiac lesions are generally less common in humans and NHPs with toxoplasmosis, and inflammation with or without cardiomyocyte necrosis is the most prevalent manifestation^9,50, as also noted in our findings. Cardiac involvement was reported in approximately 44% of free-ranging callitrichids in Rio de Janeiro State⁹, 33% of captive neotropical NHPs in the Belo Horizonte Zoological Garden^9,28, and in isolated cases among captive NHPs^21,37,38.

Severe inflammation plays a critical role in developing cardiac lesions caused by T. gondii. Experimental studies in mice have shown elevated levels of IFN-γ and TNF-α in the heart during the acute phase of infection and oxidative stress-related proteins such as COX2 during the chronic phase⁵¹. The invasion and rupture of cardiomyocyte membranes by T. gondii initiate the recruitment of antigen-presenting cells, leading to inflammation and cardiomyocyte necrosis^50,51. While these studies suggest a strong association between intramyocytic protozoans and myocarditis in humans and mice, the absence of data on inflammatory mediators like IFN-γ, TNF-α, and COX in the heart represents a key gap in understanding AFT pathophysiology in free-ranging NHPs.

IHC revealed organ-specific differences in cyst and zoite counts. The spleen showed the highest number of cysts and zoites, followed by the liver. Statistically, the spleen had significantly higher mean counts of cysts and zoites than any other organ, while the liver ranked second in zoite counts. These results underscore the importance of the spleen and liver in detecting T. gondii infection in free-ranging marmosets. Previous research on free-ranging marmosets also highlighted the liver’s role as a key organ for histopathological diagnosis of toxoplasmosis⁹.

Although AFT causes notable liver damage, the substantial presence of T. gondii forms in the spleen, as confirmed by IHC and histopathology, suggests that the spleen is critical for diagnosing toxoplasmosis. Furthermore, the strong correlation between cyst and tachyzoite counts in the spleen and liver supports their designation as preferential target organs for AFT diagnosis in free-ranging marmosets.

In contrast, the heart displayed the lowest cyst and zoite counts across histopathological and IHC evaluations, with mean zoite levels significantly lower than in other organs. This finding contrasts with a study of 16 AFT cases in marmosets, where T. gondii was readily detectable in cardiac tissue, with high concordance between zoites identified via histopathology and IHC⁹. While toxoplasmosis is recognized as a systemic disease in neotropical NHPs, these discrepancies may result from variations in host susceptibility, the virulence of pathogenic strains, sample sizes, or the number of confirmed AFT cases analyzed. Such factors likely influence the detectability of T. gondii in cardiac tissue, explaining the observed differences.

The high parasite load observed in free-ranging marmosets with AFT is a significant finding of this study. Comparative analysis revealed that the mean parasite loads in the spleen, liver, and lungs were notably higher than those in the heart. Interestingly, despite no correlation between parasitic load and cyst or zoite counts identified via IHC, organs with the highest and lowest cyst and zoite counts (spleen and heart, respectively) also exhibited the highest and lowest mean parasite loads. This pattern suggests reduced detectability of T. gondii in the heart compared to other organs.

Contrastingly, an AFT outbreak in squirrel monkeys (Saimiri sciureus) in Japan reported higher parasitic loads in the lungs, liver, and heart compared to the spleen³⁰, although the overall loads were much lower than those observed in our study. Quantifying parasitic load in NHPs with toxoplasmosis remains underexplored. Variability in parasite loads across organs may stem from differences in DNA quality from formalin-fixed paraffin-embedded tissues, smaller sample sizes, and individual susceptibility among NHPs.

Our findings also revealed significantly higher parasite loads in fatal cases of toxoplasmosis compared to non-fatal cases. This represents a novel insight, emphasizing the high lethality of the disease in free-ranging black-tufted marmosets and underscoring the severe threat toxoplasmosis poses to this population. Approximately one-fifth of infected marmosets experienced fatal outcomes, characterized by systemic lesions most prominently affecting the liver, spleen, lungs, and heart. However, it was not possible to determine specific parasite load thresholds predictive of fatal outcomes in this study.

AFT in urbanized free-ranging NHPs represents a significant ecological and public health challenge. Similar to the role of terrestrial and aquatic species as environmental sentinels for toxoplasmosis^{15,16,17,18,19}, the pronounced susceptibility of marmosets to AFT suggests they could serve as reliable indicators of the extent and risks associated with environmental contamination by T. gondii in anthropogenic environments¹⁰. Consequently, NHPs may act as valuable environmental sentinels, providing early warnings of human exposure risk, particularly for acquired, gestational, and congenital toxoplasmosis.

Multiple ecological and anthropogenic factors influence the transmission and persistence of T. gondii in the environment^52,53, particularly affecting free-ranging marmoset populations in this study. Environmental contamination by oocysts shed by domestic cats is a major driver of infection risk and the maintenance of toxoplasmosis⁵⁴. The high prevalence of the disease in marmosets suggests that anthropogenic landscapes in Central Brazil may provide favorable conditions for transmission. Despite a limited understanding of spatial and temporal variations in toxoplasmosis, urban expansion and increasing human activity may create yet unidentified conditions that sustain the parasite’s life cycle. These factors could also intensify interactions between marmosets and contaminated sources, such as domestic waste carrying T. gondii oocysts. Consequently, the growth of non-human primate (NHP) populations in these environments likely elevates the cumulative risk of infection as individuals traverse areas with varying environmental parasite loads.