Introduction

Of the 504 species of nonhuman primates currently recognized, ca. 60% are threatened (Estrada et al. 2017). The main threats to primates are degradation, fragmentation, and loss of habitat; hunting; and illegal trade. For many primates, the main threat is forest fragmentation, resulting in small populations that are restricted to isolated forest patches. Habitat loss and fragmentation diminish species diversity and ecosystem function. They may also influence pathogen–host–environment dynamics. Primates in fragmented habitats are particularly vulnerable to disease epidemics, because of their small populations (Smith et al. 2009) and because translocation and reintroduction are increasingly used to maintain their genetic diversity, promoting animal movement, which may favor disease transmission (Kock et al. 2010).

Moreover, small forest patches are often surrounded by agriculture, livestock pasture, and settlements, which increases contact between people, wildlife, and domestic animals, with the consequent possibilities of disease spillover (Mackenstedt et al. 2015; Osofsky et al. 2005; Wolfe et al. 1998).

Wild primates have a great diversity of parasites, including viruses, fungi, bacteria, and micro- and macroparasites (Nunn and Gillespie 2016). Several pathogens cause disease and promote morbidity and, in some cases, mortality, in primates. These include malaria, Chagas disease, leishmaniasis, leptospirosis, brucellosis, toxoplasmosis, and filariasis (Baitchman et al. 2006; Epiphanio et al. 2003; Percy et al. 1972; Toft and Eberhard 1998). Little is known, however, of the diversity of pathogens in wildlife populations. This is especially so in large, tropical, megadiverse countries. Factors such as population density, relative abundance, geographic distribution, and loss of species diversity can also influence the pathogen transmission cycle and disease occurrence (Cook and Karesh 2008; Suzán et al. 2012). In addition, the sex, age, and social status of individuals can influence the exposure and transmission of parasites, affecting disease occurrence in a population. Young animals, for example, are more susceptible than older ones to many infectious diseases because of low immunity (Cross et al. 2009).

Studies of pathogen occurrence in endangered populations are of great importance for conservation, and epidemiological studies of pathogens in wild animals are also important for the prevention, control, and monitoring of diseases and to develop public and animal health policies (Daszak et al. 2000; Deem et al. 2001). Connections between vertebrate species through the human–animal–ecosystem interface may have consequences for human, animal, and ecosystem health. The majority (60%) of etiological agents circulate between animals and humans (anthropozoonosis), and 72% of emerging diseases are caused by pathogens originating from wildlife (Jones et al. 2008).

Thirty-five of Brazil’s 150 primates are classified as Threatened, and 17 of these live in Brazil’s Atlantic Forest (Brazil, Ministério do Meio Ambiente 2014). Of these threatened species, the blonde capuchin (Sapajus flavius) is restricted to a few forest patches in the Atlantic Forest of Northeast Brazil in the states of Alagoas, Pernambuco, Paraíba, and Rio Grande do Norte (Fialho et al. 2014; Oliveira and Langguth 2006; Pontes et al. 2006), and classified as Endangered (Brazil, Ministério do Meio Ambiente 2014; de Oliveira et al. 2015). In 2006, the total wild population of S. flavius was estimated to be 180 individuals, living in ca. 24 subpopulations (de Oliveira et al. 2015). Subsequent studies, between 2011 and 2014, indicated a wild population of around 1000 individuals, in at least 29 subpopulations (Fialho et al. 2014; Valença-Montenegro 2011). Group sizes are 9–90 individuals (Valença-Montenegro et al. 2015).

In Paraíba state, Sapajus flavius occurs in 19 small forest fragments (Fialho et al. 2014). These remnant subpopulations face severe deforestation, both past and present, mainly as a result of cattle ranching, agriculture (sugar cane), and logging. The remaining forest fragments together comprise just 0.4% of Paraíba state (Barbosa 1996; Tabarelli et al. 2006). Deforestation and hunting are the main overt threats to S. flavius, but considering the overall low numbers, and that the populations are severely fragmented, infectious diseases may pose an additional threat, as they may increase mortality/morbidity (Osofsky et al. 2005; Smith et al. 2009).

We examined the prevalence of zoonotic infectious parasites and bacteria in Sapajus flavius living in a small, isolated forest, at an unusually high density of 12 individuals/km2, a factor that we assumed to be favorable to disease transmission. We also evaluated the influence of sex and age on risk of infection.

Methods

Study Site, Study Group, and Selected Diseases

We conducted our study in the Private Natural Heritage Reserve of Engenho Gargau, in Santa Rita municipality, 25 km from João Pessoa, the capital of Paraíba state, in Northeast Brazil (07°00′43.84′′S, 34°57′24.96′′W) (Fig. 1). The 1500-ha site is a privately owned protected area, with secondary forest in different stages of succession immersed in a matrix of sugar cane plantations. The forest is affected by hunting and firewood harvesting (Valença-Montenegro 2011). Annual mean temperature is 22–26 °C and annual mean rainfall is 1600–1800 mm (Lima and Heckendorff 1985).

Fig. 1
figure 1

The study site. The Private Natural Heritage Reserve of Engenho Gargau, Paraíba state, Brazil, is outlined in white. Note the extensive agricultural and farming activities, and the reserve’s proximity to an urban area on the right.

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Three species of primates inhabit the reserve: the common marmoset (Callithrix jacchus), the red-handed howler (Alouatta belzebul), and Sapajus flavius (Fialho and Gonçalves 2008). We estimated the population of S. flavius to be 180 individuals living in three groups (30, 60, and 90), among the largest groups ever recorded for Sapajus. S. flavius are largely frugivore-insectivores, and at this site palm fruits and sugar cane are key resources (Valença-Montenegro 2011).

We selected the following infectious agents for investigation, based on their capacity to affect or induce disease and death in primates, and their reported occurrence in domestic animals and/or humans in the area: Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii, Plasmodium spp., filarial nematodes, Leptospira spp. (24 strains), and Brucella spp..

Sample Collection

We captured 48 Sapajus flavius using Tomahawk traps, removed them from the traps using dip nets, and anesthetized them with ketamine hydrochloride (30 mg/kg) in August 2009, March 2010, September 2010, and August 2012. We carried out a clinical evaluation of each individual and collected blood samples from the femoral vein. We classed monkeys as infants, young, or adults, based on body size (Fragaszy et al. 2004). We released all individuals where we captured them on the same day or the next morning if they had not fully recovered from anesthesia before dusk.

Laboratory Analysis

We examined samples from 48 individuals: 32 males and 16 females (28 adults and 20 young). We did not test all individuals for Plasmodium and filariasis because there was insufficient blood for some analyses. We tested for antibodies against Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii, Leptospira spp. (24 serovars), and Brucella spp. using in-house diagnostic tests, and performed molecular analysis for Plasmodium spp. diagnosis. We also tested for the presence of T. cruzi, Leishmania spp., Plasmodium spp., and filaria by examining blood smears (Table I).

Table I Prevalence, tests, antigens, and primers used to evaluate the presence of selected pathogens in Sapajus flavius, in the Private Natural Heritage Reserve of Engenho Gargau, Paraíba state, Brazil, 2009–2012
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Statistical Analysis

We calculated Fisher exact 95% confidence intervals (CI) for the prevalence of each infectious disease using OpenEpi (Dean et al. 2013). We used a logistic regression to evaluate the influence of sex (female and male) and age (young and adult) on risk of infection (Dobson and Barnett 2002). We considered one model per disease (whenever the number of infected individuals exceeded one) using both predictors as factors. Whenever the predictors were significant at α = 0.1, we predicted the risk of infection, i.e., probability of subjects being infected, by sex and/or age. We conducted analyses in R, version 3.3.1 (R Core Team 2015).

Ethical Note

All procedures were in full compliance with federal permits issued by the Brazilian Government (License SISBIO 19927). The authors declare that they have no conflict of interest.

Results

Risk of Infection

All subjects showed negative results for Leptospira spp. and Brucella abortus. However, the estimated prevalence of antibodies against Leishmania spp. and Trypanosoma cruzi was 16% (8/48; 95% CI: 9–30%). Five individuals presented antibodies for both pathogens, three were positive only for Leishmania spp., and another three only for T. cruzi. We identified antibodies to Toxoplasma gondii in 12% of animals tested (6/48; 95% exact CI: 6–25%), with titers of 25 (4 individuals), 400 (1 individual), and 3200 (1 individual). Plasmodium spp. infection was present in 4% of subjects (1/24; 95% CI: 1–20%) and molecular analysis of the Plasmodium isolate showed SSU rRNA and cytochrome b sequences of Plasmodium brasilianum (GenBank KX618475 and KX618476, respectively). Blood smears were negative for Plasmodium spp., Trypanosoma spp., and Leishmania spp. parasites, but we found microfilariae in 46% of the individuals we evaluated (18/39; 95% exact CI: 30–63%). Clinical evaluation showed that all subjects in this study were apparently healthy when captured.

The predictor sex was significant only for Toxoplasma gondii (Table II), in which females had a higher chance of infection (0.25, SE = 0.11) than males (0.03, SE = 0.03), with an odds ratio of 8, i.e., chance of infection for females was eight times higher than for males. Age was not a significant factor in any infectious disease model.

Table II Results of logistic regression testing the influence of sex and age on infectious disease in Sapajus flavius in the Private Natural Heritage Reserve of Engenho Gargau, Paraíba state, Brazil, 2009–2012
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Discussion

We found that wild populations of Sapajus flavius in Engenho Gargau were infected with Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii, Plasmodium spp., and microfilariae and that S. flavius had no contact with Leptospira spp. and Brucella spp.. Females had a higher chance of T. gondii infection than males and age was not a significant factor in any infectious disease we investigated.

Our analyses indicated that the population of Sapajus flavius has had no contact with the following species of Brucella: B. abortus, B. melitensis, B. suis, B. neotomae, B. pinnipedialis, B. ceti, B. microti, and B. inopinata. Experimental studies have demonstrated that some primate species produce antibodies for, and can be affected by, these pathogens, causing disease (Mense et al. 2004; Percy et al. 1972; Schlabritz-Loutsevitch et al. 2009; Thorne 2001; Wilson 1936; Yingst et al. 2010). Brucellosis is widely distributed in bovine herds throughout Paraíba and could be transmitted to S. flavius inhabiting remnants surrounded by cattle farms (Leite et al. 2003). More studies are needed to determine the susceptibility of Neotropical primates to these pathogens.

The negative results for anti-Leptospira antibody serovars may reflect the absence of this pathogen in the population but may also be due to its virulence. Several outbreaks of leptospirosis have been described in captive primates, causing jaundice, lethargy, and abortion (Gibson 1998; Karesh et al. 1998; Leighton and Kuiken 2001). The disease is often characterized as acute, mortality is usually high, and infected individuals may die without being detected (Baitchman et al. 2006; Perolat et al. 1992). Studies in Brazil have demonstrated the occurrence of antibodies to Leptospira in Cebus and Sapajus, including S. flavius, but most of these were conducted on captive individuals or in rescue centers (Ferreira et al. 2011). Serological studies of free-ranging Neotropical primates showed positive results to different serovars, such as Pomona, Brasiliensis, Mini, Swajizak, Grippothyphosa, Sarmin, Fluminense, and Autumnalis in Cebus paella; Mangus and Fluminense in Alouatta caraya; Australis and Pomona in Callithrix penicillata; and Grippotyphosa in Ateles paniscus chamek (Dória et al. 1974; Karesh et al. 1998; Souza et al. 2006).

Our analysis showed that the chances of infection with Leishmania spp. were high for Sapajus flavius at our study site, with an infection probability of 0.56. There are autochthonous human cases of infection in the study area, and leishmaniasis, especially the cutaneous form, is considered endemic in Paraíba state (Brandão-Filho et al. 1999).

The epidemiology of Leishmania in Neotropical primates is not well studied; however, experimental studies show that Leishmania sp. can affect primates, which manifest symptoms similar to those shown by humans (Silveira et al. 1990; WHO 2010). Leishmania has been described in different primate species for Brazil and other countries, e.g., Callithrix spp., Cebus sp., Chiropotes satanas, Saguinus spp., Papio cynocephalus, Chlorocebus aethiops, and Cercopithecus mitis (Bueno 2012; Gicheru et al. 2009; Ashford 1996). Nevertheless, the effect of this pathogen on primate populations is still poorly known. So far, one study described antibodies and infection with Leishmania in Brazilian wild Sapajus using serology and molecular methods, and a report showed a fatal natural case of visceral leishmaniasis in a captive black-fronted titi monkey in Southeast Brazil (Callicebus nigrifrons), with the development of clinical signs and lesions that were typical of visceral leishmaniasis (Bueno 2012; Malta et al. 2010).

The infection rate for Trypanosoma cruzi in Sapajus flavius was 16%. Natural T. cruzi infection occurs in numerous Neotropical primate species, including Cebus. The lesions mentioned most frequently in naturally occurring T. cruzi infections in primates are myocarditis and generalized edema without necrosis or hemorrhage (Toft and Eberhard 1998). Changes in electrocardiographic patterns were reported for Cebinae (Monteiro et al. 2006; Toft and Eberhard 1998), and captive individuals in the United States have died from locally acquired T. cruzi infection (Bern et al. 2011). T. cruzi transmission can occur by the bite or oral ingestion of infected hematophagous triatomid bugs that inhabit palm trees or sugar cane (Shikanai-Yasuda et al. 1991). For example, infection in lion tamarins (Leontopithecus) in Southeast Brazil is related to oral transmission and correlates with cardiac disorders (Benchimol 2006; Monteiro et al. 2006). Sapajus flavius is a frugivore-insectivore and its diet includes a wide variety of fruits, seeds and arthropods, frogs, nestlings, and even small mammals (de Oliveira et al. 2015). Sugar cane is a fallback item in their diet (Valença-Montenegro 2011). Besides transmission by blood-sucking triatomids, the extractive and manipulative foraging typical of these monkeys indicates that they may also ingest infected triatomids. Supporting this potential source of infection, a human case of acute infection of T. cruzi was reported in Paraíba state via oral contamination due to eating cane juice contaminated with crushed infected bugs or possibly excretions from naturally infected marsupials (Shikanai-Yasuda et al. 1991).

We found the same prevalence of Trypanosoma cruzi infection for antibodies to Leishmania spp., and 62.5% of subjects (5/8) had antibodies against both Leishmania spp. and T. cruzi. This may be evidence of coinfection, or may be due to serological cross-reaction between Leishmania and other microorganisms (de Lima et al. 2010; Zanette et al. 2014). According to the Brazilian Ministry of Health Diseases Information System Notification (SINAN), 475 people in the state of Paraíba died of Chagas disease (T. cruzi infection) from 1996 to 2013; 142 were diagnosed with visceral leishmaniasis, and 319 with cutaneous leishmaniasis from 2007 to 2013 (Brazil, Ministério da Saúde 2016). These data show the circulation of these pathogens in Paraíba state, which corroborates the results mentioned in the preceding text for the population of Sapajus flavius.

We detected antibodies to Toxoplasma gondii in 12.5% of sera analyzed, and a higher chance of infection for females than males. Transmission of T. gondii can occur horizontally through ingestion of environmental oocysts in contaminated prey, soil, or water (Catão-Dias et al. 2013; Robert-Gangneux and Dardé 2012); thus this result can be explained by the monkeys’ foraging behavior. More studies are needed, however, to determine whether the sex difference relates to females having greater access to, or a greater predilection for, vertebrate hosts, or more contact with soil than males, where there is greater likelihood of contact with feline feces (felids are the only animals capable of shedding oocysts in their feces and transmitting the parasite to the environment). Cebus and Sapajus do not show high mortality from T. gondii infection, unlike callitrichids, which show acute to hyperacute disease (Catão-Dias et al. 2013). Nevertheless, Cebus and Sapajus can show mild and nonspecific clinical signs, such as abortion and premature birth, with high and persistent IgG antibody titers (Catão-Dias et al. 2013; Epiphanio et al. 2003; Ferreira et al. 2015; Resing and Oerke 2005; Santos et al. 2014; Toft and Eberhard 1998). This pathogen could therefore affect recruitment and the population growth rate directly.

Primates show three distinct responses to Toxoplasma gondii (Catão-Dias et al. 2013). Pattern I, in which the disease is markedly severe with mortality close to 100% mortality, is observed mainly in the Callitrichinae (Saguinus, Leontopithecus, and Callithrix); pattern II is characterized by severe outbreaks, with variable mortality (described for Cebidae family); and pattern III is characterized by the induction of high and persistent IgG titers, mild clinical presentation, and low mortality. Pattern III could explain our findings, especially the presence of high titers found in one individual (>3200), but we should also consider host susceptibility, contact rates, and time since exposure. Contact with T. gondii may have occurred via interactions with feline feces, because Sapajus flavius frequently goes down to the understory or to the ground during both travel and feeding (Catão-Dias et al. 2013). Wild and domestic cats can enter the forest and spread the protozoa, especially given that this area is fragmented and surrounded by agricultural farms (Molina et al. 2014). Another possibility is related to the feeding habits of S. flavius. Sapajus and Cebus have the most eclectic diets of all the Neotropical genera (Fragaszy et al. 2004). Their diets include a wide range of fruits and nuts and other plant parts, invertebrates, and small vertebrates. A study of feeding behavior of S. flavius in Gargau showed that 15% of the group’s diet was made up of prey animals, such as marsupials (Rodrigues 2013), and some small mammals act as a reservoir for T. gondii (Siqueira et al. 2013). Thus, T. gondii infection in the population of S. flavius studied was probably due to food contamination.

Infection surveillance in primates, including Neotropical species, is important for public health malaria screening and control programs (Duarte et al. 2008; Guimarães et al. 2015; Hayakawa et al. 2009). Some studies indicate that some species, principally of the genera Alouatta, Sapajus, and Cebus, can be reservoirs for malaria in extra-Amazonian regions and especially in Atlantic Forest remnants (Alvarenga et al. 2015; Fandeur et al. 2000). Our findings show that despite the low prevalence (4%) and low risk of infection by Plasmodium (0.05), P. brasilianum occurs in Sapajus flavius in Engenho Gargau, but little is known of its impact on the monkeys. In general, Plasmodium does not manifest severe clinical symptoms in primates; however, it may be debilitating and stress can lead to overt disease (Toft and Eberhard 1998). Slight anemia may be associated with low-grade parasitemia in some individuals, but they appear outwardly normal (Bueno 2012; Bueno et al. 2013; Deane 1992; Tazi and Ayala 2011; Toft and Eberhard 1998). Clinical signs reported in primates infected with malaria consist of irritability, cyclic fever, depression, listlessness, anorexia, and weight loss (Toft and Eberhard 1998). However, this pathogen may have epidemiological importance for public health, as Plasmodium species infecting primates, namely P. brasilianum and P. simium, are genetically and morphologically similar to those affecting humans, P. malariae and P. vivax, respectively (Escalante et al. 1995; Fandeur et al. 2000; Tazi and Ayala 2011). To date, the malaria cases in humans in the state of Paraíba that are reported by Brazil’s Ministry of Health are imported from endemic regions elsewhere (Brazil, Ministério da Saúde 2015). Plasmodium malariae is difficult to diagnose by examination of blood smears in humans, owing to low parasitemia and morphological similarity to P. vivax (Pina-Costa et al. 2014). Nevertheless, we found a Plasmodium-positive primate, which serves as an alert for the region and for people who live in the area, especially those in similarly fragmented and disturbed habitats.

There was a high prevalence of microfilariae infection in the population of Sapajus flavius in Engenho Gargau, with an infection risk of 0.46. More than 40 microfilariae species (Dipetalonema sp., Dirofilaria immitis, Tetrapetalonema sp., Wuchereria sp., and Edesonfilaria sp.) have been described in primates (Orihel and Seibold 1972). Of these, at least 14 species, 5 species of Dipetalonema and 9 species of Mansonella, occur in Neotropical primates, including marmosets, capuchins, squirrel monkeys, and owl monkeys (Toft and Eberhard 1998). The presence of microfilariae caused subclinical changes in infected Callithrix jacchus, despite the absence of clinical signs (Verona 2008), and some Dipetalonema species cause inflammatory responses in serous cavities (Orihel and Seibold 1972). However, few studies have examined the effect of this parasite on primates. More studies are necessary to understand which microfilaria species infect the population of S. flavius in our study site and if there is a risk for the study species or for public health.

Considering the small numbers of Sapajus flavius in the wild, and severely fragmented population, infectious diseases may pose an additional significant threat to their survival. The high density of S. flavius in this isolated small reserve (12 individuals/km2) is propitious for conservation, but may favor disease transmission. More comparative data are needed to determine how representative the incidence of pathogens in this population is for the study species, and the likelihood of an epizootic event. Knowledge of the prevalence of zoonotic infectious diseases is essential for future management actions, such as translocations and reintroductions.

In conclusion, our results confirmed the existence of microfilariae, Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp., and Plasmodium brasilianum (in order of infection risk) in Sapajus flavius in Engenho Gargau. We hope that our findings will inspire further long-term research on the clinical aspects and epidemiology of these pathogens on the demography of the few remaining populations of this species, contributing to efforts for their conservation as well to public health in the rural communities in the state of Paraíba, Brazil.

Data Availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.