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Investigation of Cryptosporidium spp. in breeding nucleus of capuchin monkeys (Sapajus spp.)

Animals of the primate order are highly susceptible to parasitic infections due to their social organization, often in groups and interaction between individuals, facilitating the transmission of pathogenic agents, and may be even more prevalent in areas modified by anthropic action. The occurrence of the protozoan Cryptosporidium spp. in wildlife animals in captivity or free-living it characterizes great importance of Public Health mainly for its zoonotic potential. This parasite has a cosmopolitan distribution, causes gastrointestinal infection, and its transmission occurs via the fecal-oral route, either through water or through contaminated food. Cryptosporidiosis affects mammals, birds, reptiles and fish, this significant zoonotic potential, in addition to being directly related to the living conditions of the host and the quality of the environment. We investigated the occurrence of Cryptosporidium spp. in primates located Research, Society and Development, v. 10, n. 11, e310101119459, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19459 2 at the Tufted Capuchin Monkey Procreation Center (School of Dentistry of Araçatuba UNESP). Fecal samples from 49 asymptomatic primates of the species Sapajus spp. species were collected below their individual cages, the soil was lined with a plastic bag, thus hindering environmental contamination. The search for oocysts was carried out using the technique of Negative Malachite Green Staining, finding negativity in all samples examined, which suggests the absence of the protozoan Cryptosporidium spp. in primates studied in the breeding center of capuchin monkeys (Sapajus spp.), noting that they were asymptomatic.

at the Tufted Capuchin Monkey Procreation Center (School of Dentistry of Araçatuba -UNESP). Fecal samples from 49 asymptomatic primates of the species Sapajus spp. species were collected below their individual cages, the soil was lined with a plastic bag, thus hindering environmental contamination. The search for oocysts was carried out using the technique of Negative Malachite Green Staining, finding negativity in all samples examined, which suggests the absence of the protozoan Cryptosporidium spp. in primates studied in the breeding center of capuchin monkeys (Sapajus spp.), noting that they were asymptomatic. Keywords: Coccidiosis; Primates; Zoonosis.

Introduction
Cryptosporidiosis, one of the main causes of diarrhea in vertebrate animals, disease caused by protozoa of the genus Cryptosporidium spp. (OIE, 2015;Madrid et al., 2015).
The most important species to public health are Cryptosporidium parvum and Cryptosporidium hominis due to their high zoonotic potential (Zahedi et al., 2016). However, 38 species of Cryptosporidium and more than 70 genotypes were identified by molecular techniques (Feng et al., 2018).
This parasite is related to cases of enterocolitis in primates (Levine, 1984). The most frequent symptoms of cryptosporidiosis are characterized by moderate to severe diarrhea with fever, vomiting and abdominal pain (Amaral et al., 2014;Kostopoulou et al., 2015).
Using Kinyoun's permanent staining technique, a 62.5% prevalence of Cryptospuridium spp infection was found in primates (Vila Alvarez, 1994). On the other hand, few studies have been conducted to examine the ecology and epidemiology of this pathogen in places with a tropical climate, in which there is a great direct or indirect interaction between domestic, wild animals and man (Parsons et al., 2015).
Due to the high prevalence of the agent in primates (Vila Alvarez, 1994)

Study location
The experiment was carried out in Capuchin monkeys (Sapajus spp.), housed at the Tufted Capuchin Monkey Procreation Center (School of Dentistry of Araçatuba -UNESP) São Paulo, Brazil.
The monkeys were kept in individual stainless steel cages, with sides of perforated coin-type plate, separated by 8 cm and one and a half meters from the floor. The bottom of the cages consisted wire mesh and the other half a continuous stainless steel plate. The treated water supply for the animals were individualized, with a continuous flow, therefore the volume not consumed by the animals was discarded in a channel located on the floor of the room.

Population
A total of 49 asymptomatic monkeys (Sapajus spp.) were part of the study, 26 females and 23 males, among them, three were young, 30 adults and 16 elderly. All were bred in the facility.

3 Ethics Committee
This study was approved by the Ethics Committee on Animal Experimentation of the Faculty of Dentistry (FOA) of the São Paulo State University (UNESP) nº 00589-2018 as well as by the Biodiversity Information and Authorization System-SISBIO nº 65508-1.
Source: Personal archive (2021). The Figure 3 illustrates a positive slide for the endoparasite Crypstosporidium spp. and Figure 4 indicates a negative lamina for the same parasite. We note that the color of the bottom of the slide is green, while the parasite stands out without color.

Results and Discussion
In our study, through the parasitological analyzes Negative Staining of Malachite Green and Kinyoun, no Cryptosporidium spp. oocysts were observed in the population of capuchin monkeys of the species Sapajus spp..
The absence of oocysts in the feces of the population studied can be explained by the circumstances to which the animals were subjected, housed individually in cages, with space between them, individual water source and continuous flow, little or no contact with the animal's own feces or of other individuals, occurring minimal anthropic contact, only for nutritional management and maintenance. Such findings suggest that direct contact is an important condition for transmission to occur, as well as the quality of the water that animals have access to.
However, animals of the Primate Order are highly susceptible to parasitic infections, due to their social organization, in groups of intense interaction between individuals, facilitating the transmission of pathogens, which may be even more prevalent in areas modified by anthropic action (Trejó-Macias et al., 2007).  There are limited studies that cross-examine the ecology and epidemiology direct or indirect contact between wild and domestic animals and humans (Parsons et al., 2015).
Nonetheless, in a study carried out using PCR (Polymerase Chain Reaction) as a diagnostic method, it was found that 4.30% of humans, 16.00% of primates and 9.60% of domestic animals were infected with Cryptosporidium. Although the condition of contact between primates and humans was varied, there was direct contact between animals (Parsons et al., 2005), showing the importance of interaction between individuals for the dissemination of the parasite.
It is important to note that the infectious evolutionary forms of Cryptosporidium spp. are highly resistant to environmental factors and can contaminate food, water and soil (Madrid et al., 2015). This fact was proven in a study in which 18 samples of four species of captive primates were analyzed by the centrifugal-flotation method with zinc sulfate. From the results obtained in this study, 10 of the animals were confirmed positive for the protozoan. The water consumed by them was also analyzed and a high number of oocysts was detected (Silva et al., 2008).
Also, in another study carried out in Paraná, there was a prevalence of 58.06% for this protozoan in the population of primates in question and, among all the pathogens researched using the modified Ziehl-Neelsen technique, Cryptosporidium presented a higher level of infection, with 49.15% of occurrence. According to the author, this result can be explained by the interaction of animals with keepers, veterinarians, park visitors and each other, in addition to the source of water and shared food (Snak et al., 2015), reinforcing its zoonotic potential as well as the environmental source of transmission and spread of this infection.

Conclusion
In our study, through the parasitological analysis of the Negative Malachite and Kinyoun Green Staining Technique, there was no positivity of Cryptosporidium spp. in the population of capuchin monkeys of the species Sapajus spp. housed in the breeding center of capuchin monkeys (Sapajus spp.). More research are encouraged, especially with wild animals in captivity or free-living to elucidate epidemiological points, transmission, zoonotic and pathogenic species causing damage to both primates and humans, since when they are in captivity the transmissibility among this public is longer range.