Gastrointestinal parasites in wild and exotic animals from a zoo in the State of Bahia, Brazil - first record

Parasitic infections can be a serious health problem for wild animals kept in captivity, however, coproparasitological assessments in Brazilian zoos are scarce and spaced. Therefore, this study aimed to evaluate the occurrence of endoparasites in the feces of wild and exotic captive animals in the zoo of Matinha Municipal Park, Itapetinga, Bahia, Brazil, the only zoo in the interior of Bahia, through the Mini-FLOTAC® technique, providing subsidies for the diagnosis and therapeutic treatment of parasitized animals. From May to August 2022, 124 stool samples from 35 species of reptiles, birds and mammals were collected. Analyzes were performed using the Mini-FLOTAC® technique in combination with Fill-FLOTAC®. The results show that 70.97% of the samples were positive for at least one gastrointestinal parasite. Birds (76.7%; 33/43) were the most parasitized animals. Twenty-seven taxa of gastrointestinal parasites were identified, whether cysts, oocysts or eggs, being 8 protozoans and 19 helminths, with a predominance of coccidia, Oxyurideae and Angusticaecum sp. for reptiles, coccidia, Ascaridia spp., Heterakis spp. and Strongyloides spp. for birds, coccidia, Ancylostomatidae, Strongylida and Strongyloides spp. for mammals. In summary, the results presented reveal the importance of periodically carrying out coproparasitological examinations in zoos, in order to subsidize interventions by the technical team to promote the health and well-being of animals. This work constitutes the first publication on the coproparasitological evaluation of animals from a zoo in the state of Bahia.


Introduction
A zoo is defined as a legal entity enterprise, consisting of a wild animals' collection kept alive in captivity or in semifreedom and exposed to public visitation, to meet scientific, conservationist, educational and sociocultural purposes. (Brasil, 2015). Zoos play an important role in welcoming and conserving endangered species or individuals unable to survive in the wild (Silva et al., 2019). Orsini and Bondan (2006) state that the long period of captivity causes functional changes, as a result of somatic (sounds, images and strange odors, among others), psychological, behavioral and mixed stressors (malnutrition, intoxication, action of infectious and parasitic agents, among others), which can make animals weakened and lacking the physical and psychological skills necessary for survival. Research, Society andDevelopment, v. 11, n. 13, e19111334959, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i13.34959 3 Parasitism can be defined as an obligate trophic association between individuals of two species in which one (the parasite) obtains its food from a living organism of another species (the host). This symbiotic relationship is very common in nature, playing an important role in ecosystems, regulating host populations, stabilizing food chains and structuring animal communities (Atkinson, 2008).
Parasitic infections can be a serious health problem for wild animals kept in captivity, and the morbidity and mortality of infections are dependent on the host species, the parasite and the parasite load, nutritional status, immunocompetence and physiological conditions of the host. The weaknesses in the proper management for each species pose a great risk to the health of the animals (Santos et al., 2015). Lima (2018) points out that environmental and ecological changes combined with the proximity between humans, domestic and wild species offer numerous opportunities for the emergence of interspecific interactions, which contribute to the spread of numerous parasitic zoonoses.
For Capasso et al. (2019) and Guo et al. (2021) animals raised in restricted environments, like zoos, are highly susceptible to gastrointestinal infection by helminths and protozoans. Zoos are environments with high contamination by parasites. These authors proved that the Mini-FLOTAC® ® technique in combination with the Fill-FLOTAC® can be used not only for the rapid diagnosis of parasitic infections in zoos, but also for monitoring control programs quickly and reliably.
Thus, the present study aimed to evaluate the occurrence of endoparasites in the feces of wild and exotic captive animals in the zoo of Matinha Municipal Park, Itapetinga, Bahia, Brazil, the only zoo in the interior of Bahia, using the Mini-FLOTAC® technique, providing subsidies for the diagnosis and therapeutic treatment of parasitized animals. It is worth mentioning that this is the first work carried out in a zoo in the Bahia state.

Study area
The Matinha Municipal Park (Figure 1), created by municipal decree nº 860 of October 11, 1973 and law nº 528 of December 19, 1991, is located in the urban perimeter of the municipality of Itapetinga, southwest of Bahia, covering 24 hectares of which 10 hectares constitute a remaining area of the Atlantic Forest Biome surrounded by the Catolé Grande River up to the bridge next to the Bus Station (Kulka, 2014).
The park aims to preserve and conserve the representation of the Atlantic Forest, serving as a refuge for many species (Itapetinga, 2004). It houses a zoo, whose squad has species of birds, mammals and reptiles from the Center for the Triage of Wild Animals -CETAS (animals that are victims of trafficking and that often are no longer able to return to nature), in addition to exotic specimens from other zoos or breeding sites, thus constituting the only environmental protection area in the municipality and the only zoo in the interior of Bahia (Freitas et al., 2007). Research, Society andDevelopment, v. 11, n. 13, e19111334959, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i13.34959

Ethical aspects
The project was submitted to the Ethics Committee on the Use of Animals in Research of the Federal University of Bahia, Campus Anísio Teixeira of the Multidisciplinary Institute in Health (UFBA) (CEUA -IMS/CAT -UFBA) and approved (Opinion No. 104/2022).

Sampling
This work is descriptive quantitative research (Dalfovo et al., 2008;Pereira et al., 2018). Thus, 124 stool samples were collected from captive animals from the zoo at Matinha Municipal Park, Itapetinga, Bahia, Brazil between May and August 2022. 15 species of birds, 14 of mammals and 6 of reptiles were sampled (Table 1). Research, Society and Development, v. 11, n. 13, e19111334959, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i13.34959 Table 1 shows the number of wild and exotic animals in captivity, with their respective orders and families, and the number of fecal samples collected.
The samples examined in this study were obtained by the keepers while cleaning the enclosures, prioritizing the collection of individual fresh fecal pellets. Each sample was defined as a sample of feces containing an amount greater than or equal to 2 grams, spontaneously eliminated by the animals, collected individually or in pools on the floor of the enclosure so as not to stress the animals. The definition of pool adopted in this work follows Fagiolini et al., (2010) and Capasso et al (2019), which consists of 2 grams of each individual fecal sample.
The fecal samples were placed in isothermal boxes (2 to 8ºC) and immediately transported to the Zoology Laboratory of the Multidisciplinary Institute in Health, Campus Anísio Teixeira of the Federal University of Bahia, in Vitória da Conquista, Bahia, for analysis.
The fecal samples were processed using the Mini-FLOTAC® technique, following all the steps and guidelines as per the original description of the technique, using two flotation solutions: FS2 (Sodium Chloride, specific gravity SG = 1,200) and FS7 (Zinc Sulfate, SG = 1,350), and each sample was analyzed twice (Cringoli et al. 2017).
The preparations were examined under a binocular Optical Microscope at 100X and 400X magnifications.
Photomicrographs and measurements of the parasitic structures were performed with the aid of a digital camera and micrometric eyepiece, respectively. Fecal samples were considered positive when at least one evolutionary form of a parasite (egg, cyst and/or oocyst) was detected (Barbosa et al., 2019).

Data analysis
Data were tabulated and analyzed using the GraphPad Prism® version 5 software. The results were expressed as the arithmetic mean number of eggs/oocysts/cysts per gram (EPG/OPG/CPG) of feces, in addition to the minimum and maximum values (Capasso et al., 2019). Prevalence was estimated by dividing the number of positive samples by the total number of samples collected from each group of animals under study (Barbosa et al., 2019).
In total, 27 taxa of gastrointestinal parasites were identified, whether cysts, oocysts or eggs, being 8 protozoans and 19 helminths (Table 2 and Figure 3). Among the protozoans, 12.5% were amoeba, 12.5% ciliated (both identified at the generic level) and the vast majority were coccidia, about 75% (5 taxa were identified to genus and the others grouped as nonsporulating coccidia). As for helminths, 10.5% were Trematoda flatworms, one of them identified at the genus level and the other only in the Trematoda class. All other helminths belonged to Nematoda, representing 89.5%. Of these, 11.8% were identified to the family, 11.8% to the order, 5.9% to the superfamily, 64.7% were identified at the genus level and 5.9% at the species level (Table 2).
and Strongyloides spp. in Artiodactyla (Table 2) Quantitatively, the parasite intensity expressed in eggs, cysts and oocysts per gram of feces (EPG, CPG and OPG) detected in the feces of reptiles from the Matinha Municipal Park is presented in Table 3. For C. carbonaria, Oxyuridae eggs form the most abundant, ranging from 10-1060 EPG. For the snakes B. constrictor and M. reticulatus oocysts of the coccidian Caryospora spp. was the most abundant parasite, ranging from 0-3500 OPG and 0-1200 EPG, respectively. In the lizard S. merianae, eggs of Strongyloides spp. were the most abundant, representing about 170 EPG (Table 3).    (Table 4).
In the feces of P. cristatus there was an absolute predominance of protozoans, reaching the highest parasitic densities among all the birds studied, being the oocysts of Eimeria spp., the most abundant, ranging from 0-44,380 OPG, followed by non-sporulated coccidia with varying densities from 0-1520 OPG. The second bird species that presented the highest densities of parasites per gram of feces was S. camelus, being Alaria sp. (0-2090 EPG) and non-sporulating coccidia (0-1200 OPG) were the most abundant parasites (Table 4).  (Table 4).

Discussion
Most fecal samples from animals from the zoo at Matinha Municipal Park, Itapetinga, Bahia were positive for gastrointestinal parasites. This same pattern was recorded in several studies with captive animals, whether in zoos or CETAS (Hofstatter & Guaraldo, 2015;Barbosa et al., 2019;Oliveira et al., 2020;Batista et al., 2021). Among the zoological groups evaluated, birds and mammals were more parasitized than reptiles. This same pattern was detected by other authors (Batista et al., 2021;Mewius et al., 2021).
The most abundant parasites in fecal samples of C. carbonaria (Oxyuridae eggs), B. constrictor and M. reticulatus snakes (Caryospora spp. oocysts) and S. merianae lizard (Strongyloides spp eggs) are commonly the most representative recorded in other works (Rataj et al., 2011;Souza et al., 2014;Rom et al., 2018). According to Ruivo (2019), the presence of oxyurids is very frequent in the lumen of the large intestine of herbivorous reptiles, being considered beneficial for the host by improving the passage of food content through the intestinal tract and contributing to the regulation of the microbiota of the cecum, through ingestion of bacteria by the parasites, however it can cause intestinal obstructions (Troiano, 2018). Infections caused by Strongyloides spp. in reptiles they can trigger asymptomatic conditions or anorexia, weight loss, lethargy, enteritis, diarrhea, urethral obstructions, nephritis, which can lead to their death (Ruivo, 2019). The genus Caryospora is found in the intestinal mucosa of snakes, lizards and turtles and its infection is usually asymptomatic (Schneller & Pantchev, 2008), but can cause destruction of the intestinal, biliary and renal epithelium with fibrosis and ulcerations (Troiano, 2018).
In most birds, coccidia were very abundant, and this pattern is commonly recorded in other studies (Hofstatter & Guaraldo, 2015;Lima et al., 2017;Oliveira et al., 2020). Coccidiosis is rare in free-ranging birds and is usually related to captive breeding, crowding or stress, where infected birds usually do not show any clinical signs in low-intensity infections, as coccidia destroy a limited number of epithelial cells, which can be replaced quickly. However, at high parasite densities, many cells are destroyed, leading to reduced food and water consumption, decreased intestinal absorption, hemorrhage, lack of appetite, weight loss, fall, loss of coordination, ruffled feathers and decreased egg production (Atkinson et al., 2008).
The parasitic intensity of coccidia recorded in fecal samples of P. cristatus was the highest when compared to all species of animals sampled in this work. Peacocks commonly have high densities of coccidia, as indicated by several studies in the literature (Rodrigues et al., 2020;Lozano et al., 2021;Yadav et al., 2021). Coccidia are spread by water and food contaminated by oocysts, affecting several species of birds and even mammals, such as man, and may be a zoonosis. Infections caused by coccidia can trigger severe damage to birds, promoting diarrhea, dehydration, apathy, reduced reproductive rate, weight loss and death (Marietto-Gonçalves et al., 2009). These results point to the need for the periodic use of anti-coccidial agents and the intensification of cleaning and disinfection of the enclosures, drinkers and feeders, in order to prevent the spread of this parasite to the zoo animals.
Samples of S. camelus, exhibited high densities of Alaria sp. and non-sporulating coccidia. Alaria sp. are trematode parasites that can cause asymptomatic conditions up to diarrhea and hematochezia (Batista et al., 2008). Its presence has already been recorded in several carnivores (canids, felids, mustelids and procyonids) (Ruas, 2005) and even birds, including S.camelus (Batista et al., 2008). Even when in large numbers, the presence of parasites in these birds may not be accompanied by characteristic clinical signs (Batista et al., 2008).
The animals of the Carnivora order studied exhibited the highest densities of parasites among all the mammals studied, with Ancylostomatidae being the most abundant for P. concolor and P. leo. This parasite was recorded by other studies that evaluated felid parasites (Srbek-Araujo et al., 2014;Gressler et al., 2016;Solórzano-García et al., 2017;Silva et al., 2021). The main adverse effects of hookworms for their hosts (humans, domestic animals and wild species) are anemia, growth retardation, secondary bacterial infections and mortality (Seguel & Gottdenker, 2017).
P. flavus showed the highest abundance of Eimeria spp. In the literature, the few studies on parasites with this mammal species generally report the presence of helminths (Taira et al., 2013;Tokiwa et al., 2014). Barbosa et al. (2019) found non-sporulating coccidian oocysts for the P. flavus sample from the Rio de Janeiro Zoo, being the record of Eimeria spp.
of the present article the first record of this coccidian genus for Jupará. The parasite recorded in the H. hydrochaeris sample (Trichostrongyloidea) was also recorded in another study carried out with capybara populations in seven cities in the state of São Paulo (Souza et al., 2021). According to Souza et al., (2021), identification at the genus or species level based only on Trichostrongyloidea eggs is impossible, however, necropsy-based studies point to the parasites Viannella hydrochoeri and Hydrochoerisnema anomalobursata as the specific trichostrongyloids of capybaras, with V. hydrochoeri the most likely parasite that affects capybaras in natural and man-made areas.
In the sample of T. terrestris, there was a record of 140 EPG of Strongylida, the only parasite found for this mammal.
These results differ from other analyzes carried out with this species, where Batista et al (2021) found only protozoans in the tapir samples, with a predominance of trophozoites and cysts of Balantidium sp., and non-sporulated oocysts of coccidia.
For Batista et al. (2021), the physical proximity of animals in zoos makes parasitic infections inevitable, which can be aggravated by the immune status of the host, whose circumstances of confinement and stress weaken the animal, thus aggravating its survival. Furthermore, some of these parasites can be zoonotic, impacting the health of zookeepers and workers (Iatta et al., 2020). Redoubled care with the hygiene and deworming of the animals are necessary in order to prevent the transmission of the parasites within the studied zoo.
The increase in the breeding stock promoted by the acquisition of new specimens and species at the zoo, may have contributed to the increase in the parasitic community, since some specimens obtained from other zoos, breeding sites and/or sorting centers were parasitized and the lack of establishment of an effective deworming and quarantine protocol can lead to contamination of individuals residing in the zoo. Oliveira et al. (2022) carried out a physical-chemical and microbiological evaluation of the water used by the animals of the zoo in the Matinha Municipal Park, being suitable for the watering of animals, but they recorded nonconformities in the microbiological parameters (Escherichia coli presence) for most of the animals' enclosures. These data indicate fecal contamination in the water of the zoo's enclosures. Therefore, we can infer that there is probably parasitic contamination in the water used for drinking, which may serve as a means of dissemination among the species that live in each enclosure. helminths were identified, with a predominance of coccidia, Oxyurideae and Angusticaecum sp. for reptiles; coccidia, Ascaridia spp., Heterakis spp. and Strongyloides spp. for birds; and coccidia, Ancylostomatidae, Strongylida and Strongyloides spp. for mammals. In summary, the results presented reveal the importance of periodically carrying out copro-parasitological examinations in zoos, in order to support interventions by the technical team to promote the health and well-being of animals.

Conclusion
It is worth mentioning that new specimens must undergo a period in quarantine, before relocating them in the enclosures, and the performance of these exams, once again, become essential for clinical diagnosis and establishment of appropriate therapeutic conduct for each case, with administration of specific antiparasitic drug for each type of parasite, whether they are protozoans or helminths. Therefore, it is evident that routine copro-parasitological assessment of captive animals in zoos effectively contributes to diagnosis and improvements in park management. This work constitutes the first publication on the coproparasitological evaluation of animals from a zoo in the Bahia state.