Intestinal parasites are often responsible for diseases in animals living in confined environments such as sanctuaries, zoological gardens and wildlife rescue centers (1). Captive animals may be more susceptible to protozoan and helminth parasites with direct life cycles, which are more prevalent and prone to disseminate in confined conditions where the animals might be more stressed due to overpopulation and malnutrition, showing clinical signs as diarrhea and dehydration, and requiring veterinary care (2, 3). Parasite transmission mainly occurs through the fecal-oral route via direct contact with infected hosts (or their fecal material), or indirectly through the ingestion of contaminated water or food (4). In a confined environment, the low hygienic measures may lead to high levels of environmental contamination, and the handlers’ movements among different premises without safe and hygiene measures may contribute to the dissemination of such parasites inside and outside the workplace. Captive non-human primates (NHPs) may act as reservoirs for zoonotic parasites and the frequent use of pharmacological treatments may lead to the selection of resistance traits (1, 5). Therefore, confined environments are of great interest for parasitological studies, involving the One-Health concept.

Parasitological investigations have been carried out worldwide in zoological parks housing NHPs. For instance, Giardia duodenalis infections were reported in several NHPs hosted in 12 zoological gardens in China (6), while in a study carried out in Malaysia in three zoos hosting 69 specimens of NHPs, there were reported 21 species of intestinal parasites with a high prevalence of nematodes like Ascaris spp. and Oesophagostomum spp., only one animal positive to Blastocystis and no observation of Giardia spp. (7). Moreover, a large survey on intestinal parasites infecting NHPs hosted in two research centers in Brazil reported a large occurrence of Balantidium coli and Entamoeba sp. among protozoans, and a general low frequency of helminths, with predominance of Trichuris trichiura (8).

In Europe, parasitological surveys on NHPs have been performed in zoological enclosures such as the Dublin Zoological Garden (Ireland) (9), the Belgrade Zoo (Serbia) (10), the Kiev Zoo (Ukraine) (11), the Brno Zoological Garden (Czech Republic) (12), the Sofia Zoo (Bulgaria) (13), the Wroclaw Zoo (Poland) (14), the Košice Zoological Garden (Slovakia) (15), among others (13, 16). Nematodes (e.g., Ascaris sp., Trichuris sp., Strongyloides sp.) are the most common parasites detected, followed by cestodes and trematodes (13). Furthermore, G. duodenalis, Cryptosporidium hominis, Blastocystis sp., and Entamoeba dispar circulation between NHPs and their zookeepers has been identified in European zoological gardens, with the confirmation of zoonotic transmission events involving Blastocystis sp. and a highly suspected zoonotic transmission of C. hominis (4). Additionally, subcutaneous Taenia crassiceps cysticercosis in a ring-tailed lemur in a Serbian zoo has been reported (17).

In Italy, some surveys on intestinal parasites infecting NHPs living in zoological gardens have been conducted so far. In central Italy, Cryptosporidium sp. and Trichuris sp. have been found infecting Lemur catta at the Giardino Zoologico of Pistoia (18), while, at the Bioparco Zoological Garden of Rome, G. duodenalis has been reported infecting L. catta, and Entamoeba spp. was diagnosed in Cercocebus torquatus, Chlorocebus aethiops, Macaca fuscata, Mandrillus sphinx, Pan troglodytes, L. catta, and Pongo pygmaeus (19). In southern Italy, Trichuris sp., Strongyloides fuelleborni, and Cryptosporidium sp. infected Papio cynocephalus at the Fasano Zoo Safari, while G. duodenalis was found infecting L. catta, Cercopithecus mona, Alouatta caraya, Nomascus concolor, Colobus guereza, and Semnopithecus entellus in a zoological garden in the Benevento province (20). Moreover, Cyclospora was detected in P. troglodytes from a wildlife animal rescue center, and in Macaca fascicularis from an experimental primate research center (21). Eight taxa of intestinal parasites (Trichuris sp., Oesophagostomum sp., Entamoeba coli, Endolimax nana, Iodamoeba bütschlii, Chilomastix mesnili, B. coli, and Blastocystis sp.) were recorded infecting M. fascicularis in a biomedical research center (22).

Concerning necropsies carried out on dead captive NHPs, Trichuris sp. from Eulemur albifrons, and Strongyloides sp. from Macaca sylvanus have been found at the Natura Viva zoo (23), and Echinococcus granulosus from L. catta at a zoo in northern Italy (24). At the Bioparco Zoological Garden of Rome, larval forms of Taenia martis from L. catta (25), and adult Trichuris sp. from L. catta, M. fuscata and C. aethiops have been reported (26, 27). Larval forms of Mesocestoides sp. from Saguinus midas were collected at a wildlife recovery center (28).

Despite their importance in public health and NHPs welfare, the currently available information on intestinal parasites infecting captive NHPs in Italy is still limited to fragmented data. Thus, here we provide a survey on intestinal parasites circulating in NHPs hosted in two wildlife rescue centers and in one zoological garden in central Italy.

Fecal samples and adult nematodes were collected during 2020–2022, from NHPs living in confined environments in Italy.

The present study investigated the presence of intestinal parasites infecting NHP species living in captivity in Italy. Based on morphological analyses from fecal samples of NHPs living at Piano dell’Abatino Park, nine parasite taxa were identified, all of them presenting direct life cycles. However, due to the sampling from premises with multiple hosts, without tracing primate individuals during defecation, the precise estimation of epizootiological parameters such as prevalence, intensity and abundance was not possible.

In this study, Balantidium/Buxtonella sp., E. coli, and I. bütschlii were the most frequently detected parasites. Most parasite taxa identified in this study have been previously reported in captive NHPs in Europe, as is the case of Trichuris sp., Oesophagostomum sp., Balantidium sp., Giardia sp., E. coli and I. bütschlii (19, 22). Giardia sp. was found in only one individual, and taking into account that this parasite is usually more frequently found in studies on NHPs in zoos, we should consider that in this case it may not be a true infection but cysts accidentally ingested from the environment. Additionally, due to the intermittent shedding of cysts, in some cases it is necessary the examination of fecal samples on consecutive days (47), and in this study no sampling on consecutive days was performed.

Balantidium/Buxtonella sp. was found infecting five of the seven NHP species sampled. Pigs are the main reservoir host of Balantidium, while rodents and NHPs may function as alternative reservoir hosts (37). Wild boars are also present at the study site, but in a small number and located in a separate facility from the NHPs. Thus, in this case swine are unlikely to participate in the transmission cycle (even if it cannot be definitively ruled out due to handler’s movements, or by the rain/wind that can easily transport the cysts from one facility to another), while wild rodents are very common within the primate enclosures. For future studies it is highly recommended the use of integrative taxonomy accounting for morphological characteristics combined with molecular approach for species identification, as it has been demonstrated how misleading the cyst morphology-based diagnostics of Balantidium sp. and Buxtonella sp. can be, leading to ambiguity in the epidemiology of these infections (38). In Italy, both Buxtonella sp. and Balantidium sp. have been reported, for instance Buxtonella sulcata infecting cattle in central Italy (39), and Balantidium coli infecting swine in the south of the country (40). Given the uncertainty in the taxonomic assignment, we have chosen to indicate this finding as Balantidium/Buxtonella sp.

Molecular testing should be also recommended for the optimal identification of D. fragilis (41). In our survey, D. fragilis-like was found in samples from M. fascicularis, and this parasite was recently reported infecting free-ranging M. fascicularis in Indonesia (42). Additionally, future molecular studies to determine the species of the strongyliform larvae found infecting S. apella and M. sylvanus are required, in particular to confirm or exclude the presence of Strongyloides sp., a zoonotic parasite of paramount relevance, reported in Italy both in dogs and humans (43). Capillaria sp. was found in one sample of a tufted capuchin monkey, however, the molecular approach for species identification gave negative results, probably due to difficulties in the genomic DNA isolation and/or PCR inhibitors. Capillaria sp. has been reported infecting different NHP species (44), including capuchin monkeys: C. capucinus in Panama (45) and C. albifrons in Ecuador (46). However, these reports were based on microscopy, thus, the use of molecular testing is also here suggested for the identification at species level to elucidate the zoonotic potential.

Trematodes, cestodes and acanthocephalans have been previously reported in free-ranging primates (48). Considering that the methods used in this study allow the detection of these parasite taxa, the lack of findings could be related to the different diets and habits of captive individuals compared to free-ranging NHPs.

Given the close phylogenetic relationship between human and NHPs, continuous parasitological surveys on captive primates should be encouraged for the monitoring of zoonotically transmitted parasites, for instance within conservation and management of threatened primate species, and in the recovery of traded NHPs. In the present study, four out of the seven NHP species under investigation are considered endangered (EN) or vulnerable (VU) by the IUCN, and while no animals hosted at Piano dell’Abatino Park showed clinical signs or symptoms of gastrointestinal origin, two animals died at the Bioparco Zoological Garden of Rome and in the CREMWA, probably because of Trichuris infection. So far, Trichuris spp. have been reported by morphological and/or molecular characterization in the following Macaca species: the Japanese macaque (26, 27, 32), the Barbary macaque (34, 35) and the long tailed macaque (22), the latter investigated only in terms of eggs presence in stool samples without any molecular characterization. Such studies revealed the presence of two separated taxonomic entities able to infect Japanese macaques living in confined environments, one specific to this host, and one shared also with other primates (26, 27, 32). Analogous molecular results were obtained also regarding the Barbary macaque hosted in the Castellar Zoo (Spain), infected by two genotypes within the T. trichiura lineage, supported also by morphological data (35).

Here we provide for the first time morphological and molecular data of T. trichiura infecting M. fascicularis, to share with the scientific community for comparative purposes. We obtained reliable data from the analyses of adult Trichuris infecting the dead long tailed macaques hosted at the CREMWA, and despite no molecular data were obtained from fecal samples from the animals hosted at Piano dell’Abatino Park, the eggs size observed in the two sample sites were overlapping, suggesting T. trichiura circulation. The long-tailed macaque from the CREMWA analyzed in the present study lived in a colony of around 30 individuals (49), thus the finding of Trichuris infection may represent a high risk for the other macaques belonging to the colony. It is also a concern, taking into account that M. fascicularis has been recently listed as an endangered species with a decreasing population trend, according to the International Union for Conservation of Nature (IUCN) (50), mainly due to the high demand in the national and international trade, and the hunting for subsistence. Moreover, there is a risk for handlers and visitors in terms of zoonotic transmission. Therefore, it is necessary the constant monitoring to trace the presence of eventual parasitic species of zoonotic interest, in both confined environments and in native areas where NHPs live near or in close contact with humans.

In conclusion, this parasitological survey revealed the presence of potentially zoonotic parasites circulating in NHPs in Italy, providing useful information for the formulation of their management and care plans, and for the elaboration of safety measures for visitors and animal keepers. Regular parasitological surveys in captive NHPs using both microscopy and molecular analyses should be recommended, in order to monitor the impact of parasitosis on the health status of captive NHPs and to properly assess the potential zoonotic transmission risk.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because Biological material taken from alive animals was not invasively collected, while material taken during necropsies was authorized by the ethical approval of the Istituto Zooprofilattico Sperimentale Lazio e Toscana.

SR: Conceptualization, Funding acquisition, Methodology, Writing – original draft. SC: Conceptualization, Formal analysis, Methodology, Writing – original draft. MMDF: Formal analysis, Writing – review & editing. CD: Resources, Writing – review & editing. FB: Supervision, Writing – review & editing. NC: Resources, Writing – review & editing. SD’A: Conceptualization, Writing – original draft, Writing – review & editing.