Prevalence of gastrointestinal parasites in captive non-human primates of zoological gardens

Introduction

In 2019, a colony of spider monkeys at a European zoo lost four juveniles within three months to overwhelming Strongyloides hyperinfection a mortality event that could have been prevented by routine faecal screening [1] . That case is far from unique. Captive non-human primates (NHPs) harbour a remarkably diverse gastrointestinal parasite fauna, a legacy of their wild ancestors' exposure combined with the amplification effects of confined living spaces, shared substrates, and group social dynamics that promote coprophagy and faecal-oral cycling [2] . The parasite assemblages found in zoo primates span both helminths and protozoa. Nematodes of the genera Strongyloides, Trichuris, and Oesophagostomum are among the most frequently reported helminth infections, capable of causing chronic diarrhoea, weight loss, anaemia, and in severe cases fatal peritonitis from nodular perforation [3] . Protozoan infections, particularly Entamoeba histolytica / dispar, Giardia duodenalis, and Balantidium coli, carry additional zoonotic significance because primate keepers working in close contact with infected animals face occupational exposure risk [4] .

Prevalence estimates from zoo primate collections vary enormously from 20% in well-resourced European facilities with concrete-floored enclosures to over 80% in tropical open-range sanctuaries with earth substrates [5, 6]. Several factors drive this variation: host taxonomy (great apes tend to harbour more diverse parasite communities than smallerbodied prosimians), enclosure design (soil floors retain infective eggs and larvae far longer than impervious surfaces), group size (larger groups amplify transmission rates through density-dependent contact), and climate (warm, humid conditions accelerate embryonation and larval development outside the host) [7] . Japan's zoological gardens house one of Asia's largest and most taxonomically diverse captive primate collections, including endangered species held under cooperative breeding programmes. Yet published parasitological data from Japanese zoos are limited to a handful of single-facility case reports, mostly from the 1990s, that did not apply modern diagnostic methods such as coproantigen ELISA for protozoa or molecular speciation [8,9] . The Kansai region home to Osaka, Kobe, and Kyoto contains three major municipal zoos whose primate collections together encompass over 30 species across all four taxonomic groups (great apes, Old World monkeys, New World monkeys, and prosimians). Surveying these three facilities simultaneously offers the opportunity to compare prevalence across different enclosure designs, management regimes, and microclimate conditions within a single geographic area. From a practical zoo management standpoint, parasitological surveillance data serve three purposes. First, they identify which host species carry the highest parasite burdens and therefore need the most frequent screening. Second, they reveal which parasite genera dominate in each facility, guiding anthelmintic and antiprotozoal drug selection. Third, they expose seasonal peaks that allow prophylactic treatment to be timed for maximum effect [10] . Without baseline data, antiparasitic interventions are necessarily reactive rather than preventive, and subclinical infections that erode body condition and reproductive success go undetected until clinical disease or death forces attention. This research was designed to fill the surveillance gap for Japanese zoo primates by addressing four objectives: (a) determine the overall and species-specific prevalence of gastrointestinal helminths and protozoa in captive NHPs across three Kansai-region zoological gardens, (b) compare prevalence among the four main primate taxonomic groups, (c) characterise seasonal fluctuations in parasite prevalence and intensity over a full calendar year, and (d) compare parasite burden across the three facilities in relation to enclosure type and management practices.

Material and Methods

Research Area Description

The three participating zoological gardens are located within a 75 km radius in Japan's Kansai region.

Material

The research population comprised 287 individually identified NHPs belonging to 34 species across four taxonomic groups: great apes (chimpanzees, Bornean orangutans, western lowland gorillas; n=43), Old World monkeys (Japanese macaques, mandrills, De Brazza's monkeys, hamadryas baboons, and others; n=113), New World monkeys (spider monkeys, capuchins, marmosets, tamarins, and others; n=82), and prosimians (ring-tailed lemurs, ruffed lemurs, slow lorises; n=49). Animals were enrolled if they had been resident at their facility for ≥90 days and had not received anthelmintic treatment within the preceding 60 days. Fresh faecal samples were collected within two hours of defaecation, labelled with individual identity (all animals were microchipped), placed in 10% formalin for preservation and a paired fresh aliquot for flotation, and transported at ambient temperature to the Osaka Research Center for Wildlife Health parasitology laboratory. Sampling occurred monthly from April 2022 through March 2023, yielding up to 12 samples per individual (total samples processed: 3,186). The research protocol was approved by the Osaka Research Center for Wildlife Health Institutional Animal Ethics Committee (Approval No. ORCWH-IACUC/2022-011, dated 8 March 2022) and endorsed by the animal welfare committees of all three participating zoos.

Methods

Each sample was processed by four complementary techniques. (1) Direct wet-mount smear: a small faecal aliquot was mixed with saline and examined at ×100 and ×400 for motile trophozoites. (2) Sodium chloride flotation (specific gravity 1.20): 3 g faeces in saturated NaCl, centrifuged at 1,500 × g for 5 min; the surface film was examined for helminth eggs. (3) Formalin-ethyl acetate sedimentation: 1 g formalin-fixed faeces processed with 10% formalin and ethyl acetate, centrifuged; the sediment was examined for heavy eggs and protozoan cysts. (4) Modified Ziehl-Neelsen staining for Cryptosporidium oocysts. Parasite identification to genus or species level followed standard morphometric keys [11] . For protozoa, Giardia and Cryptosporidium were additionally confirmed by coproantigen ELISA (IDEXX SNAP® Giardia and Cryptosporidium kits). Quantitative egg counts were performed by the McMaster technique (analytical sensitivity 50 eggs per gram, EPG). An animal was classified as positive if any parasite stage was detected in any monthly sample. Prevalence was calculated as the proportion of positive individuals. Data were analysed in R 4.3.1. Chi-square tests compared prevalence among taxonomic groups, facilities, and seasons. Logistic regression assessed the effect of host group, facility, season, sex, age class, and enclosure type on parasite positivity. Significance was set at p<0.05.

Biodiversity Assessment

Parasite species richness (number of distinct parasite taxa per individual) and Shannon diversity index (H′) were calculated for each primate taxonomic group and each facility to quantify the breadth of the parasite community. Great apes harboured the highest mean species richness (2.7±1.1 taxa per positive individual) and Shannon index (H′=1.42), followed by Old World monkeys (2.1±0.9; H′=1.28), New World monkeys (1.4±0.6; H′=0.91), and prosimians (1.1±0.4; H′=0.64). Across facilities, Osaka Tennoji Zoo showed the greatest parasite diversity (H′=1.51), consistent with its older soil-based enclosures that sustain a broader range of environmental transmission stages. Kobe Oji Zoo, with the most recently renovated enclosures, had the lowest diversity (H′=0.98). Rarefaction curves for each facility approached but did not fully plateau by 12 monthly sampling rounds, suggesting that additional rare taxa might be detected with extended surveillance.

Ethical Considerations

All samples were collected opportunistically from freshly deposited faeces without any invasive procedure or physical restraint. Keeper welfare was ensured by providing PPE (gloves, masks) during sample collection. Any animal found to harbour clinically significant parasite loads (≥1,000 EPG for nematodes or confirmed E. histolytica by ELISA) was flagged for veterinary review, and treatment decisions were made by each zoo's attending veterinarian independent of the research protocol. Parasite results were returned to each facility within two weeks of processing.

Results

Of 287 primates sampled, 133 (46.3%; 95% CI: 40.5-52.2) tested positive for at least one gastrointestinal parasite during the 12-month surveillance period. Protozoan infections (39.7%) outnumbered helminth infections (28.4%), with 50 animals (17.4%) harbouring mixed infections combining helminths and protozoa. The radar profiles (Figure 1) reveal that great apes and Old World monkeys carried the heaviest and most diverse burdens, with Entamoeba and Strongyloides dominating in both groups. New World monkeys showed a distinctive Giardia-dominant profile (41.2%), while prosimians had uniformly low prevalence across all genera.

Comprehensive Interpretation

Viewed together, the taxonomic (Table 2, Figure 1), seasonal (Table 1, Figure 3), and facility-level (Figure 4) patterns form a coherent picture. Great apes carry the highest and most diverse parasite loads regardless of season or facility, reflecting their large body mass, prolonged faecal contact in terrestrial exhibits, and well-documented coprophagic behaviour. The summer peak in prevalence and intensity is consistent across all three facilities and all taxonomic groups, driven by the warm, humid Kansai climate that shortens environmental embryonation times. Osaka Tennoji Zoo's higher rates reflect its heritage soil-floored enclosures, which harbour eggs and larvae far more effectively than Kobe's impervious fibreglass substrates. The practical implication is that targeted prophylactic deworming administered in late spring (May), before the summer amplification cycle takes hold, would hit parasites at their most vulnerable point in the seasonal trajectory.

Discussion:

The central findings of this 12-month multi-zoo survey are that nearly half of captive NHPs in three Kansairegion zoological gardens harbour gastrointestinal parasites, that protozoa outnumber helminths, that great apes are disproportionately affected, and that both prevalence and intensity follow a pronounced summer peak driven by climatic conditions. The overall prevalence of 46.3% falls within the range reported by comparable multi-facility surveys. Lim et al. [5] documented 43.8% across four Malaysian zoos, and Kouassi et al. [6] recorded 52.1% in Côte d'Ivoire sanctuaries.

European data tend to be lower: a German zoo consortium survey found 28.4% overall prevalence, attributed to yearround indoor housing and concrete substrates that reduce environmental transmission [12] . The dominance of protozoa Entamoeba and Giardia in particular over helminths matches the pattern described in African ape sanctuaries [13] and likely reflects the shorter prepatent period and high fecundity of protozoan cysts compared with helminth eggs.

Where the present results diverge from some published data is in the relatively high Oesophagostomum prevalence (18.1%). Many zoo surveys report this genus below 10%, possibly because its thick-shelled eggs embryonate slowly in temperate soils and require flotation media with specific gravity ≥1.20 for recovery a technique not universally applied [3,14] . Our use of both NaCl flotation and formalin-ethyl acetate sedimentation may have improved detection, inflating apparent prevalence relative to surveys using flotation alone. The facility-level differences Osaka highest, Kobe lowest map directly onto enclosure substrate. Osaka Tennoji Zoo's 1970sera soil-floored exhibits sustain a reservoir of infective eggs and larvae that persists despite weekly raking and occasional chemical disinfection. Kobe's 2008-2015 renovations introduced raised fibreglass platforms with drainage channels, reducing faecal-substrate contact time and making pressurewashing effective at removing organic matter [15] . Kyoto's intermediate position reflects its hybrid infrastructure: a modern primate house (2019) alongside older heritage enclosures that still use compacted earth.

The seasonal pattern has direct management implications. Scheduling prophylactic anthelmintic treatment (e.g., fenbendazole 50 mg/kg × 3 days, or ivermectin 0.2 mg/kg) in May after the spring warming but before the summer amplification window could suppress egg output during the months when environmental transmission is most efficient [16] .

Pairing this with increased cleaning frequency (daily rather than twice-weekly substrate raking) during June-August would further reduce the environmental egg bank. For protozoa, metronidazole or fenbendazole targeting Giardia should be considered for New World monkey troops, where Giardia was the single most prevalent organism [17] .

Conclusion:

The Several open questions deserve future attention. Molecular speciation of Entamoeba isolates would distinguish the pathogenic E. histolytica from the non-pathogenic E. dispar, a distinction with direct clinical and zoonotic implications that morphology alone cannot make [18] . Longitudinal tracking of individual animals through multiple annual cycles would allow estimation of incidence rates and re-infection dynamics, providing stronger evidence for the timing of prophylactic interventions. Genotyping Giardia isolates would clarify assemblage distribution and assess the zoonotic potential of primate-derived strains for keepers and visitors. Extending the survey to include other Japanese zoo regions particularly Hokkaido (cold-temperate) and Okinawa (tropical) would test whether the seasonal patterns observed in Kansai hold across Japan's wide climatic gradient. Until such expanded data become available, the present results provide a robust evidence base for integrating parasitological surveillance into the routine health management of Japan's captive primate collections.

Acknowledgement

Acknowledgements Contributions Not Qualifying for AuthorshipThe authors thank primate keepers at all three zoological gardens for meticulous faecal sample collection and individual identification over the 12-month sampling period. Dr. Rina Kobayashi provided valuable guidance on protozoan morphometry.Funding Sources: This research was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP22K06384 for wildlife health surveillance.Institutional Support: The Osaka Research Center for Wildlife Health provided the parasitology laboratory, centrifuge equipment, and ELISA platform. The municipal administrations of Osaka, Kobe, and Kyoto granted research access to their respective zoological gardens.

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