Coprological survey of gastrointestinal parasites of mammals in Dehiwala National Zoological Gardens , Sri Lanka

A cross sectional, coprological survey on gastrointestinal (GI) parasites of captive mammals in the Dehiwala National Zoological Gardens was carried out in 2014. Fresh faecal samples from all the mammal species belonging to nine orders: Primates, Carnivora, Perissodactyla, Artiodactyla, Proboscidea, Erinaceomorpha, Lagomorpha, Rodentia and Diprotodontia were analyzed. Intensity of parasite infections was determined using the McMaster technique. Of the 70 samples, 44 (62.9%) were positive for one or more GI parasites. A total of 13 types of GI parasite eggs, cysts and/or oocysts of Trichuris sp., Strongyloides sp., Toxocara sp., Spirometra sp., Moniezia sp., Nematodirus sp., Giardia sp., Blastocystis sp., Balantidium sp., Entamoeba spp., strongyle type eggs, hookworm, and coccidian oocyts were observed. The most common stage was strongyle type egg (17.1%) followed by cysts of Entamoeba spp. (14.3%). Of the infected individuals, 25% had mixed infections. A higher prevalence of helminths (81.8%) compared to protozoans (47.7%) was observed but this difference was not statistically significant (Chi square test; p>0.05). There was no significant difference in the prevalence of infection among the captive bred, imported or wild caught individuals (Chi square test; p>0.05). Mammals of seven orders were infected with GI parasites but lagomorphs and diprotodonts did not have any parasites. Among the herbivores, strongyle type, Moneizia, Entamoeba and coccidian infections were common while Nematodirus sp. in a porcupine and Spirometra sp. in a flying squirrel were rare. Common parasites of carnivores were, Toxocara and Entamoeba but Blastocystis sp. in coati was a rare infection. Trichuris and Giardia infections were common in Primates. High worm burden was evidenced in silver leaf monkey, Hamadryas baboon, African lion, black rhino, pony, porcupine and flying squirrel. Although regular deworming is carried out, results of this survey highlight the importance of faecal analysis before administering deworming and applying a more targeted approach to manage the pathogenic species. This study provides baseline data on the GI parasites of all the mammal orders at Dehiwala Zoological Gardens.


INTRODUCTION
Zoological gardens play an important role in the promotion of animal biodiversity by protecting endangered species (Kelly and English, 1997).Since animals are kept in confined areas, parasitic diseases constitute one of the major problems in zoological gardens around the world due to high environmental contamination (Rao and Acharjyo, 1984).Unlike in the wild, stress conditions caused by captivity can diminish the resistance to parasite diseases (Geraghty et al., 1982;Gracenea et al., 2002;Cordon et al., 2008).Occurrence of parasites in captive animals in zoological gardens might vary according to husbandry practices, disease prophylactic measures, parasite-host interactions and treatment administrated (Lim et al., 2008).Captive animals do not show alarming signs of parasitism, if regular de-worming practices are carried out in zoological gardens (Parsani et al., 2001).However, Parsani et al., 2001 further argue that some captive animals do show clinical signs due to parasites even if they are regularly dewormed and some will have no clinical signs even if they are never dewormed and this depends more on the parasite host interactions than deworming practices.Parasites can be brought into a zoological garden by many ways: through animal food, (contaminated fruits and vegetables, infected meat or fish, etc), intermediate and paratenic hosts (snails, ants, cockroaches and other insects, rodents, etc.), newly acquired parasitized animals and through infected zoo staff and visitors (Pencheva, 2013).
Many species of helminths and protozoans are known to infect mammals.Helminths such as Strongyloides, strongyles, Trichuris, Nematodirus, Toxocara, Moniezia and protozoan parasites such as Giardia, Balantidium, Entamoeba and coccidians are GI parasites commonly found in captive mammals worldwide.The presence of these parasites in the host may induce morbidity and even mortality (Nath et al., 2012).They can also act as the reservoir of parasites for the domestic mammals and some of these infections can be zoonoses which can spread to the humans (Bogale et al., 2014).
The Dehiwala National Zoological Gardens (referred to as Dehiwala Zoo here onwards) was established during the early years of the 20 th century and is one of the oldest zoos in Asia.It is a pioneer institute that possesses, manages and conserves wild animals and displays the animal collections to the public.The Zoo is located in the heart of Colombo city, the largest city and the commercial capital of Sri Lanka with a population of 4.6 million in the metropolitan area.Though the apparent objective of setting up this Zoo was to exhibit animals, it is now treated as an animal welfare facility, involved in educating the community and an ex situ conservation center for endangered species (Dehiwala National Zoological Garden website).Because of the space limitation, many captive animals are caged in close proximity to one another and therefore they may succumb to parasitic infections.In a previous study, faecal samples of 13 species of captive primates at Dehiwala Zoo had been examined and reported many GI parasites including many protozoans: Cryptosporidum sp., Balantidium sp., Blastocyst sp., Entamoeba sp., Giardia sp., and coccidian and nematodes: the larvae of hook worm and the eggs of Ascaris, strongyle and Trichuris (Gunasekara et al., 2012).A few years earlier in 2009, Fernando and Udagama-Randeniya studied the GI parasites and ectoparasites of captive reptiles at Dehiwala zoo.The present coprological survey was carried out to determine the GI parasites of all the mammal orders in Dehiwala Zoo.

Study site and study animals
At the time of survey, Dehiwala National Zoological Gardens comprised 70 species of mammals belonging to nine orders: 18 primates, 19 carnivores, six perissodactyles, 18 artiodactyles, two proboscideans, one lagamorph and one diprodont, one erinaceomorph and four rodent species.These mammals were distributed throughout the zoo enclosure providing maximum possible space per individual.Climatically, Dehiwala comes under the wet zone of Sri Lanka (6°51'-24°5″N and 79°52'-22°4″E) with a mean annual temperature of 27°C.
De-worming is carried out once in every three months and the food given to each animal is subjected to regular inspections by veterinary surgeons (personal communication with the Chief Veterinarian at Dehiwala Zoo).Background information about each mammal was collected using a questionnaire, which gathered information on the age, sex, physical location of the animal in the Zoo, details on deworming (last date of deworming and type of drugs given), and a brief history of the animals' origin (whether the animal was brought from another zoo/country or born at the Zoo).The physical condition (fur coat, lethargy, appetite of the animals) at the time of sampling was also noted.Samples of the mammals living in groups (eg.monkeys) were taken randomly without considering a particular individual, assuming that if there is a single infected individual in that group the others were infected as well.

Collection of samples
Fresh faecal samples from all the captive mammal species at the Dehiwala National Zoological Gardens were collected from March to October 2014.Approximately 10 g of faeces was collected in the morning about 8.00 am before the cages were cleaned by the keepers.Each animal was sampled once during the study and if they lived in groups (eg.monkeys, deer) one sample from the group was taken.For herbivores and other less aggressive animals, the faecal sample was collected directly from the rectum.Samples from carnivores and other aggressive mammals were directly picked up off the ground with the help of the caretaker.The samples were collected into small plastic seal bags.During a visit about 20 samples were collected at random whenever there was a fresh sample of a particular mammal was available and the sampling was done in every month until a final target of all the mammal species in Dehiwala Zoo was reached.Samples were brought to the laboratory in a cooler and were stored at 4°C until analysis.Processing of the samples was completed within a week in the parasitology laboratory, in the Department of Veterinary Pathobiology at the Faculty of Veterinary Medicine at University of Peradeniya using a modified salt floatation method, Sheather's sucrose floatation method, direct saline and iodine mounts.Nematode cultures were set up for some species to obtain DNA for molecular analysis to identify up to species level.

Modified salt floatation method
Three grams of faeces was measured and was taken into a 50 ml capped centrifuge tube.Then the volume was made up to 50 ml by adding 47 ml of distilled water, and mixed thoroughly using a wooden applicator.The suspension was centrifuged at 3000 g for 20 min and the supernatant was discarded.The pellet was washed twice by re-suspending in distilled water, followed by two centrifugations at 3000 g for 20 min until a clear supernatant was obtained.The pellet was emulsified with saturated salt, mixed thoroughly and was centrifuged again for another 20 min at 3000 g at room temperature.Approximately 5 ml of the top meniscus was aspirated and added to 15 ml centrifuge tube.The total volume was made up to 15 ml by adding distilled water and centrifuging for 10 min at 1370 g at room temperature.This was repeated and finally 1 ml of the suspension with the pellet at the bottom of the tube was mixed with distilled water and transferred to a 1.5 ml Eppendorf ® tube using a Pasteur pipette.Distilled water was added to make it up to 1.5 ml and the tubes were centrifuged for 10 min at 1150 g in the microcentrifuge.The supernatant was discarded and the pellet was thoroughly mixed with 0.5 ml of distilled water.Using about 0.1 ml of the suspension, each microscopic slide was prepared and covered with a cover slip without staining.Five smears were observed from each sample under the light microscope (Olympus CH 31, Philllipines).Eggs of different parasite species were identified and number of eggs in 0.5 ml was estimated and the number of eggs per gram of faeces (EPG) was calculated assuming the method had concentrated all the eggs in the 3 g of faeces into 0.5 ml.

Sheather's sucrose floatation method
Protozoan cyst and oocysts isolation was done by Sheather's sucrose floatation method.Saturated sucrose was prepared and same steps of the modified salt floatation method mentioned above were followed replacing the floatation fluid by saturated Sheather's sucrose solution.Cysts and oocysts of particular protozoan species in each sample were calculated as cysts per gram (CPG) or oocysts per gram (OPG) of faeces.

Quantitative analysis
Initially, relative estimation EPG, CPG and OPG of faeces was carried out using iodine smears and observing under the light microscope.Later, more accurate counts were taken using the McMaster technique (Wood, 1995) and results were compared with that of the modified salt floatation and Sheather's sucrose floatation methods.

Nematode cultures
Freshly harvested eggs of single infections of Toxocara sp. and Trichuris obtained from the faecal samples of the African lion (Panthera leo) and Hamadryas baboon (Papio hamadryas), respectively were cultures according to Rajapakse et al., (1992) to identify the species using DNA analysis.Eggs were stored in petri dishes in 0.1 N sulphuric acid at a depth of 0.5 cm in room temperature (25°C) to incubate.In the course of this incubation, the culture was rocked gently once a day to ensure aeration.Embryonated eggs containing infective larvae were washed three times in distilled water by centrifugation at 1150 g for 10 min to remove sulphuric and the organic matter.One ml of the suspension was transferred to five 50 ml plastic centrifuge tubes.To each tube 10 ml of saturated calcium hypochlorite solution was added at room temperature.Every five minutes, one tube with suspension was diluted to 50 ml with distilled water, in order to prevent any further de-coating action in the egg shell by the calcium hypochlorite solution.Eggs at the butt of each tube were observed under microscope to select the tube with eggs at the suitable de-coating stage with larvae coming out of eggs.Then the selected tubes were centrifuged at 1150 g for 10 min.After removal of the supernatant, suspensions were washed (1150 g for 10 min) and the hatched larvae at the butt of the tube were collected.

Molecular Identification by DNA extraction and PCR amplification
Identification of Toxocara and Trichuris was confirmed by extracting DNA from cultures or eggs in single infections.Genomic DNA was extracted using DSBIO DNA extraction kit and PROMEGA protocol following the manufacturer's instructions.The DNA obtained from eggs or larvae was re-suspended in 13 µl of distilled water.The ribosomal second internal transcribed spacer (ITS2) and the mitochondrial cytochrome oxidase subunit 1 (CO1) regions were amplified using polymerase chain reaction (PCR) for helminth parasites.Amplification reactions were performed in a final volume of 25.0 µl containing primers, deoxynucleoside triphosphates (dNTPs, 0.2 mM), Taq polymerase and aliquot of DNA template.The nematode genus specific primers used were ITS2 3S-FW:5'-CGG TGG ATC ACT CGG CTC GT-3' and CO1 FH5-FW:5'-TTT TTT GGG CAT CCT GAG GTT TAT-3'were used which amplify ribosomal DNA and mitochondrial DNA.Conditions in the PCR (Gene Amp PCR system 9700, Singapore) were as follows: initial denaturation (94°C for 3 min), followed by 35 cycles.Each cycle included a denaturation (94 °C for 1 min) annealing (50°C for 1 .5 min) and an extension (72°C for 1 min.The annealing programme was completed with a final extension (72°C for 5 min).
Amplified PCR products were separated by electrophoresis in 1.5% agarose gel stained with ethidium bromide.Then, the PCR positive samples were subjected to sequencing to identify the species of the two parasites.

Data Analysis
Prevalence of infections was calculated for each mammal species as a percentage.
The differences in the prevalence of GI infections between helminths and protozoans and the prevalence of infection among the captive bred, imported or wild caught individuals were analysed using a Chi square test in Minitab software (version 15).

Prevalence of GI parasites
Faecal samples of 70 mammal species belonging to nine orders were analysed, of which 44 (62.9%) were infected with one or more GI parasites (Table 1).Eleven individuals had mixed infection (25.1%;Tables 1 and 2).Individuals of seven orders were infected with GI parasites while the other two orders: lagomorphs and diprotodonts did not harbor any GI parasites (Table 2).Among the infected orders, all the perisodactyles, probodcideans and erinaceomorphans sampled were infected (100%), while artiodactyles had the lowest prevalence (44.4%).Overall, helminth infections were more common (81.8%) compared to the protozoan infections (47.7%;Table 2) but the difference in the infection was not statistically significant (Chi square test χ 2 = 0.078; p>0.05).Moreover, there was no significant difference in the prevalence of infection among the captive bred, imported or wild caught individuals (Chi square test χ 2 = 0.022; p>0.05).
Eggs, cysts and oocysts estimates from iodine smears, salt and sucrose floatation were comparable to those of the McMaster counts and therefore the EPG, CPG and OPG counts given in Table 1 were from the McMaster technique.Hatched out larvae of Toxocara and Trichuris from faecal cultures confirmed the eggs identified from the faecal samples of the African lion (Pantheraleo) and Hamadryas baboon (Papiohamadryas), respectively.Although data from PCR protocols confirmed the two nematode genera identified through light microscopy, the sequencing was not successful due to insufficient band size in the gel and therefore identifying the two nematodes to species level was not possible.

Types of parasites and their intensities
A total of 13 different types of species/faecal stages of parasites were identified in mammals at the Dehiwala Zoo (Figure 1).In addition, two unidentified larval stages were recorded from black rhino and flying squirrel and these could be the hatched out larvae of the nematodes infections found in these two hosts and therefore they were not considered for calculations.Seven species of Primates including Patas monkey, chimpanzee, Japanese monkey, silver leaf monkey, purple faced monkey, Hamadryas baboon and white nosed monkey were infected with Trichuris sp. and recorded low EPG of less than 1000 except Hamadryas baboon which had a high count of 5100 EPG (Table 1).According to the established infection intensity categories of World Health Organization (WHO, 1987) for soil transmitted nematodes (STNs) including Trichuris trichiura infection was defined as light (1-999 EPG) moderate (1,000-9,999) or heavy (10,000 EPG).Four species of primates were infected with Giardia: silver leaf monkey, mangabey monkey, purple faced leaf monkey and spider monkey.Of these, silver leaf monkey had a mixed infection of both Trichuris and Giardia with a CPG of 5800.Orangutan, gibbon, white handed gibbons, toque monkey, grey langur and Chinese monkey did not have any infections.The two lemur species: ringed tail and brown lemur had only strongyle type eggs at low intensities 600 or less EPG.Out of primates, only purple faced monkey and Mangabey monkey had Entamoeba infections where both had mixed infections with Giardia sp. and Trichuris sp.(Table 1).
In the carnivores, the most prevalent parasites were Toxocara and Entamoeba where five mammal species were infected with these two parasites.African lion, fishing cat, Bengal tiger, white tiger and leopard were infected with Toxocara and otter, brown bear, sloth bear, Sri Lankan palm civet and sea lion were infected with Entamoeba.Hedgehog (Order Erinaceomorpha) was also infected with Toxocara.Except in white tiger (400 EPG) all others had high intensity of Toxocara infections (>500 EPG) and African lion and fishing cat had EPG counts of 6300 and 1000, respectively.Blastocystis infection was recorded only in meerkat.Some carnivores: skunk, jackal, rusty spotted cat, Ocelot, ring tailed civet and golden palm civet did not harbor any GI parasites.
Among the herbivores, donkey, sable antelope and barking deer were infected with the tapeworm Moneizia while pony and buffalo had coccidian infections.Nematodirus sp. in porcupine (EPG= 3,300) and Spirometra sp. in flying squirrel (EPG=2,100) can be considered rare infections.Black rhino reported high intensity of strogyle type with 2300 EPG.

DISCUSSION
Results of this coprological survey show that more than half of the mammals (62.9%) at the Zoo were infected with one or more GI parasites.Similar prevalence levels of GI parasites have been reported from captive mammals in other zoos in Rangpur Recreational Garden in Bangladesh (Khatun et al., 2014) and Zoo Safari of Fasano in Italy (Fagiolini et al., 2010).A higher percentage of mammals at the Dehiwala Zoo were infected with helminths (81.8%) than protozoans (47.7%) irrespective of administration of regular deworming.The common practice at the Zoo is that all the mammals are treated with anthelmintics every three months irrespective of the parasite burden or the type of infection.Treatment should be given only to animals suffering from heavy parasitism of pathogenic species to avoid the development of drug resistance.Most animals that can tolerate existing incidental infections should be left untreated.A coprological analysis should be carried out to determine the types of parasites and the worm burden before administering the anthelmintics.Testing for infection and only treating when infection reaches a threshold would likely to reduce development of antiparasite resistance.Treatment might be on a herd basis when it comes to herbivores that occur in large herds or an individual basis especially the rare mammals.For rare species, since the number of individuals in the zoo is limited and valuable, testing and treatment on individual basis is essential.Higher helminth infections have also been reported in other zoo animals by many authors: Rangpur Recreational Garden in Bangladesh (Khatun et al., 2014) and Zoo Safari of Fasano in Italy (Fagiolini et al., 2010).Opara et al., (2010) in Nekede Owerri zoological garden in Southeast Nigeria while Varadharajan and Kandasamy (2000) in V.O.C. Park and Mini Zoo, Coimbatore.
Present study reported six nematodes: Trichuris sp., Strongyloides sp., Toxocara sp., Nematodirus sp., hookworm (Superfamily Ancylostomatidea) and strongyle type eggs (Superfamily Strongyloidea), five protozoans: Giardia sp., Blastocystis sp., Entamoeba spp., Balantidium sp. and coccidians, two cestodes: Moniezia sp. and Spirometra sp. while there were no trematode infections.A sedimentation technique has to be applied to detect the eggs of trematodes as these eggs are heavier.Trematodes require one or more intermediate host(s) for their transmission and therefore are less likely to accumulate in a captive environment (Tandon et al., 2005;Atanaskova et al., 2011).Since most of common GI protozoans and nematodes spread by the faecal-oral route, infections are spread in areas with inadequate sanitation and poor hygiene.Only Monezia infection involves an intermediate host, a mite which occurs commonly in pastures where herbivores graze.All the individuals in three orders: Perisodactyla (n=6), Erinaceomorpha (n=1) and Proboscidea (n=2) were infected with GI parasites.However, the number of individuals sampled in Erinaceomorpha and Proboscidea was low and the findings therefore cannot be generalized uncritically.
Carnivores, artiodactyles and rodents had the highest number (five each) of parasites in each order.Among the carnivores five were infected with Toxocara sp.(26.3%), which is a common parasite in felines and canines (Lim et al., 2008).Among the carnivores infected with Toxocara sp.Bengal tiger (Panthera tigris tigris), African lion (Panthera leo) and a white tiger (P.tigris tigris) were imported from other zoos while the leopard (Panthera pardus) and the fishing cat (Prionailurus viverrinus) were wild caught.It could be that these animals have all been infected from birth as Toxocara can be transmitted in the milk from mother to offspring and already had the infection before they were brought to the Zoo.However, the major route of transmission is through ingesting embryonated eggs in the environment via faecal oral route cannot be ruled out as they may have got the infections from other carnivores in the zoo as well.The close proximity of the cages of the infected animals: Bengali tiger, African lion and a white tiger could be a main reason for spreading the infection.Carnivores were also infected with Entamoeba spp.and Strongyloides sp. with a high prevalence of 26.3 % and 10.5%, respectively.The presence of synanthropic rodents such as the house mouse and the rat, which live in or near human dwellings and in the zoological gardens, is known to serve as a reservoir of several types of infections.Factors such as urbanization, overcrowded cities and inadequate sanitation have led to increasing number of these animals and consequently becoming increasingly common in transmitting diseases to humans and other animals (Brasil, 2002).
Among the 18 species of Primates investigated, high prevalence GI parasite (61.1%) was recorded.Trichuris sp. was the most prevalent (38.9%) parasite.Similar findings were obtained in the study of Dawet et al. (2013) where infection of Trichuris sp. was the most were not present in the Zoo at the time of sampling but seven other species were present, some of them were imported, captive bred or caught from the wild (Table 1).
Of the herbivores, all the perisodactyles were infected with at least a single parasite species but only eight individuals out of 18 artiodactyles (44.4%) were infected.Both these orders had Moneizia infections which is a common tapeworm in herbivores.In the life cycle of Moneizia, an oribatid mite is involved as the intermediate host.Eggs are passed out with faeces from the ruminant host along with the gravid proglottids into the soil and these eggs are eaten by oribatid mites.Grazing ruminant may ingest the mites in pastures and the eggs hatch out and develop into tapeworms in the small intestine.The black rhino (Diceros bicornis) who was pregnant during the time of sampling had a high infection of strongyle type eggs (2300 EPG) and may had been vulnerable to parasitic infections.In the case of proboscideans only two individuals: African and Asian elephants were sampled and both were found infected with three different types of parasites.Strongyles and Entamoeba sp. were found in the African elephant and unidentified protozoan cysts were found in the Asian elephant.If the food given (mostly jack leaves) has got contaminated before they were brought to the zoo, animals can be infected with the parasites after eating them.Damp and unhygienic conditions maintained in the enclosure can also increase susceptibility to the infections (Ortiz et al., 2006;Vanitha et al., 2011).
Of the four rodents examined, three were infected with GI parasites (75.0%).These rodents were not treated for worms (personal communication with a veterinarian at the Dehiwala Zoo) as they usually do not show symptoms of worm infections.But animals are more prone to parasite diseases when they are in captivity than in their natural environment.Such situations can also be dangerous to the visitors, workers and veterinarians.Lagomorphs and diprotodonts in the Dehiwala zoo did not have any GI parasites.Similarly, a study conducted in the Samsun Zoological Gardens in Turkey, lagomorphs and diprotodonts were free of parasitic infection (Gurler et al., 2010).
Mixed infections of GI parasites among the mammals at Dehiwala zoo were not very common (25.0%).Mixed infections of Trichuris sp. and Giardia sp. and Enatmoeba sp. in primates and strongyle type together with various protozoans in coati, black rhino and spotted deer were observed.
This study shows, more than half of the mammals at Dehiwala Zoo were infected with GI parasites, and some with high intensities.The presence of parasite eggs, oocysts or cysts in the faecal sample does not mean the animal is sick or will be sick nor does it mean that the animal should be treated (Wood et al., 1995).All gastrointestinal parasites are not equal; some are highly pathogenic and some are incidental.The current practice at Dehiwala Zoo is to treat every animal routinely giving anthelmintics every three months.A better control strategy should involve a more targeted approach to manage parasites effectively minimizing the pathogenic species by regular faecal examination along with administration of desired worm treatment at regular intervals.

Figure 1 :Figure 2 :
Figure 1:Helminth eggs and larvae in the faecal samples analysed from mammals in Dehiwela Zoo (A) Egg of Nematodirus sp.(B) Egg of Toxocara (C, D, E, F) Strongyle type eggs, (G) Egg of Strongyloides sp.(H) Egg of Trichuris sp.(I) Egg of Toxocara sp (J) Egg of Moniezia sp.(K) Egg of Spirometra sp.(L) Hookworm egg (M) Unidentified nematode larva in black rhino and (N) Unidentified rhabditiform larva in flying squirrel (Scale bar = 50 µm)

Table 1 :
Background information, type of gastrointestinal parasites and the intensity of infection in the mammal species at Dehiwala Zoo

Table 2 :
Prevalence of different types of gastrointestinal parasites in seven mammalian orders in the Dehiwala Zoo Trichuris infection in primates therefore can be considered light which was less than 999 EPG except in Hamadryas baboon ( 5100 EPG).Trichuris is a soil transmitted nematode where infections can easily spread through oral faecal route.Among the Primates at Dehiwala Zoo, Gunasekara et al. (2012) identified six species of protozoa: Cryptosporidum sp., Balantidium sp., Blastocyst sp., Entamoeba sp., Giardia sp., and coccidian in the chimpanzee, orang-utan, hamadryas baboon, Japanese macaque, siamang gibbon, toque monkey, grey langur, silvered leaf monkey, sooty mangabey and Formosan monkey.The helminthes they reported were hookworm larvae and eggs of Ascaris, strongyle and Trichuris .They found that toque monkey was positive for five species of GI parasites: