Gastro-intestinal Parasites of Assamese Macaque (Macaca assamensis Hodgson, 1840) in Shivapuri Nagarjun National Park, Kathmandu, Nepal

The macaque is a member of the sub family Cercopithecinae and family Cercopithecidae of primate order of mammalian class. Among five species of monkeys reported from Nepal, Assamese Macaque is one of the less common primate species. A total of 85 fresh faecal samples of Assamese Macaque (Macaca assamensis) were collected from Shivapuri Nagarjun National Park (SNNP), Kathmandu, Nepal. All the samples were macroscopically as well as microscopically examined to identify the distribution of gastrointestinal parasites using faecal floatation and Sedimentation techniques. The result revealed the distribution of three species of protozoan and seven species of helminthes parasite among Assamese Macaque of SNNP with the prevalence rate of 72.94% (62/85). Among protozoan parasites, Balantidium coli showed the highest prevalence (28.24%) followed by Entamoeba sp. (20%), and Isospora sp. (3.53%). Ascaris sp. was found to be distributed maximum (10.58%) among helminth parasites followed by Trichuris sp. (9.41%), Strongyloides sp. (8.24%), Moniezia sp. (8.24%), Oesophagostomum sp. (4.7%), Hookworm (4.7%) and Physeloptera sp.(1.17%). The present study highlights the necessity of considering parasitic diseases as one of the threat in conservation of


INTRODUCTION
Primates are the highest order of mammals which includes lemurs, monkeys, apes, human and other similar forms, typically having dexterous hands and feet, binocular vision as well as developed brain (Mary et al. 2007).The macaque is a member of the sub family Cercopithecinae and family Cercopithecidae of primate order of mammalian class.A total of five species of monkeys have been reported from Nepal: Assamese Macaque (Macaca assamensis Hodgson, 1840), Rhesus Macaque (Macaca mulatta Zimmermann, 1780), Terai Grey Langur (Semnopithecus hector Pocock, 1928), Nepal Grey Langur (Semnopithecus schistaceus Hodgson, 1840), and Himalayan Grey Langur (Semnopithecus ajax Pocock, 1928) (Jnawali et al. 2012).Among them Assamese Macaque is one of the less common primate species and is explored patchily in Nepal (Chalise 2010).Macaca assamensis are only sporadically distributed in limited areas of the Nepal, India, Bhutan, Southeast Asia, and China (Wada 2005).In Nepal it has been recorded from 380 m in Mulghat Tamor to 2350 m a.s.l. in Langtang (Chalise 2005).
Parasites play a major role in ecosystems (Esch & Fernandez 1993), host population growth and regulation (Hochachka & Dhondt 2000, Hudson et al. 1998), and community biodiversity (Hudson et al. 2002).Both helminth and protozoan parasites are common in nonhuman primates (Munene et al. 1998, Muriuki et al. 1998).Some of the parasites are considered to be nonpathogenic.However, a large number can result in physiologic disturbances, nutritional loss, or may produce lesions that result in serious debilitation, and can create opportunistic for secondary infections that may be fatal (Toft & Eberhard 1998).In monkeys, disease emergence can occur directly through human introduced diseases or indirectly through human induced changes in the disease ecology of wildlife.This study aims to determine the distribution of intestinal parasites of Assamese Macaque (Macaca assamensis).A few studies of Assamese Macaque have been done on the ecological basis (Wada 2005, Regmi & Kandel 2008, Pandey 2012) but, the information regarding the gastro-intestinal parasites of Assamese Macaque is still lacking.Hence, the present study has provided information regarding distribution of various protozoan and helminth parasites of Assamese Macaque of SNNP.

MATERIALS AND METHODS
A total of 85 faecal samples of monkeys were collected in two seasons: summer (40) and winter (45) of 2012 respectively.During the sample collection, a troop of macaques were systematically followed from dawn to dusk.About 5 gram of fresh samples was carefully collected in sterile sample collection vials with paper marking.All faecal samples were stored in vials containing 2.5% potassium dichromate solution.Before and after collection, all the faeces were examined macroscopically for consistency, presence of blood or mucus, tapeworm proglottids and adult or larval nematode.In the laboratory, the samples were microscopically examined using concentration techniques viz., faecal floatation and sedimentation.
The samples resulting from the application of these techniques were divided among three different slides, each for a different method using temporary staining (Lugol's iodine) and examined under light microscopy at 10X×10X and 40X×40X magnification.

General prevalence of intestinal parasites
Out of 85 faecal samples examined, 62 samples were found to be positive for either protozoans or helminthes or both parasites showing prevalence rate of 72.94%.The result revealed that a total of 10 different species of gastrointestinal parasites were found to be distributed among Assamese Macaque of SNNP.Distribution of helminth parasites (7 species) was found to be higher compared to the protozoan parasites (3 species).

Distribution of specific intestinal parasites
Among the three species of protozoan parasites, B. coli showed high distribution in Assamese Macaque of SNNP with prevalence rate of 28.24% followed by Entamoeba sp.(20%) and Isospora sp.(3.53%).Among the seven species of helminthes parasites reported, six species belonged to nematoda and only one species belongs to cestoda.Trematode parasite was not observed in Assamese Macaque of SNNP.Ascaris sp.(10.58%) was found to be distributed maximum among nematode parasites in these monkeys followed by distribution of Trichuris sp.

Concurrency of intestinal parasites
The result indicated that maximum number of Assamese Macaque suffered from single parasite infection, either protozoan or helminth parasites showing prevalence rate of 43.53%.15 (17.65%) samples were found to be positive for two parasites.The triple infection by intestinal parasites was 4.7% and quadruple infection of intestinal parasites in same sample was the least (1.17%).Only one sample was found infected by four different parasites.Maximum samples with multiple infections were due to combination of both protozoan and helminth parasites (Fig. 3).).This result is higher than the present study.Isospora sp. has been reported in Macaca radiata and Macaca assamensis (Varadharajan andPythal 1999, Wongsawad 2009).
The helminth parasites observed in this study were also reported in several previous studies concerning intestinal parasitic infection in Macaca radiata and Macaca mulatta (Varadharajan andPythal 1999, Eberhard et al. 2001).The prevalence rate of Ascaris sp. was reported indicating 34% from Macaca radiata (Varadharajan & Pythal 1999), followed by 10.48% from Macaca mulatta (Malla 2007) and by 7.45% from the similar species (Nepal 2010).But in the present study it was found to be 10.58% from Macaca assamensis.Oesophagostomum sp. was found to be highly prevalent in a Papio anubis (Bezjian et al. 2008) with prevalence rate of 85% followed by 20% from Assamese Macaque's (Wongsawad 2009).Some researchers of Nepal suggested low prevalence of Oesophagostomum sp. from Macaca mulatta of different areas ranging from 4-10% infection rate (Nepal 2010, Dhoubhadel 2007, Malla 2007) while in this study prevalence rate was found to be 4.7% from Macaca assamensis.Only 4.7% hookworm was observed in the study.The difference in parasite species might be due to the environmental as well as genetic and behavioural factors.Mutani et al. (2003) 2007, Varadharajan & Pythal 1999).It was found to be 13.72% from Assamese Macaque in this study.The study shows the variation of parasitic distribution as well as their prevalence according to the host as well as locality.
Oesophagostomum sp. and Strongyloides fulleborni were prevalent only in adult female guenon (Gillespie et al. 2004).This may reflect energy and nutrient stress associated with producing and raising infants.However, Strongyloides fulleborni was higher in adult male compared to adult female Red Colobus.Perhaps this reflects energy and nutrient stress associated with maintaining social dominance (Gillespie et al. 2005) which may result in an increased susceptibility to infection (Milton 1996).Dunn (1963) reported Moniezia sp. from Wild Howlers (Aloutta sp.).Although Moniezia sp. is the parasite of herbivores, its presence in the monkeys is not uncommon as the monkeys of SNNP are more closely in contact with herbivore animals of that area.It can be concluded from this study that the competitors also affect on the types and prevalence of intestinal parasites on these monkeys.

CONCLUSIONS
In conclusion the Assamese Macaque of Shivapuri Nagarjun National Park are infected with various protozoan and helminth parasites.Among them Balantidium coli was found to be highly prevalent species compared to other parasites.Test revealed that there was no significant difference in the prevalence of parasites between two seasons.The study highlights the importance of further in depth studies regarding disease aspects of monkeys and their association with their survivability in order to conserve them.

Fig. 3 .
Fig. 3. Concurrency and occurrence of intestinal parasites DISCUSSIONS Assamese Macaque is a less common primate species and is explored patchily in Nepal (Chalise 2010).It is also given the status of Endemic in distribution due to localization in Nepal only (Chalise 2005).The species is confirmed from Kimni Acham, Dadeldhura, Ramdi Palpa, Langtang NP and Helambu area, Makalu-Barun NP and Bhumlingtar, Hariharpur and Nagarjun forests of Kathmandu.They are not recorded from Tarai plain and high snowy mountains and in human settlements (Chalise 2005) although five species of monkeys have been reported from Nepal (Jnawali et al. 2012).Intestinal parasites in Assamese Macaque found in present study resemble that of the previous studies of prevalence of parasites in Macaca assamensis (Wongsawad 2009).The prevalence rate (of 93 %) reported from feral Bonnet Macaque in India (Varadharajan and Pythal 1999) was the highest prevalence of gastrointestinal parasites reported till now.Previous studies among Rhesus Monkey from different area of Nepal has shown the positivity ranging from 60-85% (Dhoubhadel 2007, Malla 2007, Nepal 2010) though prevalence rates were seen to vary according to locality.Three protozoan parasites viz: B. coli, Entamoeba sp., and Isospora sp. were observed occupying prevalence percentage of 28.24%, 20%, and 3.53% in present study.Similar protozoan parasites were also reported in Macaca assamensis (Wongsawad 2009), in free living Feral Bonnet Macaque (Macaca radiata) elsewhere in the world.B. coli was the predominent intestinal protozoan parasites, whose presence in present study was supported by previous studies 27.5 % and 62 % of balantidial infection in Macaca assamensis and Papio anubis (Wongsawad 2009, Ryan et al. 2012) whereas less prevalence of Balantidium coli was reported in the Bonnet Macaque (Varadharajan & Pythal 1999) contributing only 6%.Entamoeba sp. was found to be highly prevalent in Macaca assamensis showing documented prevalence rate of Physeloptera sp. to be 58.5% from Cercopithecus aethiops sabaeus.