Intestinal parasites and bacteria of mountain gorillas (Gorilla beringei beringei ) in Bwindi Impenetrable National Park, Uganda

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1 Primates (2005) 46:59 63 DOI /s y ORIGINAL ARTICLE Gladys Kalema-Zikusoka Æ Jessica M. Rothman Mark T. Fox Intestinal parasites and bacteria of mountain gorillas (Gorilla beringei beringei ) in Bwindi Impenetrable National Park, Uganda Received: 20 August 2002 / Accepted: 20 May 2004 / Published online: 26 August 2004 Ó Japan Monkey Centre and Springer-Verlag 2004 Abstract A survey in 1994 examined intestinal helminths and bacterial flora of mountain gorillas (Gorilla beringei beringei) in Bwindi Impenetrable National Park, Uganda. Parasites and bacteria were identified to genus in the feces of two groups of tourist-habituated and one group of non-tourist-habituated mountain gorillas. Eggs were identified as those of an anoplocephalid cestode, and nematode eggs representative of the genera: Trichuris, Ascaris, Oesophagostomum, Strongyloides, and Trichostrongylus. This is the first report of Ascaris lumbricoides-like eggs in mountain gorillas. Fecal samples (n=76) from all groups contained helminth eggs, with strongyle eggs and anoplocephalid eggs being the most common. Salmonella and Campylobacter were found in both gorilla groups. Regular long-term non-invasive fecal monitoring of the populations of mountain gorillas is essential for the prevention and identification of potential health threats by intestinal parasites and bacteria in this highly endangered subspecies. Keywords Mountain gorillas Æ Intestinal helminth parasites Æ Intestinal bacteria Æ Bwindi Impenetrable National Park Æ Uganda G. Kalema-Zikusoka Æ M. T. Fox Department of Pathology and Infectious Disease, The Royal Veterinary College, Royal College Street, London, NWI 0TU, UK Present address: G. Kalema-Zikusoka (&) Conservation Through Public Health, Plot 39 Babiiha Avenue, P.O. Box 10950, Kampala, Uganda gladys@ctph.org J. M. Rothman Department of Animal Science, Cornell University, Ithaca, NY 14853, USA Introduction Mountain gorillas (Gorilla beringei beringei) are highly endangered with approximately 300 out of the estimated 650 found in Bwindi Impenetrable National Park (BINP), and the remaining found in the Virunga Volcanoes region within the border areas of Rwanda, Democratic Republic of Congo and Uganda (Mcneilage et al. 2001). Habitat encroachment, poaching, and disease are major threats to mountain gorilla conservation (Werikhe et al. 1997). Owing to their close genetic relatedness, mountain gorillas are at risk of contracting human pathogens that may cause disease in naive hosts (Ott-Joslin 1993), with the potential of affecting their population (Grenfell and Gulland 1995). Fecal material is a potential source of disease-causing microorganisms including pathogenic parasites and bacteria. The parasite and bacterial burden in animals may increase through direct transmission and when animals are immunocompromised due to stress (Baneth et al. 1998; Sleeman et al. 2000). Mountain gorilla tourism was introduced in Rwanda in 1979 and in the Democratic Republic of Congo in 1984 (IGCP 1992). Uganda started an ecotourism program based on the Rwandan model in 1993 to generate revenue for the conservation of these highly endangered apes. Two gorilla groups, Mubare and Katendegyere, were habituated for tourism over 2 years. Establishing a sustainable ecotourism program involves the prevention of disease transmission from humans to gorillas and the minimization of disturbance of natural behavioral patterns. Tourism rules were developed by the Uganda Wildlife Authority together with supporting agencies, and include: people digging a hole 30 cm deep to defecate, maintaining a 5-m distance between tourists and gorillas, and the right of park staff to refuse entry to tourists with overt clinical signs (Macfie 1992). The aims of this study were to identify the parasites exhibiting patent infections in Ugandan mountain gorillas and establish whether such animals excrete

2 60 known bacterial pathogens (Salmonella, Shigella, Campylobacter). Methods Data were collected from late August to September (the rainy season) of 1994 at an altitude of 1,160 2,600 m (Butynski 1984). Fecal samples were collected from the night nests and fresh trails of two tourist-habituated groups (Mubare and Katendegyere) adjacent to Buhoma (1,500 m), and one non-tourist-habituated group (Rungo) adjacent to the research site of the park, Ruhija (2,300 m) at a higher altitude. Group sizes were Mubare (n=13), Katendegyere (n=7), and Rungo (n=6). During the study in Buhoma, the forest received an average daily rainfall of 1.25 cm with the wettest day receiving 3.88 cm. The mean temperature ranged from 19.8 to 27.7 C. Specific details on the history, ecology and biodiversity of the park are reported in other descriptions (Butynski 1984; Butynski and Kalina 1993; McNeilage et al. 1998). Samples used were collected less than 24 h after defecation (number of samples per group were Mubare n=28; Katendegyere n=30; and Rungo n=18). It is possible that the same individual was sampled more than once. Collected samples were kept cool and examined for parasites using the McMaster method within 24 h. Sugar solution was used to separate the eggs from fecal debris by mixing 42 ml of sugar solution with 3 g of feces, and the number of eggs in each chamber added and multiplied by a factor of 50 to determine the eggs per gram (epg) of feces (Thienpont et al. 1986). Helminth eggs were measured using a calibrated microscope eyepiece graticule. After initial examination at Buhoma, samples were subsequently preserved in 10% formalin for further examination at the Royal Veterinary College, University of London. Bacterial examination was performed using fecal swabs preserved in transport medium and refrigerated below 4 C for later examination at the Makerere University Faculty of Veterinary Medicine, Uganda. Each bacterial swab was plated out on Campylobacter selective supplement, an agar made up with a Skirrow supplement (Oxoid, UK), at 42 C for up to 72 h in a microaerophilic environment and examined microscopically. Swabs were also transferred to 10 ml of Salmonella-enriched medium, Selenite broth (Oxoid, UK), and incubated for 24 h. The broth was plated out on selective agar for Salmonella and Shigella, xylose lysine deoxycholate (LD) agar (Oxoid, UK). The LD plates were incubated for 24 h at 37 C and then analyzed for growth. Growth of typical colonies was confirmed using the Salmonella anti-sera. Shigella anti-sera was not available to confirm growth of typical colonies. Results Based on morphological characteristics, the eggs of Trichuris, Ascaris, Oesophagostomum, Strongyloides and Trichostrongylus and an anoplocephalid cestode were Fig. 1 A scattergram prepared from the dimensions of ovalshaped, thin-walled nematode eggs without polar plugs found in the feces of mountain gorillas in Bwindi Impenetrable National Park, Uganda. The numbers in the plot represent the number of eggs found with the particular measurements. The circles in the graph represent the grouping of each cluster of eggs by genus identified. A scattergram was used to differentiate recovered eggs resembling Oesophagostomum, Strongyloides and Trichostrongylus (Fig. 1). Individuals in the Mubare and Katendegyere groups had the highest nematode parasite epg counts (Table 1). The highest numbers of tapeworm epg (Fig. 2) of feces occurred in the Rungo group (Table 1). The Rungo group was the only group shedding Trichuris species eggs. Eggs of the nematode genera Oesophagostomum, Strongyloides, and Ascaris, and of an anoplocephalid cestode were present in all groups. Three fecal samples tested positive for Salmonella sp., two from the Mubare and one from the Katendegyere group (Table 2). Six fecal samples tested positive for Campylobacter sp. Discussion Habituation of gorillas and visits by tourists could cause increased disease transmission between humans and the wildlife they have come to observe and protect. Addi- Table 1 Mean intestinal parasite egg counts (eggs per gram, epg, in each genera) in tourist-habituated and non-tourist-habituated gorilla groups in Bwindi Impenetrable Forest, Uganda Group Mubare Katendegyere Rungo No. of samples/group Intestinal parasites nematodes (epg) Oesophagostomum 10,600 11, Strongyloides 1, Trichostrongylus Ascaris lumbricoides Trichuris trichuria Cestodes (epg) Anoplocephala gorillae 1,350 1,650 3,150

3 Table 2 Prevalence (% positive samples) of bacteria in Bwindi gorilla groups sampled in Bwindi Impenetrable Forest, Uganda Group Mubare Katendegyere Rungo No. of samples/group Bacteria Salmonella (%) Campylobacter (%) Fig. 2 Anoplocephala gorillae egg found in the feces of mountain gorillas in Bwindi Impenetrable National Park, Uganda tionally, the presence of human observers may put stress on populations, which may make them more susceptible to disease (Koneman et al. 1998; Baneth et al. 1998). In this survey, the gorillas habituated for tourism had higher egg counts for almost all parasites identified (Table 1). Both habituated and non-habituated gorilla groups were infected with Ascaris lumbricoides-like eggs, but only non-habituated gorillas were infected with Trichuris trichuria-like eggs. All the nematode eggs identified in the gorillas in this survey have been recorded in humans, however, there was no direct evidence of disease transmission between people and gorillas. Recent studies have suggested that humans and/or cattle may be responsible for infections of pathogenic protozoa in the population (Graczyk et al. 2002a, 2002b; Nizeyi et al. 2002a, b). The outbreak of scabies which occurred in two groups of Bwindi gorillas that range near park boundaries was likely from contact with infected humans or their clothing (Graczyk et al. 2001; Kalema-Zikusoka et al. 2002). Variables other than habituation that may affect the abundance and diversity of bacteria and parasites include: the home range of each of the groups (soil type, weather, rainfall, topography), differences in social group composition, proximity to other groups, and time of sampling. The application of long-term longitudinal studies could help to overcome some of the limitations of short surveys, but such studies are rarely performed in ecological parasitology (Bush et al. 2001). Presently, one of us (J. Rothman) is analyzing samples from a yearlong study of the parasites of individual animals in one group of Bwindi gorillas. The presence of fecal pathogens should also be correlated with overt clinical signs of diarrhea, colitis, enteritis, constipation, dehydration and listlessness (Paul-Murphy 1993; Janseen 1993). In addition to longitudinal data collection, the methodology chosen for a particular study should be reviewed considering the limitations of field conditions and transport regulations. The McMaster method, although easy to use in the field, is not as accurate for detecting and identifying parasites as other methods such as the formalin-ethyl acetate centrifugation technique and molecular techniques (Faler and Faler 1984; Thienpont et al. 1986; Bowman 1999). Flotation fluids of a higher specific gravity, such as sugar and zinc sulfate, are required to detect heavier eggs, including trematodes and cestodes (Thienpont et al. 1986; Koneman et al. 1998). Additionally, the measurement of epg should be interpreted with caution because fecal egg output varies among different parasite species, seasons of the year and intraspecific competition of parasites for resources (Soulsby 1982; Koneman et al. 1998). The eggs identified as those of an Oesophagostomum species, are morphologically indistinguishable from those of the genus Murshidia (Campana Rouget 1959). Two other trichostrongylids with eggs similar to those of Oesophagostomum species hhave been reported in the Bwindi population, Paralibyostrongylus kalinae and Hyostrongylus kigeziensis (Durrette-Dusset et al. 1992). Similarly, the eggs identified as representative of the genus Trichostongylus are indistinguishable from those of the genus Impalaia (Gibbons et al. 1977). In this study, we found eggs resembling the eggs of Ascaris lumbricoides. Although Ascaris suum is morphologically indistinguishable from A. lumbricoides, A. suum only rarely infects humans and other primates (Anderson 1995). Similarly, the polar-plugged eggs are most likely Trichuris trichuria, which typically infect humans, and not the morphologically indistinguishable Trichuris suis, which do not. The species of Strongyloides represented by the eggs found in the Bwindi gorilla feces is probably Strongyloides fulleborni, because this species typically infects primates and has been found in Bwindi and Virunga mountain gorillas (Ashford et al. 1990; 1996; Nkurunungi 1999; Sleeman et al. 2000). The other species of Strongyloides found in primates, S. stercoralis, produces larvae rather than eggs in the feces. The parasites found previously, or during this study in the Bwindi population and the Virunga mountain gorilla population are presented in Table 3. The life cycles, morphology, and history of Bwindi gorilla parasites have been reviewed by Rothman et al. (2004). There has been very little work on the bacteria found in free-ranging gorillas (Nizeyi et al. 2001). The potential for disease due to bacteria is great if pathogenic or hemolytic strains were to be introduced into these animals. Thus, it is essential to develop some baseline information on the bacterial flora that is present. In this study, we confirmed the presence of Salmonella and Campylobacter in these gorillas. A few years later, Nizeyi et al. (2001) identified infections with Salmonella, Campylobacter, and Shigella in the same gorilla population. The important question remaining is whether patho-

4 62 Table 3 Gastrointestinal parasites found in the two populations of Gorilla beringei beringei Parasite genera genic strains of these bacteria are currently present within the park. Conclusion Bwindi population a Helminths Anoplocephala gorillae Ascaris sp. Capillaria hepatica Chitwoodspirura wehri Hyostrongylus kigeziensis Hyostrongylus sp. Impalaia sp. Loa loa sp. Murshidia devians Oesophagostomum sp. Oesophagostomum stephanostomum Paralibyostrongylus kalinae Probstymaria gorillae Strongyloides fuelleborni Trichostrongylus sp. Trichuris sp. Protozoa Chilomastix mesnili Cryptosporidium parvum Encephalitozoon intestinalis Endolimax nana Entamoeba coli Entamoeba hartmanni Giardia duodenalis Giardia lamblia Iodamoeba buetscheli Virunga population b a Ashford et al. 1990; Durette-Dusset et al. 1992; Nizeyi et al. 1999; J.B. Nkurunungi, unpublished data; Graczyk et al. 2002a, b; Rothman et al b Nyeblin 1924; Redmond 1983; Hastings et al. 1992; Graczyk et al. 1999; Sleeman et al This short survey adds to our knowledge of the diversity of parasitic and bacterial flora in the endangered Bwindi gorilla population. Regular monitoring of the intestinal fauna of gorilla individuals and groups is suggested for long-term conservation of this critically endangered subspecies, and for sustainable ecotourism. Acknowledgements The authors would like to acknowledge and thank Uganda National Parks director, Dr. Eric Edroma and International Gorilla Conservation Programme (IGCP) director, Jose Kalpers for permission and support to carry out this research, and the Zebra Foundation for funding the research. The authors would also like to thank Dr. Liz Macfie, IGCP; and Bwindi Impenetrable National Park warden, Ignatius Achoka, rangers and Peace Corp volunteers; Dr. Ludwig Siefert, Walter Agai, and Kitimbo from Makerere University Veterinary Faculty; Mel Hutchinson, Dr. Andrew Rycroft and Dot Peters from the Royal Veterinary College, University of London; Dr. Jake Reynolds, World Conservation Monitoring Center; Dr. Hammerberger, North Carolina State University; Professor Vernon Reynolds, Oxford University Department of Anthropology for help with data analysis; Fiona Tripp from Olympus Cameras for the loan of a camera; and Mrs. Rhoda Kalema for logistical assistance. The authors would also like to thank Professor Dwight Bowman at Cornell University and two anonymous reviewers for editorial assistance and comments on the manuscript. References Anderson TJ (1995) Ascaris infections in humans from North America: molecular evidence for cross-infection. 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