This study focused on Oeosophagostomum sp., and more especially on O. bifurcum, as a parasite that can be lethal to humans and is widespread among humans and monkeys in endemic regions, but has not yet been documented in apes. Its epidemiology and the role played by non-human primates in its transmission are still poorly understood. O. stephanostomum was the only species diagnosed so far in chimpanzees. Until recently, O. bifurcum was assumed to have a high zoonotic potential, but recent findings tend to demonstrate that O. bifurcum of non-human primates and humans might be genetically distinct. As the closest relative to human beings, and a species living in spatial proximity to humans in the field site studied, Pan troglodytes is thus an interesting host to investigate. Recently, a role for chimpanzees in the emergence of HIV and malaria in humans has been documented. In the framework of our long-term health monitoring of wild chimpanzees from Kibale National Park in Western Uganda, we analysed 311 samples of faeces. Coproscopy revealed that high-ranking males are more infected than other individuals. These chimpanzees are also the more frequent crop-raiders. Results from PCR assays conducted on larvae and dried faeces also revealed that O. stephanostomum as well as O. bifurcum are infecting chimpanzees, both species co-existing in the same individuals. Because contacts between humans and great apes are increasing with ecotourism and forest fragmentation in areas of high population density, this paper emphasizes that the presence of potential zoonotic parasites should be viewed as a major concern for public health. Investigations of the parasite status of people living around the park or working inside as well as sympatric non-human primates should be planned, and further research might reveal this as a promising aspect of efforts to reinforce measures against crop-raiding.
The disease caused by the nodular worm Oesophagostomum bifurcum can be lethal in humans and is thus of major human health significance in certain African regions. There are still gaps in the understanding of the epidemiology of the disease, including the role of non-human primates as reservoirs of the infection. We recently conducted a survey in a community of wild chimpanzees (Pan troglodytes schweinfurthii) in Kibale National Park, Western Uganda. O. stephanostomum is so far the only species previously found in chimpanzees. A total of 311 stool samples was examined and revealed that high-ranking males are more infected than other individuals. These chimpanzees are also the more frequent crop-raiders. Moreover, we reported for the first time molecular evidence for O. bifurcum in addition to O. stephanostomum in chimpanzees. Our results raise public health concerns for a neglected infection in regions where spatial proximity between great apes and humans are increasing because of forest fragmentation.
Citation: Krief S, Vermeulen B, Lafosse S, Kasenene JM, Nieguitsila A, et al. (2010) Nodular Worm Infection in Wild Chimpanzees in Western Uganda: A Risk for Human Health? PLoS Negl Trop Dis 4(3): e630. doi:10.1371/journal.pntd.0000630
Editor: Stefan Geiger, Fundacao Oswaldo Cruz, Brazil
Received: August 18, 2009; Accepted: January 26, 2010; Published: March 16, 2010
Copyright: © 2010 Krief et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding from Museum National d'Histoire Naturelle (PPF 16) and Ecole Nationale Veterinaire d'Alfort. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Nodular worms (Oesophagostomum spp.) are commonly found as nematode parasites of pigs, ruminants and primates, including humans. In endemic foci in Africa, especially in Ghana and Togo, a high prevalence of Oesophagostomum bifurcum infection has been reported in human populations, one million being estimated at risk ,. Patients are mostly children aged <10 years . Clinical disease, due to encysted larvae, known as oesophagostomosis, sometimes leads to death –. The distinction between hookworm and nodular worms eggs is not possible  and the definitive diagnosis of oesophagostomosis in humans involved exploratory surgery or ultrasound examination. Transmission occurs through the ingestion of the infective third-stage larvae (L3) but the factors explaining such a high regional prevalence remain unknown. Eight species of Oesophagostomum have been recognized so far to occur in non-human primates . Among them, O. bifurcum, O. stephanostomum and O. aculeatum are also reported in humans . Human cases have been attributed to a zoonotic origin, non-human primates being proposed as a potential reservoir . However experimental infection of rhesus monkey (Macaca mulata) showed that O. bifurcum obtained from humans did not effectively infect monkeys . In addition, significant variations exist in lengths of adult worms isolated from humans and non-human primates . The geographic distribution in humans and some non-human primates is not overlapping , and recent molecular findings demonstrated a genetic host-affiliated sub-structuring within O. bifurcum ,. Among great apes, especially chimpanzees, bonobos and gorillas, prevalence of strongyle eggs in stools is often high and O. stephanostomum was the only species of Oesophagostomum identified so far –. However, little is known about the intensity of infection in terms of parasite load and clinical signs in great apes. It has been reported that wild apes develop clinical signs of oesophagostomosis as soon as in captivity  while the presence of parasites remains asymptomatic in wild animals. Recently fatal cases have been described in African apes from sanctuaries  and collected parasites were diagnosed as O. stephanostomum. Nevertheless, because of the phylogenetic and spatial proximity between humans and chimpanzees, potential transmission is not excluded especially in Uganda where human oesophagostomosis has been reported . Around Kibale, population density is high (up to 512 ind/km2)  and chimpanzees regularly crop-raid. Additionally recent findings confirmed that human-related diseases should be considered as a high threat for endangered apes –. As a consequence, it has been emphasized that investigations on potential cross-transmission should be reinforced. We report hereafter the results of our recent finding about nodular worm infection in wild chimpanzees (Pan troglodytes schweinfurthii) in the framework of a long-term health monitoring of the community of Kanyawara in Kibale National Park (Uganda).
Study site and study periods
The studied chimpanzees (Pan troglodytes schweinfurthhii) belonged to one community in Kibale National Park (766 km2, 0°13′–0°41′N, 30°19′–30°32′E), located in Kanyawara area. This community counted 52 chimpanzees in 2006. Ages presented are those estimated in 2006. Their home range is close to the boundary of the Park and Kanyawara chimpanzees are sometimes entering plantations for crop-raiding. Stool samples were collected from identified individuals within the minutes following defecation.
We performed analyses on two series of fecal samples (Table 1). From December 2005 to March 2006, a total of 295 fecal samples was collected from 33 chimpanzees, 17 females (13 adult females and four immature females) and 16 males (9 adult chimpanzees including five dominant individuals, four subordonate individuals and seven immature males) (set 1); coproscopy, coproculture and molecular analysis were performed on the total or parts of this set. In October 2008, 16 samples were collected from 10 identified chimpanzees, 5 females and 5 males. These samples were dried for further molecular analysis (set 2). Indeed, since coprocultures in field conditions and diagnosis of third-stage larvae (L3) are laborious and require skilled personnel for identification, we wished to test a molecular method using dried feces.
Table 1. Analyses for Oesophagostomum sp. identification from fecal samples of wild chimpanzees collected in two periods.doi:10.1371/journal.pntd.0000630.t001
For each sample of set 1 (n = 295), two grams of fresh stool were preserved in 18 mL of 10% formalin saline solution, then smears made with 50 µL of the suspension were microscopically examined. MacMaster flotation was performed at the field station on fresh stools within the day of collection. MacMaster cells were filled with one mL of filtrat of two grams of fresh stools diluted in 30 mL of magnesium sulfate. However, as electricity was not available every day, only 100 samples could be examined.
With both methods, strongyloid eggs were identified according to their size, color, shape and morula aspect (16–32 cells) and they were counted. Egg per gram (epg) counts were corrected according to the fecal consistency (ie ×2 for soft stools and ×3 for diarrheic stools) . Arithmetic corrected mean was calculated including infected and non infected samples (mean abundance). Larvae of Probstmayria sp. and larvae of unidentified species as well as ciliates were also counted during coproscopy (data not shown).
To confirm identification of strongyloid eggs in set 1, coprocultures were performed with 16 stool samples from 13 individuals (5 males, 8 females). After 10 to 15 days of incubation, larvae were collected by Baermann technique and preserved in 95% ethanol. Third-stage larvae (L3) of Oesophagostomum spp. obtained were microscopically diagnosed (long filamentous tail of the sheath, triangular intestinal cells, and length of the larvae ).
Molecular characterization was performed on samples from sets 1 and 2. With the mixture of larvae obtained from each above coproculture (n = 16 samples, set 1), DNA was prepared using Nucleo-spin Tissue (Macherey-Nagel) and ITS2 region was amplified using the primers NC1 and NC2 as described previously . ITS2 sequence of O. stephanostomum is characterized by 2 digestion sites for NLaIII while ITS2 sequence of O. bifurcum is characterized by a unique digestion site. RFLP were analyzed after digestion of the ITS2 sequence. Sequencing was performed on ITS2 sequences and compared to published data (GenBank accession numbers: AF136576 for O. stephanostomum; AF136575 and Y11733 for O. bifurcum). Another PCR test was performed from DNA obtained from the cultured samples to compare the two methods. We used a semi-nested PCR followed by direct sequencing as described before .
From set 2, 16 samples of 4 g fresh feces were stored dried on 20g of silicagel bead. Before DNA extraction, vegetal debris was removed in order to avoid PCR inhibition. DNA was extracted from 200mg of dried feces without culture by using the QIAMP DNA Stool Kit (Qiagen, Chatsworth, CA) according to instructions with the following modifications: in step 3, the suspension of 200 mg with buffer ASL was incubated overnight at 70°C and in step 12, the solution was incubated one hour with proteinase K at 70°C. Direct sequencing after PCR using NC1 and NC2 primers was performed.
Table 1 presents results obtained from the two sets of collection with the different methods of analysis.
Prevalence of strongyloid infection with egg counting
Strongyloid eggs were detected in 12% of the 295 feces examined with direct smears, that is 60% of the chimpanzees (n = 33). The arithmetic mean corrected parasite load of strongyloid eggs was 52±12 epg. The diarrheic samples had a significantly higher oesophagostomine egg counts (225 epg, n = 17) than the firm feces (19 epg, n = 217) (Kruskal-Wallis test: P<0.01). No other factor was significantly affecting egg counts by direct examination although egg counts tend to be affected by hierarchical status in males (dominants: 42 epg, n = 33, subordinates: epg: 14, n = 42).
Strongyloid eggs were detected at least once from all the chimpanzees (n = 29) with MacMaster method. The proportion of positive samples for strongyloid eggs with Mac Master flotation was 91%. The arithmetic mean corrected parasite load of strongyloids was 140±58 epg. Values of corrected epg were significantly different according to hierarchical status in males (dominants: 232 epg, n = 10, subordonates: 88 epg, n = 13; Mann-Whitney test; P value = 0.005) and fecal consistency (firm feces: 90 epg, n = 73; soft feces: 239 epg, n = 20; diarrheic feces: 414 epg, n = 7; Kruskal-Wallis test: P value = 0.021). No difference according to the sex, the age or the sampling period of the day was found.
Prevalence of Oesophagostomum spp. infection
L3 characteristic of Oesophagostomum were found after coproculture and microscopic examination in 12/16 samples from 10/13 chimpanzees (3 males; 10 females) of the set 1.
Species identification with molecular analysis
PCR-RFLP conducted on larvae from the 16 coprocultured samples (set 1) from wild chimpanzees identified O. stephanostomum and O. bifurcum. ITS2 sequence of O. stephanostomum, characterized by 2 digestion sites for NLaIII, was identified from fecal samples from 2 chimpanzees (MS, male and AL, female). ITS2 sequence of O. bifurcum, characterized by a unique digestion site, was identified from one fecal sample from one chimpanzee (AJ, male) (Fig. 1). Sequencing performed on these samples confirmed the presence of the two species. All but one samples revealed DNA sequences showing 99% of homology with Panagrolaimus sp. (AY878405 from Genbank) and one sample revealed 82–88% homology with Necator sp. (AF217891 from Genbank) nematodes. With semi-nested PCR and direct sequencing, O. stephanostomum was identified in one of the two chimpanzees positive with PCR-RFLP (MS, male) and O. bifurcum in five chimpanzees (Fig. 2). In the second set of fecal samples, which were stored dried, Oesophagostomum DNA was found in 14 of the 16 fecal samples. All the 10 chimpanzees sampled in set 2 were positive.
Figure 1. PCR-RFLP of ITS2 using the endonuclease NlaIII of DNA samples of Oesophagostomum stephanostomum (chimpanzees MS and AL) and Oesophagostomum bifurcum (chimpanzee AJ) extracted from coprocultured feces of 13 wild chimpanzees.
The 13 chimpanzees sampled are: AJ, male, 31 yr old; AL, female, 23 yr old; BB, male, 39 yr old; KK, male, 20 yrs-old; MS male 26 yr old; OG, male, 4 yr old; OM, female 1 yr old; OK, female, 11 yr old; OU, female, 26 yr old; OT, female, 7 yr old; PG, male 17 yr old; ST, male, 50 yr old; WL, female, 13 yr old. DNA from 16 samples of these chimpanzees was extracted and digested. 2 different samples from NL and from KK were analysed, one sample from OK has been tested twice. PCR products at 500 bp correspond to DNA from Panagrolaimus sp. and PCR product of the sample KK (2) to Necator -like DNA species. Control with Panagrolaimus larva (PL).doi:10.1371/journal.pntd.0000630.g001
Figure 2. Semi-nested PCR and direct sequencing on DNA extracted from 16 coprocultured samples of 13 wild chimpanzees (see identity in Figure 1).
Two different samples from NL and from KK were analysed, one sample from OK has been tested twice. Circle: Oesophagostomum stephanostomum products sequenced; square: Oesophagostomum bifurcum products sequenced. Control with Panagrolaimus larvae (PL).doi:10.1371/journal.pntd.0000630.g002
All the sequences obtained from extraction of larvae DNA and dried feces were identical between them for each of the two Oesophagostomum species. The sequences corresponding to O. stephanostomum were 100% identical to the ITS2 reference sequence of O. stephanostomum collected from a chimpanzee in Tanzania (GenBank accession number AF136576) (BLASTn). In the sequences we obtained there was no mixed sequence signals in positions 116, 176, 197 in which the reference sequence shows polymorphism. (fig. 2). Among the three sites where polymorphism is observed in ITS-2 sequences for human and monkeys for O. bifurcum (positions 56, 112, 162), there is no nucleotides equivoque in our sequences. Our sequences were identical to the ITS2 sequence of O. bifurcum collected from a human (100% of identity with GenBank accession number Y11733) and different in position 112 from the one documented in Cercopithecus mona (GenBank accession number AF136575). The species O. stephanostomum was found in 11 chimpanzees. The species O. bifurcum was found in 5 chimpanzees. One chimpanzee was co-infected by two Oesophagostomum species in the first period (AL, female) and four chimpanzees (2 females AJ and WL and two males AL and ST) were infected by the two species if we considered both periods (December 2005 to March 2006 and October 2008). Whatever the method used to identify specimens of the genus Oesophagostomum (coproculture or any molecular characterization), a total number of 26 of 32 samples (81%) were positive corresponding to 15 chimpanzees of the 18 sampled (83%).
In this study we used several methods to survey parasite status of wild chimpanzees.
We compared nodular worm eggs counts between individuals of different classes of age, sex and dominance rank. We demonstrated for the first time that wild chimpanzees could be infected by O. bifurcum. The presence of two Oesophagostomum species (O. stephanostomum and O. bifurcum) was reported in the same chimpanzees community. Our results, based on RFLP-PCR and semi-nested PCR-direct sequencing and PCR from dried stools, extend our understanding of the epidemiology of O. bifurcum, confirm accuracy of alternative method (DNA extraction from dried stools) to coproculture and raise public health awareness for a neglected disease.
However while the substantial increase of accuracy of PCR compared to coproscopy has been previously shown , technical difficulties and limitations of stool analyses and culture due to field conditions when studying wild chimpanzees have to be considered. They are overcome by using PCR directly on dried stools. Additionally, the sensitivity of molecular analysis was higher when applied directly on dried samples than samples obtained from coproculture. With both methods of coproscopy, we determined that high-ranking males in Kanyawara chimpanzee community had higher parasite burdens during the study period. Our results also provided evidence that these free-ranging chimpanzees are infected by two Oesophagostomum species (O. stephanostomum and O. bifurcum). The species O. bifurcum is responsible for human and monkeys infections and had never been characterized in wild great apes as chimpanzees so far. The species O. stephanostomum is detected in great apes and this species was recently associated with nodular lesions in chimpanzees and a gorilla from sanctuaries . Behavioral patterns of Pan troglodytes may explain that males are more infected than females by strongyloid parasites: male chimpanzees are staying all their life in their native community while females migrate. Males develop close relationships, indulging in very long grooming sessions where individuals are staying in close proximity. Our results are consistent with previous studies showing that both testosterone and cortisol were positively associated with gastrointestinal parasite infections in Kibale chimpanzees  suggesting that stress of high-ranking males may alter an efficient immune response. Additionally males are visiting plantations in the edge of the forest more frequently than females, encountering conditions favoring parasite transmission from humans and non-human primates: people being very close to the forest are usually not using latrines and monkeys in the edge such as red colobus are more infected than those from the interior, egg counts for Oesophagostomum being 10 times higher .
In spite of the severe health problem caused by oesophagostomosis to humans, epidemiology and transmission of the disease are still poorly understood . While colobus monkeys were not infected in surveys conducted in Ghana ,, in Kibale NP, primates including the arboreal red colobus (Piliocolobus tephrosceles) and black and white colobus (Colobus guereza) and more terrestrial species such as olive baboons (Papio anubis) were proved to be infected by Oesophagostomum sp. ,,. Diagnosis of the parasites species was not conducted in monkeys but previous findings suggesting no risk of infection for arboreal colobus monkeys  was not supported at the genus level in this area. The role of chimpanzees and other primates in the cycle needs thus to be further explored. Oesophagostomum bifurcum nematodes from chimpanzees may be genetically distinct from O. bifurcum nematodes from other primate species including humans as previously demonstrated ,. However, chimpanzees are more closely related to humans than non-human primates species investigated so far (colobus, baboons, patas and Mona monkeys) and investigating the genetic variability of O. bifurcum between chimpanzees and other primates would be interesting. Moreover, the home ranges of chimpanzees from Kibale NP include areas where human beings are present. Chimpanzees are visiting plantations bordering their forest home range and males, especially high-ranking males, which have higher infection level, more frequently. Humans are working or entering inside the park (researchers, research assistants, other employees from the park, poachers…). Chimpanzees are also exploiting resources also used by other non-human primates. For these reasons, even if the origin of infection is unknown, the zoonosis risk can not be excluded. Outbreaks of oesophagostomosis in human population have not been documented in the study area. However, an investigation of the parasite status in humans living in the villages surrounding the park should be planed. The presence of potentially zoonotic parasites in chimpanzees in a context where proximity between human and apes is increasing (ecotourism, crop-raiding, research…) should be viewed as a point of concern for the future of public health in this region and elsewhere. For economic and health reasons, prevention of crop-raiding programs should be reinforced.
French translation of the abstract by SK.
(0.03 MB DOC)
We are very grateful to the Uganda Wildlife Authority, the Uganda National Council for Science and Technology for permission to conduct this research and Makerere University Biological Field Station for logistical support. We deeply thank Jean-Michel Krief and Field Assistants Japan Musinguzi and Ronald Musinguzi for their help during fieldwork. We are grateful to Evelyne Heyer, Rebecca Hardin, Anton Polderman and an anonymous reviewer for their constructive comments on the manuscript.
Conceived and designed the experiments: SK AN JG. Performed the experiments: SK BV SL AN MB OB. Analyzed the data: SK BV SL OB JG. Contributed reagents/materials/analysis tools: SK JMK ML JG. Wrote the paper: SK JG. Collaborated and assisted with field studies on chimpanzees in Kibale National Park: JMK.
- 1. Polderman AM, Krepel HP, Baeta S, Blotkamp J, Gigase P (1991) Oesophagostomiasis, a common infection of man in Northern Togo and Ghana. Am J Trop Med Hyg 44: 336–344.
- 2. Storey PA, Faile G, Hewitt E, Yelifari L, Polderman AM, et al. (2000) Clinical epidemiology and classification of human oesophagotomiasis. T Roy Soc Trop Med H 94: 177–182.
- 3. Polderman AM, Blotkamp J (1995) Oesophagostomum infections in Humans. Parasitol Today 11: 451–456.
- 4. Blotkamp J, Krepel HP, Kumar V, Batea S, Van't Noordende JM, et al. (1993) Observation on the morphology of adults and larval stages of Oesophagostomum sp. isolated from man in Northern Togo and Ghana. J Helminthol 67: 49–61.
- 5. Eberhard ML, Kovacs-Nace E, Blotkamp J, Verweij JJ, Asigri VA, et al. (2001) Experimental Oesophagostomum bifurcum in monkeys. J Helminthol 75: 51–56.
- 6. de Gruijter JM, Polderman AM, Zhu XQ, Gasser RB (2002) Screening for haplotypic variability within Oesophagostomum bifurcum (Nematoda) employing a single-strand conformation polymorphism approach. Mol Cell Probes 16: 185–190.
- 7. de Gruijter JM, Ziem J, Verweij JJ, Polderman AM, Gasser RB (2004) Genetic substructuring within Oesophagostomum bifurcum (Nematoda) from human and non-human primates from Ghana based on random amplified polymorphic DNA analysis. Am J Trop Med Hyg 71: 227–233.
- 8. File SK, Mc Grew WC, Tutin CEG (1976) The intestinal parasites of a community of feral chimpanzees, Pan troglodytes schweinfurthii. J Parasitol 62: 259–261.
- 9. McGrew WC, Tutin CEG, Collins DA, File SK (1989) Intestinal parasites of sympatric Pan troglodytes and Papio spp. at two sites: Gombe (Tanzania) and Mt. Assirik (Senegal). Am J Primatol 17: 147–155.
- 10. Krief S, Huffman M, Sévenet T, Guillot J, Bories C, et al. (2005) Non invasive Monitoring of the health of Pan troglodytes schweinfurthii in the Kibale National Park, Uganda. Int J Primatol 26: 467–490.
- 11. Rousselot R, Pellissier A (1952) Esophagostomose nodulaire à Oesophagostomum stephanostomum. Pathologie du gorille et du chimpanzé. Bull Soc Path Ex 9: 569–574.
- 12. Krief S, Jamart A, Mahé S, Leendertz FH, Mätz-Rensing K, et al. (2008) Clinical and pathologic manifestation of oesophagostomosis in African great apes: does self-medication in wild apes influence disease progression? J Med Primatol 37: 188–195.
- 13. Naughton-Treves L (1998) Predicting patterns of crop damage by wildlife around Kibale National Park, Uganda. Conserv Biol 12: 156–168.
- 14. Woodford MH, Butynski TM, Karesh WB (2002) Habituating the great apes: the disease risks. Oryx 36: 153–160.
- 15. Köndgen S, Kühl H, N'Goran PK, Walsh PD, Schenk S, et al. (2008) Pandemic human viruses cause decline of endangered great apes. Curr Biol 18: 260–264.
- 16. Kaur T, Singh J, Tong S, Humphrey C, Clevenger D, et al. (2008) Descriptive epidemiology of fatal respiratory outbreaks and detection of a human-related metapneumovirus in wild chimpanzees (Pan troglodytes) at Mahale Mountains National Park, Western Tanzania. Am J Primatol 70: 755–765.
- 17. Boesch C (2008) Why do chimpanzees die in the forest? The challenges of understanding and controlling for wild ape health. Am J Primatol 70: 722–726.
- 18. Williams JM, Lonsdorf EV, Wilson ML, Schumacher-Stankey J, Goodall J, et al. (2008) Causes of death in the Kasekela chimpanzees of Gombe National Park, Tanzania. Am J Primatol 70: 766–777.
- 19. Gasser RB, Woods WG, Huffman MA, Blotkamp J, Polderman AM (1999) Molecular separation of Oesophagostomum stephanostomum and Oesophagostomum bifurcum (Nematoda: Strongyloidea) from non-human primates. Int J Parasitol 29: 1087–1091.
- 20. Verweij JJ, Wimmenhove MC, Polderman AM, Gasser R (2000) PCR assay for the specific implification of Oesophagostomum bifurcum DNA from human faeces. Int J Parasitol 30: 137–142.
- 21. Verweij JJ, Pit DS, van Lieshout L, Baeta SM, Dery GD, et al. (2001) Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples. Trop Med Int Health 6: 726–731.
- 22. Muehlenbein MP (2006) Intestinal parasite infections and fecal steroid levels in wild chimpanzees. Am J Phys Anthropol 130: 546–550.
- 23. Chapman CA, Speirs ML, Gillespie TR, Holland T, Austad KM (2006) Life on the edge: Gastrointestinal parasites from forest edge and interior primate groups. Am J Primatol 68: 397–409.
- 24. Gasser RB, de Gruijter JM, Polderman AM (2006) Insights into the epidemiology and genetic make-up of Oesophagostomum bifurcum from human and non-human primates using molecular tools. Parasitology 132: 453–460.
- 25. Gillespie TR, Greiner EC, Chapman CA (2005) Gastrointestinal parasites of the colobus monkeys of Uganda. J Parasitol 91: 569–573.
- 26. Bezjian M, Gillespie TR, Chapman CA, Greiner EC (2008) Gastrointestinal parasites of forest baboons, Papio anubis, in Kibale National Park, Uganda. J Wildlife Dis 44: 878–887.