Introduction

The microbial communities residing in the gastrointestinal tracts of primates are complex and may play important roles in health and disease. The interactions between microbial cells and primate host cells could be either commensal or parasitic and it is known that these interactions have an effect on the metabolic, developmental and immunological status of the host1,2. The compositions and constituents of these communities are influenced by several factors, such as the host diet, geography, physiology and disease state3,4.

Gorillas share a close phylogenetic relationship with humans, resulting in a high potential for pathogen exchange involving bacteria, viruses and gastrointestinal parasites5,6,7. Moreover, the presence of these pathogens in wild primates may also have negative consequences for public health and wildlife conservation management8. To date, there have been no studies examining the entire eukaryotic community residing in the intestinal tract of the gorilla; rather, most studies focus on the parasitological aspects of these eukaryotic communities using coprological studies to survey the presence of intestinal parasites in wild gorilla populations. Several such studies have been conducted in both mountain gorillas (Gorilla gorilla beringei) and western lowland gorillas (Gorilla gorilla gorilla) in different geographical locations9,10,11,12,13. The intestinal microbiota appears to be the same among social groups and individual gorillas living in the Bwindi Impenetrable Forest in Uganda and that the flora is largely dominated by entodiniomorph ciliates and helminths, while amoebae and flagellates appear to be absent9,10. The coprological studies involving western gorillas also reported many species of parasites, particularly entodiniomorph ciliates and strongylates11. Two studies examined fecal samples from western lowland gorillas living in the Dzanga-Ndoki National Park and at Bai Hokou, Central African Republic and found that most of individuals were infected with strongylates, whereas ascaroids and threadworms were only moderately present12,13. Low prevalence of Entamoeba coli, Balantidium coli and Iodamoeba butschlii have also been recorded in western lowland gorillas along with trichomonads, which were the only protozoans that were present in all gorilla age-sex classes12,13.

Despite these studies, the diversity of eukaryotic communities in primates and particularly in gorillas, remains to be elucidated, particularly with regard to intestinal fungal diversity. In addition, the morphological descriptions of these eukaryotes are typically insufficient and thus cannot be considered in taxonomical studies. In this study, we present firstly, an extensive molecular data set of the occurrence of gastrointestinal eukaryotic microbiota including some human eukaryotic pathogens in a single fecal sample from a wild western lowland gorilla from Cameroon and then followed by molecular detection of potential human eukaryotic pathogen in gastrointestinal tracts of wild population of gorillas.

Results

Eukaryotes retrieved from gut microbiota of gorilla

In total, 35 existing primer sets targeting the 18S rRNA and internal transcribed spacer (ITS) genes and other specific eukaryotic genes were used to explore the diversity of the eukaryotes that were found in a fecal sample that was obtained from a wild gorilla in Cameroon (Supplementary Tables 1). Seventeen positive PCR products were obtained. Two of them (TFR1/TFR2 and 18ScomF1/Dino18SR1) were sequenced directly as obtained sequences from these two primers yield no trouble sequences, but the remaining were problematic and were thus cloned. Overall, 1,572 clones were obtained from all of the cloning libraries that were constructed in this study (Supplementary Tables 2), resulting in the detection of 87 eukaryotic species in the fecal sample (Fig. 1). Most of the species that were present were fungi (52 species, Table 1), while 10 were identified as protozoa (Table 2), 4 as nematodes (Table 3) and 21 as plants (Table 4).

Table 1 Fungal species obtained from different cloning libraries in this study
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Table 2 Protozoal species detected by various clone libraries in this study
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Table 3 Nematode species detected by the NC1/NC2 clone library in this study
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Table 4 Plant species retrieved from the fecal sample of a wild gorilla
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Figure 1
figure 1

The eukaryotic species that were retrieved from the gut of Gorilla gorilla gorilla according to the different primers used.

A box indicates that the species was positive with the primer set used. Blue color = fungi; red color = protozoa; orange color = helminths; and green color = plants. The trees were constructed using the free software MEGA 5 and sequences that were retrieved from GenBank (ITS sequences for fungi and helminths and 18S rDNA for protozoa and plants).

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Fungal diversity

Fifty-two different species of fungi were retrieved from the gorilla fecal sample following the analysis of 428 fungal clones (accounting for 59.8% of the total species detected and 27.2% of the total clones retrieved in this study). These species were detected from different cloning libraries that were generated using various primers (Table 1, Fig. 1). Most of the detected taxa belonged to the phylum Ascomycota (36 species, 69.2% of the identified taxa), followed by taxa from the Basidiomycota (15 species, 28.8% of the detected taxa). The remaining 2% of the taxa were affiliated with the phylum Zygomycota (Table 1). Only 5 fungal species were isolated through culture-dependent methods, including Alternaria alternata, Cladosporium sp., Malassezia restricta, Malassezia globosa, and Malassezia pachydermatis.

All of the 171 sequenced clones from the fungal ITS clone library that were generated using the ITS1-F/ITS-4R primer set were related to fungal sequences that were found in the GenBank databases with the exception of one clone belonging to the Viridiplantae (green plants) (Tables 1 and 4, Fig. 1). This amplification alone allowed for the identification of 25 different fungal species (roughly half of the fungal species detected and 28.7% of all eukaryotic species recovered in this study) (Fig. 1). The majority of the fungal sequences in this clone library were of ascomycete origin (91.2% of the total number of clones and 80.8% of the species detected); the species were assigned to different taxonomic groups, including Saccharomycetales, Pleosporales, Capnodiales, Eurotiales, Hypocreales and Xylariales (Table 1). Only 6.4% of the sequences that were retrieved from this ITS library were related to the basidiomycetes, belonging to both Tremellales and Corticiales (Table 1). Our ITS results also showed the presence of a few zygomycetes-related sequences that were represented by Mortierella sp. (Table 1).

The primer set NSI/FRI, targeting the fungal 18S rRNA gene, was also used in this study, resulting in a total of 17 fungal species (16 plus one species that was retrieved from fungal ITS amplification) (Table 1, Fig. 1). In contrast to the ITS clone library, the BLAST results for the 96 clones that were obtained from this library revealed that 58 sequences (60.4%) were most closely related to the basidiomycetes, whereas 38 sequences (39.6%) were ascomycetes (Table 1).

In addition to the 41 fungal species that were recovered by the aforementioned fungal-specific primer sets, 11 were identified in other clone libraries when universal eukaryotic primers targeting 18S rDNA were used (Table 1, Fig. 1). Among these 11 species, 8 were detected using the universal eukaryotic primer set Euk1A/Euk516r.

The use of different specific and universal 18S clone libraries revealed a total of 30 fungal species in the stool sample (Fig. 1), while PCR methods using fungal ITS genes enabled the detection of 25 species (Fig. 1). Only 3 species, A. alternata, Candida rugosa and Hanseniaspora occidentalis, were detected in both the ITS and 18S amplifications, thus suggesting the complementarity of these approaches for the assessment of fungal communities in the gorilla intestinal tract.

Protozoal diversity

Ten different species of protozoans were detected in the gorilla fecal sample following both the sequencing of 246 protozoal-related clones (15.6% of all clones in this study) that were obtained from 8 different cloning libraries and the direct sequencing of the positive PCR product that were obtained with the TFR1/TFR2 primer set (Table 2, Fig. 1). Six species belonging to Ciliophora were detected from two of the cloning libraries, including Blepharocorys curvigula, Cycloposthium bipalmatum, Cycloposthium ishikawai, Parentodinium sp., Triplumaria selenica and Troglodytella abrassarti (Table 2, Fig. 1). Ninety-six sequences amplified by Leishmania specific primers were assigned to Trypanosomatidae family (Fig. 1, Table 2) as the best hit of BLAST results of these sequences was Leishmania sp. (with sequence coverage ranged between 69–79%). The remaining protozoans were identified as Blastocystis sp., Iodamoeba sp. and Tetratrichomonas buttreyi, belonging to the three taxa Stramenopiles, Amoebozoa and Parabasalia, respectively (Table 2, Fig. 1).

Helminthic diversity

Despite the use of several primers to detect the occurrences of trematodes, cestodes and nematodes in the gorilla fecal sample (Supplementary Tables 1), only the PCR amplifications using the latter primer pairs (NC1/NC2) yielded positive results; these PCR products were used to construct the nematode clone libraries (Table 3). Four nematode species, Necator americanus, Libyostrongylus douglassi, Oesophagostomum sp. and Oesophagostomum stephanostomum, were obtained from sequencing 192 positive clones in this library (Table 3, Fig. 1). The human parasitic nematode, N. americanus, accounted for 55.7% of this clone library (Table 3).

Plant diversity

Twenty-one plant species (Table 4) were retrieved in this study from 13 different cloning libraries that were generated using primer pairs targeting the 18S rRNA, ITS and chloroplast genes (Supplementary Tables 2). The plant-related clones constituted roughly 44.9% of all clones that were sequenced in this study. The majority of plant sequences that were detected in most of the cloning libraries belonged to Manilkara zapota and Musa basjoo, which comprised 28.6% and 16.7% of the total plant-related sequences, respectively (Table 4, Fig. 1). Only two plant species, Schima superba and Davidia involucrata, were detected from the chloroplast clone libraries that were constructed using the primer set rbcLZ1/rbcL19b; the 19 remaining plant species were retrieved from the 18S rRNA cloning libraries that were constructed from various 18S rRNA primer pairs (Table 4, Fig. 1).

Eukaryotic human pathogens in fecal samples of gorillas

Real time PCR examination (Supplementary Table 3) of 48 fecal samples from 21 individual of wild gorillas, sampled from Minton and Messok location in Cameroon, showed that 41 (18 gorillas, 86%) and 11 (9 gorillas, 43%) of gorillas' fecal samples harbor Oesophagostomum bifurcum and N. americanus respectively (Table 5). The results also showed that human pathogenic fungi such as Candida tropicalis, Candida parapsilosis, C. rugosa, M. restricta, M. globosa, Trichosporon spp. and Trichosporon asahii were also detected in feces of these wild animals as shown in Table 5. However, Blastocystis sp., Candida albicans, M. pachydermatis and Rhodotorula mucilaginosa were not detected in any gorillas' feces.

Table 5 Eukaryotic Human Pathogens detected by Real Time PCR in 48 fecal samples from wild gorillas (21 individuals)
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Discussion

Previous studies focusing on the detection and identification of eukaryotic communities residing in the non-human primate gastrointestinal tract analyzed the gut microbiota using the morphological features of the flora9,10,11,12,13, limiting the acquisition of knowledge regarding the real eukaryotic intestinal contents of primates. Regardless of some limitations that are associated with culture-independent methods14, these methods have recently been used to explore the eukaryotic diversity of the human gut15,16,17,18,19,20 and these techniques have played a crucial role in providing novel insight into the true diversity and composition of the gut microbiota. Because very little is known about both the diversity of eukaryotic organism occurring in the digestive tract of non-human primates and the presence of potential pathogens in their guts, two general approaches were used in our investigation; firstly, culture dependent and independent methods were carried out in a single stool sample from gorilla then additional stool samples of wild gorillas were screened for pathogens by specific PCRs.

Fungal diversity and detection of human fungal pathogens

Culture-dependent approaches limited the number of fungi that were isolated, revealing less fungal diversity because of difficulties that were encountered in their growth using routine laboratory culture21. In contrast, the culture-independent methods were more effective and revealed a larger diversity of fungi16.

Although the molecular-based methods potentially detected a wide range of fungi in this study, but it is still difficult to predict whether they are the real symbiotic fungal components of gut in these animal or potential environmental fungal contaminants. The immediate collection of feces from animal in forest is a complicate process especially with these endangered wild animals and therefore the difficulties in preventing direct contact of feces bulk with its surrounding environment during and after defecation time are a major drawback in such studies. Moreover, the nature of some ascomycetous and basidiomycetous species that were detected in this study are considered to be saprobes and thus they possibly representing environmental fungal contaminants as they are often found in association with plants, animals and their interfaces22. For example several members of Saccharomycetales such as Hanseniaspora spp., Saccharomycopsis crataegensis, Pichia spp., Issatchenkia spp. and Candida quercitrusa regard as plant associated fungi and many of them described as common fermentative spoilage yeast23,24,25,26,27. Candida entomophila characterized by its ability to ferment glucose and D-xylose therefore it usually has been isolated from wood-inhabiting insects and decaying wood28. Furthermore, some members of orders Eurotiales, Hypocreales, Xylariales and Pleosporales that have been identified in this study are regarded as the most common environmental fungi and they abundantly occur in forest soils or on fading leaves of herbaceous and woody plants such as cosmopolitan genus of Penicillium (Eurotiales) which has been found to play important role as decomposers of organic materials in soil29, several species of Bionectria (Hypocreales) which can be found as common saprophytes on dead broad-leaf trees in forest30, along with members of the genus Xylaria (Xylariales) that usually occur as saprobes or as parasite on flowering plants in lowland forests31 and finally some species of Pleosporales that occur as saprobic fungi on decaying leaf or animal dung32.

Another explanation for occurrence of these fungi in feces of gorillas is the fact that these apes are herbivore in their behavior and they feed on different parts of plants in which different fungi coexist. Therefore these fungi could also represent transient contaminants in the gut of gorillas. Moreover, several of the ascomycetous yeast that were detected in this study, such as Candida orthopsilosis, C. rugosa, C. tropicalis and Galactomyces geotrichum, have been previously described in the human gut15,19,33. Some of the basidiomycetous yeasts that were detected in our sample, such as the Malassezia and Trichosporon species, are regarded as human pathogens34,35. The remaining basidiomycetous fungi that were identified in our study including the saprotrophics, wood decomposers and symbiotic fungi, such as Termitomyces, which is generally regarded as a nutritional source for termites36 that are in turn regarded as a source of food and particularly of protein and vitamins, for wild gorillas37. Finally, using real-time PCR, many human fungal pathogens were detected in gorilla feces including species in the genera Candida, Malassezia and Trichosporon with high prevalence of C. tropicalis (81%), M. globosa (43.5%) and C. parapsilosis (38%) through our survey (Table 5).

Protozoal Diversity

Numerous studies have been performed on the intestinal parasites of wild non-human primate species, especially on gorillas9,10,11,12,13; however, this study, to the best of our knowledge, is the first molecular study attempting to detect both parasitic and commensal protozoans in the gastrointestinal tract of the wild western lowland gorilla. In the present study, the majority of the intestinal protozoa that were detected in the fecal sample belonged to four different phyla: the Ciliophora, Amoebozoa, Parabasalia and Stramenopiles. The most prevalent protozoal species that were found in this study were ciliates; approximately 6 species were detected in the fecal sample, which is in agreement with results from previous studies that identified entodiniomorph ciliates in the majority of fecal samples that had been collected from wild western lowland gorillas at Bai Hokou, Dzanga-Ndoki National Park (Central African Republic) and the Lopé Reserve (Gabon)11,12,13. However, our results conflict with those from the study by Modry et al.38, in which T. abrassarti was the sole entodiniomorph ciliate that was detected in captive western lowland gorillas at the Prague Zoo in the Czech Republic. T. abrassarti has also been morphologically observed in fecal specimens from wild lowland gorillas39, where it appears to play an important role in digestion because of its ability to ferment polysaccharides in the hindguts of primates40.

Our study described the first report of B. curvigula, Parentodinium sp. and Cycloposthiidae species, including C. bipalmatum, C. ishikawa and T. selenica in the gastrointestinal tract of the wild gorilla.

Members of the Amoebozoa were detected at low frequencies despite the use of many primer sets that target the 18S rRNA genes of the major groups in this phylum (Supplementary Tables 1). Only Iodamoeba spp. were found in the present study. The low abundance of amoebae reported here agrees with studies by Freeman et al.13 and Lilly et al.12, both of whom reported the low prevalence of amoebae in fecal samples of wild lowland gorillas. However, our results disagree with the study by Sleeman et al.10, who reported the high prevalence of amoebae in mountain gorillas. Blastocystis sp. was retrieved from the intestinal tract of one gorilla in this study. Screening the 48 samples from 21 individual of gorillas has demonstrated that this protozoan is not frequent within western lowland gorillas in Cameroon (Table 5). Blastocystis can be found in both humans and nonhuman primates41,42. In humans, it appears to be a causative agent of irritable bowel syndrome (IBS) in certain circumstances41, while in non-human primates, its role remains unclear.

Either none or very few enteric flagellate protozoa have been observed in the fecal samples of wild gorillas9,10,11,12,13; additionally, only some members of the trichomonads and Giardia sp. have been recorded in both captive and wild gorillas, respectively10,43. These previous studies are in partial agreement with our study, in which only T. buttreyi, a trichomonad, was detected. T. buttreyi has also been detected in ruminant feces and it appears to be harmless to its host44.

Helminthic diversity and detection of human nematode pathogens

We attempted to determine the presence of trematodes, cestodes and nematodes in the fecal samples of the gorillas. Only parasites belonged to the phylum Nematoda were identified; we did not report the presence of any other groups of helminths. Previous studies that were conducted in wild lowland gorilla populations in Gabon and the Central African Republic showed also an absence of cestodes and a scarcity of trematodes in fecal samples11,12,13.

The presence of nematode species, such as O. stephanostomum, in the intestinal tract of gorillas has been morphologically confirmed by Sleeman et al.10. Oesophagostomum spp. can also infect ruminants, pigs and monkeys45. Some reports have described human infections with Oesophagostomum spp., particularly in northern Togo and Ghana where they have been known to cause serious health problems45,46. Another nematode that was recorded in our study was N. americanus, which is an obligate hookworm parasite that is responsible for most common chronic infections in humans, particularly in areas of rural poverty in the tropics and subtropics47. This hookworm is generally transmitted through contact with contaminated soil and resides in the intestinal tract of its host47. The hookworm's infection with N. americanus has been described previously in intestinal tract of both mountain gorillas (G. g. beringei) inhabiting at Bwindi Impenetrable National Park, South West Uganda48 and western lowland gorillas (G. g. gorilla) residing at Dzanga-Sangha Protected Areas, Southwest of Central African Republic49. Finally, an ostrich-specific nematode (L. douglassi) was also detected in our sample. As this parasite commonly infects the ostrich proventriculus and can cause libyostrongylosis, which has a high mortality rate among juvenile birds50, the detection of this nematode in gorilla's feces in this study could be resulted either from environmental contamination or consumption of contaminated food items. Analyzing of more fecal samples is needed to further explaining the presence of this parasite in feces of this animal. In our survey, high percentage of both human parasitic worms; O. bifurcum and N. americanus (86% and 43% respectively) was discovered in the stool samples of western lowland gorillas from Cameroon (Table 5).

Residual plants in gut of gorilla

Gorillas are largely herbivorous and consume a wide variety of plant species (between 50 and 300)51,52. Studies of western gorillas have shown that fruit is an essential part of their diet53,54, but they also eat leaves, shoots, flowers and the woody parts of plants51,52. In this study, we detected 21 different plant species in the fecal sample of a wild gorilla collected around the village of Minton. Primer sets targeting both 18S rRNA and the chloroplast rbcL genes were used to identify the residual plant species in the gorilla feces. Unexpectedly, only two phylotypes of plants were detected when the primer targeting the chloroplast rbcL gene was used. These results are in agreement with those of Bradle et al.55, who also detected few plants in western gorilla feces using the same primer set, which preferentially amplifies DNA from chloroplast-rich tissues, such as leaves or stems, rather than fruits, flowers and seeds55. Not surprisingly, roughly 19 plant species were co-amplified along with other eukaryotes in this study when universal eukaryotic primers targeting 18S rRNA were used. These plant species belong to different families that may be consumed by wild western lowland gorillas.

In conclusions, this is the first study to characterize fecal eukaryotic diversity, including fungi, in non-human primates using a comprehensive extended molecular analysis. The multiple primer set approach used herein enabled us to recover a high diversity of eukaryotes from the intestinal tract of the wild lowland gorilla, which may include human pathogens as revealed by our real-time PCR assessments in gorillas' feces. Although the detection of fungi species should be interpreted cautiously because the possibility of environmental contamination, the presence of human parasites in gorillas should be viewed as an important public health concern, particularly for surrounding rural villages where habitat overlap is frequent. Additional studies from other geographic locations and using the methodological strategy presented here are required for detailed descriptions of the occurrences and abundances of eukaryotes, including pathogens, in the guts of non-human primates, which have until now been poorly described.

Methods

Source of fecal samples

A total of 48 fecal samples were collected from 21 individual western lowland gorillas (G. g. gorilla) in this study (Supplementary Tables 4). One fecal sample was collected in a site near Minton village which located in south-central Cameroon and was used in this study for exploring the occurrence of gut eukaryotes in gorilla intestinal tract through using PCR-based amplification using various primers, followed by cloning and sequencing, while the rest of 47 fecal samples were collected from different sites around Messok village which located in the south-east Cameroon and were used in this study for investigating the presence of human eukaryotic pathogens in gut of gorillas. The sample collection protocol was described previously56. The GPS position, time and date were recorded for all samples. The fecal samples were preserved in RNAlater (Ambion, Austin, TX) and kept at room temperature at base camps for less than 3 weeks then transported to a central laboratory and kept at −80°C.The collection of the fecal samples was approved by the Ministry of Scientific Research and Innovation of Cameroon. No other permit was required for the described field as this research was non-invasive work and the collection of the samples from soil did not disrupt the wild fauna.

DNA extraction

Total DNA was extracted from the frozen fecal samples using a modification of the Qiagen stool procedure and the Qiamp® DNA Stool Mini Kit (Qiagen, Courtaboeuf, France)16. The inner part of the fecal bulk was used for extraction to avoid as much as possible an eventual contamination with soil organisms and/or environmental species during collection as previously described by14. Aliquots of 200 mg of this part were added into tubes containing a 200 mg mixture of 0.1, 0.5 and 2 mm zirconium beads and 1.5 ml of ASL buffer (Qiagen). The samples were mixed vigorously by agitation in a FastPrep BIO 101 agitator (Qbiogene, Strasbourg, France) at 3,200 rpm for 90 seconds. Agitation was followed by heating at 95°C for 10 min to increase both the yield of DNA and proteinase K digestion before the DNA was bound to a column, washed and eluted in TE buffer.

Genomic amplification

All universal and specific eukaryotic primers targeting both the ITS and 18S rRNA genes that were used in this study were adopted from previously published studies (Supplementary Tables 2). The 50 µL PCR reaction mixture contained 5 µL of dNTPs (2 mM of each nucleotide), 5 µL of DNA polymerase buffer (Qiagen, Courtaboeuf, France), 2 µl of MgCl2 (25 mM), 0.25 µL of HotStarTaq DNA polymerase (1.25 U) (Qiagen, Courtaboeuf, France), 1 µL of each primer and 5 µL of DNA. The PCR cycling conditions for all amplifications were as follows: 1 cycle at 95°C for 15 min, 40 cycles at 95°C for 0.5 min, 48–60°C for 0.5–2 min (Supplementary Tables 2) and 72°C for 1–2 min, followed by a final cycle at 72°C for 5 min. All amplifications were performed in a PCR system 2720 thermal cycler (Applied Biosystems, Courtaboeuf, France). Amplification products were visualized on a 1.5% agarose gel that was stained with ethidium bromide and viewed under a UV light source. The PCR products were purified using the Nucleo-Fast® 96 PCR Kit (Marcherey-Nagel, Hoerdt, France) according to the manufacturer's instructions.

Cloning, Sequencing and phylogenetic analyses

The cloning and sequencing reactions were performed as previously described16. The PCR products were cloned separately using the pGEM® -T Easy Vector System Kit (Promega, Madison, USA). Aliquots (150 µl) of cell suspensions were plated onto LB (Luria-Bertani Broth) agar plates that were supplemented with ampicillin (100 mg/mL), X-GAL (80 mg/mL) and IPTG (120 mg/mL) and the plates were incubated overnight at 37°C. Positive clones were suspended in 25 µL of distilled water and stored at −20°C. The presence of the insert was confirmed by PCR amplification using the M13 forward (5′-GTAAAACGACGGCCAG-3′) and M13 reverse (5′-AGGAAACAGCTATGAC-3′) primers (Eurogentec, Seraing, Belgium). The purified PCR products were sequenced in both directions using the BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing products were run on an ABI PRISM 3130 automated sequencer (Applied Biosystems). Finally, intestinal eukaryotes were identified by comparing the resulting sequences with those that were deposited in GenBank using the basic local alignment search tool (BLAST), which is available at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/). Phylogenetic analyses were performed using MEGA5.04 and a distance matrix neighbor-joining (NJ) approach57.

Real-Time PCR Assay for Detection of human pathogen

Primers and probes specific to some human eukaryotic pathogens were used as described previously (Supplementary Table 3). For the primers and probes used for first time in this study, sequences corresponding to each species were collected in GenBank and aligned using multiple sequence alignment ClustalW2 and the PRIMER 3 software58 was used to design primer sets in the conserved regions of aligned sequences. The specificity of each primer was tested using the basic local alignment search tool (BLAST), which is available at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/). The real-time PCR reactions were conducted using 25 µL total volumes and analyzed for 44 cycles using a CFX96™ Real-Time PCR Detection System (BIO-RAD, Life Science, Marnes-la-Coquette, France) following methods recommended by the manufacturer. Amplification reactions were done as follows: 95°C for 15 min, 60°C for 0.5 min and 72°C for 1 min.

Species confirmation and Microsatellite analyses

The DNA was extracted from gorilla fecal samples in order to determine the number of individuals that carrying human eukaryotic pathogens. Total of 48 samples were genotyped at 7 polymorphic loci (D18s536, D4s243, D10s676, D9s922, D2S1326, D2S1333 and D4S1627) as described previously56. The gender of gorillas was determined by amplification of a region of the amelogenin gene that contains a deletion in the X, but not the Y chromosome56. To exclude the allelic dropout, all loci were amplified four times. Aliquot 1 µL of PCR products was mixed with 10 µL of formamide and 0.25 µL of the ladder marker (ROX GeneScan 400HD, Applied Biosystem), The resulting amplifications were analyzed by 3130xl Genetic Anlayser sequencer (Applied Biosystem, Foster City,CA). Amplification products were visualized and sized using Genemapper 3.7 software (Applied Biosystems).

Culturing and identification of fungal species

The fecal samples were serially diluted and six-fold dilutions were spread onto potato dextrose agar (Fluka® Analytical, France), Czapek dox agar (Fluka® Analytical, France) and Dixon agar. The plates were incubated aerobically at room temperature. The colonies exhibiting different morphologies were restreaked to obtain pure cultures. The fungi were amplified using fungal primers (ITS1-F/ITS-4R) and identified as previously described16.

Nucleotide sequence accession numbers

All sequences obtained in this work have been deposited in GenBank database with the accession numbers JX158488 to JX159965.