Progress in understanding the phylogeny of the Plasmodium vivax lineage.

There has been some controversy about the evolutionary origin of Plasmodium vivax, particularly whether it is of Asian or African origin. Recently, a new malaria species which closely related to ape P. vivax was found in chimpanzees, in addition, the host switches of P. vivax from ape to human was confirmed. These findings support the African origin of P. vivax. Previous phylogenetic analyses have shown the position of P. vivax within the Asian primate malaria parasite clade. This suggested an Asian origin of P. vivax. Recent analyses using massive gene data, however, positioned P. vivax after the branching of the African Old World monkey parasite P. gonderi, and before the branching of the common ancestor of Asian primate malaria parasites. This position is consistent with an African origin of P. vivax. We here review the history of phylogenetic analyses on P. vivax, validate previous analyses, and finally present a definitive analysis using currently available data that indicate a tree in which P. vivax is positioned at the base of the Asian primate malaria parasite clade, and thus that is consistent with an African origin of P. vivax.

Potential zoonotic pathogens hosted by endangered bonobos.

Few publications, often limited to one specific pathogen, have studied bonobos (Pan paniscus), our closest living relatives, as possible reservoirs of certain human infectious agents. Here, 91 stool samples from semicaptive bonobos and bonobos reintroduced in the wild, in the Democratic Republic of the Congo, were screened for different infectious agents: viruses, bacteria and parasites. We showed the presence of potentially zoonotic viral, bacterial or parasitic agents in stool samples, sometimes coinfecting the same individuals. A high prevalence of Human mastadenoviruses (HAdV-C, HAdV-B, HAdV-E) was observed. Encephalomyocarditis viruses were identified in semicaptive bonobos, although identified genotypes were different from those identified in the previous fatal myocarditis epidemic at the same site in 2009. Non-pallidum Treponema spp. including symbiotic T. succinifaciens, T. berlinense and several potential new species with unknown pathogenicity were identified. We detected DNA of non-tuberculosis Mycobacterium spp., Acinetobacter spp., Salmonella spp. as well as pathogenic Leptospira interrogans. Zoonotic parasites such as Taenia solium and Strongyloides stercoralis were predominantly present in wild bonobos, while Giardia lamblia was found only in bonobos in contact with humans, suggesting a possible exchange. One third of bonobos carried Oesophagostomum spp., particularly zoonotic O. stephanostomum and O. bifurcum-like species, as well as other uncharacterized Nematoda. Trypanosoma theileri has been identified in semicaptive bonobos. Pathogens typically known to be transmitted sexually were not identified. We present here the results of a reasonably-sized screening study detecting DNA/RNA sequence evidence of potentially pathogenic viruses and microorganisms in bonobo based on a noninvasive sampling method (feces) and focused PCR diagnostics.

Notes on Morphology and Life History of Probstmayria gombensis (Nematoda: Cosmocercoidea: Atractidae), Parasitic in Eastern Chimpanzees, Pan troglodytes schweinfurthii, in Bulindi, Uganda.

Probstmayria gombensis File, 1976 (Nematoda: Cosmocercoidea: Atractidae) individuals discharged in the feces of eastern chimpanzees, Pan troglodytes schweinfurthii, in Bulindi, Uganda, were examined morphologically. Adults and fourth-stage larvae, all females, found in the feces, and the third-stage larvae excised from the uterus of the gravid females were described. By close observation of the molting worms, it was considered that the uterine third-stage larvae attain molting phase, and then are laid to become fourth-stage larvae. Nutrients required for larval development in the uterus seem to be supplied by the mother after the eggshell is formed. After some growth in the host intestine, the fourth-stage larvae undergo the final molt to the adult stage. The genital primordium was very small in the early fourth-stage larvae but rapidly developed with embryonation in the pre-molt and molting phases. Such precocity suggests parthenogenetic reproduction without insemination by males. This style may enhance rapid autoinfection in the host intestine under the condition of male worm scarcity. Several ellipsoidal pseudocoelomocytes with granules of unknown function were found ventral to the intestine of the adults, fourth-stage larvae, and third-stage larvae.

Ape Origins of Human Malaria.

African apes harbor at least twelve Plasmodium species, some of which have been a source of human infection. It is now well established that Plasmodium falciparum emerged following the transmission of a gorilla parasite, perhaps within the last 10,000 years, while Plasmodium vivax emerged earlier from a parasite lineage that infected humans and apes in Africa before the Duffy-negative mutation eliminated the parasite from humans there. Compared to their ape relatives, both human parasites have greatly reduced genetic diversity and an excess of nonsynonymous mutations, consistent with severe genetic bottlenecks followed by rapid population expansion. A putative new Plasmodium species widespread in chimpanzees, gorillas, and bonobos places the origin of Plasmodium malariae in Africa. Here, we review what is known about the origins and evolutionary history of all human-infective Plasmodium species, the time and circumstances of their emergence, and the diversity, host specificity, and zoonotic potential of their ape counterparts.

Sympatric western lowland gorillas, central chimpanzees and humans are infected with different trichomonads.

We investigated intestinal trichomonads in western lowland gorillas, central chimpanzees and humans cohabiting the forest ecosystem of Dzanga-Sangha Protected Area in Central African Republic, using the internal transcribed spacer (ITS) region and SSU rRNA gene sequences. Trichomonads belonging to the genus Tetratrichomonas were detected in 23% of the faecal samples and in all host species. Different hosts were infected with different genotypes of Tetratrichomonas. In chimpanzees, we detected tetratrichomonads from 'novel lineage 2', which was previously reported mostly in captive and wild chimpanzees. In gorillas, we found two different genotypes of Tetratrichomonas. The ITS region sequences of the more frequent genotype were identical to the sequence found in a faecal sample of a wild western lowland gorilla from Cameroon. Sequences of the second genotype from gorillas were almost identical to sequences previously obtained from an anorexic French woman. We provide the first report of the presence of intestinal tetratrichomonads in asymptomatic, apparently healthy humans. Human tetratrichomonads belonged to the lineage 7, which was previously reported in domestic and wild pigs and a domestic horse. Our findings suggest that the ecology and spatial overlap among hominids in the tropical forest ecosystem has not resulted in exchange of intestinal trichomonads among these hosts.

Ancient Introgression between Two Ape Malaria Parasite Species.

The Laverania clade comprises the human malaria parasite Plasmodium falciparum as well as at least seven additional parasite species that infect wild African apes. A recent analysis of Laverania genome sequences (Otto TD, et al. 2018. Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria. Nat Microbiol. 3: 687-697) reported three instances of interspecies gene transfer, one of which had previously been described. Generating gene sequences from additional ape parasites and re-examining sequencing reads generated in the Otto et al. study, we identified one of the newly described gene transfers as an assembly artifact of sequences derived from a sample coinfected by two parasite species. The second gene transfer between ancestors of two divergent chimpanzee parasite lineages was confirmed, but involved a much larger number of genes than originally described, many of which encode exported proteins that remodel, or bind to, erythrocytes. Because successful hybridization between Laverania species is very rare, it will be important to determine to what extent these gene transfers have shaped their host interactions.

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.

Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum.

Exploring interactions between Blastocystis sp., Strongyloides spp. and the gut microbiomes of wild chimpanzees in Senegal.

BACKGROUND: Gut parasites exert an important influence on the gut microbiome, with many studies focusing on the human gut microbiome. It has, however, undergone severe richness depletion. Hygienic lifestyle, antimicrobial treatments and altered gut homeostasis (e.g., chronic inflammation) reduce gut microbiome richness and also parasite prevalence; which may confound results. Studying species closely related to humans could help overcome this problem by providing insights into the ancestral relationship between humans, their gut microbiome and their gut parasites. Chimpanzees are a particularly promising model as they have similar gut microbiomes to humans and many parasites infect both species. AIMS: We study the interaction between gut microbiome and enteric parasites in chimpanzees. Investigating what novel insights a closely related species can reveal when compared to studies on humans. METHODS: Using eighty-seven faecal samples from wild western chimpanzees (Pan troglodytes verus) in Senegal, we combine 16S rRNA gene amplicon sequencing for gut microbiome characterization with PCR detection of parasite taxa (Blastocystis sp., Strongyloides spp., Giardia duodenalis, Cryptosporidium spp., Plasmodium spp., Filariae and Trypanosomatidae). We test for differences in gut microbiota ecosystem traits and taxonomical composition between Blastocystis and Strongyloides bearing and non-bearing samples. RESULTS: For Blastocystis, twelve differentially abundant taxa (e.g., Methanobrevibacter), including Prevotella and Ruminococcus-Methanobrevibacter enterotype markers, replicate findings in humans. However, several richness indices are lower in Blastocystis carriers, contradicting human studies. This indicates Blastocystis, unlike Strongyloides, is associated to a "poor health" gut microbiome, as does the fact that Faecalibacterium, a bacterium with gut protective traits, is absent in Blastocystis-positive samples. Strongyloides was associated to Alloprevotella and five other taxonomic groups. Each parasite had its unique impact on the gut microbiota indicating parasite-specific niches. Our results suggest that studying the gut microbiomes of wild chimpanzees could help disentangle biological from artefactual associations between gut microbiomes and parasites.

Entamoeba histolytica infection in humans, chimpanzees and baboons in the Greater Gombe Ecosystem, Tanzania.

Entamoeba histolytica is an enteric parasite that infects approximately 50 million people worldwide. Although E. histolytica is a zoonotic parasite that has the potential to infect nonhuman primates, such transmission is poorly understood. Consequently, this study examined whether E. histolytica is present among humans, chimpanzees and baboons living in the Greater Gombe Ecosystem (GGE), Tanzania. The primary aims were to determine patterns of E. histolytica infection in a system with human-nonhuman primate overlap and to test associations between infection status and potential risk factors of disease. Entamoeba spp. occurred in 60.3% of human, 65.6% of chimpanzee and 88.6% of baboon samples. Entamoeba histolytica occurred in 12.1% of human, 34.1% of chimpanzee and 10.9% of baboon samples. Human E. histolytica infection was associated with gastrointestinal symptoms. This was the first study to confirm the presence of E. histolytica in the GGE. The high sample prevalence of E. histolytica in three sympatric primates suggests that zoonotic transmission is possible and stresses the need for further phylogenetic studies. Interventions targeting better sanitation and hygiene practices for humans living in the GGE can help prevent E. histolytica infection in humans, while also protecting the endangered chimpanzees and other primates in this region.

Diversity of Entamoeba spp. in African great apes and humans: an insight from Illumina MiSeq high-throughput sequencing.

Understanding the complex Entamoeba communities in the mammalian intestine has been, to date, complicated by the lack of a suitable approach for molecular detection of multiple variants co-occurring in mixed infections. Here, we report on the application of a high throughput sequencing approach based on partial 18S rDNA using the Illumina MiSeq platform. We describe, to our knowledge, for the first time, the Entamoeba communities in humans, free-ranging western lowland gorillas and central chimpanzees living in the Dja Faunal Reserve in Cameroon. We detected 36 Entamoeba haplotypes belonging to six haplotype clusters, containing haplotypes possessing high and low host specificity. Most of the detected haplotypes belonged to commensal Entamoeba, however, the pathogenic species (Entamoeba histolytica and Entamoeba nuttalli) were also detected. We observed that some Entamoeba haplotypes are shared between humans and other hosts, indicating their zoonotic potential. The findings are important not only for understanding the epidemiology of amoebiasis in humans in rural African localities, but also in the context of wild great ape conservation.

Oesophagostomiasis in non-human primates of Gombe National Park, Tanzania.

Oesophagostomum sp. is a parasitic nematode that frequently infects wild chimpanzees. Although nodular lesions are commonly associated with infection, some wild chimpanzee populations seem to tolerate Oesophagostomum nodular lesions while those at Gombe and other sites suffer from associated morbidity and mortality. From August 2004 to December 2013, we examined demographic (i.e., age, sex) and individual correlates (i.e., fecal consistency, Oesophagostomum egg production) to Oesophagostomum-associated pathology in 14 individually recognized chimpanzees at Gombe Stream National Park, Tanzania. In addition, we characterized Oesophagostomum-associated pathology in 14 individual sympatric primates including baboons, colobus, and cercopithecid monkeys. In five chimpanzees, there was no evidence of any significant underlying disease aside from oesophagostomiasis to explain the thin condition or diarrhea. All five of these chimpanzees had moderate to numerous parasitic nodules. In general, nodules were more numerous in older chimpanzees. Three of four chimpanzees with the highest average Oesophagostomum egg counts in feces collected during the year prior to their death had numerous parasitic nodules at necropsy. In contrast, the four chimpanzees with the lowest egg counts had only moderate numbers of nodules. No association (P = 0.74) was noted between frequency of diarrhea in the year prior to death and the number of nodules noted at necropsy. Nodules were also present in all baboons examined documenting pathology associated with Oesophagostomum infection in wild baboons. In contrast, no lesions were noted in colobus or cercopithecid monkeys, although it is uncertain if they are infected as no fecal studies have been completed in these species to date at Gombe. Sequence of DNA isolated from nodules in chimpanzees matched (99%) Oesophagostomum stephanostomum. Further research is needed to identify the types of Oesophagostomum causing lesions in baboons and to determine if baboons suffer from these infections. Am. J. Primatol. 80:e22572, 2018. © 2016 Wiley Periodicals, Inc.

Plasmodium reichenowi EBA-140 merozoite ligand binds to glycophorin D on chimpanzee red blood cells, shedding new light on origins of Plasmodium falciparum.

BACKGROUND: All symptoms of malaria are caused by the intraerythrocytic proliferation of Plasmodium merozoites. Merozoites invade erythrocytes using multiple binding ligands that recognise specific surface receptors. It has been suggested that adaptation of Plasmodium parasites to infect specific hosts is driven by changes in genes encoding Plasmodium erythrocyte-binding ligands (EBL) and reticulocyte-binding ligands (RBL). Homologs of both EBL and RBL, including the EBA-140 merozoite ligand, have been identified in P. falciparum and P. reichenowi, which infect humans and chimpanzees, respectively. The P. falciparum EBA-140 was shown to bind human glycophorin C, a minor erythrocyte sialoglycoprotein. Until now, the erythrocyte receptor for the P. reichenowi EBA-140 remained unknown. METHODS: The baculovirus expression vector system was used to obtain the recombinant EBA-140 Region II, and flow cytometry and immunoblotting methods were applied to characterise its specificity. RESULTS: We showed that the chimpanzee glycophorin D is the receptor for the P. reichenowi EBA-140 ligand on chimpanzee red blood cells. CONCLUSIONS: We propose that the development of glycophorin C specificity is spurred by the P. falciparum lineage. We speculate that the P. falciparum EBA-140 evolved to hijack GPC on human erythrocytes during divergence from its ape ancestor.

Gastrointestinal parasite infections and self-medication in wild chimpanzees surviving in degraded forest fragments within an agricultural landscape mosaic in Uganda.

Monitoring health in wild great apes is integral to their conservation and is especially important where they share habitats with humans, given the potential for zoonotic pathogen exchange. We studied the intestinal parasites of wild chimpanzees (Pan troglodytes schweinfurthii) inhabiting degraded forest fragments amid farmland and villages in Bulindi, Uganda. We first identified protozoan and helminth parasites infecting this population. Sixteen taxa were demonstrated microscopically (9 protozoa, 5 nematodes, 1 cestode, and 1 trematode). DNA sequence analysis enabled more precise identification of larval nematodes (e.g. Oesophagostomum stephanostomum, O. bifurcum, Strongyloides fuelleborni, Necator sp. Type II) and tapeworm proglottids (genus Bertiella). To better understand the ecology of infections, we used multidimensional scaling analysis to reveal general patterns of association among parasites, climate, and whole leaf swallowing-a prevalent self-medicative behaviour at Bulindi linked to control of nodular worms (Oesophagostomum spp.). Prevalence of parasites varied with climate in diverse ways. For example, Oesophagostomum sp. was detected in faeces at higher frequencies with increasing rainfall but was most clearly associated with periods of low temperature. Certain parasites occurred together within chimpanzee hosts more or less frequently than expected by chance. For example, the commensal ciliate Troglodytella abrassarti was negatively associated with Balantidium coli and Oesophagostomum sp., possibly because the latter taxa make the large intestine less suitable for T. abrassarti. Whole leaves in faeces showed independent associations with the prevalence of Oesophagostomum sp., Strongyloides sp., and hookworm by microscopic examination, and with egestion of adult O. stephanostomum by macroscopic inspection. All parasites identified to species or genus have been reported in wild chimpanzees inhabiting less-disturbed environments than Bulindi. Nevertheless, several disease-causing taxa infecting these chimpanzees are potentially transmissible between apes and humans (e.g. rhabditoid and strongyle nematodes), underscoring the importance of identifying and reducing risks of pathogen exchange in shared landscapes.

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

Molecular features of hookworm larvae (Necator spp.) raised by coproculture from Ugandan chimpanzees and Gabonese gorillas and humans.

Species composition of Necator hookworms was surveyed in (i) Ugandan chimpanzees living around farms and villages at Bulindi, (ii) Gabonese gorillas under habituation in Moukalaba-Doudou National Park (MDNP), and (iii) Gabonese villagers living adjacent to MDNP. Internal transcribed spacers (ITS) of rDNA and partial cytochrome c oxidase subunit 1 (Cox1) gene of mtDNA were analyzed from larvae obtained by coproculture. Three ITS types (I, II and III) and three Cox1 haplotype groups (A, B and C) were demonstrated. ITS type I and Cox1 haplotype group A, representing Necator americanus, were demonstrated in the hookworm larvae from Gabonese gorillas and humans, but not from Ugandan chimpanzees. Type II and haplotype groups B and C, presumably representing N. gorillae, were found in larvae from Ugandan chimpanzees and Gabonese gorillas and humans. These features were overall similar with those found previously in the Central African Republic. Meanwhile, type III was proven in a larva from a Gabonese gorilla as the first demonstration from a non-human primate. Cox1 haplotypes obtained from Ugandan chimpanzees formed a subgroup within group B, presumably reflecting dispersal and diversification processes of the apes.

The host specificity of ape malaria parasites can be broken in confined environments.

Recent studies have revealed a large diversity of Plasmodium spp. among African great apes. Some of these species are related to Plasmodium falciparum, the most virulent agent of human malaria (subgenus Laverania), and others to Plasmodium ovale, Plasmodium malariae and Plasmodium vivax (subgenus Plasmodium), three other human malaria agents. Laverania parasites exhibit strict host specificity in their natural environment. Plasmodium reichenowi, Plasmodium billcollinsi, Plasmodium billbrayi and Plasmodium gaboni infect only chimpanzees, while Plasmodium praefalciparum, Plasmodium blacklocki and Plasmodium adleri are restricted to gorillas and Plasmodium falciparum is pandemic in humans. This host specificity may be due to genetic and/or environmental factors. Infrastructures hosting captive primates, such as sanctuaries and health centres, usually concentrate different primate species, thus favouring pathogen exchanges. Using molecular tools, we analysed blood samples from captive non-human primates living in Gabon to evaluate the risk of Plasmodium spp. transfers between host species. We also included blood samples from workers taking care of primates to assess whether primate-human parasite transfers occurred. We detected four transfers of Plasmodium from gorillas towards chimpanzees, one from chimpanzees to gorillas, three from humans towards chimpanzees and one from humans to mandrills. No simian Plasmodium was found in the blood samples from humans working with primates. These findings demonstrate that the genetic barrier that determines the apparent host specificity of Laverania is not completely impermeable and that parasite exchanges between gorillas and chimpanzees are possible in confined environments.

Schistosoma mansoni in Gabon: Emerging or Ignored?

Schistosomiasis affects millions of people across Africa. We detected eggs of Schistosoma mansoni in western lowland gorilla and central chimpanzee fecal samples in Loango National Park, Gabon. We analyzed nuclear and mitochondrial DNA, namely internal transcribed spacer and cytochrome c oxidase subunit 1 fragments, and the resulting maximum likelihood phylogenetic analyses and haplotype network of the ITS and COI, respectively, showed that the samples from gorillas and chimpanzees clustered clearly within the S. mansoni clade. This is the first confirmed record of S. mansoni from Gabon, which urges surveillance in the area and prompts questions regarding the extent of zoonotic transmission and the clinical impact.

Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild-Living Chimpanzees and Gorillas.

Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide, is only distantly related to other human malaria parasites and has thus been placed in a separate subgenus, termed Laverania Parasites morphologically similar to P. falciparum have been identified in African apes, but only one other Laverania species, Plasmodium reichenowi from chimpanzees, has been formally described. Although recent studies have pointed to the existence of additional Laverania species, their precise number and host associations remain uncertain, primarily because of limited sampling and a paucity of parasite sequences other than from mitochondrial DNA. To address this, we used limiting dilution polymerase chain reaction to amplify additional parasite sequences from a large number of chimpanzee and gorilla blood and fecal samples collected at two sanctuaries and 30 field sites across equatorial Africa. Phylogenetic analyses of more than 2,000 new sequences derived from the mitochondrial, nuclear, and apicoplast genomes revealed six divergent and well-supported clades within the Laverania parasite group. Although two of these clades exhibited deep subdivisions in phylogenies estimated from organelle gene sequences, these sublineages were geographically defined and not present in trees from four unlinked nuclear loci. This greatly expanded sequence data set thus confirms six, and not seven or more, ape Laverania species, of which P. reichenowi, Plasmodium gaboni, and Plasmodium billcollinsi only infect chimpanzees, whereas Plasmodium praefalciparum, Plasmodium adleri, and Pladmodium blacklocki only infect gorillas. The new sequence data also confirm the P. praefalciparum origin of human P. falciparum.

Strongyloides infections of humans and great apes in Dzanga-Sangha Protected Areas, Central African Republic and in degraded forest fragments in Bulindi, Uganda.

DNA sequence analysis was carried out on Strongyloides spp. larvae obtained from fecal samples of local humans, a wild western lowland gorilla (Gorilla gorilla gorilla) and a central chimpanzee (Pan troglodytes troglodytes) inhabiting Dzanga-Sangha Protected Areas (DSPA), Central African Republic, and eastern chimpanzees (Pan troglodytes schweinfurthii) living in degraded forest fragments on farmland in Bulindi, Uganda. From humans, both Strongyloides fuelleborni and Strongyloides stercoralis were recorded, though the former was predominant. Only S. fuelleborni was present in the great apes in both areas. Phylogenetic analysis of partial mtDNA cytochrome c oxidase subunit 1 gene (Cox1) and comparison of 18S rDNA hyper variable region IV (HVR-IV) sequences implied that in DSPA S. fuelleborni populations in humans differ from those in the nonhuman great apes.

Molecular identification of Entamoeba species in savanna woodland chimpanzees (Pan troglodytes schweinfurthii).

To address the molecular diversity and occurrence of pathogenic species of the genus Entamoeba spp. in wild non-human primates (NHP) we conducted molecular-phylogenetic analyses on Entamoeba from wild chimpanzees living in the Issa Valley, Tanzania. We compared the sensitivity of molecular [using a genus-specific polymerase chain reaction (PCR)] and coproscopic detection (merthiolate-iodine-formaldehyde concentration) of Entamoeba spp. We identified Entamoeba spp. in 72 chimpanzee fecal samples (79%) subjected to species-specific PCRs for six Entamoeba species/groups (Entamoeba histolytica, Entamoeba nuttalli, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli and Entamoeba polecki ST2). We recorded three Entamoeba species: E. coli (47%), E. dispar (16%), Entamoeba hartmanni (51%). Coproscopically, we could only distinguish the cysts of complex E. histolytica/dispar/moshkovskii/nuttalli and E. coli. Molecular prevalence of entamoebas was higher than the prevalence based on the coproscopic examination. Our molecular phylogenies showed that sequences of E. dispar and E. coli from Issa chimpanzees are closely related to sequences from humans and other NHP from GenBank. The results showed that wild chimpanzees harbour Entamoeba species similar to those occurring in humans; however, no pathogenic species were detected. Molecular-phylogenetic methods are critical to improve diagnostics of entamoebas in wild NHP and for determining an accurate prevalence of Entamoeba species.