Anoplocephalid tapeworms in mountain gorillas (Gorilla beringei beringei) inhabiting the Volcanoes National Park, Rwanda.

Cestodes of the family Anoplocephalidae parasitize a wide range of usually herbivorous hosts including e.g. rodents, ungulates, primates, elephants and hyraxes. While in some hosts, the epidemiology of the infection is well studied, information is lacking in others. In this study of mountain gorillas in the Virunga Massif, an extensive sample set comprising adult cestodes collected via necropsies, proglottids shed in feces, and finally, fecal samples from both night nests and identified individuals were analysed. Anoplocephala gorillae was the dominant cestode species detected in night nest samples and individually known gorillas, of which only 1 individual hosted a Bertiella sp. It was shown that the 2 species can be distinguished through microscopy based on egg morphology and polymerase chain reaction (PCR) assays for diagnostics of both species were provided. Sequences of mitochondrial (cox 1) and nuclear (ITS1, 18S rDNA, 28S rDNA) markers were used to evaluate the phylogenetic position of the 2 cestodes detected in mountain gorillas. Both types of fecal samples, from night nests and from identified individuals, provided comparable information about the prevalence of anoplocephalid cestodes, although the analysis of samples collected from identified gorilla individuals showed significant intra-individual fluctuation of A. gorillae egg shedding within a short period. Therefore, multiple samples should be examined to obtain reliable data for wildlife health management programmes, especially when application of anthelmintic treatment is considered. However, while A. gorillae is apparently a common symbiont of mountain gorillas, it does not seem to impair the health of its host.

Novel insight into the genetic diversity of strongylid nematodes infecting South-East and East Asian primates.

With many non-human primates (NHPs) showing continued population decline, there is an ongoing need to better understand their ecology and conservation threats. One such threat is the risk of disease, with various bacterial, viral and parasitic infections previously reported to have damaging consequences for NHP hosts. Strongylid nematodes are one of the most commonly reported parasitic infections in NHPs. Current knowledge of NHP strongylid infections is restricted by their typical occurrence as mixed infections of multiple genera, which are indistinguishable through traditional microscopic approaches. Here, modern metagenomics approaches were applied for insight into the genetic diversity of strongylid infections in South-East and East Asian NHPs. We hypothesized that strongylid nematodes occur in mixed communities of multiple taxa, dominated by Oesophagostomum, matching previous findings using single-specimen genetics. Utilizing the Illumina MiSeq platform, ITS-2 strongylid metabarcoding was applied to 90 samples from various wild NHPs occurring in Malaysian Borneo and Japan. A clear dominance of Oesophagostomum aculeatum was found, with almost all sequences assigned to this species. This study suggests that strongylid communities of Asian NHPs may be less species-rich than those in African NHPs, where multi-genera communities are reported. Such knowledge contributes baseline data, assisting with ongoing monitoring of health threats to NHPs.

Identification of potentially zoonotic parasites in captive orangutans and semi-captive mandrills: Phylogeny and morphological comparison.

Cysts and trophozoites of vestibuliferid ciliates and larvae of Strongyloides were found in fecal samples from captive orangutans Pongo pygmaeus and P. abelii from Czech and Slovak zoological gardens. As comparative material, ciliates from semi-captive mandrills Mandrillus sphinx from Gabon were included in the study. Phylogenetic analysis of the detected vestibuliferid ciliates using ITS1-5.8s-rRNA-ITS2 and partial 18S ribosomal deoxyribonucleic acid (rDNA) revealed that the ciliates from orangutans are conspecific with Balantioides coli lineage A, while the ciliates from mandrills clustered with Buxtonella-like ciliates from other primates. Morphological examination of the cysts and trophozoites using light microscopy did not reveal differences robust enough to identify the genera of the ciliates. Phylogenetic analysis of detected L1 larvae of Strongyloides using partial cox1 revealed Strongyloides stercoralis clustering within the cox1 lineage A infecting dogs, humans, and other primates. The sequences of 18S rDNA support these results. As both B. coli and S. stercoralis are zoonotic parasites and the conditions in captive and semi-captive settings may facilitate transmission to humans, prophylactic measures should reflect the findings.

Gastrointestinal symbiont diversity in wild gorilla: A comparison of bacterial and strongylid communities across multiple localities.

Western lowland gorillas (Gorilla gorilla gorilla) are Critically Endangered and show continued population decline. Consequently, pressure is mounting to better understand their conservation threats and ecology. Gastrointestinal symbionts, such as bacterial and eukaryotic communities, are believed to play vital roles in the physiological landscape of the host. Gorillas host a broad spectrum of eucaryotes, so called parasites, with strongylid nematodes being particularly prevalent. While these communities are partially consistent, they are also shaped by various ecological factors, such as diet or habitat type. To investigate gastrointestinal symbionts of wild western lowland gorillas, we analysed 215 faecal samples from individuals in five distinct localities across the Congo Basin, using high-throughput sequencing techniques. We describe the gut bacterial microbiome and genetic diversity of strongylid communities, including strain-level identification of amplicon sequence variants (ASVs). We identified strongylid ASVs from eight genera and bacterial ASVs from 20 phyla. We compared these communities across localities, with reference to varying environmental factors among populations, finding differences in alpha diversity and community compositions of both gastrointestinal components. Moreover, we also investigated covariation between strongylid nematodes and the bacterial microbiome, finding correlations between strongylid taxa and Prevotellaceae and Rikenellaceae ASVs that were consistent across multiple localities. Our research highlights the complexity of the bacterial microbiome and strongylid communities in several gorilla populations and emphasizes potential interactions between these two symbiont communities. This study provides a framework for ongoing research into strongylid nematode diversity, and their interactions with the bacterial microbiome, among great apes.

A Cross-Sectional Study on the Occurrence of the Intestinal Protist, Dientamoeba fragilis, in the Gut-Healthy Volunteers and Their Animals.

Dientamoeba fragilis is a cosmopolitan intestinal protist colonizing the human gut with varying prevalence depending on the cohort studied and the diagnostic methods used. Its role in human health remains unclear mainly due to the very sporadic number of cross-sectional studies in gut-healthy populations. The main objective of this study was to expand knowledge of the epidemiology of D. fragilis in gut-healthy humans and their animals. A total of 296 stool samples from humans and 135 samples from 18 animal species were analyzed. Using qPCR, a prevalence of 24% was found in humans in contrast to conventional PCR (7%). In humans, several factors were found to influence the prevalence of D. fragilis. A more frequent occurrence of D. fragilis was associated with living in a village, traveling outside Europe and contact with farm animals. In addition, co-infection with Blastocystis spp. was observed in nearly half of the colonized humans. In animals, D. fragilis was detected in 13% of samples from eight species using qPCR. Our molecular phylogenies demonstrate a more frequent occurrence of Genotype 1 in gut-healthy humans and also revealed a likely a new protist species/lineage in rabbits related to D. fragilis and other related organisms.

Fecal glucocorticoids and gastrointestinal parasite infections in wild western lowland gorillas (Gorilla gorilla gorilla) involved in ecotourism.

Wildlife ecotourism can offer a source of revenue which benefits local development and conservation simultaneously. However, habituation of wildlife for ecotourism can cause long-term elevation of glucocorticoid hormones, which may suppress immune function and increase an animal's vulnerability to disease. We have previously shown that western lowland gorillas (Gorilla gorilla gorilla) undergoing habituation in Dzanga-Sangha Protected Areas, Central African Republic, have higher fecal glucocorticoid metabolite (FGCM) levels than both habituated and unhabituated gorillas. Here, we tested the relationship between FGCM levels and strongylid infections in the same gorillas. If high FGCM levels suppress the immune system, we predicted that FGCM levels will be positively associated with strongylid egg counts and that gorillas undergoing habituation will have the highest strongylid egg counts, relative to both habituated and unhabituated gorillas. We collected fecal samples over 12 months in two habituated gorilla groups, one group undergoing habituation and completely unhabituated gorillas. We established FGCM levels and fecal egg counts of Necator/Oesophagostomum spp. and Mammomonogamus sp. Controlling for seasonal variation and age-sex category in strongylid infections we found no significant relationship between FGCMs and Nectator/Oesophagostomum spp. or Mammomonogamus sp. egg counts in a within group comparison in either a habituated group or a group undergoing habituation. However, across groups, egg counts of Nectator/Oesophagostomum spp. were lowest in unhabituated animals and highest in the group undergoing habituation, matching the differences in FGCM levels among these gorilla groups. Our findings partially support the hypothesis that elevated glucocorticoids reduce a host's ability to control the extent of parasitic infections, and show the importance of non-invasive monitoring of endocrine function and parasite infection in individuals exposed to human pressure including habituation process and ecotourism.

Mapping gastrointestinal gene expression patterns in wild primates and humans via fecal RNA-seq.

BACKGROUND: Limited accessibility to intestinal epithelial tissue in wild animals and humans makes it challenging to study patterns of intestinal gene regulation, and hence to monitor physiological status and health in field conditions. To explore solutions to this limitation, we have used a noninvasive approach via fecal RNA-seq, for the quantification of gene expression markers in gastrointestinal cells of free-range primates and a forager human population. Thus, a combination of poly(A) mRNA enrichment and rRNA depletion methods was used in tandem with RNA-seq to quantify and compare gastrointestinal gene expression patterns in fecal samples of wild Gorilla gorilla gorilla (n = 9) and BaAka hunter-gatherers (n = 10) from The Dzanga Sangha Protected Areas, Central African Republic. RESULTS: Although only a small fraction (< 4.9%) of intestinal mRNA signals was recovered, the data was sufficient to detect significant functional differences between gorillas and humans, at the gene and pathway levels. These intestinal gene expression differences were specifically associated with metabolic and immune functions. Additionally, non-host RNA-seq reads were used to gain preliminary insights on the subjects' dietary habits, intestinal microbiomes, and infection prevalence, via identification of fungi, nematode, arthropod and plant RNA. CONCLUSIONS: Overall, the results suggest that fecal RNA-seq, targeting gastrointestinal epithelial cells can be used to evaluate primate intestinal physiology and gut gene regulation, in samples obtained in challenging conditions in situ. The approach used herein may be useful to obtain information on primate intestinal health, while revealing preliminary insights into foraging ecology, microbiome, and diet.

Genetic diversity of primate strongylid nematodes: Do sympatric nonhuman primates and humans share their strongylid worms?

The close phylogenetic relationship between humans and nonhuman primates (NHPs) can result in a high potential for pathogen exchange. In recent decades, NHP and human interactions have become more frequent due to increasing habitat encroachment and ecotourism. Strongylid communities, which include members of several genera, are typically found in NHPs. Using optimized high-throughput sequencing for strain-level identification of primate strongylids, we studied the structure of strongylid communities in NHPs and humans co-habiting a tropical forest ecosystem in the Central African Republic. General taxonomic assignment of 85 ITS-2 haplotypes indicated that the studied primates harbour at least nine genera of strongylid nematodes, with Oesophagostomum and Necator being the most prevalent. We detected both host-specific and shared strongylid haplotypes. Skin-penetrating Necator gorillaehaplotypes were shared between humans and gorillas but Necator americanus were much more restricted to humans. Strongylid communities of local hunter-gatherers employed as trackers were more similar to those of gorillas compared to their relatives, who spent more time in villages. This was due to lower abundance of human-origin N. americanus in both gorillas and trackers. Habituated gorillas or those under habituation did not show larger overlap of strongylids with humans compared to unhabituated. We concluded that the occurrence of the human-specific strongylids in gorillas does not increase with direct contact between gorillas and humans due to the habituation. Overall, our results indicate that the degree of habitat sharing between hosts, together with mode of parasite transmission, are important factors for parasite spillover among primates.

Gut microbiome composition of wild western lowland gorillas is associated with individual age and sex factors.

OBJECTIVES: Environmental and ecological factors, such as geographic range, anthropogenic pressure, group identity, and feeding behavior are known to influence the gastrointestinal microbiomes of great apes. However, the influence of individual host traits such as age and sex, given specific dietary and social constraints, has been less studied. The objective of this investigation was to determine the associations between an individual's age and sex on the diversity and composition of the gut microbiome in wild western lowland gorillas. MATERIALS AND METHODS: Publicly available 16S rRNA data generated from fecal samples of different groups of Gorilla gorilla gorilla in the Central African Republic were downloaded and bioinformatically processed. The groups analyzed included habituated, partially habituated and unhabituated gorillas, sampled during low fruit (dry, n = 28) and high fruit (wet, n = 82) seasons. Microbial community analyses (alpha and beta diversity and analyses of discriminant taxa), in tandem with network-wide approaches, were used to (a) mine for specific age and sex based differences in gut bacterial community composition and to (b) asses for gut community modularity and bacterial taxa with potential functional roles, in the context of seasonal food variation, and social group affiliation. RESULTS: Both age and sex significantly influenced gut microbiome diversity and composition in wild western lowland gorillas. However, the largest differences were observed between infants and adults in habituated groups and between adults and immature gorillas within all groups, and across dry and wet seasons. Specifically, although adults always showed greater bacterial richness than infants and immature gorillas, network-wide analyses showed higher microbial community complexity and modularity in the infant gorilla gut. Sex-based microbiome differences were not evident among adults, being only detected among immature gorillas. CONCLUSIONS: The results presented point to a dynamic gut microbiome in Gorilla spp., associated with ontogeny and individual development. Of note, the gut microbiomes of breastfeeding infants seemed to reflect early exposure to complex, herbaceous vegetation. Whether increased compositional complexity of the infant gorilla gut microbiome is an adaptive response to an energy-limited diet and an underdeveloped gut needs to be further tested. Overall, age and sex based gut microbiome differences, as shown here, maybe mainly attributed to access to specific feeding sources, and social interactions between individuals within groups.

Plasmodium ovale wallikeri in Western Lowland Gorillas and Humans, Central African Republic.

Human malaria parasites have rarely been reported from free-ranging great apes. Our study confirms the presence of the human malaria parasite Plasmodium ovale wallikeri in western lowland gorillas and humans in Dzanga Sangha Protected Areas, Central African Republic, and discusses implications for malaria epidemiology.

Impact of stress on the gut microbiome of free-ranging western lowland gorillas.

Exposure to stressors can negatively impact the mammalian gastrointestinal microbiome (GIM). Here, we used 454 pyrosequencing of 16S rRNA bacterial gene amplicons to evaluate the impact of physiological stress, as evidenced by faecal glucocorticoid metabolites (FGCM; ng/g), on the GIM composition of free-ranging western lowland gorillas (Gorilla gorilla gorilla). Although we found no relationship between GIM alpha diversity (H) and FGCM levels, we observed a significant relationship between the relative abundances of particular bacterial taxa and FGCM levels. Specifically, members of the family Anaerolineaceae (ρ=0.4, FDR q=0.01), genus Clostridium cluster XIVb (ρ=0.35, FDR q=0.02) and genus Oscillibacter (ρ=0.35, FDR q=0.02) were positively correlated with FGCM levels. Thus, while exposure to stressors appears to be associated with minor changes in the gorilla GIM, the consequences of these changes are unknown. Our results may have implications for conservation biology as well as for our overall understanding of factors influencing the non-human primate GIM.

Gastrointestinal protists and helminths of habituated agile mangabeys (Cercocebus agilis) at Bai Hokou, Central African Republic.

Infectious diseases including those caused by parasites can be a major threat to the conservation of endangered species. There is thus a great need for studies describing parasite infections of these species in the wild. Here we present data on parasite diversity in an agile mangabey (Cercocebus agilis) group in Bai Hokou, Dzanga-Sangha Protected Areas (DSPA), Central African Republic. We coproscopically analyzed 140 mangabey fecal samples by concentration techniques (flotation and sedimentation). Agile mangabeys hosted a broad diversity of protistan parasites/commensals, namely amoebas (Entamoeba spp., Iodamoeba buetschlli), a Buxtonella-like ciliate and several parasitic helminths: strongylid and spirurid nematodes, Primasubulura sp., Enterobius sp., and Trichuris sp. Importantly, some of the detected parasite taxa might be of potential zoonotic importance, such as Entamoeba spp. and the helminths Enterobius sp., Trichuris sp., and strongylid nematodes. Detailed morphological examination of ciliate cysts found in mangabeys and comparison with cysts of Balantioides coli from domestic pigs showed no distinguishing structures, although significant differences in cyst size were recorded. Scanning or transmission electron microscopy combined with molecular taxonomy methods are needed to properly identify these ciliates. Further studies using molecular epidemiology are warranted to better understand cross-species transmission and the zoonotic potential of parasites in sympatric non-human primates and humans cohabiting DSPA.

Correction to: Do habituation, host traits and seasonality have an impact on protist and helminth infections of wild western lowland gorillas?

Affiliation of Klára J. Petrzelková was incorrectly assigned as 2, 9, 10 in the original version of this article when in fact it should have been 3, 9, 10. Correct affiliations are presented here.

Diversity of Mammomonogamus (Nematoda: Syngamidae) in large African herbivores.

Four species of Mammomonogamus are known from large African herbivores. A recent study demonstrated that a single Mammomonogamus species was shared by both western lowland gorillas (Gorilla gorilla gorilla) and African forest elephants (Loxodonta cyclotis) in Central African Republic, suggesting lower species diversity than previously described in literature. We examined more than 500 fecal samples collected from sympatric African forest elephants, western lowland gorillas, and African forest buffaloes (Syncerus caffer nanus) at four study sites across Central Africa and examined them by coproscopic methods to detect Mammomonogamus eggs, which were found at three of the study sites. Subsequently, sequences of 18S rDNA, 28S rDNA, and cox1 amplified from individual eggs were analyzed. Phylogenetic analyses of both nuclear and mitochondrial DNA revealed two clades: one formed by sequences originating from Gabonese buffaloes and the other comprising gorillas and elephants. The gorilla-elephant clade was further differentiated depending on the locality. We show the existence of at least two distinct species of Mammomonogamus, M. loxodontis in elephants and gorillas and M. nasicola in buffaloes. The available information on Mammomonogamus in African herbivores is reviewed.

Assessing Host-Virus Codivergence for Close Relatives of Merkel Cell Polyomavirus Infecting African Great Apes.

UNLABELLED: It has long been hypothesized that polyomaviruses (PyV; family Polyomaviridae) codiverged with their animal hosts. In contrast, recent analyses suggested that codivergence may only marginally influence the evolution of PyV. We reassess this question by focusing on a single lineage of PyV infecting hominine hosts, the Merkel cell polyomavirus (MCPyV) lineage. By characterizing the genetic diversity of these viruses in seven African great ape taxa, we show that they exhibit very strong host specificity. Reconciliation analyses identify more codivergence than noncodivergence events. In addition, we find that a number of host and PyV divergence events are synchronous. Collectively, our results support codivergence as the dominant process at play during the evolution of the MCPyV lineage. More generally, our results add to the growing body of evidence suggesting an ancient and stable association of PyV and their animal hosts. IMPORTANCE: The processes involved in viral evolution and the interaction of viruses with their hosts are of great scientific interest and public health relevance. It has long been thought that the genetic diversity of double-stranded DNA viruses was generated over long periods of time, similar to typical host evolutionary timescales. This was also hypothesized for polyomaviruses (family Polyomaviridae), a group comprising several human pathogens, but this remains a point of controversy. Here, we investigate this question by focusing on a single lineage of polyomaviruses that infect both humans and their closest relatives, the African great apes. We show that these viruses exhibit considerable host specificity and that their evolution largely mirrors that of their hosts, suggesting that codivergence with their hosts played a major role in their diversification. Our results provide statistical evidence in favor of an association of polyomaviruses and their hosts over millions of years.

No impact of strongylid infections on the detection of Plasmodium spp. in faeces of western lowland gorillas and eastern chimpanzees.

BACKGROUND: Although a high genetic diversity of Plasmodium spp. circulating in great apes has been revealed recently due to non-invasive methods enabling detection in faecal samples, little is known about the actual mechanisms underlying the presence of Plasmodium DNA in faeces. Great apes are commonly infected by strongylid nematodes, including hookworms, which cause intestinal bleeding. The impact of strongylid infections on the detection of Plasmodium DNA in faeces was assessed in wild, western, lowland gorillas from Dzanga Sangha Protected Areas, Central African Republic and eastern chimpanzees from Kalinzu Forest Reserve, Uganda. METHODS: Fifty-one faecal samples from 22 habituated gorillas and 74 samples from 15 habituated chimpanzees were analysed using Cytochrome-b PCR assay and coprological methods. RESULTS: Overall, 26.4% of the analysed samples were positive for both Plasmodium spp. and strongylids. However, the results showed no significant impact of intensity of infections of strongylids on detection of Plasmodium DNA in gorilla and chimpanzee faeces. CONCLUSION: Bleeding caused by strongylid nematode Necator spp. cannot explain the presence of Plasmodium DNA in ape faeces.

Host specificity and basic ecology of Mammomonogamus (Nematoda, Syngamidae) from lowland gorillas and forest elephants in Central African Republic.

Syngamid strongylids of the genus Mammomonogamus undoubtedly belong among the least known nematodes with apparent zoonotic potential and the real diversity of the genus remains hard to evaluate without extensive molecular data. Eggs of Mammomonogamus sp. are frequently found in feces of African forest elephants (Loxodonta cyclotis) and western lowland gorillas (Gorilla gorilla gorilla) in Dzanga-Sangha Protected Areas. Using sedimentation-based coproscopic techniques, we found the eggs of Mammomonogamus in 19·7% elephant and 54·1% gorilla fecal samples with 8-55 and 1-24 eggs per gram of fecal sediment for elephants and gorillas, respectively. We used a combination of light microscopy, scanning electron microscopy and analysis of cytochrome c oxidase subunit I (cox1) and a partial sequence of 18S rDNA isolated from single eggs to test the hypothesis of possible Mammomonogamus conspecificity in gorillas and elephants. Whereas 18S rDNA sequences were identical in both gorillas and elephants, we distinguished seven different haplotypes within the cox1. Two haplotypes were found in both gorillas and elephants suggesting sharing of Mammomonogamus. Assignment of the parasite to M. loxodontis is proposed. Provided sequences represent the first genomic data on Mammomonogamus spp.

Do habituation, host traits and seasonality have an impact on protist and helminth infections of wild western lowland gorillas?

Increased anthropogenic activity can result in parasite exchanges and/or general changes in parasite communities, imposing a health risk to great apes. We studied protist and helminth parasites of wild western lowland gorilla groups in different levels of habituation, alongside humans inhabiting Dzanga-Sangha Protected Areas in the Central African Republic. Faeces were collected yearly during November and December from 2007 to 2010 and monthly from November 2010 to October 2011. Protist and helminth infections were compared among gorilla groups habituated, under habituation and unhabituated, and the effect of host traits and seasonality was evaluated. Zoonotic potential of parasites found in humans was assessed. No significant differences in clinically important parasites among the groups in different stages of habituation were found, except for Entamoeba spp. However, humans were infected with four taxa which may overlap with taxa found in gorillas. Females were less infected with spirurids, and adults had higher intensities of infection of Mammomonogamus sp. We found seasonal differences in the prevalence of several parasite taxa, but most importantly, the intensity of infection of unidentified strongylids was higher in the dry season. This study highlights that habituation may not necessarily pose a greater risk of protist and helminth infections in gorilla groups.

Multiple Cross-Species Transmission Events of Human Adenoviruses (HAdV) during Hominine Evolution.

Human adenoviruses (HAdV; species HAdV-A to -G) are highly prevalent in the human population, and represent an important cause of morbidity and, to a lesser extent, mortality. Recent studies have identified close relatives of these viruses in African great apes, suggesting that some HAdV may be of zoonotic origin. We analyzed more than 800 fecal samples from wild African great apes and humans to further investigate the evolutionary history and zoonotic potential of hominine HAdV. HAdV-B and -E were frequently detected in wild gorillas (55%) and chimpanzees (25%), respectively. Bayesian ancestral host reconstruction under discrete diffusion models supported a gorilla and chimpanzee origin for these viral species. Host switches were relatively rare along HAdV evolution, with about ten events recorded in 4.5 My. Despite presumably rare direct contact between sympatric populations of the two species, transmission events from gorillas to chimpanzees were observed, suggesting that habitat and dietary overlap may lead to fecal-oral cross-hominine transmission of HAdV. Finally, we determined that two independent HAdV-B transmission events to humans occurred more than 100,000 years ago. We conclude that HAdV-B circulating in humans are of zoonotic origin and have probably affected global human health for most of our species lifetime.

A comparative molecular survey of malaria prevalence among Eastern chimpanzee populations in Issa Valley (Tanzania) and Kalinzu (Uganda).

BACKGROUND: Habitat types can affect vector and pathogen distribution and transmission dynamics. The prevalence and genetic diversity of Plasmodium spp. in two eastern chimpanzee populations-Kalinzu Forest Reserve, Uganda and Issa Valley, Tanzania-inhabiting different habitat types was investigated. As a follow up study the effect of host sex and age on infections patterns in Kalinzu Forest Reserve chimpanzees was determined. METHODS: Molecular methods were employed to detect Plasmodium DNA from faecal samples collected from savanna-woodland (Issa Valley) and forest (Kalinzu Forest Reserve) chimpanzee populations. RESULTS: Based on a Cytochrome-b PCR assay, 32 out of 160 Kalinzu chimpanzee faecal samples were positive for Plasmodium DNA, whilst no positive sample was detected in 171 Issa Valley chimpanzee faecal samples. Sequence analysis revealed that previously known Laverania species (Plasmodium reichenowi, Plasmodium billbrayi and Plasmodium billcollinsi) are circulating in the Kalinzu chimpanzees. A significantly higher proportion of young individuals were tested positive for infections, and switching of Plasmodium spp. was reported in one individual. Amongst the positive individuals sampled more than once, the success of amplification of Plasmodium DNA from faeces varied over sampling time. CONCLUSION: The study showed marked differences in the prevalence of malaria parasites among free ranging chimpanzee populations living in different habitats. In addition, a clear pattern of Plasmodium infections with respect to host age was found. The results presented in this study contribute to understanding the ecological aspects underlying the malaria infections in the wild. Nevertheless, integrative long-term studies on vector abundance, Plasmodium diversity during different seasons between sites would provide more insight on the occurrence, distribution and ecology of these pathogens.