Three new Prototapirella species, Opisthotrichum janus, and Troglocorys cava add to Entodiniomorphida (Ciliophora, Trichostomatia) diversity in mountain gorillas in Rwanda.

Recently, a high number of Entodiniomorphida species was discovered in Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda compared to other primates. Thirteen species of five genera (Troglocorys, Gorilloflasca, Prototapirella, Troglodytella, and Opisthotrichum) were identified with 10 species in Gorilloflasca, Prototapirella, and Troglodytella being host-specific, to our current knowledge. The remaining three species have been described for other herbivorous mammals; Prototapirella gorillae for western lowland gorillas; Troglocorys cava for western lowland gorillas, chimpanzees, and guinea baboons; Opisthotrichum janus in the family Ophryoscolecidae is regarded as specific to African antelopes. Here, we show the first indication that wild herbivorous hindgut fermenting mammals might have rumen ophryoscolecids in the intestine sympatric with their native intestinal ciliates. We also describe three new Prototapirella species, P. sabyinyo, P. sulcata, and P. curiosa. The somatic ciliary zones of the three new species are atypical of Prototapirella while their buccal infraciliature is not different from that of other Prototapirella species.

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.

Resurrection of the ancestral RH5 invasion ligand provides a molecular explanation for the origin of P. falciparum malaria in humans.

Many important infectious diseases are the result of zoonoses, in which pathogens that normally infect animals acquire mutations that enable the breaching of species barriers to permit the infection of humans. Our understanding of the molecular events that enable host switching are often limited, and yet this is a fundamentally important question. Plasmodium falciparum, the etiological agent of severe human malaria, evolved following a zoonotic transfer of parasites from gorillas. One gene-rh5-which encodes an essential ligand for the invasion of host erythrocytes, is suspected to have played a critical role in this host switch. Genome comparisons revealed an introgressed sequence in the ancestor of P. falciparum containing rh5, which likely allowed the ancestral parasites to infect both gorilla and human erythrocytes. To test this hypothesis, we resurrected the ancestral introgressed reticulocyte-binding protein homologue 5 (RH5) sequence and used quantitative protein interaction assays to demonstrate that this ancestral protein could bind the basigin receptor from both humans and gorillas. We also showed that this promiscuous receptor binding phenotype of RH5 was shared with the parasite clade that transferred its genome segment to the ancestor of P. falciparum, while the other lineages exhibit host-specific receptor binding, confirming the central importance of this introgression event for Plasmodium host switching. Finally, since its transfer to humans, P. falciparum, and also the RH5 ligand, have evolved a strong human specificity. We show that this subsequent restriction to humans can be attributed to a single amino acid mutation in the RH5 sequence. Our findings reveal a molecular pathway for the origin and evolution of human P. falciparum malaria and may inform molecular surveillance to predict future zoonoses.

Cryptosporidium and giardia species in newly and previously habituated gorillas and nearby water sources in Bwindi Impenetrable National Park, Uganda.

INTRODUCTION: cryptosporidium and giardia are of great one health significance and major cause of protozoan diarrhea in humans and primates; they are found in the faecal matter of animals and humans and also in contaminated water and soil as well. Therefore, we aimed at establishing the prevalence and shedding intensity of faecal Cryptosporidium and giardia in the Newly Habituated Mountain Gorillas (NHMG) and Previously Habituated Mountain Gorillas (PHMG) and in selected water sources within the gorilla home ranges in the month of January 2018. METHODS: we conducted a cross sectional study in the southern sector of Bwindi Impenetrable National Park where a total of 56 faecal samples from both NHMG (34) and PHMG (22) and 30 water samples were purposively collected. Faecal and water samples were transported in a cooler box at 4ºC to Makerere University Parasitology Laboratory for analysis. The samples were analyzed using modified Ziehl-Neelsen technique and Ether concentration method for Cryptosporidium and giardia respectively. RESULTS: the prevalence of cryptosporidium was established as 13 (59.1%), 15 (44.1%) and 7 (23.3%) in PHMG, NHMG and water respectively. The mean concentration of the oocysts per gram was 222±52.9 in PHMG, 174±41.5 in NHMG and 31±13.2 in water. The prevalence of giardia was 3 (13.6%), 4 (11.8%) and 3 (10%) in PHMG, NHMG and water respectively. The mean concentration of the oocysts per gram was 34±19.9 in PHMG, 25±12.4 in NHMG and 5±2.9 in water. There was no significant difference in both the prevalence of cryptosporidium (p>0.05) and giardia (p>0.05) in the PHMG and NHMG. This indicates that there is high risk of cross infection among the gorillas within the forest sharing similar home ranges. CONCLUSION: the park authorities should ensure that procedures for proper waste disposal while in the forest are properly followed, water drawn for drinking from the forest should be avoided. Further research should be carried out to identify whether the strains of the parasites found in water or other animals in the forest are the same with strains in gorilla dung in order to confirm cross infection.

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.

Three new Troglodytella and a new Gorilloflasca ciliates (Entodiniomorphida) from mountain gorillas (Gorilla beringei beringei) in Rwanda.

Four new species, Troglodytella gracilis, T. virunga, T. dolium, and Gorilloflasca longior were described from the mountain gorillas in Rwanda. The three Troglodytella species share a retractable adoral ciliary zone, four non-retractable ciliary arches, two broad skeletal plates, a skeletal rod plate, numerous longitudinal cortical grooves, and four contractile vacuoles. The anterior ciliary arch is the longest of the four arches, extending transversely on the left body surface. T. gracilis and T. virunga are characterized by an elongated body, a tail flap, and a wedge-shaped macronucleus. T. virunga can be distinguished from T. gracilis by a trifurcate dorsal lobe, three right lobes, and two ventral spines. T. dolium has a barrel-shaped body and a rod-shaped or boomerang-shaped macronucleus. The buccal infraciliature of the three Troglodytella is composed of an adoral polybrachykinety, a perivestibular polybrachykinety, and paralabial kineties. G. longior has a long flask-shaped body, a vestibulum, a frontal lobe, a posterior cavity, an ellipsoidal or peanut-shaped macronucleus, a contractile vacuole, non-retractable adoral and vestibular ciliary zones in buccal area, a cavity ciliary zone, and two longitudinal dorsal ciliary zones. The infraciliature of G. longior is the same as that of G. africana.

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.

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.

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.

Molecular characterisation of protist parasites in human-habituated mountain gorillas (Gorilla beringei beringei), humans and livestock, from Bwindi impenetrable National Park, Uganda.

BACKGROUND: Over 60 % of human emerging infectious diseases are zoonotic, and there is growing evidence of the zooanthroponotic transmission of diseases from humans to livestock and wildlife species, with major implications for public health, economics, and conservation. Zooanthroponoses are of relevance to critically endangered species; amongst these is the mountain gorilla (Gorilla beringei beringei) of Uganda. Here, we assess the occurrence of Cryptosporidium, Cyclospora, Giardia, and Entamoeba infecting mountain gorillas in the Bwindi Impenetrable National Park (BINP), Uganda, using molecular methods. We also assess the occurrence of these parasites in humans and livestock species living in overlapping/adjacent geographical regions. RESULTS: Diagnostic PCR detected Cryptosporidium parvum in one sample from a mountain gorilla (IIdA23G2) and one from a goat (based on SSU). Cryptosporidium was not detected in humans or cattle. Cyclospora was not detected in any of the samples analysed. Giardia was identified in three human and two cattle samples, which were linked to assemblage A, B and E of G. duodenalis. Sequences defined as belonging to the genus Entamoeba were identified in all host groups. Of the 86 sequence types characterised, one, seven and two have been recorded previously to represent genotypes of Cryptosporidium, Giardia, and Entamoeba, respectively, from humans, other mammals, and water sources globally. CONCLUSIONS: This study provides a snapshot of the occurrence and genetic make-up of selected protists in mammals in and around BINP. The genetic analyses indicated that 54.6% of the 203 samples analysed contained parasites that matched species, genotypes, or genetic assemblages found globally. Seventy-six new sequence records were identified here for the first time. As nothing is known about the zoonotic/zooanthroponotic potential of the corresponding parasites, future work should focus on wider epidemiological investigations together with continued surveillance of all parasites in humans, other mammals, the environment, and water in this highly impoverished area.

Gorilloflasca africana n.g., n.sp., (Entodiniomorphida) from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda.

A new entodiniomorphid ciliate species, Gorilloflasca africana n. g., n. sp. was described from the Virunga mountain gorillas, Gorilla beringei beringei, in Rwanda. It is characterized by a flask-shaped body, a long tubular vestibulum, a round frontal lobe, a large posterior cavity, an ellipsoidal or peanut-shaped macronucleus and a single contractile vacuole. G. africana has the adoral and the vestibular ciliary zones in the buccal area. The adoral ciliary zone is non-retractable, encircling the vestibular opening. The vestibular ciliary zone extends posteriorly in the vestibulum. The somatic ciliary zones are the cavity ciliary zone in the posterior cavity along the ventral side of its opening and two longitudinal ciliary zones on the dorsal body surface. The buccal infraciliary bands of G. africana are a C-shaped adoral polybrachykinety, a stick-shaped vestibular kinety band, and paralabial kineties. The anterior region of the vestibular kinety band is composed of short kineties whereas, kineties in the remaining region are longitudinal. The somatic infraciliary bands are a cavity polybrachykinety and two longitudinal polybrachykineties. Gorilloflasca is a member of the family Blepharocorythidae based on the non-retractable adoral ciliary zone, the frontal lobe, the large posterior cavity and the vestibular longitudinal kineties.

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.

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.

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.

Unravelling the Laverania.

How did an ape-infecting Plasmodium species jump to a human host?

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.

Prototapirella ciliates from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda with the descriptions of two new species.

The morphology of Prototapirella fosseyi n. sp., P. rwanda n. sp. and P. gorillaeImai, Ikeda, Collet, and Bonhomme, 1991 in the Entodiniomorphida were described from the mountain gorillas, Gorilla beringei beringei, in Rwanda. The ciliates have a retractable adoral ciliary zone, four non-retractable ciliary tufts in four caudalia, and one broad skeletal plate beneath the body surface. P. rwanda has a dorsal lobe and ventral lobes in two rows whereas P. fosseyi has no lobes. These two new species have an elongated body, a flat tail flap leaning to the ventral, a macronucleus with a tapering anterior end, a round posterior end and a shallow depression on the dorsal side, a micronucleus lying near the anterior end of macronucleus, a thin left region of the skeletal plate, a distinct skeletal rod plate, and four contractile vacuoles. P. gorillae has some variations in the nuclei and the skeletal plate. The infraciliary bands of three Prototapirella species were the same as some Triplumaria species; a C-shaped adoral polybrachykinety, a slender perivestibular polybrachykinety, and paralabial kineties in their retractable adoral ciliary zone and short lateral polybrachykineties in their four caudalia. The perivestibular polybrachykinety is joined only to the right end of adoral polybrachykinety.

Amoebic meningoencephalitis and disseminated infection caused by Balamuthia mandrillaris in a Western lowland gorilla (Gorilla gorilla gorilla).

CASE DESCRIPTION A 22-year-old male gorilla (Gorilla gorilla gorilla) housed in a zoo was evaluated for signs of lethargy, head-holding, and cervical stiffness followed by development of neurologic abnormalities including signs of depression, lip droop, and tremors. CLINICAL FINDINGS Physical examination under general anesthesia revealed a tooth root abscess and suboptimal body condition. A CBC and serum biochemical analysis revealed mild anemia, neutrophilia and eosinopenia consistent with a stress leukogram, and signs consistent with dehydration. Subsequent CSF analysis revealed lymphocytic pleocytosis and markedly increased total protein concentration. TREATMENT AND OUTCOME Despite treatment with antimicrobials, steroids, and additional supportive care measures, the gorilla's condition progressed to an obtunded mentation with grand mal seizures over the course of 10 days. Therefore, the animal was euthanized and necropsy was performed. Multifocal areas of malacia and hemorrhage were scattered throughout the brain; on histologic examination, these areas consisted of necrosis and hemorrhage associated with mixed inflammation, vascular necrosis, and intralesional amoebic trophozoites. Tan foci were also present in the kidneys and pancreas. Immunohistochemical testing positively labeled free-living amoebae within the brain, kidneys, eyes, pancreas, heart, and pulmonary capillaries. Subsequent PCR assay of CSF and frozen kidney samples identified the organism as Balamuthia mandrillaris, confirming a diagnosis of amoebic meningoencephalitis. CLINICAL RELEVANCE Infection with B mandrillaris has been reported to account for 2.8% of captive gorilla deaths in North America over the past 19 years. Clinicians working with gorillas should have a high index of suspicion for this diagnosis when evaluating and treating animals with signs of centrally localized neurologic disease.