Detection of DNA of filariae closely related to Mansonella perstans in faecal samples from wild non-human primates from Cameroon and Gabon.

BACKGROUND: The Onchocercidae is a family of filarial nematodes with several species of medical or veterinary importance. Microfilariae are found in the blood and/or the dermis and are usually diagnosed in humans by microscopy examination of a blood sample or skin biopsy. The main objectives of this study were to evaluate whether filariae DNA can be detected in faecal samples of wild non-human primates (NHPs), whether the detected parasites were closely related to those infecting humans and whether filarial DNA detection in faeces is associated with co-infections with nematodes (Oesophagostumum sp. and Necator sp.) known to cause blood loss while feeding on the host intestinal mucosa. METHODS: A total of 315 faecal samples from 6 species of NHPs from Cameroon and Gabon were analysed. PCRs targeted DNA fragments of cox1 and 12S rDNA genes, to detect the presence of filariae, and the internal transcribed spacer 2 (ITS2), to detect the presence of Oesophagostomum sp. and Necator sp. infections. RESULTS: Among the 315 samples analysed, 121 produced sequences with > 90% homology with Onchocercidae reference sequences. However, 63% of the 12S rDNA and 78% of the cox1 gene sequences were exploitable for phylogenetic analyses and the amplification of the 12S rDNA gene showed less discriminating power than the amplification of the cox1 fragment. Phylogenetic analyses showed that the cox1 sequences obtained from five chimpanzee DNA faecal samples from Gabon and two from Cameroon cluster together with Mansonella perstans with high bootstrap support. Most of the remaining sequences clustered together within the genus Mansonella, but the species could not be resolved. Among the NHP species investigated, a significant association between filarial DNA detection and Oesophagostomum sp. and Necator sp. infection was observed only in gorillas. CONCLUSIONS: To our knowledge, this is the first study reporting DNA from Mansonella spp. in faecal samples. Our results raise questions about the diversity and abundance of these parasites in wildlife, their role as sylvatic reservoirs and their potential for zoonotic transmission. Future studies should focus on detecting variants circulating in both human and NHPs, and improve the molecular information to resolve or support taxonomy classification based on morphological descriptions.

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.

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.

Humans and great apes cohabiting the forest ecosystem in central african republic harbour the same hookworms.

BACKGROUND: Hookworms are important pathogens of humans. To date, Necator americanus is the sole, known species of the genus Necator infecting humans. In contrast, several Necator species have been described in African great apes and other primates. It has not yet been determined whether primate-originating Necator species are also parasitic in humans. METHODOLOGY/PRINCIPAL FINDINGS: The infective larvae of Necator spp. were developed using modified Harada-Mori filter-paper cultures from faeces of humans and great apes inhabiting Dzanga-Sangha Protected Areas, Central African Republic. The first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA and partial cytochrome c oxidase subunit 1 (cox1) gene of mtDNA obtained from the hookworm larvae were sequenced and compared. Three sequence types (I-III) were recognized in the ITS region, and 34 cox1 haplotypes represented three phylogenetic groups (A-C). The combinations determined were I-A, II-B, II-C, III-B and III-C. Combination I-A, corresponding to N. americanus, was demonstrated in humans and western lowland gorillas; II-B and II-C were observed in humans, western lowland gorillas and chimpanzees; III-B and III-C were found only in humans. Pairwise nucleotide difference in the cox1 haplotypes between the groups was more than 8%, while the difference within each group was less than 2.1%. CONCLUSIONS/SIGNIFICANCE: The distinctness of ITS sequence variants and high number of pairwise nucleotide differences among cox1 variants indicate the possible presence of several species of Necator in both humans and great apes. We conclude that Necator hookworms are shared by humans and great apes co-habiting the same tropical forest ecosystems.

Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis.

BACKGROUND: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. METHODS: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. CONCLUSION: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

Identification of hookworm (Necator americanus) antigens and their translation in vitro.

During in vitro culture adult (day 35) Necator americanus synthesise a wide range of protein species many of which are excreted or secreted into the culture medium. Both post infection (day 117) hamster sera and sera from infected humans precipitate antigens of 15, 30, 33, 44, 46 and 69 kDa although individual human sera exhibit some variability in absolute specificity. In immunoblotting experiments antigens of 33 kDa are routinely recognised by human sera although two-dimensional gel analysis suggests that more than one polypeptide is involved. RNA isolated from adult worms direct the in vitro synthesis of numerous polypeptides possessing antigenic determinants recognised by sera from infected hamsters and humans. Post-translational modification of N. americanus encoded polypeptides is not, therefore, a prerequisite for antigenicity.