Adult hookworms (Necator spp.) collected from researchers working with wild western lowland gorillas

Background: In general, studies on the diversity of strongylid nematodes in endangered host species are complicated as material obtained by non-invasive sampling methods has limited value for generic and species identification. While egg morphology barely allows assignment to family, the morphology of cultivated infective third stage larvae provides a better resolution at the generic level but cannot be used for exact species identification. Morphology-based taxonomic approaches greatly depend on the examination of adult worms that are usually not available. Methods: Hookworm parasites in two European researchers, who participated in gorilla research in the Central African Republic, were expelled after anthelmintic treatment to the faeces, collected and morphologically examined. A male worm discharged naturally from a wild bonobo (Pan paniscus) in Congo was also examined for comparison. Results: Two species of Necator were identified in researchers' faecal material: Necator americanus (Stiles, 1902) and N. gorillae Noda & Yamada, 1964; the latter species differed in having a smaller body, smaller buccal cavity and shorter spicules with spade-shaped membranes situated distally. Males of N. gorillae also possessed unusual cuticular thickenings on the dorsal side of the prebursal region of the body. These characters, shared with the male worm from the bonobo, correspond well to the description of N. gorillae described from gorillas in Congo. Conclusions: Based on the morphology of the hookworms recovered in this study and previous molecular analyses of larvae developed from both humans and western lowland gorillas (Gorilla gorilla gorilla) from this locality, we conclude that the researchers became infected with gorilla hookworms during their stay in the field. This is the first report of infection with a Necator species other than N. americanus in humans

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

Comparison of ITS-2 sequences of hookworms from apes and humans from Dzanga Sangha Protected Areas.

<p>Host and accession number in DNA database are given in parentheses. Dots indicate homologous nucleotides with <i>N. americanus</i> (<i>N. a.</i>) from Guatemala (AF217891); dash indicates absence of nucleotide. Major indels are shaded.</p

Map of study site in Dzanga-Sangha Protected Areas, Central African Republic.

<p>Dark grey – Dzanga and Ndoki Sectors of the protected Dzanga Ndoki National Park; light grey - Dzanga Sangha Dense Forest Special Reserve; 1 – location of villages Bayanga, Mossapoula and Yandumbé; M – research camp Mongambe; B – research camp Bai Hokou.</p

Amplification of partial ITS-1 region using specific primers.

<p>Primers AmerF3 and AmerR2 only amplified specific band of about 250-1, while no clear band was formed for types II and III. CongF and Nem5.8R resulted in formation of specific band of about 650 bp for types II and III, while prominent bands were not formed for type I.</p

Amino acid substitutions found among the haplotypes of partial <i>cox1</i> gene.

<p>* No. of haplotypes.</p

Evolutionary relationships of <i>Necator</i> spp. from apes and humans in Dzanga-Sangha Protected Areas.

<p>Tree was reconstructed by Neighbor-Joining method on mtDNA <i>cox1</i> sequences each with 669 nucleotides. Host and accession number in DNA database in parentheses are given at each branch. Humans are CAR people except for two Europeans marked with asterisks, who acquired the infection in CAR. Bootstrap values larger than 50 are shown.</p

Comparison of ITS-1 sequences of hookworms from apes and humans from Dzanga Sangha Protected Areas.

<p>Host and accession number in DNA database are given in parentheses. Dots indicate homologous nucleotides with <i>N. americanus</i> (<i>N. a.</i>) from Guatemala (AF217891); dash indicates absence of nucleotide. Major indels are shaded.</p

Restriction enzyme digestion of DNA sequences amplified with Civ18S1500F and Nem5.8R.

<p>By digestion with <i>Alu</i> I, two bands were formed for type I in area from 200 to 500 bp, while only one band was formed for types II and III. By digestion using <i>Hinc</i> II, two bands of about 300 and 500 bp were formed for type I, while only one band similar to original band was visible for types II and III.</p