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Nematodes belonging to the genus Oesophagostomum frequently infect wild chimpanzees (Pan troglodytes) across widely separated field sites. Nodular lesions (granulomas) containing Oesophagostomum are commonly seen in the abdomen of infected chimpanzees post-mortem. At Taï National Park, Côte d'Ivoire, previous studies have identified larvae of a variety of Oesophagostomum spp. in wild chimpanzee stool, based on sequencing of larval DNA, and nodular lesions associated with Oesophagostomum, identified morphologically to the genus level but not sequenced. Here we present three recent cases of parasitic granulomas found post-mortem in chimpanzees at Taï. We complement descriptions of gross pathology, histopathology and parasitology with PCR and sequencing of DNA isolated from the parasitic nodules and from adult worms found inside the nodules. In all three cases, we identify Oesophagostomum stephanostomum as the causative agent. The sequences from this study were identical to the only other published sequences from nodules in nonhuman primates-those from the wild chimpanzees of Gombe, Tanzania., (© 2024 The Author(s). American Journal of Primatology published by Wiley Periodicals LLC.)

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.

Nodular roundworms ( Oesophagostomum spp.) are frequent parasites of the large intestine in several mammal species including humans and pigs, and their study often requires the use of infective larvae produced using several coproculture techniques. However, there is no published comparison of techniques to determine which yields the highest number of larvae. This study compares the number of larvae recovered from coprocultures made with charcoal, sawdust, vermiculite, and water in an experiment repeated twice using feces from a sow naturally infected with Oesophagostomum spp. at an organic farm. A higher number of larvae were recovered from coprocultures using sawdust relative to other types of media used, and this was consistent across the two trials. The use of sawdust to culture Oesophagostomum spp. larvae is rarely reported and our study suggests it can yield higher numbers relative to other media.

Helminths of the genus Oesophagostomum cause enteric diseases and affect domestic animals such as pigs. The aim of this study was to explore the species composition and genetic diversity of Oesophagostomum spp. infecting pigs in close contact with humans in the state of Piauí, Brazil. Eighty-seven fecal samples were collected for parasitological tests and molecular analysis. Through microscopy, the overall positivity rate for strongyliform eggs was 81.6% among the pigs studied. Forty-two strongyliform egg samples were subjected to PCR and six cox1 sequences (637 bp) were identified for the genus Oesophagostomum. The sequences were identified as Oesophagostomum dentatum, O. quadrispinulatum and O. columbianum. In the phylogenetic tree and haplotype network, 89 sequences were separated into seven clusters, which also included reference sequences from GenBank. Oesophagostomum dentatum and O. quadrispinulatum were seen to be closely related species and formed a monophyletic group related to O. aculeatum. Oesophagostomum columbianum showed similarity with sequences from parasites infecting small ruminants and the clade was positioned closer to O. bifurcum. High interspecific diversity was found and intraspecific diversity varied according to the species. This was the first study to characterize Oesophagostomum DNA sequences obtained from pigs in Brazil.

Parasitic nematodes of Oesophagostomum spp., commonly known, as 'nodular worms' are emerging as the most widely distributed and prevalent zoonotic nematodes. Oesophagostomum infections are well documented in African non-human primates; however, the taxonomy, distribution and transmission of Oesophagostomum in Asian non-human primates are not adequately studied. To better understand which Oesophagostomum species infect Asian non-human primates and determine their phylogeny we analysed 55 faecal samples from 50 orangutan and 5 gibbon individuals from Borneo and Sumatra. Both microscopy and molecular results revealed that semi-wild animals had higher Oesophagostomum infection prevalence than free ranging animals. Based on sequence genotyping analysis targeting the Internal transcribed spacer 2 of rDNA, we report for the first time the presence of O. aculeatum in Sumatran apes. Population genetic analysis shows that there is significant genetic differentiation between Bornean and Sumatran O. aculeatum populations. Our results clearly reveal that O. aculeatum in free-ranging animals have a higher genetic variation than those in semi-wild animals, demonstrating that O. aculeatum is circulating naturally in wildlife and zoonotic transmission is possible. Further studies should be conducted to better understand the epidemiology and dynamics of Oesophagostomum transmission between humans, non-human primates and other wild species and livestock in Southeast Asia.

Anthelmintic resistance (AR) against gastrointestinal nematodes (GINs) of sheep and goats is a global concern. To address the problem, this study assessed the status of AR in different government and private sheep and goat farms in Bangladesh. We conducted fecal egg count reduction test (FECRT) and Egg hatch assay (EHA) experiments. For the detection of resistant larvae, pooled fecal samples from treated and non-treated groups were subjected to coproculture. Furthermore, 195 adult Haemonchus parasites were genotyped to ascertain benzimidazole (BZ) resistance allele from seven topographic zones of Bangladesh using allele specific PCR (AS-PCR). In FECRT, the percentage reduction along with 95% confidence intervals indicated that GINs were resistant to albendazole (ABZ), levamisole (LEV) and ivermectin (IVM). Coproculture revealed that Haemonchus spp., Oesophagostomum spp. and Trichostrongylus spp. were resistant to anthelmintics. ABZ resistance was also confirmed by in vitro EHA in all the farms except the private goat farm in Mymensingh. The genotype frequencies were 6% for homozygous resistant (rr), 59% for heterozygous (rS) and 35% for homozygous susceptible (SS) among different topographic zones. The allelic frequency of the mutation conferring resistance (r) ranged from 25% to 47% signifying resistance to BZ in nematodes of sheep/goats. The genotype frequencies (rr, rS and SS) and allelic frequencies (r and S) varied significantly (p˂0.05) in different zones in Bangladesh. Overall, the data suggest an alarming condition created by multiple AR in Bangladesh., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Standard diagnostic methods currently in use for the identification of helminth infections in ruminants are based on the morphological analysis of immature and adult stages of parasites. This paper describes a method for the semiquantitative identification of nematodes, mainly Trichostrongyloidea, at species-level resolution. The method is based on amplification and fragment analysis followed by minisequencing of the ITS-2 region (internal transcribed spacer 2) of the ribosomal DNA of parasite eggs or larvae. This method allows for the identification of seven genera (Chabertia, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, Teladorsagia, and Trichostrongylus) and 12 species (Chabertia ovina, Cooperia curticei, Cooperia punctata, Cooperia oncophora/Cooperia surnabada, Haemonchus contortus, Haemonchus placei, Haemonchus longistipes, Oesophagostomum asperum, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei, and Trichostrongylus colubriformis) of infectious nematodes of domestic ruminants. The concordance between the morphological and molecular analyses in the detection of genera ranged from 0.84 to 0.99, suggesting the proposed detection method is specific, semiquantitative, less laborious, and highly cost-efficient.

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., (© 2019 John Wiley & Sons Ltd.)

The naturalization of alien Reeves's muntjacs (Muntiacus reevesi) on Izu-Oshima Island, Tokyo, Japan, has proceeded intensively over the last five decades. To clarify whether the gastrointestinal helminths of these animals were brought from their original endemic area or were newly acquired in Japan, 32 Reeves's muntjacs trapped on the island were parasitologically examined. In addition to Gongylonema pulchrum in the oesophagus (34.4% prevalence), Chabaudstrongylus ninhae (Dróżdż, 1967) (Trichostrongylidae: Cooperiinae) and Oesophagostomum muntiacum Jian, 1989 (Chabertiidae: Oesophagostominae) were prevalent in the small (28.1%) and large (46.9%) intestines, respectively. For the first time, these trichostrongylid or chabertiid worms were genetically characterized based on partial nucleotide sequences of the nuclear ribosomal RNA gene (rDNA) and mitochondrial DNA cytochrome c oxidase subunit 1 gene (cox-1), and the phylogenetic relationships with other members of their family were explored. Since these two intestinal nematode species are inherent in muntjacs, this study demonstrates a new distribution of exotic helminth species in Japan in accordance with the naturalization of alien mammalian hosts. The molecular genetic data collected here could assist the taxonomic assessment of morphological variants in different Muntiacus spp. and/or of different geographical origins. Furthermore, our data may help to define the phylogenetic relationships among such isolates.

The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 μM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 μM and 10 μM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 μM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The transmission of zoonoses by wildlife, including elephants, is a growing global concern. In this study, we screened for helminth infections among Asian wild elephants (Elephas maximus) of the Salakpra Wildlife Sanctuary, Kanchanaburi, Thailand. Elephant faecal samples (45) were collected from the sanctuary grounds during January through November 2013 and assayed individually using the tetranucleotide microsatellite technique. Microscopic examination indicated a high prevalence of strongylids (93.0%) and low prevalences of trichurids (2.3%) and ascarids (2.3%). To identify the strongylid species, small subunit (SSU) rDNA sequences were amplified from copro-DNA and compared with sequences in GenBank. The generated SSU-rDNA sequences comprised five distinct haplotypes that were closely related to Oesophagostomum aculeatum. A phylogenetic analysis that incorporated related nematodes yielded a tree separated into two main clades, one containing our samples and human and domestic animal hookworms and the other consisting of Strongyloides. The present results indicate that O. aculeatum in local elephants is a potential source of helminthiasis in human and domestic animals in this wild-elephant irrupted area.

The present study examined sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1), and internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) among Oesophagostomum asperum isolates from goats in Hunan Province, China. A portion of the cox1 (pcox1), nad1 (pnad1) genes and the ITS (ITS1+5.8S rDNA+ITS2) rDNA were amplified by polymerase chain reaction (PCR) separately from adult O. asperum individuals and the representative amplicons were subjected to sequencing from both directions. The lengths of pcox1, pnad1 and ITS rDNA were 366 bp, 681 bp and 785 bp, respectively. The A+T contents of gene sequences were 71.5-72% for pcox1, 73.7-74.2% for pnad1 and 58-58.8% for ITS rDNA. Intra-specific sequence variations within O. asperum were 0-1.6% for pcox1, 0-1.9% for pnad1 and 0-1.7% for ITS rDNA, while inter-specific sequence differences among members of the genus Oesophagostomum were significantly higher, being 11.1-12.5%, 13.3-17.7% and 8.5-18.6% for pcox1, pnad1 and ITS rDNA, respectively. Phylogenetic analyses using combined sequences of pcox1 and pnad1, with three different computational algorithms (Bayesian inference, maximum likelihood and maximum parsimony), revealed distinct groups with high statistical support. These findings demonstrated the existence of intra-specific variation in mtDNA and rDNA sequences among O. asperum isolates from goats in Hunan Province, China, and have implications for studying molecular epidemiology and population genetics of O. asperum.

Many infectious diseases caused by eukaryotic pathogens have a devastating, long-term impact on animal health and welfare. Hundreds of millions of animals are affected by parasitic nematodes of the order Strongylida. Unlocking the molecular biology of representatives of this order, and understanding nematode-host interactions, drug resistance and disease using advanced technologies could lead to entirely new ways of controlling the diseases that they cause. Oesophagostomum dentatum (nodule worm; superfamily Strongyloidea) is an economically important strongylid nematode parasite of swine worldwide. The present article reports recent advances made in biology and animal biotechnology through the draft genome and developmental transcriptome of O. dentatum, in order to support biological research of this and related parasitic nematodes as well as the search for new and improved interventions. This first genome of any member of the Strongyloidea is 443 Mb in size and predicted to encode 25,291 protein-coding genes. Here, we review the dynamics of transcription throughout the life cycle of O. dentatum, describe double-stranded RNA interference (RNAi) machinery and infer molecules involved in development and reproduction, and in inducing or modulating immune responses or disease. The secretome predicted for O. dentatum is particularly rich in peptidases linked to interactions with host tissues and/or feeding activity, and a diverse array of molecules likely involved in immune responses. This research progress provides an important resource for future comparative genomic and molecular biological investigations as well as for biotechnological research toward new anthelmintics, vaccines and diagnostic tests., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Nodular Oesophagostomum genus nematodes are a major public health concern in some African regions because they can be lethal to humans. Their relatively high prevalence in people has been described in Uganda recently. While non-human primates also harbor Oesophagostomum spp., the epidemiology of this oesophagostomosis and the role of these animals as reservoirs of the infection in Eastern Africa are not yet well documented., Methodology/principal Findings: The present study aimed to investigate Oesophagostomum infection in terms of parasite species diversity, prevalence and load in three non-human primates (Pan troglodytes, Papio anubis, Colobus guereza) and humans living in close proximity in a forested area of Sebitoli, Kibale National Park (KNP), Uganda. The molecular phylogenetic analyses provided the first evidence that humans living in the Sebitoli area harbored O. stephanostomum, a common species in free-ranging chimpanzees. Chimpanzees were also infected by O. bifurcum, a common species described in human populations throughout Africa. The recently described Oesophagostomum sp. found in colobine monkeys and humans and which was absent from baboons in the neighboring site of Kanyawara in KNP (10 km from Sebitoli), was only found in baboons. Microscopic analyses revealed that the infection prevalence and parasite load in chimpanzees were significantly lower in Kanyawara than in Sebitoli, an area more impacted by human activities at its borders., Conclusions/significance: Three different Oesophagostomum species circulate in humans and non-human primates in the Sebitoli area and our results confirm the presence of a new genotype of Oesophagostomum recently described in Uganda. The high spatiotemporal overlap between humans and chimpanzees in the studied area coupled with the high infection prevalence among chimpanzees represent factors that could increase the risk of transmission for O. stephanostomum between the two primate species. Finally, the importance of local-scale research for zoonosis risk management is important because environmental disturbance and species contact can differ, leading to different parasitological profiles between sites that are close together within the same forest patches.

We explored molecules involved in in vitro exsheathment of Oesophagostomum dentatum L3s using a proteomic-transcriptomic-bioinformatic approach. Analysis of L3s before, during and after exsheathment identified 11 proteins that were over-expressed exclusively during exsheathment. These proteins (including key enzymes, heat shock, structural and nematode-specific proteins) were inferred to be involved in development, metabolism, structure, motility and/or host-parasite interactions. Some of these molecules represented homologues linked to entry into and exit from the dauer stage in the free-living nematode Caenorhabditis elegans. The approach established here provides a basis for investigations of ecdysis in other strongylid nematodes., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

In the present study, the internal transcribed spacers (ITS) of ribosomal DNA (rDNA) of Oesophagostomum asperum and O. columbianum were amplified and sequenced. The ITS-1, 5.8S and ITS-2 rDNA sequences of O. asperum were 374 bp, 153 bp and 259 bp in length, respectively, and the corresponding sequences of O. columbianum were 259, 153 and 218 bp in length, respectively. Sequence differences in the ITS-1 and ITS-2 rDNA between the two Oesophagostomum species were 9.5-10.2% and 12.7-13.9%, respectively. Sequence differences in the ITS-1 and ITS-2 rDNA among members of the genus Oesophagostomum were 2.5-11.6% and 6.8-22.3%, respectively. Based on genetic markers in the ITS rDNA, an effective polymerase chain reaction (PCR) approach was developed to differentiate O. columbianum from O. asperum with a sensitivity of 0.2 ng/μl DNA. Since accurate characterization of parasites at different taxonomic levels is essential for population genetic studies and control of parasitosis, the present findings have important implications for studying epidemiology, taxonomy and population biology, as well as for the control of oesophagostomiasis.

Introduction: Soil-transmitted helminths (STHs) are a major health concern in tropical and sub-tropical countries. Oesophagostomum infection is considered endemic to West Africa but has also been identified in Uganda, East Africa, among primates (including humans). However, the taxonomy and ecology of Oesophagostomum in Uganda have not been studied, except for in chimpanzees (Pan troglodytes), which are infected by both O. bifurcum and O. stephanostomum., Methods and Findings: We studied Oesophagostomum in Uganda in a community of non-human primates that live in close proximity to humans. Prevalence estimates based on microscopy were lower than those based on polymerase chain reaction (PCR), indicating greater sensitivity of PCR. Prevalence varied among host species, with humans and red colobus (Procolobus rufomitratus) infected at lowest prevalence (25% and 41% by PCR, respectively), and chimpanzees, olive baboons (Papio anubis), and l'hoest monkeys (Cercopithecus lhoesti) infected at highest prevalence (100% by PCR in all three species). Phylogenetic regression showed that primates travelling further and in smaller groups are at greatest risk of infection. Molecular phylogenetic analyses revealed three cryptic clades of Oesophagostomum that were not distinguishable based on morphological characteristics of their eggs. Of these, the clade with the greatest host range had not previously been described genetically. This novel clade infects humans, as well as five other species of primates., Conclusions: Multiple cryptic forms of Oesophagostomum circulate in the people and primates of western Uganda, and parasite clades differ in host range and cross-species transmission potential. Our results expand knowledge about human Oesophagostomum infection beyond the West African countries of Togo and Ghana, where the parasite is a known public health concern. Oesophagostomum infection in humans may be common throughout Sub-Saharan Africa, and the transmission of this neglected STH among primates, including zoonotic transmission, may vary among host communities depending on their location and ecology.

The characteristics of the intergenic spacer rDNAs (IGS rDNAs) of Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical locations in Mainland China were determined, and the phylogenetic relationships of the two species were reconstructed using the IGS rDNA sequences. The organization of the IGS rDNA sequences was similar to their organization in other eukaryotes. The 28S-18S IGS rDNA sequences of both O. dentatum and O. quadrispinulatum were found to have variable lengths, that is, 759-762 bp and 937-1128 bp, respectively. All of the sequences contained direct repeats and inverted repeats. The length polymorphisms were related to the different numbers and organization of repetitive elements. Different types and numbers of repeats were found between the two pig nodule species, and two IGS structures were found within O. quadrispinulatum. Phylogenetic analysis showed that all O. dentatum isolates were clustered into one clade, but O. quadrispinulatum isolates from different origins were grouped into two distinct clusters. These results suggested independent species and the existence of genotypes or subspecies within pig nodule worms. Different types and numbers of repeats and IGS rDNA structures could serve as potential markers for differentiating these two species of pig nodule worms.

Oesophagostomum spp., commonly known as 'nodule worms', is one of the important emerging nematode zoonoses worldwide. In the present study, we sequenced the complete mitochondrial (mt) genomes of two small ruminant nodule worms, Oesophagostomum asperum and Oesophagostomum columbianum, and compared them with that of pig nodule worms (Oesophagostomum dentatum and Oesophagostomum quadrispinulatum). The complete mt genomes of O. asperum andO. columbianum were 13,672 and 13,561 bp, respectively. Both mt genomes were circular, and consisted of 36 genes, including 12 genes coding for proteins, 2 genes for rRNA, and 22 genes for tRNA. The gene content and arrangement are identical to that of pig nodule worms. The availability of full mtDNA sequences of O. asperum and O. columbianum provide useful information for studying population genetics of Oesophagostomum spp., molecular epidemiology and control of O. asperum and O. columbianum infection in small ruminants., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Gene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods., Results: An improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest-neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes., Conclusions: Our reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.

In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy)-propane (EPNP) significantly inhibited (≥90%) larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase) inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways.

The present study identified and characterized new major sperm protein (MSP) genes from the two nodule worms Oesophagostomum dentatum and Oesophagostomum quadrispinulatum collected from pigs in China. Total genomic DNA was extracted individually from 10 male nematode samples representing O. dentatum, and 4 male nematode samples representing O. quadrispinulatum. A pair of primers (OMSP1F/MSP1R) was designed based on the MSP gene sequences of Ascaris suum and O. dentatum available in GenBank, and used to amplify the MSP genes from the two porcine nodule worms. The PCR products were purified and subsequently cloned into pGEM-T Easy vector. Recombinants were identified by PCR and sequenced. Sequence analysis revealed that there were two different types of MSP sequences in O. dentatum and O. quadrispinulatum, one contained intron, and the other did not. The lengths of the MSP sequences containing introns were 433 bp or 439 bp in O. dentatum, and 436 bp, 439 bp or 446 bp in O. quadrispinulatum, containing 1 or 2 introns. Five and three new members of the MSP multigene family were identified in O. dentatum and O. quadrispinulatum in this study, respectively. The MSP sequences without introns were 381 bp in length, and can be deduced into 126 amino acids. The sequences of MSP genes containing introns seem to be more conserved than those without introns. The identities of deduced amino acid sequences of the MSP genes containing introns were 96.0-100% within and between the two nodule worms, and were 81.1-93.7% compared with other published MSP sequences of the representative nematodes. The present study identified new MSP genes with introns from O. dentatum and O. quadrispinulatum for the first time. The identification and characterization of newly described MSP genes from O. dentatum and O. quadrispinulatum have implications for further studies of molecular biology and reproduction control of Oesophagostomum spp., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3-5.2% for pcox1, 0-4.9% for pnad1, and 0-7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7-13.4% for pcox1, 11-14.6% for pnad1, and 14.9-18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.

In the present study, the complete mitochondrial DNA (mtDNA) sequences of the pig nodule worm Oesophagostomum quadrispinulatum were determined for the first time, and the mt genome of Oesophagostomum dentatum from China was also sequenced for comparative analysis of their gene contents and genome organizations. The mtDNA sequences of O. dentatum China isolate and O. quadrispinulatum were 13,752 and 13,681 bp in size, respectively. Each of the two mt genomes comprises 36 genes, including 12 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, but lacks the ATP synthetase subunit 8 gene. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T are 75.79% and 77.52% for the mt genomes of O. dentatum and O. quadrispinulatum, respectively. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), all revealed that O. dentatum and O. quadrispinulatum represent distinct but closely-related species. These data provide novel and useful markers for studying the systematics, population genetics and molecular diagnosis of the two pig nodule worms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In strongylid roundworms, such as Oesophagostomum dentatum (porcine nodule worm), some sex-specific genes are likely to be associated with parasite maturation, development and reproduction. In this study, an analysis of transcription of the two sex-specific genes (vit and msp) encoding vitellogenin and major sperm protein of O. dentatum, respectively, revealed that adult females transcribed vit and adult males msp at high levels, in contrast to immature larval stages and pre-adult worms from in vitro cultures for which no transcription of vit or msp was detected. The analysis showed that neither presence nor absence of the heterologous sex, nor the duration of infection, was central to vit or msp transcription. In small or "virgin" adults, no or only low-level transcription of vit and msp was detectable. We hypothesize that the transcription of the sex-specific genes is linked to endogenous factors, such as size, maturation of the reproductive organs and/or fitness of the worms, and not to exogenous influences. The maturation of worms appears to be linked, to some extent, to the expression of the genes studied herein., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

Oesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal deoxyribonucleic acid (DNA) of the porcine nodule worms Oesophagostomum dentatum and O. quadrispinulatum, a pair of specific primers (OdspF/OdspR2) for O. dentatum and a pair of specific primers (OqspF/OqspR) for O. quadrispinulatum were designed and used to develop a multiplex polymerase chain reaction (PCR) assay for the identification and differentiation of the two porcine nodule worms. This approach allowed the specific identification and differentiation of O. dentatum and O. quadrispinulatum, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amount of DNA detectable using this multiplex PCR assay was 0.1 ng for both O. dentatum and O. quadrispinulatum. The identity of 53 porcine nodule worms collected from pigs from different geographical localities in mainland China was ascertained as O. dentatum or O. quadrispinulatum, respectively, by this multiplex PCR method. This multiplex PCR assay is useful for the simultaneous identification of eggs of O. dentatum and O. quadrispinulatum and should provide a useful tool for the diagnosis and molecular epidemiological investigation of Oesophagostomum spp. infection in pigs.

A multiplex real-time PCR was developed and evaluated for the simultaneous detection of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples. Using well-defined control samples (N = 150), known positive fecal samples (N = 50), and fecal samples from an area in Ghana where human infections with all 3 nematode species are endemic (N = 339), the method proved to be highly specific and sensitive. Cycle threshold (Ct) values, reflecting parasite-specific DNA load, showed significant correlation with the intensity of infection as measured by microscopy using Kato-Katz fecal smears or by species specific third-stage larval count after coproculture. The multiplex real-time PCR described combined with the simple fecal sample collection procedure and the potential for high throughput makes this approach a powerful diagnostic tool to study species-specific transmission patterns of human hookworm-like infections. Moreover, this procedure facilitates monitoring of intervention programs and allows species-specific detection of treatment failure following rounds of mass treatment.

In the present study, samples of Oesophagostomum spp. collected from pigs from different geographical localities in mainland China were characterized genetically by polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) and restriction fragment length polymorphism (PCR-RFLP) techniques using genetic markers in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The second internal transcribed spacer (ITS-2) was amplified from 51 individual nodule worms by PCR, and the amplicons were analyzed by SSCP. With the exception of slight microheterogeneity, SSCP analyses displayed two distinct banding profiles that allowed the identification of all Oesophagostomum spp. samples examined into two groups, the first one represented O. dentatum, and the second one may represent O. quadrispinulatum. Then, the entire ITS was amplified from individual samples, and the amplicons were digested with restriction endonuclease Pst I. The results of RFLP analyses were consistent with that of SSCP. Sequence analysis of ITS rDNA supported the identification and differentiation of Chinese Oesophagostomum spp. samples into two species, namely, O. dentatum and O. quadrispinulatum. These PCR-based approaches provide useful complementary tools to traditional methods for the accurate identification of Oesophagostomum spp. (irrespective of developmental stage) and have implications for studying the ecology and population genetic structures of these parasites and for the prevention and control of the diseases they cause.

There are substantial gaps in the knowledge of the molecular processes of development and reproduction in parasitic nematodes, despite the fact that understanding such processes could lead to novel ways of treating and controlling parasitic diseases, through blocking or disrupting key biological pathways. Biotechnological advances through large-scale sequencing projects, approaches for the analysis of differential gene and protein expression and functional genomics (e.g., double-stranded RNA interference) now provide opportunities to investigate the molecular basis of developmental processes in some parasitic nematodes. The porcine nodule worm, Oesophagostomum dentatum (order Strongylida), may provide a platform for testing the function of genes from this and related nematodes, given that this species can be grown and maintained in culture in vitro for periods longer than other nematodes of the same order. In this article, we review relevant biological, biochemical and molecular biological and genomic information about O. dentatum and propose that the O. dentatum - pig system provides an attractive model for exploring molecular developmental and reproductive processes in strongylid nematodes, leading toward new intervention methods and biotechnological outcomes.

Material and Methods: Large intestines of five slaughtered sows, aging 10 months to 3 years and originating from different herds of southern Poland, were examined parasitologically according to an agar-gel method. The intestines were uncoiled and divided into three sections: section 1--caecum and the first 0-20% part of colon, section 2-20--60% of large intestine and section 3--60-100% length of gut. Adult worms of Oesophagostomum were differentiated on the basis of species and sex, using the shape of buccal capsule and oesophagus, the length of male spicules, and the distance from vulva to anus as well as from anus to the tip of tail of females., Results: In the case of two sows, in 30% of the contents from the section 1 of large intestines a total of 10 specimens of Oesophagostomum quadrispinulatum (Marcone, 1901) Alicata, 1935 were found. This is a new nematode species in the parasitic fauna of Poland and the present record enlarges its geographical range. The infection with O. quadrispinulatum seems to be more dangerous from that of O. dentatum--the most common porcine nodular worm.

Full-length genes representing different isoforms of the ubiquitin-conjugating enzyme UBC-2 were isolated from Oesophagostomum dentatum, cloned and sequenced. The alignment of their sequences (designated Od-ubc-2.1 to Od-ubc-2.3) revealed nucleotide variation at three positions within the predicted open reading frame of 444 bp. Substitutions were at positions 141 (A<-->G), 142 (A<-->G) and 296 (T<-->C). Both former substitutions resulted in amino acid changes from a glycine residue to an arginine residue, whereas the latter resulted in a change from isoleucine to threonine. Comparison of predicted OD-UBC-2 with UBC-2 (protein) homologues/orthologues from 12 other species representing nematodes, Drosophila melanogaster, Saccharomyces cerevisiae, mice and humans revealed identities between species varying from 77 to 100% at the amino acid level, and motifs associated with protein conformation and function were identified. While the function of a representative ubc-2 gene from O. dentatum could not be established in C. elegans, it is likely to play a key role in the catabolism of proteins and in the development of O. dentatum.

In the present study, we constructed gender-enriched cDNA libraries for the adult stage of the parasitic nematode Oesophagostomum dentatum (order Strongylida) using suppressive-subtractive hybridization (SSH), sequenced clones from the female-library and male-library (480 from each) and conducted bioinformatic and microarray analyses of the expressed sequence tags (ESTs). In total, 873 ESTs (440 male and 433 female) were obtained, achieving a sequencing success of 91%The nucleotide sequences reported in this article (Tables 1-5) have been deposited in the EMBL, GenBank and DDJB databases under the Accession nos. AM157797-AM158083. Microarray analyses of 516 unique ESTs representing both gender-enriched libraries revealed differential hybridization for 391 of them (75.8%). Of these, 220 (56.3%) had significantly greater signal intensities in the female than in the male, and 154 (70%) of these were predicted to have homologues in C. elegans. These homologues were predicted to be involved in key biological processes, including embryonic nutrition, gametogenesis, molecular binding/transport or metabolism, nucleic acid synthesis and function, and signal transduction. Of the 171 ESTs with statistically higher signal intensities in male O. dentatum, 43.8% had homologues in C. elegans. These homologues included major sperm proteins (MSPs) or MSP-like molecules, keratin-like molecules, molecules involved in metabolism, PDZ domain-containing proteins, sugar binding proteins, protein kinases, serine proteases or protease inhibitors, molecules involved in proteolysis and other proteins, such as enzymes and various putative proteins. Of the 287 ESTs (from both gender-enriched cDNA libraries) with no known homologues in C. elegans, 50 (17.4%) had homologues in other nematodes, 8 had homologues in various other organisms and 104 (36.2%) had no homology to any sequence in current gene databases. The present study lays a foundation for the isolation and molecular, biochemical and functional characterization of selected genes from the gender-enriched cDNA archives established for O. dentatum.

The nodule worm Oesophagostomum bifurcum (Nematoda: Strongylida) is a parasite of major human health importance predominantly in northern Togo and Ghana. Currently, it is estimated that 0.25 million people are infected with this nematode, and at least 1 million people are at risk of infection. Infection with this parasite causes significant disease as a consequence of encysted larvae in the wall of the large intestine. In spite of the health problems caused by O. bifurcum, there have been significant gaps in the knowledge of the biology, transmission and population genetics of the parasite. This review provides an account of some recent insights into the epidemiology and genetics of the parasite from human and non-human primate hosts in specific regions of Africa using molecular tools. Recent research findings are discussed mainly in relation to non-human primates being reservoirs of infection, and the consequences for the prevention and control of oesophagostomiasis in humans are briefly discussed.

Ternidens deminutus (Strongylida) is a parasitic nematode infecting non-human and human primates in parts of Africa, Asia and the Pacific islands. The present study genetically characterized T. deminutus and defined genetic markers in nuclear ribosomal DNA (rDNA) as a basis for developing molecular-diagnostic tools. The sequences of the second internal transcribed spacer (ITS-2) of rDNA were determined for adult specimens of T. deminutus (Nematoda: Strongylida: Oesophagostominae) from the Olive baboon and the Mona monkey. The length and G+C content of the ITS-2 sequences was 216 bp and approximately 43%, respectively. While there was no sequence variation among individual T. deminutus specimens from the baboon, 6 (2.8%) nucleotide differences were detected in the ITS-2 between the parasite from baboon and that of the Mona monkey, which is similar to the difference (3.2%) between 2 other species of Oesophagostominae (Oesophagostomum bifurcum and O. stephanostomum) from non-human primates, suggesting significant population variation or the existence of cryptic (i.e. hidden) species within T. deminutus . Pairwise comparisons of the ITS-2 sequences of the 2 operational taxonomic units of T. deminutus with previously published ITS-2 sequences for selected members of the subfamilies Oesophagostominae and Chabertiinae indicated that species from primates (including those representing the subgenera Conoweberia and Ihleia) are closely related, in accordance with previous morphological studies. The sequence differences (27-48.3%) in the ITS-2 between the 2 taxonomic units of T. deminutus and hookworms (superfamily Ancylostomatoidea) enabled their identification and delineation by polymerase chain reaction (PCR)-based mutation scanning. The genetic markers in the ITS-2 provide a foundation for improved, PCR-based diagnosis of T. deminutus infections and for investigating the life-cycle, transmission patterns and ecology of this parasite.

An AFLP approach was established to investigate genetic diversity within Oesophagostomum bifurcum (order Strongylida) from human and non-human primates. Evaluation of different combinations of restriction enzymes (n = 8) and primers (n = 29) demonstrated that the use of HindIII/BglII digested templates and primers with the selective nucleotides + AG/ +AC, respectively, was the most effective for the analysis of O. bifurcum DNA. A total of 63 O. bifurcum adults from human, Patas monkey, Mona monkey and Olive baboon hosts from different geographical regions in Ghana were subjected to analysis using this method. Cluster analysis revealed 4 genetically distinct groups, namely O. bifurcum from the Patas monkey (I), from the Mona monkey (II), from humans (III) and from the Olive baboon (IV). These findings were concordant with those achieved previously using RAPD analysis and supports population genetic substructuring within O. bifurcum according to host species. The results demonstrated the effectiveness of the present AFLP method for establishing genetic variation within O. bifurcum, and indicates its applicability to other parasitic nematodes of human and/or veterinary health importance.

Major sperm protein (msp) genes were isolated from complementary (cDNA) and genomic DNA libraries prepared from the parasitic nematode, Oesophagostomum dentatum, characterized at the nucleotide and amino acid (aa) levels, and their expression was investigated. Three different msp cDNA and 2 genomic sequences were determined, each with an open reading frame (ORF) of 381 nucleotides. Nucleotide variation was detected at 30 positions in the ORF among all 5 sequences. Conceptual translation of the full-length msp sequences inferred 4 different MSPs each of 126 aa. These predicted MSPs differed at aa positions 15 (serine <----> threonine), 101 (alanine <----> glycine), 103 (glutamine <----> leucine) and 126 (proline <----> leucine). Southern blot analysis of O. dentatum genomic DNA, digested separately with various restriction endonucleases, displayed multiple (n = 7-13) bands for each enzyme, providing support for a multigene family. Also, at the genomic level, sequence tracts consistent with a 'substitute' TATA box sequence motif were identified within a region (-1 to -123 nt) preceding the 2 msp genes. In contrast to other species of nematode investigated to date, no GATA transcription factor binding motif was detected immediately upstream of the msp coding region. Real-time PCR analysis demonstrated that msp mRNA was expressed exclusively in the males of both fourth-stage larvae (L4s) and adults of O. dentatum (raised in pigs after intragastric inoculation). The magnitude of expression in male O. dentatum raised in pigs in the presence of female worms was the same as in males in the absence of females. Comparative analyses showed aa sequence conservation among MSPs from various nematodes, suggesting similar functional roles for these proteins.

Random amplified polymorphic DNA (RAPD) was used to study genetic variation within Oesophagostomum bifurcum in Ghana. Four different decamer primers were used for the amplification of DNA from individual O. bifurcum adults (n = 41) from humans and non-human primates (including the Mona monkey, Patas monkey and Olive baboon) from different geographic regions. Analysis of the amplicons from all 41 nematodes by high resolution, denaturing polyacrylamide gel electrophoresis defined a total of 326 informative RAPD bands. Cluster analysis of the RAPD data (based on pairwise comparison of banding profiles) showed that O. bifurcum from humans was genetically distinct from O. bifurcum from the Mona and Patas monkeys, and from the Olive baboon. These findings clearly demonstrate the existence of population genetic substructuring within O. bifurcum from different primate hosts in Ghana, and raise interesting questions about host specificity, epidemiology (e.g., zoonotic transmission), and ecology of the different genotypes of O. bifurcum.

Understanding reproductive and developmental processes of socioeconomically important parasitic nematodes is of fundamental scientific interest and could have important implications for developing novel methods for parasite control via the disruption or interruption of such processes. Central to investigating reproductive molecular biology is the identification and characterisation of genes with sex-specific expression profiles. However, there is currently a paucity of information on such genes and their expression patterns in parasitic nematodes. This article describes recent progress on the characterisation of sex-specific genes from a parasitic nematode of veterinary importance, and discusses the fundamental scientific and applied implications of this work.

A male-specifically expressed sequence tag was used as a probe to screen adult male Oesophagostomum dentatum (Nematoda; Strongylida) gene libraries. The cDNA clones isolated coded for a serine/threonine protein phosphatase with approximately 85% identity to two Caenorhabditis elegans proteins implicated in reproduction. However, the genomic structures for the two species were distinct, in that the O. dentatum gene contained seven introns, whereas the C. elegans homologues contained three (two of which were conserved between the two nematodes). The promoters of all three nematode genes contained two putative GATA motifs separated by six to seven nucleotides and located within 100 nucleotides of the predicted transcriptional start site. RNA interference (RNAi) experiments in C. elegans, targetting the two homologues, revealed a consistent reduction in the number of progeny produced by treated worms, indicating a functional role in reproduction. Expression of green fluorescent protein, directed by the putative promoters for the C. elegans phosphatase genes, was analysed in transgenic C. elegans. The present results suggest that there is a significant degree of conservation between O. dentatum and C. elegans in the features and function of the serine/threonine protein phosphatase characterised, which should have implications for detailed investigations into molecular reproductive processes of some parasitic nematodes.

A cDNA was isolated from an adult male Oesophagostomum dentatum gene library by screening with a male-specific, partial expressed sequence tag (EST) probe identified previously using a differential display technique. The full-length cDNA of 642 bp included 5' and 3' untranslated regions of 44 and 121 nucleotides, respectively, and encoded a predicted protein with a putative 18 amino acid signal sequence and a mature polypeptide of 14.7 kDa comprising approximately 15% cysteine residues. The amino acid sequence showed similarity with a number of proteins from Caenorhabditis elegans, parasitic nematodes, insects and amphibia, all of which contain a trypsin inhibitor-like cysteine-rich domain. A 3-dimensional structure model constructed for the O. dentatum protein (designated OdmCRP) inferred that it is composed of 2 domains, each with 5 disulfide bonds, which are indicative of the Ascaris family of serine protease inhibitors. These findings indicate that OdmCRP, with 2 structural domains relating to functionally active sites, is a new member of this inhibitor family.

Genetic markers in the mitochondrial genome have proven useful for population genetic studies because of their maternal inheritance and relatively high evolutionary rates. In this study, we exploited the high resolution capacity of PCR-coupled single-strand conformation polymorphism (SSCP) to screen for sequence variation in part of the cytochrome c oxidase subunit 1 gene (p cox 1) among individuals of the parasitic nematode, Oesophagostomum bifurcum from human or Mona monkey hosts from Africa. SSCP analysis revealed distinct profiles among some of the individuals, and subsequent sequence analysis of representative samples defined 10 different haplotypes. For comparative purposes, the p cox 1 sequences for representatives of four other species of Oesophagostomum from livestock were included. While there were high levels (11.5-13.7%) of sequence difference among the latter species, there was no fixed nucleotide difference between O. bifurcum individuals from humans and those from monkeys. The data support the proposal that O. bifurcum from the two primate hosts represents a single species and that the haplotypic variability in p cox 1 represents population variation. The results reinforce the usefulness of the SSCP-sequencing approach for studying genetic variation in nematode populations using mitochondrial markers.

Degenerated primers were used to amplify DNA fragments of the triosephosphate isomerase (TPI) gene from complementary DNA (cDNA) and from genomic DNA of two species of porcine gastrointestinal nematodes, Oesophagostomum dentatum and O.quadrispinulatum. Polymerase chain reaction (PCR) fragments amplified from cDNA were 520 bp in size for both species, while genomic fragments were 1,035 bp for O. dentatum (GC-content: 45%) and 1,331 bp for O. quadrispinulatum (44%). Sequence analyses revealed blocks of high homology in the exons interrupted by more variable parts in the intron regions. Five exons were predicted from the genomic sequences in the conserved regions which corresponded to the respective cDNA sequences with 6% interspecific differences. The predicted protein sequences (161 amino acids) were 98% similar between the species and showed 71% similarity to the putative protein of Caenorhabditis elegans. As a housekeeping gene, TPI could be amplified from cDNA of both infectious third-stage larvae and adults. Interspecific variations in the non-coding regions allow the PCR-based differentiation of the two Oesophagostomum spp.

Information about genetic changes during the selection process could indicate mechanisms underlying the spread of resistance to anthelmintic drugs. For clarification of the role of the Mpi locus encoding mannose-phosphate isomerase enzyme in determining resistance, genotyping of Oesophagostomum dentatum strains was performed using an isoelectrofocusing technique. In levamisole- and pyrantel-selected strains the allele associated with resistance has probably been found. Significant values for genetic differentiation between treated and untreated strains of common origin were recorded by F(st) indices (theta = 0.078; P = 0.0008). The specific genomic makeup of a flubendazole-resistant strain, which did not correlate with that of the remaining isolates, might be ascribed to a different action of the anthelmintic or different environmental conditions under which resistance against this drug arose. The absence of heterozygotes in male populations indicated an XX/X0 system of sex determination for the Mpi locus, thus providing a greater potential for the development of resistance. A possible involvement of alleles linked with mannose-phosphate isomerase in alterations of membrane receptors that can be associated with resistance against imidothiazole-based drugs is discussed.

In light of the lack of molecular data on the sexual differentiation, maturation and interaction of parasitic nematodes of livestock, the present study investigated sex-specific gene expression in the nodule worm, Oesophagostomum dentatum (Strongylida). Using the technique of RNA arbitrarily-primed polymerase chain reaction (RAP-PCR), 31 expressed sequence tags (ESTs) differentially-displayed between the sexes were cloned. Northern blot analysis proved ten ESTs to be expressed exclusively in males (adults and fourth-stage larvae), while two were expressed solely in female stages. None of the ESTs were expressed in infective third-stage larvae. Sequence analysis and subsequent database searches revealed two male-specific ESTs to have significant similarity to Caenorhabditis elegans (predicted) proteins, a protein containing an EGF-like cysteine motif and a serine/threonine phosphatase. Another two male-specific ESTs had similarity to non-nematode sequences. The two female-specific ESTs had similarity to vitellogenin-5 and endonuclease III (predicted) from C. elegans. The remaining ESTs had no similarity to any nucleic acid or protein sequences contained in the databases. The isolation and characterisation of sex-specific ESTs from O. dentatum provides a unique opportunity for studying the reproductive biology of parasitic nematodes at the molecular level, with a view toward novel approaches for parasite control.

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodule worm) is of major human health significance in northern Togo and Ghana where the human hookworm, Necator americanus, also exists at high prevalence. Accurate diagnosis of O. bifurcum infection in humans is central to studying the epidemiology and controlling the parasite. To overcome limitations of current copro-diagnostic methods, we have developed an alternative, molecular approach. Utilising genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal DNA, we have established a two-step, semi-nested PCR method for the specific amplification of minute amounts (fg) of O. bifurcum DNA from human faecal samples. Using a panel of 155 well-defined faecal and DNA samples, the assay achieved a sensitivity of 94.6% and a specificity of 100%. This PCR assay will be useful for the diagnosis of O. bifurcum infection and as a molecular tool for elucidating the epidemiology of human oesophagostomiasis.

The glucose-6-phosphate dehydrogenase (G6PD, EC.1.1.1.49), glucose phosphate isomerase (GPI, EC.5.3.1.9), and malate dehydrogenase (MDH, EC.1.1.1.37) isoenzymatic patterns of Chabertia ovina were determined by starch-gel electrophoresis. The G6PD and GPI isoenzymatic patterns were characterized by the existence of three phenotypes: (1) a single and slow anodic band, (2) a single and fast anodic band, and (3) a large spot matching its migration with bands 1 and 2. These three phenotypes may be explained as the existence of only one gene locus for the G6PD and GPI in C. ovina. Allelic frequencies and the Hardy-Weinberg test were determined. This test indicated that the population was not in Hardy-Weinberg equilibrium. The MDH isoenzymatic pattern of C. ovina was characterized by the presence of two bands with anodic and cathodic migration. Furthermore, comparative isoenzyme studies were carried out between Oesophagostomum venulosum and C. ovina. The different G6PD, GPI, and MDH isoenzymatic patterns observed for the two species allowed us to distinguish them and, therefore, to use isoenzymatic patterns as a diagnostic tool to discriminate these species.