Systematic relationships of some members of the genera Oesophagostomum and Chabertia (Nematoda: Chabertiidae) based on ribosomal dna sequence data

doi:10.1016/S0020-7519(98)00144-1

International Journal for Parasitology

Volume 28, Issue 11, 1 November 1998, Pages 1781-1789

https://doi.org/10.1016/S0020-7519(98)00144-1 Get rights and content

Abstract

The present study characterised seven species of the Chabertiidae (Nematoda: Strongyloidea) belonging to either the subfamily Oesophagostominae (Oesophagostomum radiatum, Oesophagostomum venulosum, Oesophagostomum dentatum, Oesophagostomum quadrispinulatum, Oesophagostomum columbianum, Oesophagostomum bifurcum) or to the subfamily Chabertiinae (Chabertia ovina) by their second internal transcribed spacer rDNA sequence, assessed the extent of intraspecific variation and interspecific differences in the sequence, and inferred the phylogenetic relationship of C. ovina with respect to members of the Oesophagostominae. In both the phenetic and cladistic analyses of the sequence data, Chabertia was nested within Oesophagostomum, suggesting either that the species examined represent members of the same genus, or alternatively, that Oesophagostomum may represent more than one genus.

Introduction

Various studies have examined the evolutionary history of the parasitic strongylid nematodes based on morphological data and relationships with their hosts 1, 2, 3, 4, 5, 6, 7. However, for many groups of parasitic nematodes, their relatively simple body plan and limited morphological differentiation between related taxa provide a poor basis upon which to construct phylogenies. Such is the case for strongylid nematodes (order Strongylida), where the proposed phylogenetic relationships of different superfamilies and families based on morphological characters are somewhat speculative [7]. At the subfamily level, for example, conflicting phylogenetic relationships have been proposed for different subfamilies of the Trichostrongylidae 4, 5, 8 and the Chabertiidae 2, 9.

The family Chabertiidae (Strongyloidea) is characterised by the possession of four branches to the dorsal ray and a J-shaped ovejector [2]. It includes two subfamilies, the Chabertiinae and the Oesophagostominae. The former occurs in rumi- nants and rodents and is characterised by a large subglobular buccal capsule but lacking a cervical groove, while the latter occurs in ruminants, pigs, rodents and primates and possesses a small cylindrical buccal capsule and a cervical groove in some genera [2]. Developmental studies have shown that fourth-stage larvae of Oesophagostomum venulosum have subglobular buccal capsules, a characteristic feature of the Chabertiinae, while the adult has a cylindrical buccal capsule [10], the presence of a globular buccal capsule being a plesiomorphic character in cladistic analyses of the Strongyloidea 2, 11. It has been suggested that more work is required to evaluate the importance of buccal capsule size and shape [4], while the cervical groove at the level of the excretory pore occurs only in the tribe Oesophagostominea 1, 2.

Phylogenetic studies of the Chabertiidae utilising host preference and morphological characters of the parasites have regarded the Chabertiinae and Oesophagostominae as separate but related subfamilies 1, 2, 4, 9, with the Oesophagostominae derived from the Chabertiinae. Studies examining the evolutionary relationships of subgenera within the genus Oesophagostomum 3, 12, including species that infect ruminants, African suids, rodents and primates, however, have suggested an origin in suids, but have not included genera of the Chabertiinae as potential outgroups 1, 7. Thus, the phylogenetic relationships of species within the Chabertiinae and Oesophagostominae are not yet fully resolved. Using DNA sequence data, Chilton and co-workers [11] established that the two subfamilies were sister taxa and were related to the Phascolostrongylinae and Cloacininae, both occurring in Australian marsupials.

Recent studies have demonstrated that the sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) allows the unequivocal identification of a range of strongyloid nematode species, irrespective of developmental stage (e.g. 11, 13, 14, 15, 16, 17, 18, 19, 20, 21), and that phylogenetic relationships can be inferred from ITS-2 sequence data sets [11]. The aims of the present study were to characterise some members of the Chabertiidae by their ITS-2 rDNA sequence, to assess the extent of intraspecific variation and interspecific differences in the sequence, and to infer the phylogenetic relationship of Chabertia ovina with respect to members of the Oesophagostominae using ITS-2 sequence data.

Section snippets

Parasite material

Seven species of parasitic nematode belonging to the Chabertiidae (comprising the Chabertiinae and Oesophagostominae) were used for this study (Table 1). The nematode samples represented different life-cycle stages and originated from a variety of hosts from different geographical locations (Table 1). Adult worms were removed from the large intestine at necropsy, washed repeatedly in physiological saline and frozen at −70°C until required for DNA isolation. For some species, monospecific

Intraspecific variation

No intraspecific variation was detected between the two Australian isolates of Oesophagostomum radiatum (Oer2 and Oer5), despite their different life-cycle stages. Three Danish isolates of Oesophagostomum dentatum, representing two pools of third-stage larvae (Oed11 and Oed13) and an adult worm (Oed17), all had the same ITS-2 sequence, with one polymorphic site (see Fig. 2

. Alignment of the second internal transcribed spacer rDNA sequences of seven species of the family Chabertiidae with that of

Discussion

The ITS-2 sequence data have defined accurate genetic markers for the identification of species within the Chabertiidae. Given that the level of intraspecific variation in the ITS-2 sequence is low and that there is currently no evidence for significant variation among different developmental stages (eggs, larvae and adults) of a species (e.g. 13, 14, 16), these markers are of significance for the development of PCR-based diagnostic systems for the identification of nematode eggs in faecal

Acknowledgements

Project support was provided by the Australian Research Council, the Department of Industry, Science and Tourism and the Collaborative Research Program of the University of Melbourne (R.G.). Neil Chilton is the recipient of an ARC Australian Research Fellowship. Lisa Newton was the recipient of postgraduate scholarship from the Department of Veterinary Science (The University of Melbourne) and a J.D. Smyth Travel Award from the Australian Society for Parasitology. The authors are very grateful

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Concerning O. venulosum and C. ovina specimens, identification was easier since all our samples corresponded to morphological descriptions and all sequences were homologous with sequence from GenBank. Diverse studies have demonstrated that the internal transcribed spacer regions (ITS1 and ITS2) of ribosomal DNA provide reliable genetic markers for a specific identification of a number of parasite groups as rDNA maintains sequence homogeneity within a species (Andree et al., 1999; Campbell et al., 1995; Elbadri et al., 2002; Ellis et al., 1986; Newton et al., 1998; Wu et al., 2005). Here, our study confirms the relevance for this region ITS2 for an accurate taxonomic classification.
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KF364623–KF364632). These ten sequence types were aligned over 243 positions with ITS-2 reference sequences for H. contortus (n = 13) and H. placei (n = 5) (Stevenson et al., 1995; Cerutti et al., 2010; Brasil et al., 2012; Gharamah et al., 2012) and for Bunostomum phlebotomum, Nematodirus rupicaprae, Oesophagostomum columbianum and Trichostrongylus axei (‘outgroups’) (n = 4) (Hoste et al., 1995; Newton et al., 1998; Gasser et al., 1999; Jex et al., 2009) and subjected to phylogenetic analyses. The analyses unequivocally identified H. placei and H. contortus with strong nodal support (posterior probability values: 0.8–1.00%; bootstrap values: 80–98%) (Fig. 2).
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quadrispinulatum, and O. asperum were grouped with C. ovina in a sister clade. These results were similar to phylogenetic trees based on nuclear ITS-2 and 28S rDNA sequences (Newton et al., 1998; Dorris et al., 1999; Gouÿ de Bellocq et al., 2001). All these findings indicated complex phylogenetic relationships of Oesophagostomum spp. in sheep and goats.
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We evaluated a combined microscopic-molecular approach for the diagnosis of key strongylid infections in sheep using panels of well-defined control and test samples. The method established is based on the separation of nematode eggs from faecal samples using a salt flotation procedure, the extraction and column-purification of genomic DNA, followed by real-time PCR and melting-curve analysis. Specific and semi-quantitative amplification from (a minimum of 0.1–2.0 pg) genomic DNA of Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp., Cooperia oncophora, Oesophagostomum columbianum, Oesophagostomum venulosum or Chabertia ovina is achieved using a specific, forward oligonucleotide primer located in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) together with a conserved reverse primer in the large subunit of rDNA. Using a panel of well-defined genomic DNA samples from eggs from sheep monospecifically infected with H. contortus or Te. circumcincta, there was a correlation between cycle threshold (Ct) values in the PCR and numbers of egg per gram of faeces, thus allowing the semi-quantitation of parasite DNA in faeces. The findings of the present study indicate that a microscopic-molecular approach provides a useful tool for diagnosis, for epidemiological and ecological surveys as well as for integration into parasite monitoring, drug resistance (i.e. ‘egg count reduction’) testing or control programmes, particularly following semi- or full-automation.

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^fn1: Note: Nucleotide sequence data reported in this paper are available in the embl, GenBank^TM and DDJB databases under the accession numbers Y10790, Y11735, Y11736, Y11733, Y10789, AJ006149 and AJ006150.

^fn2: Present address: Macfarlane Burnet Centre for Medical Research, Po Box 254, Fairfield, Victoria 3078, Australia.

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Systematic relationships of some members of the genera Oesophagostomum and Chabertia (Nematoda: Chabertiidae) based on ribosomal dna sequence datafn1

Abstract

Introduction

Section snippets

Parasite material

Intraspecific variation

Discussion

Acknowledgements

Adv Parasitol

Int J Parasitol

Int J Parasitol

Int J Parasitol

Int J Parasitol

Int J Parasitol

Int J Parasitol

Int J Parasitol

Int J Parasitol

Mol Cell Probes

Mol Cell Probes

Int J Parasitol

Int J Parasitol

Int J Parasitol

Mol Phylogenet Evol

Mol Biochem Parasitol

Nematode parasites of mammals. Am Zool