Ian Beveridge

Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea)

The nucleotide sequence of the second internal transcribed spacer (ITS-2) from ribosomal DNA has been determined for 3 members of the Hypodontus macropi species complex. Sequences were compared from nematodes collected from 3 species of Australian macropodid marsupial, Petrogale persephone, Macropus robustus robustus and Thylogale billardierii. The ITS-2 of each operational taxonomic unit ranged from 287 to 292 bases in length, and had a GC content of 36.6-40.1%. Differences in nucleotide sequence between nematodes from the different host species ranged from 25.0% to 28.3%. The data suggest that H. macropi from P. persephone represents a different species to those in M. r. robustus and T. billardierii. The unique feature of this study is that it represents a comparison of the ribosomal DNA sequences of nematode species which are morphologically indistinguishable but which have been demonstrated to be genetically distinct (i.e. cryptic) species based on electrophoretic data. The results also demonstrate further that morphological characters alone are often not adequate for species recognition. Differences between these 3 species of H. macropi in their recognition sites for restriction endonucleases, indicates that a PCR-RFLP approach could be used, in conjunction with allozyme electrophoresis, to establish how many species are present within the H. macropi complex.

Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation

The accurate analysis of genetic variation has major implications in many areas of biomedical research, including the identification of infectious agents (such as parasites), the diagnosis of infections, and the detection of unknown or known disease-causing mutations. Mutation scanning methods, including PCR-coupled single-strand conformation polymorphism (SSCP), have significant advantages over many other nucleic acid techniques for the accurate analysis of allelic and mutational sequence variation. The present protocol describes the SSCP method of analysis, including all steps from the small-scale isolation of genomic DNA and PCR amplification of target sequences, through to the gel-based separation of amplicons and scanning for mutations by SSCP (either by the analysis of radiolabeled amplicons in mutation detection enhancement (MDE) gels or by non-isotopic SSCP using precast GMA gels). The subsequent sequence analysis of polymorphic bands isolated from gels is also detailed. The SSCP protocol can readily detect point mutations for amplicon sizes of up to 450–500 bp, and usually takes 1–2 days to carry out. This user-friendly, low-cost, potentially high-throughput platform has demonstrated the utility to study a wide range of pathogens and diseases, and has the potential to be applied to any gene of any organism.

Differences in the second internal transcribed spacer (ribosomal DNA) between five species of Trichostrongylus (Nematoda: Trichostrongylidae)

The second internal transcribed spacer (ITS-2) of the ribosomal DNA of 5 species of Trichostrongylus has been sequenced. The ITS-2 of the 5 species was 237 or 238 bases in length, and had a GC content of approximately 30%. No evidence of intraspecific variation was detected in the ITS-2 sequence of T. colubriformis, T. vitrinus or T. retortaeformis, irrespective of the life cycle stage examined. There was evidence, however, of variation at five positions in the ITS-2 sequence of T. vitrinus samples and at one position in T. axei, indicating intra-individual variation in the sequence of different copies of the ribosomal DNA. Nonetheless, there were consistent sequence differences between the five Trichostrongylus species examined. The level of interspecific differences in nucleotide sequence was low (1.3-7.6%), with the species infecting birds (T. tenuis) being genetically more different to the four species found in mammals. Some of the nucleotide differences between species occurred at the recognition sites of endonucleases, which makes them of important diagnostic value for species identification. Also of significance are the recognition sites for several enzymes located within the regions of sequence homology for the five species of Trichostrongylus. These may prove useful in distinguishing between genera of trichostrongyle nematodes.

The prevalence and epidemiology of gastrointestinal parasites of horses in Victoria, Australia

A quantitative post mortem study of 150 horses from Victoria was conducted to determine the prevalence and epidemiology of gastrointestinal parasites. A total of 42 species of metazoan parasite was found. The following species of non-cyathostome parasite were found (% prevalence): Trichostrongylus axei (51%); Habronema muscae (13%); H. majus (2%); Draschia megastoma (5%); Gastreophilus intestinalis (81%); G. nasalis (29%); Parascaris equorum (5%); Anoplocephala perfoliata (29%); Fasciola hepatica (0.7%); Oxyuris equi (7%); Strongylu vulgaris (23%); S. edentatus (23%); S. equinus (3%); Craterostomum acuticaudatum (7%); Triodontophorus serratus (8%); T. tenuicollis (8%); T. brevicauda (3%). Ninety-five per cent of horses were infected with gut-wall encysted stages of cythostomes with a mean intensity of 113,000 larvae per horse. Ninety-three per cent of all horses harboured adult cyathosome worms; 24 species representing 6 genera were found. The 3 most prevalent species were Cylicostephanus longiburstatus (76%); Cyathostomum catinatum (68%) and Cylicocyclus nassatus (54%). Seventeen species of strongyle were present in high abundance, which allowed their site distribution in the large intestine to be determined. Twelve species preferred the large colon to the small colon and caecum, and the remaining 5 species preferred the caecum. Statistical analysis of the parasitological data set allowed effects of sex, age, type, and physical condition of the horse as well as the season and environment on the prevalence and mean intensity of infection to be determined.

Anoplocephala perfoliata of horses--significant scope for further research, improved diagnosis and control.

Anoplocephala perfoliata is the commonest tapeworm parasite of horses and is incriminated as a significant cause of clinical disease (e.g., ileocaecal intussusception, caeco-caecal intussusception and/or caecal perforation), particularly in horses chronically infected with large numbers of worms. The high prevalence (approximately 20-80%) of the parasite in some countries suggests an increased risk of clinical cases. In spite of research, there is still a paucity of information regarding the pathogenesis of the disease, the epidemiology of the parasite in different geographical regions and there are significant limitations with the diagnosis of infection. The present article provides an account of the biology, epidemiology and pathogenic effects of A. perfoliata, the diagnosis of infection and treatment. It highlights some gaps in knowledge of the parasite and the disease it causes, and suggests opportunities for future research and prospects for improved diagnosis, prevention and control.

Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock -Bionomic and biotechnological implications

Parasitic nematodes of livestock have a major economic impact worldwide. In spite of the health problems caused by nematodes and advances toward the development of vaccines and new therapeutic agents against some of them, relatively limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of parasitic nematodes of livestock are central to their effective control, particularly given the current, serious problems with anthelmintic resistance in nematode populations. Traditional diagnostic techniques have considerable limitations, and there have been some advances toward the development of molecular-diagnostic tools. This article provides a brief account of the significance of parasitic nematodes (order Strongylida), reviews the techniques that have been evaluated or used for diagnosis and describes developments in polymerase chain reaction (PCR)-based methods for the specific diagnosis of nematode infection/s and the genetic characterisation of the causative agents. The advances made in recent years provide a solid foundation for the development of practical, highly sensitive and specific diagnostic tools for epidemiological investigations and for use in control programmes.

Phylogenetic relationships of Australian strongyloid nematodes inferred from ribosomal DNA sequence data

The sequence of the second internal transcribed spacer of the ribosomal DNA was determined for the following strongyloid nematodes: Cylicocyclus insignis, Chabertia ovina, Oesophagostomum venulosum, Cloacina communis, Cloacina hydriformis, Labiostrongylus labiostrongylus, Parazoniolaimus collaris, Macropostrongylus macropostrongylus, Macropostrongylus yorkei, Rugopharynx australis, Rugopharynx rosemariae, Macropostrongyloides baylisi, Oesophagostomoides longispicularis and Paramacropostrongylus toraliformis, and compared with published sequences for species of Strongylus and for Hypodontus macropi. The resultant phylogenetic trees supported current hypotheses based on morphological evidence for the separation of the families Strongylidae and Chabertiidae, but did not support the separation of the endemic Australian genera as a distinctive clade within the Chabertiidae. The implications of this finding for the phylogenetic origins of the Australian strongyloids are discussed.

Goat–Nematode interactions: think differently

Goats (caprine) and sheep (ovine) are infected with the same principal gastrointestinal nematode (GIN) species, which provoke similar pathological changes and economic consequences. However, until now, the majority of data on host-parasite interactions have been accumulated from ovine studies. This article aims to emphasize the need for specific caprine studies. It is hypothesized that, owing to divergent evolutionary processes, sheep and goats have developed two different strategies to regulate GIN infections, respectively, based on immune response versus feeding behavior. Generation of additional comparative data should result in a better understanding of the possible trade-offs between these two basic regulatory processes. Goat studies should also help to avoid past errors in the control of GIN species owing to the lack of relevant information.

Evolution of the trypanorhynch tapeworms: Parasite phylogeny supports independent lineages of sharks and rays

Trypanorhynch tapeworms (Platyhelminthes: Cestoda) are among the most diverse and abundant groups of metazoan parasites of elasmobranchs and are a ubiquitous part of the marine food webs that include these apex predators. Here we present a comprehensive analysis of their phylogeny, character evolution and host associations based on 10years of sampling effort, including representatives of 12 of 15 and 44 of 66 currently recognized trypanorhynch families and genera, respectively. Using a combination of ssrDNA and lsrDNA (Domains 1-3) for 79 and 80 taxa, respectively, we maintain one-to-one correspondence between molecules and morphology by scoring 45 characters from the same specimens used for sequencing, and provide museum vouchers for this material. Host associations are examined through likelihood-based ancestral character state reconstructions (ACSRs) and by estimating dates of divergence using strict and relaxed molecular clock models in a Bayesian context. Maximum parsimony and Bayesian inference analyses of rDNA produced well-resolved and strongly supported trees in which the trypanorhynchs formed two primary lineages and were monophyletic with respect to the diphyllidean outgroup taxa. These lineages showed marked differences in their rates of divergence which in turn resulted in differing support and stability characteristics within the lineages. Mapping of morphological characters onto the tree resulting from combined analysis of rDNA showed most traits to be highly plastic, including some previously considered of key taxonomic importance such as underlying symmetries in tentacular armature. The resulting tree was found to be congruent with the most recent morphologically based superfamily designations in the order, providing support for four proposed superfamilies, but not for the Tentacularioidea and Eutetrarhynchoidea. ACSRs based on the combined analysis of rDNA estimated the original hosts of the two primary parasite lineages to be alternatively rajiform batoids and carcharhiniform sharks. This fundamental split provides independent support for rejecting the notion that rays are derived sharks, and thus supports the most recent molecular phylogenies of the Neoselachii. Beyond the basal split between shark- and ray-inhabiting lineages, no pattern was found to suggest that the trypanorhynchs have closely tracked the evolutionary histories of these host lineages, but instead, it appears that host-switching has been common and that the subsequent evolution of the parasites has been ecologically driven primarily through overlap in the niches of their shark and ray hosts. Using a relaxed molecular clock model calibrated by means of host fossil data, the ray-inhabiting lineage is estimated to have diversified around the Jurassic-Cretaceous boundary, whereas the shark-inhabiting lineage is estimated to have diversified later, in the Middle Cretaceous. Although the large error associated with the estimated divergence dates prevents robust conclusions from being drawn, the dates are nevertheless found to be consistent in a relative sense with the origins of their major hosts groups. The erection and definition of the suborders Trypanobatoida and Trypanoselachoida, for the major clades of trypanorhynchs parasitizing primarily rays and sharks, respectively, is proposed for the two primary lineages recovered here.

The origins and evolutionary expansion of the Strongylida (Nematoda)

The Strongylida are thought to have arisen from free-living rhabditoid nematodes, but the relationships between the major groupings within the Strongylida, the Strongylina, the Metastrongylina, Trichostrongylina and the Ancylostomatina are far from clear in spite of the abundance of morphological data now available for analysis. Evolutionary mechanisms including co-evolution, host switching, host dispersal, use of intermediate hosts, various sites of localisation within the definitive host and modifications of life-cycle strategies appear to have been utilised in the expansion of the Strongylida, with different mechanisms predominating in different families or superfamilies. Co-evolution appears to have been a major mode of evolution in the Strongylina, in contrast to the Trichostrongylina, which have used host dispersal and host-switching to great advantage. The phylogeny of the Ancylostomatina shows little association with host evolution, but does match the feeding preferences of the hosts. The Metastrongylina have utilised intermediate hosts and life cycle modifications including a shift to extra-intestinal sites as major means of diversification, in contrast to the other sub-orders. The review, while indicating much progress in our understanding of the phylogeny of the Strongylida, also reveals that enormous gaps still exist, and emphasises the tentative nature of many of the phylogenetic hypotheses tendered to date.