Biomedical subjects

N B Chilton

Publications and source records attributed to N B Chilton.

At least 55 records · Page 3Linked to original sources

PubMed1998

Differences in the 5.8S rDNA sequences among ascarid nematodes.

Sequences of the 5.8S rDNA were obtained for 14 species of nematode from different superfamilies and families within the order Ascaridida. All sequences were 157 bp in length. Sequence differences among species ranged from 0 to 18 bp (0-11.5%). A phenetic analysis of the sequence data groups the 14 taxa into their respective superfamilies and families, but does not discriminate fully at the subfamily level. A phylogenetic analysis of the sequence data failed to resolve the evolutionary relationships at the superfamily level. The 5.8S gene may be useful for phylogenetic studies of the phylum Nematoda at the ordinal level.

Animals↗

PubMed1998

A comparison of the first internal transcribed spacer of ribosomal DNA in seven species of Trichostrongylus (Nematoda: Trichostrongylidae).

The first internal transcribed spacer (ITS-1) of the ribosomal DNA of seven species of Trichostrongylus was sequenced. The length of ITS-1 in the different species varied from 387 to 390 bases. The G + C content of the ITS-1 sequences were approximately 42%. Little or no intraspecific variation was detected in the three species. Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus vitrinus, for which multiple isolates from different geographical regions were sequenced. In contrast, the level of ITS-1 sequence differences between species ranged from 1.3% to 5.7%. The greatest sequence differences were detected between T. tenuis, the parasite species which infects birds and the six species found in mammals. Some of the nucleotide differences occurred at sites corresponding to recognition sites for restriction endonucleases. These results are compared with previous data obtained for the second internal transcribed spacer (ITS-2). The ITS-1 data indicate that this region of rDNA may also be useful for systematic studies in trichostrongylid nematodes.

Animals↗

PubMed1998

Common secondary structures for the second internal transcribed spacer pre-rRNA of two subfamilies of trichostrongylid nematodes.

Sequences of the second internal transcribed spacer ribosomal DNA for the parasitic trichostrongylid nematodes Trichostrongylus probolurus, Trichostrongylus rugatus and Camelostrongylus mentulatus were compared with previously published sequences for five other species within the genus Trichostrongylus. The secondary structures of the second internal transcribed spacer pre-rRNA for these nematodes were predicted using an energy minimisation method. The results indicate that a common secondary structure of the second internal transcribed spacer of these nematodes is maintained despite distinct differences in primary sequence between species. Sequence differences among Trichostrongylus species ranged from 1.3 to 7.6%, but each species differed by 22-26% in sequence when compared with C. mentulatus which belongs to a different subfamily.

Animals↗

PubMed1998

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

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.

Animals↗

PubMed1998

Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal DNA sequences.

Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (approximately 34-45%) and second (approximately 50-53%) internal transcribed spacers among the species was greater than in the 5.8S gene (approximately 3-5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.

Animals↗

PubMed1998

Molecular characterization of a Toxocara variant from cats in Kuala Lumpur, Malaysia.

The ascaridoid nematode of cats from Kuala Lumpur, Malaysia, previously identified morphologically as Toxocara canis, was characterized using a molecular approach. The nuclear ribosomal DNA (rDNA) region spanning the first internal transcribed spacer (ITS-1), the 5.8S gene and the second internal transcribed spacer (ITS-2) was amplified and sequenced. The sequences for the parasite from Malaysian cats were compared with those for T. canis and T. cati. The sequence data showed that this taxon was genetically more similar to T. cati than to T. canis in the ITS-1, 5.8S and ITS-2. Differences in the ITS-1 and ITS-2 sequences between the taxa (9.4-26.1%) were markedly higher than variation between samples within T. canis and T. cati (0-2.9%). The sequence data demonstrate that the parasite from Malaysian cats is neither T. canis nor T. cati and indicate that it is a distinct species. Based on these data, PCR-linked restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) methods were employed for the unequivocal differentiation of the Toxocara variant from T. canis and T. cati. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and zoonotic potential of this parasite.

Animals↗

PubMed1997

Polymerase chain reaction-linked single-strand conformation polymorphism of ribosomal DNA to fingerprint parasites.

To overcome limitations of the morphological identification of some parasites (and their different developmental stages) to species, we have established a polymerase chain reaction-linked single-strand conformation polymorphism technique (PCR-SSCP) utilizing the second internal transcribed spacer (ITS-2) of ribosomal (r)DNA. This spacer was specifically chosen in the study of strongylid and ascarid nematodes because it is known to provide reliable species markers. The ITS-2 from individual parasites was amplified by PCR, then denatured and subjected to electrophoresis on a mutation detection enhancement (MDE) (nondenaturing) gel matrix. PCR-SSCP analysis showed that the single-strand ITS-2 patterns produced allowed the accurate identification of species. The method also allowed the display of (low-level) variation in patterns within some species between different geographical isolates. These findings demonstrate the usefulness of PCR-SSCP of ITS-2 for the identification of nematode species and indicate its potential for resolving variation in the ITS-2 sequence within species.

Animals↗

PubMed1997

Differentiation of Oesophagostomum bifurcum from Necator americanus by PCR using genetic markers in spacer ribosomal DNA.

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana where Necator americanus (human hookworm) also exists at high prevalence. However, very little is known about the transmission patterns of O. bifurcum, partly due to the difficulty in differentiating O. bifurcum from N. americanus at some life-cycle stages using morphological features. To overcome this limitation, a molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to the regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR assays were developed for the specific amplification of DNA of O. bifurcum or N. americanus, which have the potential to confirm the identity of eggs from faeces and larvae from the intestine or environment. The application of species-specific PCR has important implications for studying the epidemiology and population biology of O. bifurcum.

Animals↗

PubMed1997

Rapid PCR-based delineation of the porcine nodular worms, Oesophagostomum dentatum and O. quadrispinulatum.

At some stages of development, it is impossible to identify the porcine nodular worms Oesophagostomum dentatum and O. quadrispinulatum to the species level using morphological parameters. A molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed to overcome this limitation. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR procedures were developed for the specific amplification of DNA of O. dentatum or O. quadrispinulatum, which are now used routinely to monitor the purity of larval cultures and to confirm the identity of larvae derived from the intestine or faeces. The application of specific PCR has major implications for studying the population biology of nodular worms in the pig model.

Animals↗

PubMed1997

PCR-based methods for identification of potentially zoonotic ascaridoid parasites of the dog, fox and cat.

Genomic DNA was extracted from ascaridoid nematodes collected from dogs, foxes and cats. A region spanning the second internal transcribed spacer (ITS-2) of the ribosomal DNA of each sample was amplified by PCR. Representative ITS-2 products for each nematode species (Toxocara canis, Toxocara cati and Toxascaris leonina) were sequenced. Restriction sites were identified for use as genetic markers in a PCR-linked RFLP assay. The three species could be differentiated from each other and from other ascaridoids that may be found in human tissues by use of two endonucleases, HinfI and RsaI. Primers were designed to unique regions of the ITS-2 sequences of the three species for use in diagnostic PCR procedures and primer sets evaluated against panels of homologous and heterologous DNA samples. Results suggest that both methods are good candidates for further development for the detection and/or identification of ascaridoid larvae in human tissues.

Animals↗

PubMed1997

The 5.8S rDNA sequences of 18 species of bursate nematodes (order Strongylida): comparison with rhabditid and tylenchid nematodes.

The 5.8S rDNA sequences of 18 species of bursate nematode were 153 bp in length and had a G + C content of 47-50%. Interspecific differences in rDNA sequence among the bursate nematodes were low (0-5.2%), but the extent of the sequence differences between the parasitic bursate nematodes, a free-living rhabditid nematode and 2 species of parasitic tylenchid nematodes (12-33%) suggests that the 5.8S rRNA gene may provide useful phylogenetic information with respect to the relationships of the different orders within the phylum Nematoda.

Animals↗

PubMed1997

Molecular delineation of Cylicocyclus nassatus and C. ashworthi (Nematoda:Strongylidae).

The nucleotide sequences of the first internal transcribed spacer (ITS-1), 5.8S gene and second internal transcribed spacer (ITS-2) of ribosomal DNA have been determined for Cylicocyclus nassatus, C. ashworthi and C. insignis. Pairwise comparisons revealed sequence differences between the taxa ranging from 3.8 to 6.2% for the ITS-2 and 2.2-2.7% for the ITS-1. For the ITS-1, the level of the sequence difference between C. ashworthi and C. nassatus (2.2%) was equivalent to that between C. nassatus and C. insignis (2.2%), indicating that C. ashworthi and C. nassatus represent separate species. Theoretical restriction maps were constructed from the sequence data, and a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-linked RFLP) technique was established to unequivocally distinguish C. ashworthi from C. nassatus.

Animals↗

PubMed1997

Evidence for hybridisation between Paramacropostrongylus iugalis and P. typicus (Nematoda:Strongyloidea) in grey kangaroos, Macropus fuliginosus and M. giganteus, in a zone of sympatry in eastern Australia.

Specimens of Paramacropostrongylus iugalis and P. typicus, collected from eastern (Macropus giganteus) and western (M. fuliginosus) grey kangaroos in New South Wales and Queensland, were examined morphologically and electrophoretically at 4 enzyme loci previously demonstrated to be diagnostic between the 2 species. Collections of P. iugalis from M. giganteus from outside the zone of sympatry of the 2 kangaroo species conformed electrophoretically and morphologically with previous studies. Within the zone of sympatry, the 2 nematode species were distinguishable electrophoretically, with most P. iugalis occurring in M. giganteus and all P. typicus occurring in M. fuliginosus. Some specimens of P. iugalis were identified in M. fuliginosus and, in both host species, nematodes were encountered with electrophoretic profiles intermediate between P. iugalis and P. typicus. The frequent occurrence in these specimens of heterozygotes suggested that the genetic barriers between the 2 nematode species were not complete and that genetic interchange (i.e. hybridisation) was occurring.

Alleles↗

PubMed1997

An electrophoretic analysis of patterns of speciation in Cloacina clarkae, C. communis, C. petrogale and C. similis (Nematoda:Strongyloidea) from macropodid marsupials.

An electrophoretic study was conducted on Cloacina clarkae, C. communis, C. petrogale and C. similis based on 19 enzyme loci. C. communis was widely distributed in Macropus robustus, showing some genetic variation among populations but occasionally switching to other macropodid hosts (M. agilis, M. antilopinus). C. similis occurred in members of the Petrogale penicillata complex, Macropus dorsalis and Thylogale billardierii, but showed no evidence of genetic differentiation in spite of its occurrence in different host species and in geographically distinct regions of Australia. C. clarkae from Macropus eugenii was genetically indistinguishable from C. similis and was considered synonymous with it. C. petrogale occurred in a similarly diverse range of hosts and geographical regions to C. similis, but was represented electrophoretically as 4 distinct genetic species, 1 in Petrogale assimilis, a second in P. lateralis purpureicollis, a third in Macropus parryi in Queensland and a fourth in M. eugenii in South Australia. Although the host and geographical ranges of C. similis and C. petrogale are analogous, the genetic uniformity of the former and diversity of the latter illustrate the incomplete understanding we have of the immediate causes of speciation in nematodes.

Alleles↗

PubMed1997

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.

Animals↗

PubMed1997

Echinococcus granulosus myophilin--relationship with protein homologues containing "calponin-motifs".

Myophilin, a smooth-muscle protein of the tapeworm Echinococcus granulosus, was recently postulated to be a member of the calponin family of proteins. A detailed genetic analysis revealed that 17 proteins had significant homology with the amino-acid sequence of the N-terminal region of myophilin and/or possessed one or more "calponin-motifs". Comparison of the amino-acid sequences of the N-terminus showed that the homologous proteins clustered into distinct groups based on the number of calponin-motifs. The calponin-motif of myophilin was genetically more similar to that present in the muscle protein mp20 of Drosophila melanogaster than to those in any other homologous proteins of vertebrates. The existence of a distinct motif which is "conserved" in other proteins across a range of species suggests an important functional role for the motif.

Amino Acid Sequence↗

PubMed1997

Characterization of Oesophagostomum bifurcum and Necator americanus by PCR-RFLP of rDNA.

Esophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana, where Necator americanus (human hookworm) also exists at high prevalence. Yet, very little is known about the transmission patterns of O. bifurcum, which is in part due to the difficulties in diagnosis and in differentiating some life-cycle stages of O. bifurcum from N. americanus using morphological features. As a first step toward developing a molecular-diagnostic assay, it was evaluated whether ribosomal (r)DNA could provide genetic markers for the identification of O. bifurcum and N. americanus to species. Internal transcribed spacer rDNA (plus flanking and intervening sequences) was analyzed by polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) using several restriction endonucleases. The analysis showed that there was no detectable intraspecific difference in the size of the PCR products among multiple samples, that there was a consistent size difference in the products (of 110 bp or 350 bp, depending on region amplified) between the species, and that there was no significant variation in restriction patterns within each species. These results indicate that the rDNA spanning the internal transcribed spacers provides useful genetic markers for the identification of O. bifurcum and N. americanus to species, which has important implications for developing PCR-based tools to study the epidemiology and population biology of O. bifurcum.

Animals↗

PubMed1996

Species markers for equine strongyles detected in intergenic rDNA by PCR-RFLP.

Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosomal DNA was amplified from genomic DNA by polymerase chain reaction (PCR) using conserved primers, digested separately with six restriction endonucleases (AluI, BfaI, CfoI, Hae III, VSpI and XbaI) and the fragments separated by agarose gel electrophoresis. The PCR products of the three Strongylus species were approx. 90-100 bp smaller in size compared with those of the other 13 species. The PCR-RFLP analysis of the rDNA region spanning the first and second internal transcribed spacers plus the 5.85 rDNA gene (ITS+) produced characteristic patterns for each of the 16 species examined, and no variation in RFLP patterns was detected within the species Cy. catinatum, where multiple isolates were analysed. The study demonstrates that the internal transcribed spacer sequences provide genetic markers for the species identification of a range of equine strongyles. These markers will be of use for the identification of egg and larval stages, where morphological characters alone are unreliable. The results also indicate that the spacer sequences will be of use to study the systematics of equine strongyles.

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