Biomedical subjects

R B Gasser

Publications and source records attributed to R B Gasser.

At least 91 records · Page 5Linked to original sources

PubMed1999

Scanning for nucleotide variations in mitochondrial DNA fragments of Schistosoma japonicum by single-strand conformation polymorphism.

In this study, we employed a mutation scanning approach for the direct visual display of genetic variability in mitochondrial DNA (mtDNA) fragments within and among populations of Schistosoma japonicum from the People's Republic of China. Fragments of the NADH dehydrogenase 1 gene (ND1) and the cytochrome c oxidase subunit I (COI) were individually amplified from parasite DNA by polymerase chain reaction (PCR), denatured and subjected to single-strand conformation polymorphism (SSCP) analysis. Using ND1 and COI fragments, individuals representing different genotypes could be readily identified based on characteristic and reproducible SSCP profiles. The results demonstrated the usefulness of SSCP for the direct visual display of low-level sequence variation in mtDNA of S. japonicum prior to DNA sequence analysis. This approach has important implications for studying the genetic structure and biology of S. japonicum populations, and for analysing the inheritance of mitochondrial DNA.

Animals↗

PubMed1999

Species-specific amplification by PCR of ribosomal DNA from some equine strongyles.

The first and second internal transcribed spacer sequences of 28 morphologically-defined species of horse strongyle were characterized, and specific oligonucleotide primers were designed for some species based on the nucleotide differences. Utilizing these primers, a PCR approach was developed for the specific amplification of ribosomal DNA of Strongylus vulgaris, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicostephanus longibursatus or Cylicostephanus goldi. The method allowed the species-specific amplification of parasite DNA derived from faecal samples and/or copro-cultures, demonstrating the potential of the approach for the diagnosis of equine strongyloidosis. The establishment of this PCR assay also has implications for studying the biology and epidemiology of equine strongyles and anthelmintic resistance using faecal egg count reduction tests.

Animals↗

PubMed1998

Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism.

Echinococcosis, a disease caused by infection with the larval stage of a tapeworm parasite of the genus Echinococcus, is of major socio-economic importance, and studying genetic variability within and between Echinococcus populations has important implications for disease control and epidemiology. Various DNA approaches have been used to study Echinococcus genetics, but most methods do not allow the accurate display or definition of mutational/allelic variation. To overcome this limitation, we established a mutation scanning approach. Single-strand conformation polymorphism (SSCP) of two different enzymatically amplified mitochondrial (mt) DNA regions was evaluated using seven different genotypes of Echinococcus (defined as G1, G4, G6, G8, O, V and M2). The NADH dehydrogenase 1 gene (ND1) or the cytochrome c oxidase subunit 1 (CO1) were amplified by polymerase chain reaction from parasite DNA, denatured and directly subjected to electrophoresis in a non-denaturing gel matrix. Each of the seven genotypes examined could be delineated from one another based on characteristic and reproducible banding patterns. The results demonstrate the usefulness of SSCP for the direct visual display of sequence variation in mtDNA of Echinococcus without the need for DNA sequencing or restriction analyses, and indicate its potential for studying allelic variability in a range of other genes.

Animals↗

PubMed1998

A mutation scanning approach for the identification of hookworm species and analysis of population variation.

To overcome limitations in the morphological identification of different developmental stages of hookworms to species, we have established a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR-SSCP) utilizing the internal transcribed spacers (ITS) of ribosomal (r)DNA. These spacers were specifically chosen because they provide reliable species markers for strongylid nematodes. ITS spacers were amplified by PCR from DNA derived from individual parasites of seven species of hookworm, then denatured and subjected to electrophoresis in a mutation detection enhancement (MDE) (non-denaturing) gel matrix. PCR SSCP analysis showed that the single-strand ITS patterns produced allowed the unequivocal identification of all species. The method also allowed the direct display of sequence variation within some species where multiple individual worms were examined. These findings demonstrate the usefulness of the SSCP approach for hookworm identification, the detection of population variation and the direct display of sequence variation in rDNA.

Ancylostomatoidea↗

PubMed1998

Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA-intercalating agent.

This study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold-supplemented gels. The latter was achieved because of differences (approximately 0.1-8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11-39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR-amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.

Animals↗

PubMed1998

Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.

Animals↗

PubMed1998

Analysis of sequence homogenisation in rDNA arrays of Haemonchus contortus by denaturing gradient gel electrophoresis.

Testing different theories of concerted evolution experimentally has been hampered mainly due to the lack of appropriate model systems and technical limitations. In this study, we employed a denaturing gradient gel electrophoresis (DGGE) approach for the display and definition of nucleotide variations in the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of the parasitic nematode, Haemonchus contortus. The ITS-2 was amplified from individual adult nematodes by PCR and subjected to DGGE. Of the 94 individuals (representing nine different populations) analysed, 13 different DGGE profiles were displayed. Eighteen bands representing those profiles were excised and sequenced. Sequencing defined 13 different types of ITS-2 with 12 nucleotide variations (4 transitions, 5 transversions, 1 insertion and 2 deletions) which could be related to particular positions of the predicted secondary structure for the ITS-2 pre-rRNA. The results showed that individuals of interbreeding populations of H. contortus can have rDNA arrays that are partially or fully homogenised for different sequence variants (despite interindividual variation), suggesting that the homogenisation process is driven mainly by intrachromosomal exchange. The findings also demonstrated the capacity of the DGGE-sequencing strategy to quantify the frequency of ITS-2 sequence types within individual nematodes from different populations without the need for cloning or Southern blot procedures. This has important implications for studying the mechanisms of sequence homogenisation in rDNA and pre-rRNA processing as well as for elucidating speciation events and population differentiation at the molecular level.

Animals↗

PubMed1998

PCR-SSCP of rDNA for the identification of Trichinella isolates from mainland china.

A polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis of the expansion segment 5 (domain IV) of the large subunit of ribosomal DNA was employed to characterize seven isolates of Trichinella from China (A-G), including six of pig origin from regions in Dengxian (A), Tianjin (B), Harbin (D), Baoshan (E), Xinye (F) and Xian (G), and one of canine origin from Changchun (C). Isolates A, D, E, F and G were classified as Trichinella spiralis based on SSCP patterns, while the patterns for isolates B and C were consistent with those of Trichinella nativa or Trichinella T6. The results were supported by random amplified polymorphic DNA (RAPD) analysis using five decamer primers and were in accordance with ecological information for the isolates. Single strand conformation polymorphism results also allowed the direct display of mutational sequence variation in the expansion segment among the five isolates of T. spiralis from China, indicating its usefulness for studying population variation within that species.

Animals↗

PubMed1998

Specific amplification of Necator americanus or Ancylostoma duodenale DNA by PCR using markers in ITS-1 rDNA, and its implications.

Necator americanus and Ancylostoma duodenale are the two most important species of human hookworm, and occur in sympatry over much of their distribution. The specific diagnosis of hookworm infections is central to control. Diagnosis currently relies on the detection of hookworm eggs in human faeces and/or the specific identification of larvae by 'copro-culture' combined with microscopic examination. However, the eggs of the two species are morphologically indistinguishable, and the procedure of copro-culture is tedious and time-consuming to carry out. To work toward overcoming these limitations, a molecular approach utilizing genetic markers in the first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was established. The ITS-1 sequences of both hookworm species were determined, and specific oligonucleotide primers designed to regions of major sequence difference between the species were evaluated in polymerase chain reaction (PCR). Using a range of control samples, the primers allowed the specific identification of as little as 10 pg DNA of A. duodenale or N. americanus. The findings indicate clearly the potential for specific PCR to confirm the identity of eggs from faeces and larvae from the environment or host tissues. This should have important implications for studying fundamental aspects relating to anthelmintic efficacy and the epidemiology of hookworms.

Ancylostoma↗

PubMed1998

Genetic markers for strongylid nematodes of livestock defined by PCR-based restriction analysis of spacer rDNA.

Twenty-four species of parasitic nematode (order Strongylida) from sheep, goats, cattle or pigs were characterised using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). The ribosomal (r)DNA region spanning the first internal transcribed spacer (ITS-1), 5.8S rRNA gene and the second internal transcribed spacer (ITS-2) (designated ITS) was amplified from genomic DNA by polymerase chain reaction (PCR), digested separately with four restriction endonucleases (RsaI, HinfI, DraI or NlaIII) and the fragments separated by agarose gel electrophoresis. The PCR products amplified from all species appeared as a single band of approximately 870 bp in size, except for Ostertagia ostertagi whose product was approximately 1250 bp. The PCR-RFLP analysis of ITS revealed characteristic restriction patterns for all species, except for C. surnabada and C. oncophora which had identical patterns. The study demonstrated that ITS contains useful genetic markers for the identification of a range of strongylid nematodes of livestock. These markers should be of use in specific PCR assays for the identification of developmental stages of the parasites where morphological characters are unreliable.

Animals↗

PubMed1998

Genetic evidence indicating that Cooperia surnabada and Cooperia oncophora are one species.

Sequences of the second internal transcribed spacer (ITS-2) of ribosomal DNA were determined for the trichostrongylid nematodes Cooperia surnabada and Cooperia oncophora, to test the hypothesis that they represent one species. Also included for comparison were other morphologically distinct species within the genus, namely Cooperia punctata and Cooperia curticei. There were no differences in the consensus ITS-2 sequences between C. oncophora and C. surnabada, whereas each taxon differed from C. punctata and C. curticei by 1.7% and 4.1%, respectively. Also, C. punctata differed from C. curticei by 5.0%. Based on these results and the DNA studies of other trichostrongylid species, it is proposed that C. oncophora and C. surnabada represent a single species.

Animals↗

PubMed1998

Differences in the second internal transcribed spacer of four species of Nematodirus (Nematoda: Molineidae).

Genetic differences among Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus and Nematodirus battus in the nucleotide sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA ranged from 3.9 to 24.7%. Pairwise comparisons of their ITS-2 sequences indicated that the most genetically similar species were N. spathiger and N. helvetianus. N. battus was the most genetically distinct species, with differences ranging from 22.8 to 24.7% with respect to the other three species. Some of the nucleotide differences among species provided different endonuclease restriction sites that could be used in restriction fragment length polymorphism studies. The ITS-2 sequence data may prove useful in studies of the systematics of molineid nematodes.

Animals↗

PubMed1998

Necator americanus (Nematoda: Ancylostomatidae) from Africa and Malaysia have different ITS-2 rDNA sequences.

The nucleotide sequences of the second internal transcribed spacer of rDNA were determined for adult worms of Necator americanus originating from Togo (Africa) and Sarawak (Malaysia). The length of the sequences of specimens from Togo (325 bp) were shorter than those from Sarawak (327 bp). There were six fixed genetic differences in the aligned sequences of N. americanus from Sarawak and Togo, excluding one or two polymorphic sites within the sequence of N. americanus from each geographical region. These findings suggest that there is either population variation in the sequence of N. americanus, or that N. americanus from the two countries may represent genetically distinct but morphologically similar (i.e. cryptic) species, however, comparison of the sequence differences among other hookworm species supports the latter conclusion.

Africa↗

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

What's in that band?

The analysis of molecular variation in parasites has important implications for studying gene function and organisation, taxonomy, phylogeny and population genetics. Polymerase chain reaction-based mutation scanning methods can have significant advantages over some currently used DNA approaches for the analysis of allelic and mutational sequence variation in parasites. The present report describes briefly the principles of some of these methods, examines some of their advantages and disadvantages, and indicates their potential for applications in parasitology.

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

Dideoxy fingerprinting: application to the genotyping of Echinococcus.

Dideoxy fingerprinting is an efficient method for the detection of sequence variation in PCR-amplified DNA segments. It is a hybrid between single-strand conformation polymorphism and dideoxy sequencing, employing only one dideoxynucleotide in the sequencing reaction. Herein, we report the application of dideoxy fingerprinting to genetically type cestodes of the genus Echinococcus, utilising the mitochondrial cytochrome c oxidase subunit I as the gene sequence for analysis. All of the seven genotypes (G1, G4, G6, G8, O, V and M2) examined could be readily differentiated from one another by their characteristic and reproducible dideoxy fingerprinting profiles. Only subtle variation in profiles was detected among some of the eight isolates representing genotype G1, and no variation was detected between two samples of genotype G4 and of genotype M2. The capacity of dideoxy fingerprinting to detect all nucleotide variations over 150-250bp fragments indicates that it should be possible to distinguish among all of the genotypes of Echinococcus thus far described. Although employed herein to display sequence variation in the cytochrome c oxidase subunit I of Echinococcus, dideoxy fingerprinting could be used for the high-resolution analysis of nucleotide variations in other parasite genes, without the need for DNA sequencing. This has important implications for studying the genetic structure of parasite populations.

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↗