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

Gasser, Robin B.; Stevenson, Lisa A.; Chilton, Neil B.; Nansen, P.; Bucknell, D.G.; Beveridge, Ian

doi:10.1006/mcpr.1996.0050

Cited by 48 publications

(24 citation statements)

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“…To create tools that would enable identification of pre-parasitic stages, molecular approaches have been developed to identify egg and larval stages of the most common species. Initially, first and second internal transcribed spacer (ITS) sequences were characterized [16], and specific oligonucleotide primers designed for some common species [22]. The method allowed the species-specific amplification of parasite DNA derived from faecal samples.…”

Section: The Development Of Species-specific Dna Probesmentioning

confidence: 99%

Recent developments in research into the Cyathostominae and Anoplocephala perfoliata

et al. 2004

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-Intestinal helminths are an important cause of equine disease. Of these parasites, the Cyathostominae are the commonest group that infect horses. These nematodes consist of a complex tribe of 51 species, although individual horses tend to harbour 10 or so common species, in addition to a few rarer species. The Cyathostominae can be extremely pathogenic, and high levels of infection result in clinical symptoms ranging from chronic weight loss to colic, diarrhoea and death. As part of their life cycle, immature cyathostomins penetrate the large intestinal wall, where they can enter a state of inhibited larval development. These larvae can exist in this state for months to years, after which they subsequently re-emerge. If larvae re-emerge in large numbers (i.e. several million), severe pathological consequences ensue. The inhibited larvae are also relatively refractory to several of the currently available anthelmintics, so that horses treated previously with anthelmintics can still carry life-threatening burdens of these parasitic stages. Little is known about the cyathostomin larvae during their mucosal phase, and current research efforts are focused on investigating the biology of these stages. Much of the research described here highlights this area of research and details studies aimed at investigating the host immune responses that the mucosal larvae invoke. As part of this research effort, molecular tools have been developed to facilitate the identification of larval and egg stages of cyathostomins. These molecular tools are now proving very useful in the investigation of the relative contributions that individual, common cyathostomin species make to the pathology and epidemiology of mixed helminth infections. At the more applied level, research is also in progress to develop an immunodiagnostic test that will allow numbers of mucosal larvae to be estimated. This test utilises antigen-specific IgG(T) serum antibody responses as markers of infection. As anthelmintic resistance will be the major constraint on the future control of the Cyathostominae, researchers are now actively investigating this area and studies aimed at elucidating the molecular mechanisms of drug resistance are described. Another parasite which has assumed a clinically important role in horses is the tapeworm, Anoplocephala perfoliata. This parasite is prevalent world-wide and has been shown to be a significant cause of equine colic. Because previous methods of estimating the infection intensity of tapeworm were inaccurate, recent research has been directed at developing an immunodiagnostic ELISA for these cestodes. Specific IgG(T) responses to antigens secreted by adult tapeworms have been shown to provide a reasonable indication of infection intensity. An ELISA based on these responses is now commercially available. The steps involved in the development of this ELISA are described here. In addition to these recent advances in research, this review also outlines the principle areas for future research into these important equin...

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Section: The Development Of Species-specific Dna Probesmentioning

confidence: 99%

Recent developments in research into the Cyathostominae and Anoplocephala perfoliata

et al. 2004

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“…Genomic DNA isolated from individual worms of two taxa of Cylicocyclus was analysed in order to test the reproducibility of the taxonomic and phylogenetic analysis based on the RAPD-PCR results. Earlier, often only single individuals/specimens of small strongyles, although from a range of species, had been included in phylogenetic studies based on ITS-2 sequence variation (Gasser et al 1996;Hung et al 1999;Hung et al 2000, McDonnell et al 2000. Previous taxonomic analysis of parasites by RAPD-PCR demonstrated that this method was suitable for the comparison of closely related species (Costa et al 2001;Hampl et al 2001;Pavlicek et al 1999).…”

Section: Discussionmentioning

confidence: 96%

“…For example, Gasser et al (1996) delineated 11 small strongyle species based on the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) sequences. Small strongyle species were also compared using PCR single-stranded conformation polymorphism technology (Gasser and Monti 1997).…”

Section: Introductionmentioning

confidence: 99%

Microchip capillary electrophoresis-based genetic comparison of closely related cyathostomin nematode parasites of horses using randomly amplified polymorphic DNA polymerase chain reaction

Posedi

Drögemüller²,

Schnieder³

et al. 2004

Parasitol Res

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The microchip-based capillary electrophoresis technology represents a valuable recent development for the analysis of complex DNA banding patterns. We have used this technology for the differentiation of the closely related cyathostomin species Cylicocyclus elongatus and C. insigne from the horse. We found that the Agilent 2100 bioanalyser in combination with the DNA 7500 Lab Chip were suited to perform a phylogenetic DNA fingerprinting analysis of the parasite species studied. The analysis of the electrophoretic data was optimised and it was possible to resolve a phylogenetic tree where all 12 individual worms of the two Cylicocyclus species studied were assigned to their species as determined by microscopic identification based on morphological traits. Thus, our data indicated that the procedure described here provides an additional powerful tool that can be employed for species delineation of closely related strains or species, such as the two taxa of Cylicocyclus investigated in the present study. Furthermore, by determining the second internal transcribed spacer region of three and nine individual worms for C. elongatus and C. insigne, respectively, low intraspecific variations of only up to 0.3% were demonstrated.

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“…They cannot be classified to species as preparasitic stages, and there is a limited number of morphological features that allow identification of the parasitic larval stages (Dvojnos and Harcenko, 1990). Recently, molecular methods have been used to allow study at the individual species level (Gasser et al, 1996;Kaye et al, 1998;Hung et al, 1999;Hodgkinson et al, 2001). To investigate the relative importance of individual species, we have developed species-specific DNA probes for the identification of six common cyathostomins, Cylicocyclus ashworthi, Cylicocyclus insigne, Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus goldi and Cylicostephanus longibursatus.…”

Section: Introductionmentioning

confidence: 99%

A PCR–ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis

Hodgkinson

Lichtenfels

Mair

et al. 2003

International Journal for Parasitology

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We report the use of six oligoprobes designed from intergenic spacer region sequences to identify fourth-stage larvae (L4) of the tribe Cyathostominae. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed as a positive control to identify all these members of the Cyathostominae. The intergenic spacer region was amplified by PCR using conserved primers. Initially, three oligoprobes were used in Southern blot analysis. To facilitate high-throughput identification, these and a further four oligoprobes were developed for use in a PCR -ELISA. All probes were validated for their ability to detect cyathostomin PCR products in the PCR -ELISA, using DNA from morphologically identified adult parasites. Initially, 712 L4 were isolated from the diarrhoeic faeces from horses (n ¼ 17) with clinical larval cyathostominosis. PCR products from 522 of these L4 were subjected to analysis, with 413 L4 being identified as one of the aforementioned species. With reference to individual species analysis, 28.5% of the 522 L4 were identified as C. longibursatus, 25.7% as C. nassatus, 15.9% as C. ashworthi, 7.3% as C. goldi and 1.7% as C. catinatum. No L4 were identified as being C. insigne species. When L4 within faeces from individual horses were compared, no sample was found to comprise parasites of one species. The least number of species identified in a single sample was two. This study suggests that clinical larval cyathostominosis is predominantly caused by mixed-species infections. q

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

Cited by 48 publications

References 0 publications

Recent developments in research into the Cyathostominae and Anoplocephala perfoliata

Recent developments in research into the Cyathostominae and Anoplocephala perfoliata

Microchip capillary electrophoresis-based genetic comparison of closely related cyathostomin nematode parasites of horses using randomly amplified polymorphic DNA polymerase chain reaction

A PCR–ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis

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