Seasonal development of Cyathostominae larvae on pasture in a northern temperate region of the United Kingdom.

A study following the development of Cyathostominae from egg to the infective larval third stage was conducted from April to December 2001 in west central Scotland. Duplicate samples (1 kg) of naturally infected faeces were placed on a 78 cm2 plot each week on a cyathostomin-free pasture. Subsamples of the grass surrounding the faecal plot were collected weekly on four occasions and the number of larvae obtained determined. Few larvae were recovered in the first week of development of individual plots, followed by a rise in the numbers of larvae in second, third and fourth weeks of development of each sample. The climatic conditions were seen to have an effect on the rate of development. Specifically, from multilevel, multivariable linear regression models it was evident that the factors associated with numbers of infective larvae recovered from pasture were the time since the faeces samples were laid down, the average temperature and rainfall during the previous week, as well as the interaction between temperature and rainfall. Conversely, from the model, the number of larvae recovered from pasture was associated with neither the number of eggs within the faeces samples placed on the plots nor with the viability of these eggs.

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

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=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.

Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses.

Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-specific. The fifth probe recognised all 16 cyathostomin species but did not bind to members of the genus Strongylus. Furthermore, these probes were used to identify individual infective L3, eggs and L4 indicating that they will be invaluable to furthering the study of the epidemiology and pathogenesis of these important equine nematodes.

From long range mapping to sequence-ready contigs on human chromosome 6.

Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. The strategy we are following involves establishing a high density framework map of the order of 15 markers per Megabase using radiation hybrid (RH) mapping. The markers are then used to identify large-insert genomic bacterial clones covering the chromosome, which are assembled into sequence-ready contigs by restriction enzyme fingerprinting and sequence tagged site (STS) content analysis. Contig gap closure is performed by walking experiments using STSs developed from the end sequences of the clone inserts.