Comparative sequence analysis of the intergenic spacer region of cyathostome species

The ribosomal DNA intergenic spacer was amplified by the polymerase chain reaction from 16 cyathostome species using primers derived from conserved regions within the flanking 18S and 26S rRNA genes. This generated a 1.5-2.5 kb fragment which was sequenced from five species. The areas covering the 26S and 18S rRNA genes were more than 99% similar among the five species. Furthermore, in all species there existed a highly conserved region of approximately 380 bp at the 3' end of the intergenic spacer. Subsequently, two cyathostome-specific primers were designed to amplify a smaller, more variable region of the intergenic spacer. Eleven further species were amplified using these primers and analysis showed that sequence similarities varied from 40 to 97% between species. The sequence information obtained in this study is being used to develop a PCR-based assay for the differentiation of preparasitic stages of cyathostomes.