Cloning and characterization of ribosomal RNA genes from three species of Haemonchus (Nematoda: Trichostrongyloidea) and identification of PCR primers for rapid differentiation.

Abstract

Genomic DNAs prepared from adult worms of Haemonchus contortus, Haemonchus placei, and Haemonchus similis were used to clone and map complete ribosomal RNA gene repeats from each species. The lengths of the repeating units were estimated at 7.2, 7.2, and 7.4 kb, respectively, with a second and distinct repeat 6.9 kb in length identified from H. contortus only. Restriction enzyme recognition sites within all ribosomal RNA genes were fully conserved except for the loss of a SalI site within the large subunit rRNA gene of the 6.9-kb H. contortus repeat where only minor differences were observed in the restriction enzyme recognition sequences within the external spacer DNAs. Sequence analysis of the subcloned small subunit ribosomal DNAs from each species demonstrated 100% conservation within the 1758-bp fragments with only limited sequence variability observed within the adjacent 5' external-transcribed spacer. Enzymatic amplification of external spacer sequences using primers complementary and proximal to the 3'-end of the large subunit and the 5'-end of the small subunit rDNAs enabled rapid differentiation of individual worms of H. contortus from H. placei by utilizing the size variability within this region of the repeat.