The establishment and evaluation of molecular procedures for the characterisation of taeniid cestodes

Genetic heterogeneity between and within species of parasites is now well documented. In recent years, the application of DNA analytical procedures has greatly enhanced our understanding of relationships between parasite species and has provided a more reliable means of differentiation at the specific and intraspecific levels. However, the application of DNA analysis for the identification and characterisation of cestode parasites has been limited. The present study is concerned with the establishment and application of molecular techniques for characterising taeniid cestodes in the genera Taenia and Echinococcus. The isolation of undegraded DNA is a prerequisite to DNA analysis. A rapid and reproducible procedure based on selective precipitation of nucleic acid by a quarternary ammonium compound, cetyltrimethyl ammonium bromide, was developed for isolating total genomic DNA from cestode tissue. With this procedure, restriction endonuclease analysis of DNA could be carried out within two hours from the start of DNA isolation. The technique was equally applicable to plasmid and mitochondrial DNA. Restriction endonuclease analysis of total genomic DNA was found to be of limited value for the identification and differentiation of cestode parasites. This was due-to poor resolution and the lack of distinctive repetitive DNA. An alternative approach was to carry out restriction endonuclease analysis of the smaller extrachromosomal genome in mitochondria (mitochondrial DNA; mtDNA). Distinct mtDNA restriction banding patterns were obtained at the species level and evidence of intraspecific variation was also shown between certain isolates of Taenia hydatigena. However, only very minute quantities of mtDNA could be isolated from cestode tissue. This inherent disadvantage subsequently led to the construction of a mitochondrial DNA probe. DNA probes were also constructed using repetitive ribosomal DNA sequences and total genomic DNA. These three types of DNA probes were subsequently evaluated for their ability to differentiate between closely related species and intraspecific variants of cestode parasites. Using recombinant DNA techniques, the 17.6 kb mitochondrial genome of Taenia hydatigena was cloned into plasmid pBR322 in Escherichia coli. Characterisation of the cloned mtDNA by restriction mapping provided some structural details of the mitochondrial genome which are essential for future in vitro manipulations. An 11.0 kb ribosomal DNA sequence of Echinococcus granulosus was also cloned into plasmid pBR322 in E. coli. A restriction map was constructed and the ribosomal RNA genes located within the cloned sequence. The organisation of the small and large RNA genes were shown to be essentially the same as in other eukaryotes. A simple and rapid hybridisation protocol was established for the evaluation of the biotinylated DNA probes. Hybridisation analysis indicated poor sensitivity for the biotinylated mtDNA probes. This is probably because of the low copy number of mtDNA. In contrast, repetitive DNA sequences were readily detected by biotinylated ribosomal DNA and total genomic DNA probes. Interspecific and intraspecific differences were detected in cestode parasites by Southern blot hybridisations using the three types of DNA probes. Furthermore, morphologically identical taeniid eggs from different species were differentiated using a simple dot blot procedure with total genomic DNA probes. In future studies, the cloned mitochondrial genome of T. hydatigena could be used as a model for in vitro investigations into the functional aspects of respiratory metabolism of cestode parasites. Since the mitochondrial genome is rapidly evolving, it could also be used for phylogenetic studies to determine lineages among cestode parasites. In addition, the use of biotinylated ribosomal DNA and total genomic DNA as DNA probes could be extented to detect genetic variants of cestode parasites not only within a research laboratory but also in the field