DNA typing of the human small intestinal protozoan parasite Giardia lamblia
At present there is no satisfactory means of typing strains of Giardia lamblia which can explain the broad range of clinical symptoms seen in giardiasis or which can identify genotypes in epidemiological studies. This thesis attempts to address these problems by developing DNA based typing systems sensitive enough to be able to identify many different Giardia genotypes and which may be applied to the organisms found in clinical samples. Four different techniques were assessed for their ability to identify multiple polymorphic loci in the Giardia genome which may be used to genotype and identify isolates of Giardia and upon which the future development of PCR-typing protocols may be based. These techniques included RFLP analysis, random amplified polymorphic DNA (RAPD) analysis, M13 DNA fingerprinting and minisatellite DNA fingerprinting. Minisatellite DNA fingerprinting proved to be the most discriminatory, recognising many hypervariable loci within the Giardia genome which proved useful for in vitro studies on genotypic heterogeneity within Giardia isolates. This approach would require further development in order to be used on in vivo infections where it could directly assess the relationship between genotype and pathogenicity. Therefore the variable repeats recognised on Giardia fingerprints were sought by constructing and screening a Giardia genomic DNA cosmid library. Once cloned these repeats would form the basis of sensitive and specific PCR-based fingerprinting protocols ideal for typing large numbers of infections. The repeat sequences cloned in this way turned out to be Giardia variable surface protein genes with short, imperfect tandem repeats in their 3' flanking DNA. This work has important implications for the future development and use of fingerprinting techniques on Giardia and may be useful in the study of chromosome rearrangement in Giardia which is likely to be involved in surface antigen switching.