PhDAt 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
