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The development of PCR-based DNA fingerprinting tools for studying genetic variation in Giardia duodenalis

Hopkins, Richard M. (1997) The development of PCR-based DNA fingerprinting tools for studying genetic variation in Giardia duodenalis. PhD thesis, Murdoch University.

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The aim of this study was to investigate the usefulness of PCR-based DNA fingerprinting approaches to address problems regarding the genetic characterisation of Giardia duodenalis isolates.

A PCR-based method for genotyping Giardia cysts purified directly from faecal samples was devised using a polymorphic region near the 5’ end of the small subunit (SSU) rDNA gene. Isolates were collected from humans and dogs living in the same isolated Aboriginal communities where Giardia infections are highly endemic. Analysis revealed distinct differences between the genotypes of Giardia isolates recovered from different hosts suggesting that zoonotic transmission occurs infrequently between these two groups. It was argued that host adaptation of particular genotypes could account for the apparent lack of cross-infection.

Analysis of complete SSU rDNA sequences was used to examine the phylogenetic structure of the G. duodenalis morphological group. The use of PCR to amplify SSU rDNA gene sequences facilitated the analysis of isolates recovered from a diverse range of human and animal hosts, some of which had previously proven difficult to characterise. Comparison of SSU rDNA revealed five distinct genetic groups. The majority of human and animal isolates were separated into two SSU rDNA groups, the division between which corresponded exactly to the major genetic assemblages established using isoenzyme electrophoresis. Within each rDNA group, isolates exhibited very high levels of sequence similarity. These results confirmed there is a fundamental genetic division between the major isoenzyme assemblages and suggested that isolates which make up each genetic assemblage represent highly conserved phylogenetic groups.

Complete SSU rDNA sequence analysis also recognised an additional three genetic groups, all of which consisted of Giardia isolates recovered from animal hosts. Isolates from Australian dogs and rats were separated into distinct genetic lineages, whereas isolates from a goat and a cat formed a third rDNA genetic group more closely related to the majority of human and animal isolates. These findings suggest that whereas some Giardia genotypes are capable of infecting a number of host species, others may be rigidly host specific.

For fine-scale resolution of Giardia genotypes, the polymorphic potential of the noncoding rDNA intergenic spacer (IGS) was examined. When used in combination with restriction fragment length polymorphism (RFLP) analysis, PCR amplification of the IGS was found to be at least as sensitive as isoenzyme electrophoresis in terms of its ability to discriminate between individual Giardia isolates. Furthermore, IGS-PCR analysis recognised the same conserved genetic groups of Giardia isolates as those established using isoenzyme electrophoresis and SSU rDNA sequence analysis.

Much of the heterogeneity associated with the IGS region was due to individual Giardia isolates harbouring populations of intergenic spacers that varied in both length and sequence. However, the presence of multiple IGS classes did not affect the stability of DNA fingerprinting profiles. No evidence for variability in banding profiles was observed between parent and cloned Giardia isolates which suggests that populations of trophozoites are homogeneous with regard to the types of IGS-classes they maintain. Similarly, stable IGS-PCR RFLP fingerprints were observed in isolates which had been cultured over periods of several years.

The IGS-PCR fingerprinting strategy was successfully applied to faecally purified cyst samples recovered from Aboriginal communities in which Giardia was endemic. These results provided an insight into different aspects of Giardia transmission. For example, the potential for intrafamilial transmission was highlighted by the fact that Giardia samples collected from four family members at the same time all contained identical genotypes. Furthermore, the same genotype present in the family group was distributed amongst other individuals of similar age living in the same as well as different communities, possibly indicative of an outbreak of transmission. IGS-PCR analysis was also able to detect the presence of mixed infections and showed that, in some individuals the genotype of Giardia isolates changed over time, whereas in others the same genotype was maintained.

Analysis of hypervariable DNA sequences revealed that polymorphic minisatellite loci are found in abundance in the Giardia genome whereas microsatellites occur infrequently. The potential for an individual minisatellite locus to be used as a DNA fingerprinting marker was evaluated using the PCR. The results revealed that minisatellite amplification was only possible with a subset of Giardia genotypes and that limited size polymorphisms were evident. The inability to amplify PCR products from all Giardia isolates suggests that it may be necessary to design a panel of microsatellite markers in order to fingerprint a broad range of Giardia genotypes.

The results of this study demonstrate the potential for PCR-based DNA fingerprinting tools to examine the transmission dynamics of Giardia in endemic areas. The IGS-PCR fingerprinting tool in particular, offers the capacity for fine-scale resolution of Giardia genotypes without the need for in vitro cultivation. This should enable the movement of individual isolates within a defined geographical area to be tracked. Application of the IGS-PCR fingerprinting tool to field-based studies should contribute significantly to our understanding of the epidemiology of Giardia infections.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Science
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Thompson, Andrew and Groth, David
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