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Phenotypic characteristics of differing genetic groups of Giardia duodenalis and their implication for species identification

Binz, Nicolette (1996) Phenotypic characteristics of differing genetic groups of Giardia duodenalis and their implication for species identification. PhD thesis, Murdoch University.

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Abstract

Although it has long been acknowledged that the Giardia duodenalis group of isolates is very diverse and heterogeneous, we still lack comprehensive studies which compare groups of isolates from a selected geographical area on the basis of both genotypic and phenotypic characteristics. In the present study, twelve isolates (eleven from Western Australia, one from the USA) were selected based on the genetic diversity they exhibited upon isoenzyme analysis. Prior to any characterisation, the isolates were all cloned and a cloned cell line was subsequently selected at random for further characterisation. RAPD-PCR verified their distinctive groupings and showed that both parent and clone retained their basic classification and that none of the cloned cell lines had become cross contaminated despite more than three years of continuous in vitro cultivation.

Isolates were first verified as being members of the duodenalis morphological group on the basis of Filice’s (1952) system which used the shape of the median body, to order isolates as belonging to one of three morphological groups. The usefulness of Filice’s (1952) system in further subdividing the duodenalis morphological group was also tested. Although his system holds true in differentiating between the three morphological groups of G. duodenalis, G. agilis and G. muris, it failed to resolve the complex of isolates constituting G. duodenalis any further.

Morphological variation at the ultrastructural level has recently been reintroduced as a taxonomic tool in Giardia research and caudal flagellar and total trophozoite length were used to characterise the isolates chosen for the present study. Intraspecific variation within the G. duodenalis group was found to extend into the morphological characters tested here. Trophozoite length varied from 13.5|im (PIcl0) to 18.4|im (BAH 12cl4) and caudal flagellar length ranged from 4.6|im (PlclO) to 16.1pm (BAH 33c7). UPGMA analysis grouped isolates similarly to previously obtained isoenzyme electrophoretic data.

Prior analysis of growth dynamics in isolates of G. duodenalis had shown significant differences between two genetically distinct isolates (Binz etal, 1992) and these results were confirmed and extended during the present study. Mean generation times obtained for the twelve isolates studied varied from 6.6 hours (PlclO) to 24.5 hours (BAH 33c7). Scheffe’s post hoc test identified four distinct groups among the isolates examined. These results have implications not only for the epidemiology of infection but also for the suggested artificial laboratory induced selection of some genotypes over others by the currently used in vitro culture system.

At the commencement of this study only tentative data were available on the characteristics of the dual nuclei of Giardia trophozoites, and the DNA content of individual nuclei. Again, significant intraspecific variation was demonstrated among the isolates studied and DNA content was found to vary from 0.060pg per trophozoite (BAH 33c7) to 0.165pg per trophozoite (BAH 39c9), representing a 2.75 fold difference. Scheffe’s post hoc test identified six distinct groups. Although the data in context with previously published information permitted speculation about ploidy levels in G. duodenalis, the question of genome size could not be resolved.

The extensive heterogeneity demonstrated across the characters examined in the present study reflected that observed in most previous studies on G. duodenalis. Isoenzyme electrophoretic data in particular led Meloni etal. (1988a) and Andrews et al. (1989) to speculate about the presence of a complex of cryptic species within the duodenalis morphological group. However, no study to date has addressed the problem of species identification within this heterogeneous assemblage of isolates, nor has any attempted to collect data to solve the question of how many species there are.

The identification of species is a multi-step process and in the first instance requires the selection of an appropriate species concept and a case for the adoption of the evolutionary species concept was made. The delimitation of species within this framework requires organisms first to be grouped phylogenetically, preferably using DNA sequence or isoenzyme data, and these groups must then be ranked into the same or different species, measuring genetic differences in biologically meaningful characters. Taking into consideration all the data generated during the present study, the value of these genotypic and phenotypic characters in the identification of species within the G. duodenalis morphological group was examined. Growth dynamics and DNA content were found to be more useful in distinguishing groups of isolates than both trophozoite and caudal flagellar length. Although no species could be identified using the current set of characters, the study did demonstrate that some characters were more appropriate for delimiting species than others. Should the evolutionary species concept be adopted for the delimitation of species within the G. duodenalis morphological group, then there is a definite need to design studies in such a way that the data can be applied to the problem of species identification within the group, and biological characters of medical importance may provide the kind of data needed to rank groups of genetically differentiated isolates into species.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary Studies
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: repository@murdoch.edu.au. Thank you.
Supervisor(s): Thompson, Andrew and Lymbery, Alan
URI: http://researchrepository.murdoch.edu.au/id/eprint/52237
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