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Item Differentiation of strains of Giardia intestinalis by identification of restriction fragment length polymorphims (RFLP's) and the construction of a gene library [microform] : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1993) Campbell, Shalome AnittaGiardia intestinalis is a flagellate protozoan which infects the gastrointestinal tract of humans and other mammals such as cats, dogs and farm animals. The organisms involved have been assigned to a single species, which might be taken to suggest the absence of host specificity, yet there is little epidemiological evidence to suggest that human infections are derived from nonhuman sources. This suggests some degree of host specificity which in turn implies the existance of different strains of Giardia intestinalis. Consequently, two related questions of public health interest can be raised: do animal strains of Giardia intestinalis infect humans and if not, how can cysts of human and animal origin detected in water samples be distinguished? If all or even some strains from animals fail to infect humans then it is probable that water may often be unnecessarily condemned as unsuitable for human use. Conversely, if animal strains infect humans then exclusion of animals from water catchment areas would be desirable. To clarify this situation, it is desirable to be able to distinguish individual strains of G. intestinalis and this thesis represents a preliminary attempt to do so using the techniques of molecular genetics. Initial experiments attempted to detect Restriction Fragment Length Polymorphisms (RFLP's) produced by digests of total genomic DNA using a range of restriction endonucleases. The results were difficult to interpret because an excessive number of bands were produced. However, some denser bands, probably representing repetitive DNA sequences, were relatively well resolved and could allow comparisons between strains to be made. This repetitive DNA is GC-rich and was separated from most of the nonrepetitive genomic DNA by CsCl centrifugation in the presence of Hoescht 33258 stain. Using this approach, GC-rich fractions of DNA from several strains of G. intestinalis were compared using a variety of restriction endonucleases. Most did not reveal differences but digestion with some restriction endonucleases revealed minor differences. This demonstrated the potential usefulness of this approach in distinguishing between strains of Giardia but the procedure was laborious and required large amounts of DNA which could only be produced by the culturing of organisms in bulk. This is not yet possible in the case of many strains of G. intestinalis, so it was concluded that an alternative approach using digests of total genomic DNA followed by electrophoresis, Southern blotting and hybridisation with specific DNA probes would represent both a better theoretical and practical approach. A desirable preliminary step to facilitate this approach is the production of a Giardia gene library and the latter part of this thesis describes this process. The major problem encountered with the production of the library was that the DNA produced by a range of extraction methods was sheared and was present only in low concentration. This problem was traced to the presence of an excessive amount of polysaccharide which appeared to be strongly associated with unsheared DNA and hence caused it to be trapped at the interphase during phenol/chloroform extractions. As only the supernatant was retained, the high molecular weight DNA was largely lost during such extraction steps. This difficulty was overcome by precipitating the DNA with isopropanol at an early stage in the extraction method. This reagent does not precipitate the polysaccharides present in the cell lysate so that these are removed with the supernatant. The precipitated DNA was redissolved and, following conventional purification procedures, represented a high yield of unsheared DNA. Using this DNA, a library was made using LambdaGEM-11 Xho Half-site arms TM (Promega). Experiments showed that the library is representative of the genome of G. intestinalis and exceeded by 6-fold the number of clones required for a 99.9% probability that any particular sequence of interest is present. The availability of such a library should permit the selection of suitable clones for use as probes to hybridise with total genomic digests to reveal differences between strains of G. intestinalis. The ability to distinguish strains would allow investigations of host specificity which may have implications for the formulation of testing procedures designed to prevent human infections of G. intestinalis but avoiding the unnecessary condemnation of water supplies containing Giardia strains that do not infect humans. The development of strain-specific probes would also serve as a useful epidemiological tool in tracing the source of infection within a community.Item The development of techniques to distinguish species and strains of giardia : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1995) Farrant, Kirsty JaneWater supplies, in some rural areas of New Zealand, contain Giardia cysts. This is assumed to make the water unsuitable for human consumption. G. intestinalis and/or G. muris cysts may be present but are not distinguished by the standard test. G. muris infects rodents only so it is not infectious for humans. However G. intestinalis infects humans and a wide range of animals, but it is unclear if the strains which infect animals also infect humans. If G. intestinalis strains are host-specific, then since water in rural areas may contain cysts derived only from animal species it would follow that the water (even if G. muris and/or G. Intestinalis cysts were found) may not be infectious for humans. Investigation of host-specificity of G. intestinalis would be facilitated by a reliable test to distinguish strains of the organism and this thesis investigates the use of PCR for this purpose. A series of random primers were investigated for their ability to distinguish strains of G. intestinalis when used with a variety of PCR protocols. We found that several of these primers (especially GC50 at an annealing temperature of 35°C, and GC80 at an annealing temperature of 35°C) had the potential to distinguish strains. The differences seen were not large but this may be because some of the isolates were clonally related. Consequently we concluded that further modifications and extensions of PCR when applied to human and animal strains should distinguish strains and may have the potential to address the question of host-specificity. The major aim of the thesis however was to produce primers which when used in the PCR are capable of distinguishing G. muris from G. intestinalis. The same approach,ie the use of a random primer, was used to distinguish G. muris from G. intestinalis. Clear differences were seen but the non-specificity of the random primer would allow the organisms to be reliably distinguished only in the absence of other organisms. To avoid this lack of specificity an amplified band produced with G. muris DNA but not with G. intestinalis DNA was sequenced and a primer pair was selected. These primers were, in principle, long enough (21-mer and 23-mer) to be specific for the target DNA and were chosen so as to have matched melting temperatures. The selected primer pair amplified a sequence 307bp long, and the primer sequences were specific for the target species, namely G. muris. Thus in our hands using PCR this primer pair amplified DNA from the available strains of G. muris but failed to amplify DNA from any of seven G. intestinalis strains. Further work is required to establish both an optimal method for lysing cysts and to estimate the minimum number of cysts required to ensure that DNA is available for amplification. However, the availability of the G. muris -specific primers, along with the recently developed genus and G. intestinalis -specific primers should allow us to undertake investigations of water supplies to see if G. muris, G. intestinalis or both species are present. In the case of a small rural supply it would seem reasonable to accept the potability of water supplies containing G. muris only, as long as assurance could be given that G. intestinalis was not present.Item The occurrence of Giardia in cats and dogs in New Zealand and subsequent isolation and differentiation of strains : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology, Massey University, New Zealand(Massey University, 1988) Tonks, Michael CraigGiardiasis, a debilitating diarrhoea that affects many people every year is caused by the ubiquitous protozoan parasite Giardia intestinalis (syn lamblia, duodenalis) . This parasite infects and causes disease in birds and animals as well as man and has no known host specificity. Dogs and cats are some of the animals infected by Giardia and due to their close association with man, may be carrier sources of human giardiasis. In an attempt to discover a relationship between man and these animals, a survey of the level of Giardia infection in cats and dogs in both Hamilton and Palmerston North, New Zealand, was undertaken. Percentages of 25% and 8% for dogs and 3% and 7% for cats respectively were obtained. Statistically the level of infection in Hamilton was higher than that of Palmerston North. In both cities the sex and breed of the animals showed no correlation to infection although animals less than 3 years old were more likely to be infected. Clinical manifestations of giardiasis were observed but did not significantly correlate with the presence of Giardia and were not necessarily caused by the Giardia when present. To further enhance the relationship hypothesized it was attempted to culture the Giardia from the cats and dogs and relate them to cultured human isolates. Our attempts were unsuccessful and from 91 samples only 8 human strains from 5 geographical areas were isolated. These isolations were made by both in vitro and in vivo techniques that both yielded 7% sample to culture success. The isolated Giardia strains plus a control culture, Bris/83/HEPU/106 supplied by Boreham, Australia, were compared by growth rate and sodium dodecylsuphate polyacrylamide gel electrophoresis (SDS-PAGE) . Both these tests showed the similarity of these strains. The average growth rate was 0.09 ± 0.01 hours-1 and no strain varied from the statistical mean. In relation to the total protein banding patterns measured by SDS-PAGE, the isolates varied, at most, by one or two bands. An isolate of G. muris extracted from a naturally infected mouse and a human isolate that was extracted from an experimentally infected mouse were also compared by SDS-PAGE to a cultured isolate. The results showed many different bands between all three samples and suggests that an adaption, or selection, of the Giardia must take place when it is cultured. If this is so, then perhaps the emphasis put on strain variation of cultured Giardia trophozoites is to be questioned.Item Computational identification of four spliceosomal snRNAs from the Deep-Branching Eukaryote Giardia intestinalis(PloS ONE, 2008) Chen XS; White WT; Collins LJ; Penny DRNAs processing other RNAs is very general in eukaryotes, but is not clear to what extent it is ancestral to eukaryotes. Here we focus on pre-mRNA splicing, one of the most important RNA-processing mechanisms in eukaryotes. In most eukaryotes splicing is predominantly catalysed by the major spliceosome complex, which consists of five uridine-rich small nuclear RNAs (U-snRNAs) and over 200 proteins in humans. Three major spliceosomal introns have been found experimentally in Giardia; one Giardia U-snRNA (U5) and a number of spliceosomal proteins have also been identified. However, because of the low sequence similarity between the Giardia ncRNAs and those of other eukaryotes, the other U-snRNAs of Giardia had not been found. Using two computational methods, candidates for Giardia U1, U2, U4 and U6 snRNAs were identified in this study and shown by RT-PCR to be expressed. We found that identifying a U2 candidate helped identify U6 and U4 based on interactions between them. Secondary structural modelling of the Giardia U-snRNA candidates revealed typical features of eukaryotic U-snRNAs. We demonstrate a successful approach to combine computational and experimental methods to identify expected ncRNAs in a highly divergent protist genome. Our findings reinforce the conclusion that spliceosomal small-nuclear RNAs existed in the last common ancestor of eukaryotes.Item Non-protein-coding-RNA processing in the deep-branching protozoan parasite Giardia intestinalis : a thesis presented in partial fulfilment of the requirements for the degree of PhD in Molecular Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2008) Chen, Xiaowei[Abstract not supplied]