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Item Uncovering the genetic diversity of Giardia intestinalis in isolates from outbreaks in New Zealand(BioMed Central Ltd, 2022-12) Ogbuigwe P; Biggs PJ; Garcia-Ramirez JC; Knox MA; Pita A; Velathanthiri N; French NP; Hayman DTSBACKGROUND: Giardia intestinalis is one of the most common causes of diarrhoea worldwide. Molecular techniques have greatly improved our understanding of the taxonomy and epidemiology of this parasite. Co-infection with mixed (sub-) assemblages has been reported, however, Sanger sequencing is sometimes unable to identify shared subtypes between samples involved in the same epidemiologically linked event, due to samples showing multiple dominant subtypes within the same outbreak. Here, we aimed to use a metabarcoding approach to uncover the genetic diversity within samples from sporadic and outbreak cases of giardiasis to characterise the subtype diversity, and determine if there are common sequences shared by epidemiologically linked cases that are missed by Sanger sequencing. METHODS: We built a database with 1109 unique glutamate dehydrogenase (gdh) locus sequences covering most of the assemblages of G. intestinalis and used gdh metabarcoding to analyse 16 samples from sporadic and outbreak cases of giardiasis that occurred in New Zealand between 2010 and 2018. RESULTS: There is considerable diversity of subtypes of G. intestinalis present in each sample. The utilisation of metabarcoding enabled the identification of shared subtypes between samples from the same outbreak. Multiple variants were identified in 13 of 16 samples, with Assemblage B variants most common, and Assemblages E and A present in mixed infections. CONCLUSIONS: This study showed that G. intestinalis infections in humans are frequently mixed, with multiple subtypes present in each host. Shared sequences among epidemiologically linked cases not identified through Sanger sequencing were detected. Considering the variation in symptoms observed in cases of giardiasis, and the potential link between symptoms and (sub-) assemblages, the frequency of mixed infections could have implications for our understanding of host-pathogen interactions.Item The human antibody response to Giardia intestinalis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1991) Chan, Judy Lai PengGiardiasis is usually diagnosed in the laboratory by microscopic examination of faeces for the presence of cysts and / or trophozoites. However in principle, it is possible that Giardia infection could be diagnosed serologically. To investigate this possibility an Enzyme Immunoassay (EIA) was developed using Giardia-specific mouse serum as antibody. The efficiency of different Giardia antigen preparations to detect antibody in this test was investigated. The antigens included live trophozoites, frozen and thawed trophozoites, sonicated trophozoites, trophozoite membranes and cysts. Antibody titres were low and no marked differences were detected when the four different antigens were compared. However, since following a natural infection the immune response to surface proteins probably predominates, we concluded that live trophozoites or cysts represented the most appropriate antigens to use in an EIA test to detect Giardia-specific antibody in human serum. Live trophozoites adsorbed to polystyrene microtitre wells were removed by the washing procedure, thus giving an insensitive test and inconsistent results. This problem was overcome by precoating the microtitre wells with poly-I-lysine following which trophozoites and cysts adhered to the wells strongly enough to resist the washing procedures. The EIA test was optimised with Giardia specific mouse serum as antibody and the same system was used to detect antibody in human serum. lll IgG, IgM, and IgA antibody were assayed in "current infection", "convalescent" and "negative control" human sera. IgG antibody titres were slightly elevated in "convalescent" sera as compared to the other two groups. IgM antibody titres were slightly elevated m "current infection" sera and IgA antibody levels were not found to be elevated. Since IgM antibody is present early in an infection but does not persist, its presence or absence could, m principle, be used to distinguish a current from a previous infection of Giardia. However, only slightly elevated levels of Giardia-specific IgM antibody in "current infection" sera were detected in our tests so this approach to diagnosis will need further development if it is to be used for diagnostic purposes. Giardia IS not an invasive orgamsm and it is possible that some antigens may play a major role in eliciting an immune response in humans. Thus, potential Giardia antigens were investigated by "immunoblotting" total Giardia proteins with human sera from clinically diagnosed cases of giardiasis. It was found that the human antibody response to Giardia vanes between individuals and many Giardia proteins reacted with the Immune human sera. However, IgG antibody found m many of the serum samples reacted with a 200 kDa, 62 kDa and 42 kDa protein. IgA and IgM antibodies also reacted with a 62 kDa protein which may be similar to a 55 kDa structural protein (tubulin) found in Giardia.Item Giardia intestinalis : aerobic metabolism and physiology of in vitro growth : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in microbiology at Massey University, Palmerston North, New Zealand(Massey University, 1988) Miller, Sally JayneG. intestinalis; the causative agent of giardiasis, parasitises a number of vertebrates including man; and has a worldwide distribution. Although giardiasis is now a widely recognised public health concern; little is known of its aetiological agent. This is primarily due to the fact that a protocol for the routine axenic cultivation of this intestinal parasite was not available until 1976. With the advent of in vitro cultivation, an increasing number of reports have outlined the in vitro growth requirements of G. intestinalis; however; the physiology and unique metabolism of this protozoan still require further clarification. Utilising two strains of G. intestinalis (Bris/83/HEPU/106 and Hast/87/MUGU/68), the influences of envirornnental factors such as pH and temperature on axenic culture growth were investigated. Variations in both temperature and pH were shown to effect the in vitro growth rate of the two strains examined. Growth of Bris/83/HEFU/106 was markedly impaired at non-optimal temperature; (optimal growth of Bris/83/HEPU/106 and Hast/87/MUGU/68 occurred at 37°c); while growth of Hast/87/MUGU/68 continued, at a reduced rate, over a wider, non-optimal temperature range (30-40°c). Both strains exhibited marked pH optima for culture growth (pH 6. 75-7.50) with a rapid decline in culture growth rates outside these pH levels. Clonal growth of G. intestinalis trophozoites in semi-solid agarose has been utilised in the past as an assay of trophozoite viability in vitro. The suitability of such an assay for use during this study was investigated for both Bris/83/HEPU/106 and Hast/87/MUGU/68. Over the range of agarose concentrations examined, the colony fonning efficiency (CFE) of both strains was extremely variable. While Hast/87jMUGU/68 was better adapted to growth in agarose medium, with CFE of up to 60% recorded; these rates of clonal growth were often not reproducible, as the growth of trophozoite colonies remained inconsistent despite duplication of all assays. The thiol reducing agent L-cysteine, has been reported to be a specific growth requirement of G. intestinalis in vitro. The correlation between reducing conditions and the growth and attachment of Bris/83/HEPU/106 and Hast/87jMUGU/68 in culture, was investigated as trophozoites were exposed to a range of L-cysteine concentrations in TYl-S-33 growth medium. Enhanced growth of experimental cultures was directly related to increases in L cysteine concentration and corresponding decreases in the 0-R Potential of growth medium. Culture growth occurred at a maximal rate where the concentration of L-cysteine in growth medium exceeded 0.15% w/v. All cultures failed to grow in the absence of L cysteine. Trophozoite attachment in culture was most rapid during the 30-90 minutes following culture establishment. Under elevated L-cysteine concentrations (0.15-0.25% w/v) this attachment reached maximal levels (85-95%). In the absence of L-cysteine, attachment of trophozoites in culture continued, but at a markedly reduced rate. The oxygen sensitivity of G. intestinalis trophozoites was investigated in TYl-S-33 utilising a protocol developed during this study, where the exposure of trophozoites to dissolved oxygen was directly controlled through adjustment of oxygen flow into growth medium. Bris/83/HEPU/106 and Hast/87jMUGU/68 trophozoites displayed a similar degree of oxygen sensitivity at 37°c. A slow decline in culture viability was recorded upon exposure of trophozoites to 4.0- 6.0 ppm dissolved oxygen in growth medium. At 8.0 ppm; exponential killing of trophozoites was preceded by a 'lag phase' of 3-4 hours duration. In contrast; the killing of cultures commenced almost immediately after exposure of trophozoites to 12.0 ppm dissolved oxygen. At temperatures below 37°c (20°c and 3o0 C) , Bris/83/HEPU/106 exhibited a reduced sensitivity to elevated dissolved oxygen levels in TYl-S-33, as both the T1/2 of killing, and the lag phases preceding this killing were extended. The basis for the observed 'temperature-dependant' oxygen sensitivity of G. intestinalis is not known. Oxygen consumption by G. intestinalis has recently been reported by several workers; however; there is still very little known of the metabolic role of 'active respiration' in this 'aerotolerant anaerobe'. Consumption of oxygen by Hast/87jMUGU/68 in PBS was demonstrated. using a Model 97-08 Oxygen Electrode. Dissolved oxygen was removed from PBS by trophozoites at a rate of 3.2-5.3 10- 9 ppm/cell/hr. This oxygen consumption was inhibited. up to 50% by the flavoantagonist, Quinacrine dihydrochloride, at concentrations of 250-1000 µg/ml in PBS solution. Tbe concentrations of Quinacrine which were inhibitory to oxygen consumption by trophozoites over a 5 hour period were well in excess of the Quinacrine MLC (Minimum Lethal Concentration).Item Viability of Giardia intestinalis cysts : assessing viability under environmental conditions : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 1998) Kakubayashi, MichikoMuch work has been put into the detection and monitoring of Giardia, but once found, it is not easy to tell whether the cysts are viable and thus infective. There are fluorescently labelled monoclonal antibody kits which can be used to identify Giardia, but are the Giardia cysts viable? Excystation has been the main method used to determine the viability of cysts. This is quite unreliable as varying excystation conditions seem to be required for different strains of cysts. Using samples of fresh cysts, certain batches consistently measured 80-95% viable, while others resulted in viability measurements of 0-10%. The cysts themselves displayed the normal morphology of viable cysts. The assumption that partially excysted trophozoites as well as completely excysted trophozoites are viable may also lead to over-estimation of viable cyst numbers. Another commonly used method for estimating the viability of Giardia is staining with vital dyes, in particular the combination of fluorescein diacetate (FDA) and propidium iodide (PI). These also gave unexpected results where none of the cysts in a fresh sample stained with FDA, which usually stains viable cysts. An alternative dye, 4',6-diamidino-2-phenylindole (DAPI) was used in the place of FDA. The combination of DAPI and PI showed viabilities of 85.7% for cyst samples. This correlated well with 88% viability using excystation. Using the DAPI/PI combination, the viability of G. intestinalis cysts over time was monitored under different temperature conditions, and in sea water. Temperature was quite significant in the viability of the cysts – cysts stored at 4°C remained viable for 62 days, while those stored at 25°C were non-viable after 5 days. Sea-water had an immediately lethal effect on the G. intestinalis cysts, with all cysts non-viable after 45 minutes. Giardia intestinalis trophozoites can be cultured in the laboratory. By the addition of bile to the growth media, it is possible to transform these into cysts. Over the course of four days in encystation media, a large proportion of the trophozoites in the culture were converted into cysts, 3.5 X 10⁵ cysts/ml from an initial trophozoite concentration of 7.2 X 10⁵ organisms/ml. However, the cysts generated from the strains of G. intestinalis used were completely non-viable, compared with viability rates for fresh in vivo cysts of 80-95%. A population of hamsters was found to be carrying a Giardia which seemed different to recognised species. An analysis was carried out by PCR and sequencing of sections of the ribosomal DNA of this Giardia. Through this it was found to be closely related to Giardia muris, but perhaps not as closely related as to be a species of G. muris, possibly a sub-species. The rDNA analysis used may be very useful in typing other strains and species of Giardia.Item Genotyping of human and animal isolates of Giardia intestinalis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 2002) Kwan, Errol StephenGiardia intestinalis is an important protozoan parasite that infects humans and animals. It has been suggested that cattle may be a major source of human Giardia infection so a dairy farming region of New Zealand was investigated. This thesis uses three molecular methods to genotype G. intestinalis isolates obtained from human and animal faecal specimens collected in the Waikato region of New Zealand, to determine if giardiasis is a zoonotic disease. Random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) fingerprinting techniques were initially assessed for their ability to genotype G. intestinalis isolates. "Clear cut" evidence of zoonosis could not be established by either method, due to a low sample number. To determine the stability of the G. intestinalis genome an axenic culture of G. intestinalis trophozoites was stressed with toxic levels of metronidazole and the survivors, following a number of passages, were examined using AFLP and RAPD analysis. The DNA fingerprints were compared to those of the original wild-type with the results being indicative of an unstable G. intestinalis genome. A third molecular method was employed, which amplifies a portion of the tandemly repeated ribosomal DNA (rDNA). Each cyst contains 512 head to tail tandem repeat copies of the rRNA gene made up of both conserved and variable regions. The use of nested primers increased the sensitivity and specificity of the PCR reaction allowing the amplification of a 505bp rDNA fragment. DNA sequence analysis and alignment of the amplified products facilitated the comparison of G. intestinalis isolates. The relationship of the sequence data was generated and displayed using Splitstree software indicating that zoonosis did occur.Item Differentiation of human and calf isolates of Giardia intestinalis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1999) Hunt, Cynthia LeeTraditionally farm runoff has been blamed for the contamination of aquatic waterways with Giardia cysts especially during the natural calving seasons. But despite Giardia intestinalis being one of the most commonly acquired waterborne gastrointestinal parasites in humans little is known about the extent of G.intestinalis transmission between humans and animals throughout a defined geographical region, This study examines the characterisation of human, calf and laboratory adapted isolates of G.intestinalis. Specific amplification primers were developed to target a section of the ribosomal DNA (rDNA) unit. This locus is considered to be rapidly evolving and therefore suitable for use in the elucidation of phylogenetic relationships between G.intestinalis isolates. The isolates characterised were collected in the Waikato district from naturally infected humans and calves throughout 1998 but especially during the spring calving season of August and September. Giardia from calves from a second province as well as laboratory adapted isolates cultured from a variety of hosts were also surveyed. Sequence analysis of human, calf and laboratory adapted G.intestinalis isolates showed the presence of three distinct groups. All calf G.intestinalis isolates clustered together despite differences in the collection time and site. The human G.intestinalis isolates split into two clusters, corresponding to recognised 'Polish' and 'Belgian' subtypes. Surprisingly the laboratory adapted isolates grouped with the human 'polish' subtype despite striking differences in isolate origin. The current data strongly suggests that host specific G.intestinalis strains are present in the environment. Cross-transmission has so far not been detected. The occurrence of isolate specific rDNA sequences enabled the development of diagnostic polymerase chain reaction (PCR) amplification primers. In conjunction with the existing primers these allow the identification of human specific Giardia as well as differentiating between the 'Belgian' and 'polish' subtypes. These primers offer the ability to quickly and economically identify potential sources of human giardiasis in the environment. Using such molecular tools may lead to an overall decrease in human giardiasis resulting from environmental contamination sources.Item Eukaryotic signature proteins : guides to pathogenic eukaryotic parasites : a thesis presented in partial fulfilment of the requirements of the degree of PhD in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2012) Han, JianEukaryotic Signature Proteins (ESPs) are proteins that delineate the eukaryotes from the archaea and bacteria. They have no homologues in any prokaryotic genome, but their homologues are present in all main branches of eukaryotes. ESPs are thus likely to have descended from ancient proteins that have existed since the first eukaryotic cell. This project looks at ESPs of some eukaryotic parasites and human (Homo sapiens) as their host organism and focuses on Giardia lamblia, a fresh water pathogenic basal eukaryote. The ESP datasets from Giardia and two other parasites, Trichomonas vaginalis and Plasmodium falciparum, as well as the host human were calculated in light of available genomic data and the datasets contained a range of proteins associated with membrane, cytoskeleton, nucleus and protein synthesis. ESPs have great potential in phylogenetic studies since these proteins are present in all eukaryotes and are expected to have a slow and constant rate of evolution. Phylogenetic analyses were performed on the 18 eukaryotic organisms including some basal eukaryotes, and also for mammals, using orthologues of the all ESPs from these organisms. Strategies such as concatenating sequences and constructing consensus networks were tested to evaluate their potential with large numbers of ESP alignments. The results were promising, and ESPs hold great potential for their use in future phylogenetic analyses of eukaryotes. RNA interference is hypothesised to be an ancient mechanism for gene regulation and like the ESPs, it is typically found in all main branches of eukaryotes. High throughput sequencing data from Giardia and Trichomonas small RNAs (15-29mers) were re-analysed showing two length peaks for Giardia RNAs: a “larger peak” and an “ultra small peak”, the former of which is likely to be the product of the enzyme Dicer, which processes miRNA. The “ultra small peak” but not the “larger peak” was also found in Trichomonas. The two peaks possibly represent two different mechanisms of RNA interference (RNAi) in these parasites, but analysis of potential target sites from the Dicer-processed RNAs has not yet shown any indication that ESPs are regulated any differently from other parasite proteins. Sugar metabolic pathways including glycolysis and citric acid cycle were searched for ESPs, this was done to determine the relationship between the conservation of eukaryotic metabolic pathways and conservation of individual proteins. However no ESPs were identified from these pathways because Giardia has enzymes that show more similarity to those from prokaryotes than eukaryotes. These enzymes are significantly different from that of the host‟s, and these alternative enzymes offer potential as novel drug targets. In addition, ESPs that are present from host but lost in some parasites were analysed, and these ESPs are involved in many understudied pathways. It is these differences which can provide a guide in determining which pathways we should examine when designing drug targets. Overall, numerous proteomic similarities and differences in ESPs were identified between host and parasite. These proteins show potential for future evolutionary studies, and will guide future directions in ancestral eukaryotic regulation and metabolism.