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    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 DTS
    BACKGROUND: 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.
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    The role of protozoan genetic diversity in human disease : implications for the epidemiology of cryptosporidiosis and giardiasis in New Zealand : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Ogbuigwe, Paul Chiamaka
    Cryptosporidium and Giardia are two common causes of diarrhoea in humans and livestock and responsible for multiple outbreaks of gastroenteritis every year in New Zealand and around the globe. Despite their prevalence, there are few effective therapies or vaccines, against either parasite. This is largely due to the difficulty of manipulating these parasites in vitro. The understanding of the epidemiology of this parasite in New Zealand is incomplete, due to the presence of multiple dominant subtypes of each parasite within samples from the same outbreak. In this thesis, new techniques are employed to investigate the genetic diversity of these parasites within hosts and develop an in vitro assay for comparing the infectivity of multiple subtypes of Cryptosporidium. Current methods for the purification of Giardia cysts from faecal samples do not adequately remove debris from the sample and produce low numbers of purified cysts. This hampers molecular techniques that benefit from uncontaminated samples resulting in the use of expensive methods like immunomagnetic separation. Here, a novel method for the purification of cysts from faecal samples was developed, which produced purified oocysts with negligible debris and a 10-fold increase in yield over current techniques. Epidemiological and molecular investigations of past giardiasis and cryptosporidiosis outbreaks in New Zealand have highlighted inconsistent results, where epidemiologically linked cases can have different dominant subtypes identified through Sanger sequencing. Here, amplicon-based metabarcoding was utilised to resolve Giardia and Cryptosporidium outbreak epidemiology in New Zealand. Human faecal samples from past outbreaks previously classified using Sanger sequencing were analysed using next-generation sequencing. This strategy uncovered significant within-host diversity and identified potential emerging subtypes of Cryptosporidium that could have public health significance in the future. Analysis of diversity within outbreaks provided previously unidentified genetic links between samples from the same outbreak. Previous studies show that people experience different symptoms depending on the subtype of Cryptosporidium they are infected with. Also, the dominant subtypes of the parasite in a region, like the USA and Australia, have changed multiple times within the past 20 years. This suggests there are differences in infectivity between subtypes, but further analysis of this problem has been hampered by the lack of adequate cell culture systems that allow the complete development of the parsite in vitro. To better understand the differences in infectivity between subtypes of Cryptosporidium, an analysis of the expression of Cryptosporidium genes in the COLO-680N cell line at multiple timepoints during infection was carried out using the NanoString nCounter analysis system. This was done to investigate whether differences in gene expression could account for differences in infectivity. Furthermore, utilising flow cytometry a system was developed capable of identifying and quantifying infection in infected cells with and without the use of a fluorescent antibody. A novel signal was identified in the near-infra red range that was specific to Cryptosporidium infection and showed better signalling characteristics than the fluorophore.
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    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 Peng
    Giardiasis 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.
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    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 Stephen
    Giardia 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.
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    Assessment of methods for determining viability of Giardia spp. in freshwater and seawater : a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Massey University
    (Massey University, 1993) Donaghy, Morgan John
    The determination of viability of Giardia spp. cysts is difficult. At present the problem is that once detected it is not known whether this cyst is viable and therefore potentially infective for humans. This study assesses two methods; Nomarski Differential Interference Contrast Microscopy (Nomarski DIC) and Fluorogenic Dyes (Fluorescein Diacetate and Propidium Iodide) as they compare to the current benchmark for viability Excystation. In vivo Giardia muris and Giardia intestinalis cysts were assessed for viability at time intervals and different temperatures in two separate inactivation systems; chlorine at standard municipal treatment levels and seawater with a view to their use in routine viability testing of cysts detected in environmental samples. G. muris was trialled as it was thought it may prove to be a good model for G. intestinalis. The effect of seawater as an environmental inactivation system is important due to current domestic waste disposal practises ie. sea disposal of treated waste. These effects were assessed in this study by Excystation, Nomarski DIC and Fluorogenic Dyes. Seawater has a cysticidal effect on Giardia cysts. This is due mainly to osmotic and alkaline nature of seawater. Nomarski DIC when compared to Excystation, has a limited capacity for determining viability of cysts from freshwater, seawater and chlorine inactivation systems. Fluorogenic Dyes seem more suited to determination of viability of cysts isolated from fresh and untreated waters.
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    Giardia in New Zealand animals : prevalence, viability in the environment and preservation : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Microbiology at Massey University
    (Massey University, 1993) Marino, Michelle Rose
    Little is known about the epidemiology of giardiasis in New Zealand. Most interest has been focused on the occurrence of Giardia cysts in the water ways of this country and in particular in municipal water supplies. Little is known about the occurrence of asymptomatic or even symptomatic giardiasis in people as this is not a notifiable disease, even less is known about giardiasis in domestic or wild animals. Giardia can now be cultured in the laboratory. G. intestinalis may be cultured in vitro while G. muris is often cultured in the mouse. G. intestinalis cysts when harvested are often only a portion of the total cells present with trophozoites and incompletely formed cysts being a larger portion of the cell population. For ease of counting cysts it was desirable to destroy the trophozoites and incompletely formed cysts. On average 64% of the trophozoites can be destroyed when harvested cultures are incubated in double distilled water overnight. Trophozoites were found to remain present in the water for weeks when stored at 4°C. Sonication destroyed trophozoites within two minutes while incompletely formed cysts persisted in suspension with completely formed cysts. The only way to quickly and easily destroy trophozoites and incompletely formed cysts was to incubate the cell suspension in 0.1% SDS for approximately two minutes and then wash the cysts remaining by slow centrifugation. The result is a clean suspension of non-viable completely formed cysts. New Zealand animals shown to harbour the parasite Giardia include farm animals; cattle, sheep, dogs and chickens, of importance to anyone using animal manure on their gardens especially on vegetables that do not require cooking before consumption. Domestic animal wastes can enter the water supplies on farms and get into rivers that supply town water supplies. Wild animals infected with Giardia studied in more detail were the possum, house mouse and ship rat. These animals may be a reservoir for contaminating water ways in less populated areas of New Zealand though they were more likely to just maintain the infection within their own population due to little contact with running water. Other wild animals defecating near water ways could serve as sources of infection. Little is known about the zoonotic potential of Giardia found in animals. In past trials with Giardia cysts from beavers were shown to infect 2 out of 3 people. Dogs and cats have been implicated as possible sources of household infections and it is recommended when treating a family for giardiasis to also treat household pets. Giardia intestinalis cysts cultured in vitro are commonly used for experimental work due to the ease of harvesting large numbers. It was not known if the in vitro cultures truly reflected the characteristics of Giardia isolates from people and animals. Morphologically, few cysts harvested from the flask are elliptical in shape, most being round. It is thought in vitro culturing is highly selective and resulting cultures would only represent a small portion of the wild population. G. intestinalis cysts cultured in vitro were compared to G. intestinalis isolated from human faeces and G. muris cultured in the mouse. Cysts could not survive in the absence of water. Laboratory trials found that Giardia cysts were able to survive and remain viable for months in cold water (4°C) and for shorter periods of time at higher storage temperatures. Cysts suspended in water free of faecal matter were viable and detectable for a longer period of time than those cysts exposed to faecal matter. Cysts incubated at 4°C had the best survival rate with respect to viability and the length of time cysts were present. Preservation of cysts in 10% formalin did not necessarily prolong the length of time Giardia cysts could be stored for. Cysts stored in water at 4°C survived for as many months as cysts fixed in 10% formalin. Giardia cysts studied in the laboratory are often fixed in 10% formalin or Schaudin's fixative (PVA) to enable long term storage of specimens in suspension or on slides. Using Schaudin's fixative, Giardia cysts were destroyed and the internal morphology of the cysts was greatly distorted. Fixing with osmium tetroxide was not found to distort internal morphology as no difference in morphology was viewed under Nomarski optics between viable organisms and those fixed with osmium tetroxide.
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    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 Craig
    Giardiasis, 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.
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    Enhanced surveillance of potentially foodborne enteric disease within a New Zealand public health service : thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies in Public Health at Massey University, Palmerston North, New Zealand
    (Massey University, 2009) Shadbolt, Tui Louise
    An enhanced notified enteric disease surveillance trial began on 1 July 2007 and continued until 30 June 2008. The aim of the trial was to measure the quality, timeliness and completeness of data collected and submitted by a regional Public Health Service (PHS) to the Institute of Environmental Science and Research Limited (ESR), via the national disease database (EpiSurv) for notified cases of enteric diseases. The trial evaluated two different methods of data collection: postal questionnaires and telephone interviews. Telephone interview techniques were used to improve the contact rate, timeliness and completeness of data gathered from all notified cases of campylobacteriosis in the Manawatu, Horowhenua and Tararua regions. The target set for the project was to achieve a 95% contact rate with 90% full completion of all EpiSurv data fields. For all notified cases of campylobacteriosis a 97% contact rate was achieved in a time frame of between zero to 20 days (three day median) and completeness of all the EpiSurv case report fields ranged between 96 – 100% in the final data. Prior to the commencement of the study, between 1 July 2004 to 30 June 2005, MidCentral PHS (MCPHS) made contact with around 58% of all notified cases of campylobacteriosis and 77% of all other notified enteric disease cases1 . A short pre-screen mail questionnaire, with reply-paid envelope, was sent to all notified cases of cryptosporidiosis, giardiasis, salmonellosis and yersiniosis in the MCPHS regions. EpiSurv case report fields were completed using information supplied in the returned questionnaires. Return rate, timeliness, and completeness were compared with the telephone interview group. Fifty three percent of cases we attempted to contact via mail questionnaire responded within two to 63 days (six day median) and completeness of all the EpiSurv case report fields ranged between 81 – 100%. In addition, we monitored the newly introduced ESR Early Aberration Reporting System (EARS) flags for increased levels of disease compared to historical disease rates, and assessed its usefulness as a tool to identify potential outbreaks in the region. While no outbreaks that had not already been identified by PHS staff were found by monitoring the EARS system, EARS has become an important tool in the MCPHS for comparing our rates of disease with bordering PHSs. EARS also provided a good quick reference tool for media enquiries and the graphs produced in EARS have been well utilised as visual aids for training and seminars presented during the trial period. The results of the surveillance trial initiatives were compared to the rest of New Zealand (NZ) over the same time frame and with a comparable, medium-sized, PHS. While the results of the telephone interviews from the MCPHS trial were close to the comparable PHS, they were significantly higher than for the rest of NZ. The postal questionnaires achieved a lower contact rate than the comparable PHS but similar to the rest of NZ. However, the quality of data gathered in the returned MCPHS postal questionnaire was significantly higher in most fields. Additional analysis was undertaken which indicated that those cases living in higher deprivation and rural areas were less likely to respond to a postal questionnaire. An over-representation of common enteric disease notifications from rural areas in the MCPHS was also highlighted by our research. This trial has shown the effectiveness of utilising telephone interviews and telemarketing techniques for gathering timely and complete data for human enteric disease surveillance within the MCPHS. It has also demonstrated that a short pre-screen questionnaire can be effective in collecting good quality data needed to complete the standard EpiSurv case report form.