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Item Population structure and pathogen interaction of Escherichia coli in freshwater: Implications of land-use for water quality and public health in Aotearoa New Zealand.(John Wiley & Sons, Inc., 2024-08-02) Cookson AL; Devane M; Marshall JC; Moinet M; Gardner A; Collis RM; Rogers L; Biggs PJ; Pita AB; Cornelius AJ; Haysom I; Hayman DTS; Gilpin BJ; Leonard MFreshwater samples (n = 199) were obtained from 41 sites with contrasting land-uses (avian, low impact, dairy, urban, sheep and beef, and mixed sheep, beef and dairy) and the E. coli phylotype of 3980 isolates (20 per water sample enrichment) was determined. Eight phylotypes were identified with B1 (48.04%), B2 (14.87%) and A (14.79%) the most abundant. Escherichia marmotae (n = 22), and Escherichia ruysiae (n = 1), were rare (0.68%) suggesting that these environmental strains are unlikely to confound water quality assessments. Phylotypes A and B1 were overrepresented in dairy and urban sites (p < 0.0001), whilst B2 were overrepresented in low impact sites (p < 0.0001). Pathogens ((Salmonella, Campylobacter, Cryptosporidium or Giardia) and the presence of diarrhoeagenic E. coli-associated genes (stx and eae) were detected in 89.9% (179/199) samples, including 80.5% (33/41) of samples with putative non-recent faecal inputs. Quantitative PCR to detect microbial source tracking targets from human, ruminant and avian contamination were concordant with land-use type and E. coli phylotype abundance. This study demonstrated that a potential recreational health risk remains where pathogens occurred in water samples with low E. coli concentration, potential non-recent faecal sources, low impact sites and where human, ruminant and avian faecal sources were absent.Item 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 ChiamakaCryptosporidium 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.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 Methods of testing for giardia in water : a thesis presented in partial fulfilment of the requirements for the degree Master of Science in Microbiology at Massey University(Massey University, 1995) Hastie, Joanna ClareSince the 1960's when the first waterborne outbreaks of Giardia were reported in America, it has been recognised as a disease causing organism. From these outbreaks the USA Environmental Protection Agency (EPA) developed a method for testing large volumes of water for Giardia cysts, this was adapted into the 16th edition of the Standard Methods. To test the method cultured cysts were required for spiked trials. A published method of encystation by Schupp et al (1988) was investigated as a potential source of cysts. Morphologically correct cysts were gained in the greatest number at 37°C over 72 hours at a bile concentration of 5g/l. Using cultured cysts and cysts from animals and water, viability and the least number needed to iniate a culture were assessed. When 10 of the cysts produced in vitro were excysted it was possible to obtain a culture. For cysts from animal and water origins at levels up to 10,000 cysts, it was not possible to obtain cultures. Variations of the Standard Method of water testing for Giardia had been reported by different laboratories. We investigated the sensitivity of this method using some of the reported variations such as staining on a membrane filter, the use of monoclonal antibody stains and methods of washing cysts free of the sampling core. We found the method could detect to the 5 x 10 2 cysts/5001 of water, a recovery of 10%. The recoveries obtained over a range of cysts spiked was between 10-40%. An alternative method to sampling and processing the sample was tangential filtration. Four tangential filtration units were compared to the concentration techiques of centrifugation and sedimentation (these were those used in the Standard Method). The tangential filtration units were found not to be as sensitive as centrifugation and sedimentation. They also presented difficulties with particulate matter or sediment. When compared to the sampling method, the unit was unable to concentrate the 5001 of tap water due to the sediment levels. Staining methods were evaluated. Slide staining was compared to staining on a filter, the filter method was found to give a better recovery. Comparison between commercially available monoclonal antibody stain, a polyclonal antibody stain and Lugols iodine stain, found that the monoclonal and polyclonal antibody stains lead to easier identification by illuminating the cyst (it still had to be checked for internal morphology) than the iodine stain. The monoclonal antibody stains were found to be more specific than the polyclonal stain. Methods of inactivating the antigens recognised by the monoclonal antibody stain persist so cross contamination between samples was investigated. Hypochlorite concentrations of 4% and higher over 20 minutes were found to inactivate the antigen recognised. Other chemicals were compared but none were found to inactivate the antigen. A study of a family infected with Giardia was undertaken, to test methods used in the laboratory and study modes of transmission. Giardia cysts were found in the river that supplied the farm tank but not in the tank itself. The house tank also tested negative for Giardia. The family had young children attending school and playgroup, person to person transmission may also have been involved. Animals on the farm had positive tests for Giardia.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 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 JohnThe 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.Item Prevalence of selected infectious diseases in Samoan dogs : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University, Manawatu, New Zealand(Massey University, 2013) Carslake, Rosalind JaneSamoa has a tropical island climate ideally suited to many infectious diseases, and vectors for some infectious diseases are known to be present. Dogs are very commonly owned in Samoa with 88% of households owning an average of two dogs. Many canine infectious diseases are zoonotic and there is limited preventative medicine available for dogs in Samoa. There are very few studies into the presence of zoonotic pathogens in Samoa or other South Pacific islands, and the role of dogs as a reservoir for zoonotic diseases is unknown. The prevalence of selected infectious diseases was evaluated in 242 dogs undergoing surgical sterilisation in Samoa in July 2010 and August 2011. Data were obtained from dogs’ owners by interview, including age, environment and any previous preventative medication. Serum and faecal samples were collected, and the skin examined for external parasites. Seroprevalence of Leishmania infantum, Anaplasma phagocytophilum, Ehrlichia canis, Borrelia burgdorferi and Dirofilaria immitis were assessed using point of care qualitative ELISA assays. Faecal flotation was performed on fresh faecal samples to screen for intestinal parasites. Ninety-three faecal samples were also tested for Giardia and Cryptosporidium spp. The median age of dogs was one year, with a range of four months to eight years and 73.3% were male. The vast majority of dogs were owned, the remaining were stray animals. Prevalence of D. immitis was 46.8% and A. phagocytophilum seroprevalence was 8.4%. All serum samples tested negative for E. canis, B. burgdorferi and L. infantum. Prevalence of hookworm was 92.6%. Trichuris vulpis, Dipylidium caninum, Toxocara canis and Capillaria spp. were also detected. Prevalence of Giardia spp. was 29.0% while no Cryptosporidium was detected. Fleas were found on 83.7% of the dogs, ticks on 42.1% and lice on 8.1%. Identified ticks were Rhipicephalus sanguineus, with no Ixodes spp. found. The results indicate a very high prevalence of hookworm, D. immitis, and external parasites in Samoan dogs. This study provides valuable information on canine health and suggests dogs could play a role in the spread of some zoonoses in Samoa. Further studies are required to review the public health implications of this study.