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
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Date
2021
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Massey University
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Abstract
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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Cryptosporidiosis, Giardiasis, New Zealand, Epidemiology, Cryptosporidium, Giardia, Variation (Biology)