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Factors Influencing Overland Mobility of Cryptosporidium Oocysts

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Factors Influencing Overland Mobility of Cryptosporidium Oocysts Factors Influencing Overland Mobility of Cryptosporidium Oocysts A dissertation submitted by Christine E. Kaucner In fulfilment for the requirements of the degree of Master of Science (MSc.) Centre for Water and Waste Technology School of Civil and Environmental Engineering The University of New South Wales January 2007 ABSTRACT The mechanisms responsible for overland transport of faecal pathogens, particularly Cryptosporidium oocysts, from animal sources to water bodies are not fully understood. Surface properties of microbes, such as electrostatic charge and hydrophobicity, are thought to contribute to their aggregation and attachment to solid surfaces. There is conflicting evidence that methods used to purify Cryptosporidium oocysts from faecal material may affect the oocyst surface, leading to biased conclusions from transport studies. By studying oocyst surface properties, aggregation and soil attachment, this thesis addressed whether oocyst purification methods influence overland transport studies, and whether oocysts are likely to be associated with particles during transport. When using the microbial adhesion to hydrocarbon (MATH) assay with octane, oocyst hydrophobicity was shown to be method and isolate dependent, with oocysts displaying moderate to high hydrophobicity in 0.01 M KNO3. There was no observed attachment, however, to the hydrophobic octyl-SepharoseTM bead ligands when using the same suspension solution. Oocyst age did not appear to influence their hydrophobicity. A small but statistically significant proportion of oocysts displayed a net negative surface charge as observed by their attachment to an anion exchange ligand (DEAE). There was no difference in hydrophobicity or surface charge observed between purified oocysts and oocysts that had been extracted without the use of harsh chemicals and solutions with dehydrating properties. Purified oocysts did not aggregate at pH values between 3.3 and 9.0, nor in solutions lower than 0.59 M in ionic strength at a pH 2.7 which is approaching the reported isoelectric point of oocysts. This finding suggests that oocysts may not form aggregates under general environmental conditions. The association of purified oocysts with soil particles was observed in settling columns. Attachment to soil particles was not conclusive since the settling of the soil particles may have entrained single oocysts. Nonetheless, approximately 27% of oocysts were estimated to be unbound to soil or associated with small soil particles. Hence models for oocyst overland transport should consider a significant fraction as single entities or associated with soil particles less than about 3 m in size. i ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ………………………………. Date ………………………………. ii ACKNOWEDGMENTS First and foremost I must acknowledge the support of my supervisors, Professor Nicholas Ashbolt and Dr. Cheryl Davies. I thank them for their guidance, and for not giving up on me when life, or work, overcame my part-time study endeavours. Next I wish to thank those involved with the AwwaRF-CRCWQT project. There were many who worked on the project, but in particular I’d like to thank Dr. Christobel Ferguson (Ecowise Environmental) who was the project manager. Thanks also go to the staff from the Centre for Water and Waste Technology who supported this project, in particular Dr. Gautam Chattopadhyay, Lynette Menzies and Robbie Smith. I am thankful to the Cooperative Research Centre for Water Quality and Treatment for the support they have provided me. Including me under the umbrella of their organisation and providing financial support for overseas travel is gratefully acknowledged. From BTF Decisive Microbiology I would like to thank Dr. Graham Vesey for supplying the Iowa strain of Cryptosporidium oocysts, and also allowing me the use of a flow cytometer. Thanks go to Jin Chung (also a UNSW PhD student) for her help with flow cytometry. Thanks also to Associate Professor Justin Brookes (Adelaide University) who answered many questions about Stoke’s Law, Stephen Burgun who allowed me access to Arthursleigh Farm for soil collection, the Leppington Pastoral Company for permitting collection of faecal material from their calves and Dr. Michael Storey (Sydney Water) for proof reading this thesis. To my parents; I thank them for all their support over the years, both financial and otherwise. Thanks also to Gabriel and Julie Dayeh who have become my surrogate Sydney family. And a million thanks go to Andrew for his patience and understanding while I completed this work. And finally, I’d like to dedicate this thesis to my sister who was always proud of me and my achievements. I know she would have also been proud of this work. iii iv TABLE OF CONTENTS Abstract.................................................................................................................................. i Acknowedgements............................................................................................................... iii Table of Contents ................................................................................................................. v Index of Figures.................................................................................................................... x Index of Tables .................................................................................................................. xiii Abbreviations .................................................................................................................... xiv Chapter One Introduction.................................................................................................. 1 Chapter Two Background.................................................................................................. 4 2.1 Introduction.............................................................................................................. 4 2.2 Cryptosporidium Background.................................................................................. 7 2.2.1 Brief history of Cryptosporidium............................................................................... 7 2.2.2 Cryptosporidium in the environment ......................................................................... 9 2.2.3 Cryptosporidium oocyst viability and infectivity .................................................... 13 2.2.4 Cryptosporidium oocyst survival ............................................................................. 15 2.3 Oocyst Isolation from Faecal Material................................................................. 16 2.4 Surface Chemistry.................................................................................................. 19 2.4.1 Colloidal chemistry .................................................................................................. 21 2.4.1.1 DLVO theory............................................................................................... 23 2.4.1.2 The van der Waals interaction...................................................................... 24 2.4.1.3 The electrical double layer interaction......................................................... 25 2.4.2 Measurement of surface chemistry .......................................................................... 26 2.4.2.1 Microbial adhesion to hydrocarbons (MATH) ............................................ 28 2.4.2.2 Hydrophobic interaction chromatography (HIC)......................................... 29 2.4.2.3 Contact angle................................................................................................ 31 2.4.2.4 Atomic force microscopy............................................................................. 31 2.4.2.5 Salting-out.................................................................................................... 31 2.4.2.6 Surface charge.............................................................................................. 32 v 2.4.3 Cryptosporidium surface properties......................................................................... 33 2.4.3.1 Oocyst hydrophobicity..................................................................................33 2.4.3.2 Oocyst surface charge ...................................................................................34 2.5 Aggregation and Attachment.................................................................................36 2.5.1 Particle aggregation..................................................................................................36 2.5.2 Oocyst attachment and aggregation ......................................................................... 37 2.6 Sedimentation Kinetics...........................................................................................39 2.6.1 Gravitational
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