Investigation of Environmental Reservoirs of Strongyloides stercoralis as Targets for Utilising Nematophagous Fungi as Potential Biocontrol Agents A thesis submitted for the degree of Doctor of Philosophy Tara A. Garrard, Bachelor of Medical Science, Bachelor of Science (Hons) November 2018 College of Science and Engineering Flinders University of South Australia Australia TABLE OF CONTENTS Contents THESIS ABSTRACT .......................................................................................................... i ACKNOWLEDGEMENTS ............................................................................................... iii DEDICATION ................................................................................................................. v LIST OF FIGURES .......................................................................................................... vi LIST OF TABLES .......................................................................................................... xiii 1. Chapter One: General Introduction ..................................................................... 1 1.1. Human strongyloidiasis: identifying knowledge gaps, with emphasis on environmental control ............................................................................................. 3 1.1.1. Abstract .................................................................................................. 3 1.1.2. Key learning points ................................................................................. 4 1.1.3. Key Words .............................................................................................. 5 1.1.4. Introduction ........................................................................................... 5 1.1.5. Methods ................................................................................................. 7 1.1.6. Conclusion ............................................................................................ 23 1.1.7. References ............................................................................................ 24 1.2. Biocontrol for environmental control of the human parasite, Strongyloides stercoralis: A necessary next step .......................................................................... 40 1.2.1. Abstract ................................................................................................ 40 1.2.2. Introduction ......................................................................................... 41 1.2.3. Nematophagus fungi and biocontrol in agricultural crops .................. 44 1.2.4. Human health and biocontrol .............................................................. 45 1.2.5. Strongyloides stercoralis ...................................................................... 46 1.2.6. Use of biocontrol and Strongyloides species ....................................... 48 1.2.7. Conclusion ............................................................................................ 51 1.2.8. Keywords .............................................................................................. 51 1.2.9. References ............................................................................................ 51 1.3. Research aims and directions ...................................................................... 58 2. Chapter Two: Use of real-time PCR for detection of Strongyloides stercoralis in environmental samples .............................................................................................. 60 2.2. Introduction ................................................................................................. 60 2.2.1. Environmental samples ........................................................................ 60 2.3. Real-time PCR analysis method ................................................................... 62 2.3.1. Sample storage ..................................................................................... 62 2.3.2. DNA extraction ..................................................................................... 62 2.3.3. Real-time PCR analysis of soil and canine faecal samples from the Northern Territory .............................................................................................. 62 2.3.4. Samples tested ..................................................................................... 65 2.4. Results ......................................................................................................... 65 2.5. Issues and pathways forward ...................................................................... 69 2.5.1. Degradation of DNA in environmental samples .................................. 69 2.5.2. Sample collection issues....................................................................... 70 2.5.3. Future Pathways ................................................................................... 71 2.6. References ................................................................................................... 71 3. Chapter Three: Isolation and characterisation of nematophagous fungi from Northern Territory soils .............................................................................................. 72 3.1. Introduction ................................................................................................. 72 3.1.1. Biological control methods .................................................................. 73 3.1.2. Parasitic nematodes and nematophagous fungi ................................. 74 3.2. Isolation or identification of nematophagous fungi from soil methods ..... 77 3.2.1. Culture methods................................................................................... 77 3.2.2. Molecular methods .............................................................................. 79 3.2.3. Ethics of augmentation biocontrol method in Indigenous communities 81 3.3. Aims and Objectives .................................................................................... 83 3.4. Methods ...................................................................................................... 83 3.4.1. Media and reagents ............................................................................. 83 3.4.2. Free-living nematode used in isolation ................................................ 86 3.4.3. Soil Samples .......................................................................................... 87 3.4.4. Plate Method for the isolation of Paecilomyces lilacinus from soils ... 87 3.4.5. Sprinkle plate technique ...................................................................... 88 3.4.6. Method positive control....................................................................... 89 3.4.7. Assessment of cultures isolated from sprinkle plate technique .......... 89 3.4.8. Viability stains ...................................................................................... 90 3.5. Results ......................................................................................................... 90 3.5.1. Northern Territory soils ........................................................................ 90 3.5.2. Paecilomyces lilacinus selective media .............................................. 108 3.5.3. Positive control................................................................................... 111 3.6. Discussion .................................................................................................. 115 3.7. Assessment of fungal cultures isolated from the sprinkle plate technique 115 3.7.1. Microscopy – Dissecting microscope ................................................. 116 3.7.2. Microscopy - Compound microscope ................................................ 117 3.7.3. Presence of nematophagous fungi in the sample? ............................ 125 3.7.4. Fungal species that are not strictly nematophagous but display similar properties ......................................................................................................... 126 3.7.5. Other potential detection methods ................................................... 129 3.7.6. Use of isolates in further work with Strongyloides spp. .................... 130 3.8. References ................................................................................................. 131 4. Chapter Four: Nematophagous fungi efficacy in varying substrates and nutrient levels ......................................................................................................................... 136 4.1. Introduction ............................................................................................... 136 4.2. Methods .................................................................................................... 140 4.2.1. Soil Nutrient analysis .......................................................................... 140 4.2.2. Nematophagous efficacy.................................................................... 142 4.2.3. Statistical analysis .............................................................................. 143 4.3. Results ....................................................................................................... 143 4.3.1. Nematode survival and nematophagous efficacy.............................. 143 4.3.2. Nutrient Analysis ................................................................................ 146 4.4. Discussion .................................................................................................