Identification of Giardia duodenalis in Environmental and Animal Samples in Scotland with Development of Novel Approaches of Filtration Elution Ben Horton MSc by Research Institute of Biological Chemistry, Biophysics and Bio-engineering. Heriot-Watt University September 2016 “The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information contained in it must acknowledge this thesis as the source of the quotation or information." Abstract Giardia duodenalis is a waterborne flagellated protozoan parasite known to cause substantial cases of disease throughout the world. The parasite is argued to be zoonotic, and as such consumption of water contaminated by animal faeces containing parasite cysts is thought to lead to human infection. Infection is skewed towards the developing world, but outbreaks do occur within the developed world. This project had two aims: to identify the prevalence of G. duodenalis within a range of Scottish samples, both faecal and water, and to develop a novel method for the elution of G. duodenalis cysts from filter matrixes by incorporating megasonic sonication into a pre-existing method – the FiltaMax system. Molecular techniques found that water samples within this study were mostly negative for parasite DNA, however faecal samples were often positive, with animal samples testing sporadically positive throughout the study. A novel methodology for filter matrix elution of G. duodenalis cysts was developed and proven to be comparable to current leading filtration methods. This megasonic method also boasted significant advantages over the FiltaMax system: such as reduction in labour involved, substantially reduced damage of the parasite during elution and future automation is a possibility. 2 Acknowledgements I would like to thank both of my supervisors, Dr Frank Katzer and Dr Helen Bridle for all of their support, expertise and advice which was given throughout the duration of this research project. I would also like to thank Moredun Scientific who made this project a possibility via both funding and allocation of time within my working week to complete project work. Finally, my thanks also go to staff within the protozoology division of The Moredun Research Institute for their helpful nature during this project, particularly Clare Hamilton, Jackie Thompson and Paul Bartley. Contents 1. Chapter 1 – Introduction ..................................................................... 1 1.1 Giardia Infection, Epidemiology and Assemblages ........................... 1 1.2 Giardiasis in the UK ............................................................................. 3 1.3 Nomenclature history of Giardia species ............................................ 7 1.4 Giardia duodenalis – Assemblages and Sub-Assemblages .............. 10 1.4.1 Sub-Assemblages of Giardia duodenalis and the debate for further .................. speciation. ......................................................................................................... 11 1.5 The Morphology and Lifecycle of Giardia duodenalis..................... 13 1.5.1 Cyst Morphology .............................................................................................. 14 1.5.2 Trophozoite Morphology .................................................................................. 15 1.5.3 Lifecycle of Giardia duodenalis ....................................................................... 16 1.6 Filtration Methodologies for Waterborne Pathogens……………19 1.7 Zoonotic Giardiasis – Risk or no risk? ............................................. 20 1.7.1 Livestock and Farmyard Animals as Risk of Zoonotic Infection ...................... 21 1.7.2 Companion Animals and Risk of Zoonotic Infection ........................................ 25 1.7.3 Wildlife and Risk of Zoonotic Infection ............................................................ 26 1.7.4 Level of Threat of Zoonotic Giardia Assemblages to Public Health................ 27 1.8 Infection, Diagnosis and Treatment of Giardia duodenalis in Humans ...................................................................................................... 29 1.8.1 Giardiasis and Infection in Humans ................................................................ 29 1.8.2 Diagnostic Methods for G. duodenalis Infection ............................................. 31 1.8.3 Treatment of Giardiasis .................................................................................... 33 1.9 Project Brief, Outline and Aim .......................................................... 34 2 2. Chapter 2 – Development of Novel Approaches of Giardia duodenalis Filtration Elution using Megasonic Sonication. .................. 36 2.1 Waterborne Disease and Water Regulations ................................... 36 2.1.1 Current Filtration of Giardia duodenalis from Water Sources ....................... 37 2.2 Megasonic Sonication and Incorporation into Giardia duodenalis Filtration .................................................................................................... 39 2.3 Materials and Methodologies ............................................................. 41 2.3.1 Viability Assessment of Megasonic Exposed Cysts .......................................... 41 2.3.2 Direct Membrane Seeding ................................................................................ 43 2.3.3 Seeding into Increased Eluting Volume (1200ml PBST) .................................. 45 2.3.4 Seeding into Sponge Filter Matrices ................................................................ 46 2.3.5 A Complete Procedure of G. duodenalis Elution using Megasonic .................... Sonication ......................................................................................................... 46 2.4 Results and Discussion........................................................................ 47 2.4.1 Viability Assessment of Megasonic Exposed Cysts .......................................... 47 2.4.2 Megasonic Elution from Directly Seeding Membranes.................................... 48 2.4.3 Seeding into Increased Eluting Volume (1200ml PBST) .................................. 50 2.4.4 Megasonic Elution from Sponge Filter Matrices ............................................. 53 2.4.5 A Complete Procedure of Giardia duodenalis Elution using Megasonic Sonication .................................................................................................................... 55 2.5 Conclusion ........................................................................................... 58 3. Chapter 3 – Identification of G. duodenalis in Environmental Samples in Scotland .................................................................................. 60 3.1 Environmental Contamination of Giardia duodenalis within Scotland ...................................................................................................... 60 3.2 Materials and Methodologies ............................................................. 62 3.2.1 DNA Samples .................................................................................................... 62 3 3.2.2 DNA Standards (Plasmids) .............................................................................. 62 3.2.3 PCR (qPCR and n-PCR) Methods .................................................................... 64 3.3 Results and Discussion of Environmental DNA Screening............. 71 3.3.1 Detection of G. duodenalis in Scottish Water .................................................. 71 3.3.2 Cattle Faecal DNA Sample Results .................................................................. 75 3.3.3 Conclusion ........................................................................................................ 80 4. Chapter 4 – General Discussion ........................................................ 82 4.1 Discussion of Overall Results ............................................................. 82 5. Appendices ........................................................................................... 85 5.1 Appendix A: Scottish Water Sample Listings. ...................................................... 85 5.2 Appendix B: Cattle Sample Results & Relation to Cattle Age (weeks 0-9) ......... 90 5.3 Appendix C: Paper to be submitted to Water Research Journal ......................... 96 5.4 Appendix D: Supplementary Information .......................................................... 118 5.4.1 Images of G. duodenalis Cysts ............................................................................. 118 5.4.2 Diagrams of Filta-Max™ Standard Methodology ................................................... 119 6. Thesis References .............................................................................. 119 4 1. Chapter 1 – Introduction 1.1 Giardia Infection, Epidemiology and Assemblages Giardia as a genus consists of several distinct species which are all known to have specific host ranges, with several being very narrow and others being widespread in terms of host range. Specifically, only G. duodenalis is of concern with regards to public health, as only this species is known to infect humans. However, it is also the species of the genus with the widest host range, thus has the potential to be zoonotic. The answer to the question, “is this parasite zoonotic”, has been a cause of much debate since the discovery of G. duodenalis as an intestinal protozoon of man (Feng & Xiao, 2011). Infection with the