This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Alzaylaee, Hind A O Title: Molecular approaches to improve understanding of the distribution of human-infecting Schistosoma species General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. 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Molecular approaches to improve understanding of the distribution of human-infecting Schistosoma species Hind Alzaylaee A dissertation submitted to the University of Bristol in accordance with the requirements of the degree of Doctor of Philosophy (PhD) in the Faculty of Life Sciences School of Biological Sciences, March 2020 Word Count: 36,090 i Abstract It is estimated that over 200 million people are at risk from schistosomiasis, a Neglected Tropical Disease caused by Schistosoma trematode parasites that has significant health and socioeconomic consequences. To enable the successful control and eradication of this disease, it is crucial to develop and employ accurate diagnostics of Schistosoma trematodes in the natural environment. The overarching aims of the work for this thesis were i) to assess the potential of environmental DNA (eDNA)-based approaches to assess the distribution and abundance of schistosomes, and ii) to investigate the utility of eDNA to enable a broader understanding of the ecology of the freshwater communities where schistosomes are found. Environmental DNA-based tests for the human-infecting schistosomes S. mansoni and S. haematobium are reported. These are shown to exhibit a high-level of species specificity, especially the S. mansoni assay, and shown to be capable of detecting schistosomes in natural freshwater environments. A novel eDNA xenomonitoring approach to detect the schistosome infection in intermediate host snails is reported. The results correspond strongly with those obtained from direct tests of snails infection status from molecular analyses of their tissue samples, suggesting eDNA-based xenomonitoring could be a powerful surveillance tool for assessing infection status of host snail populations. A metabarcoding study of biological diversity within African freshwaters is reported. The results highlight the potential of eDNA metabarcoding for the characterization of metazoan communities from different habitats, but clearly demonstrate the need for very high levels of sequence read coverage and/or taxon-specific primers to more fully describe the biological communities present. Overall, the results presented support the concept that eDNA- based approaches are useful for monitoring schistosome presence in endemic freshwaters, potentially enabling enhanced detection of schistosomiasis infection risk, and evaluating the success of interventions to control and eliminate this disease. ii Dedication I dedicate my thesis and PhD journey to my loving parents. I am extremely grateful for the unconditional love and support they have shown me, as well as the countless sacrifices they have made to support my development. In fact, without their compassion and encouragement, this achievement would not have been possible. Long may their love cultivate my success. I would have been very happy if I could share this moment with my father and older brother but, tragically, they passed away during my PhD study. It was a particularly hard time for me, but I am convinced that their souls are always by my side. I am absolutely certain that I have made them both proud and I will not forget them in my prayers. I would also like to thank my family, brothers and sisters for their support, prayers and for not leaving my side. I also dedicate my work to my dear husband and would like to specially thank him for his love, faith, understanding, acceptance, prayers and constant support to pursue my dreams. He unconditionally supports me in all my endeavours and motivates me to do better. Without his patience for the past two years, I would not have been to complete my PhD. I would further like to thank him for teaching me to believe in God, in myself, and in my dreams. iii Acknowledgments I wish to express sincere gratitude to my research supervisor, Prof. Martin Genner, for giving me the opportunity to carry out my PhD research under his kind supervision. His constant encouragement and valuable guidance have been instrumental for carrying out this research project. He taught me the methodology I used as well as the best ways to carry out and present one's research with clarity and impact. I also express my thanks to Prof. Eric Morgan for his help, guidance and knowledge. Both of them have been crucially enabling for broadening my knowledge by attending courses, workshops and conferences. I am grateful to my sponsors, Princess Nourah Bint Abdul Rahman University and Saudi Arabia Culture Bureau in UK, for funding my scholarship and giving me the opportunity to study abroad. I extend a special thanks to Dr. Rupert Collins for helping me in the laboratory and providing essential advice during my research process, as well Prof Richard Wall for his support. I would like to thank Prof. Russell Stothard, Dr. Gabriel Rinaldi, Dr. Benjamin Ngatunga, Dr Asilatu Shechonge and Zikmund Bartonicek for providing me with the samples for my study. I am thankful to my research group, especially Carlos for his help, as well as Pavrally, Girardo and Sandra. Dr. Martha Betson was kind enough to let me train in her lab at University of Surrey and taught me the necessary molecular skills for my lab work, for which I am very grateful. I thank my friend Maryam, who was always there when I needed and made the PhD journey more enjoyable, and Mona and Shatha, with whom I have had many informative and motivating conversations with. iv Author’s Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: DATE: 02.03.2020 v Table of Contents Abstract ………………………………………………………………………………………………………………………………… ii Dedication …………………………………………………………………………………………………………………………….. iii Acknowledgements ………………………………………………………………………………………………………………. i v Author’s declaration ……………………………………………………………………………………………………………… v Table of contents ………………………………………………………………………………………………………………….. vi List of figures ………………………………………………………………………………………………………………………… x List of tables …………………………………………………………………………………………………………………………. xii Abbreviations………………………………………………………………………………………………………………………… xiii Publications …………………………………………………………………………………………………………………………… xv Chapter 1: General Introduction………………………………………………………………………………………… 1 1.1 Schistosomiasis …………………………………………………………………………………………………………..…… 2 1.2 The biology of schistosomes and their host species ………………………………………………………… 2 1.2.1 Species and their definitive host …………………………………………………………………………………… 2 1.2.2 Schistosoma (life cycle) ………………………………………………………………………………………………… 3 1.2.3 Intermediate host – freshwater snails ………………………………………………………………………….. 5 1.3 The distribution of Schistosoma and the epidemiology of schistosomiasis ………………………. 15 1.4 Pathology and morbidity of schistosomiasis …………………………………………………………............ 18 1.4.1 Cercarial dermatitis ………………………………………………………………………………………………………. 19 1.4.2 Acute schistosomiasis (Katayama syndrome) ……………………………………………………………….. 19 1.4.3 Chronic schistosomiasis ………………………………………………………………………………………………… 20 1.5 Treatment of schistosomiasis ………………………………………………………………………………………….. 22 1.6. Control strategies …………………………………………………………………………………………………………… 24 1.7 Diagnosis of schistosome presence in the natural environment ………………………………………. 27 vi 1.7.1 Molecular techniques …………………………………………………………………………………………........... 28 1.7.2 Environmental DNA ……………………………………………………………………………………………………… 28 1.8 Aims ………………………………………………………………………………………………………………………………… 35 Chapter 2: Schistosoma species detection by environmental DNA assays in African 36 freshwaters ……………………………………………………………………………………………………………………… Abstract ……………………………………………………………………………………………………………………………….. 37 2.1 Introduction ……………………………………………………………………………………………………………………. 38 2.2 Materials and Methods