Surveillance of Microbial Pathogens in the Umgeni River, Durban South Africa ________________________________________________________________________ Atheesha Singh Submitted in fulfilment of the academic requirements for the degree of Doctor of Philosophy in the Discipline of Microbiology; School of Life Sciences, College of Agriculture, Engineering and Science at the University of KwaZulu-Natal, Westville Campus. As the candidate’s supervisor, I have approved this thesis for submission. Signed: Name: Professor Johnson Lin Date: i PREFACE The experimental work described in this dissertation was carried out in the Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Durban, South Africa from March 2010 to November 2012, under the supervision of Professor Johnson Lin. These studies represent original work by the author and have not otherwise been submitted in any form for any degree or diploma to any tertiary institution. Where use has been made of the work of others it is duly acknowledged in the text. ii COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 1 - PLAGIARISM I, Mrs. Atheesha (Ganesh) Singh, declare that 1. The research reported in this thesis, except where otherwise indicated, is my original research. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signed ………………………………………… Atheesha (Ganesh) Singh (204007017) Declaration Plagiarism 22/05/08 FHDR Approved iii COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 2 - PUBLICATIONS DETAILS OF CONTRIBUTION TO PUBLICATIONS that form part and/or include research presented in this thesis (include publications in preparation, submitted, in press and published and give details of the contributions of each author to the experimental work and writing of each publication) Publication List Publication 1 Title: Waterborne Human Pathogenic Viruses of Public Health Concern Authors: Atheesha Ganesh and Johnson Lin Journal: International Journal of Environmental Health Research Published: online 2013- http://dx.doi.org/10.1080/09603123.2013.769205 Contributions: Miss Atheesha Ganesh performed the writing of the review article. Professor Johnson Lin performed the editing of the article and submission to the journal. The article forms part of Chapter One of the thesis. Publication 2 Title: Water Quality Indicators: Bacteria, Coliphages, Enteric Viruses Authors: Johnson Lin and Atheesha Ganesh Journal: International Journal of Environmental Health Research Published: online 2013- http://dx.doi.org/10.1080/09603123.2013.769201 Contributions: Both authors contributed equally to the writing of the review article. Professor Johnson Lin submitted the article to the journal. The article forms part of Chapter One of the thesis. Publication 3 Title: Canonical Correspondence Analysis (CCA) for the microbiological, physico-chemical and heavy metal assessment of the Umgeni River-South Africa Authors: Atheesha Ganesh and Johnson Lin Journal: Water SA Published: Submitted Revision Contributions: Miss Atheesha Ganesh performed all experimental work and the writing of the article. Professor Johnson Lin performed the editing of the article and submission to the journal. The article forms Chapter Two of thesis. iv Publication 4 Title: Microbial Pathogens in the Umgeni River- WRC Report No. K8/990 Authors: Johnson Lin, Atheesha Ganesh and Moganavelli Singh Journal: Water Research Commission- Technical Report Published: In Press Contributions: Miss Atheesha Ganesh performed all experimental work and the writing of the report. Professor Johnson Lin performed the editing of the report and submission to the journal. Dr Singh assisted in tissue culture experimentation. The technical forms part of chapter One, Two, Three and Four of the thesis. Publication 5 Title: Detecting the Presence of Virus-like Particles (VLPs) from the Umgeni River-South Africa Authors: Atheesha Ganesh and Johnson Lin Journal: Clean-Soil, Air, Water Published: Submitted Revision Contributions: Miss Atheesha Ganesh performed all experimental work and the writing of the article. Professor Johnson Lin performed the editing of the article and submission to the journal. The article forms chapter three of the thesis. Publication 6 Title: Detection and Quantification of Hepatitis B Viruses in the Umgeni River-South Africa Authors: Atheesha Ganesh and Johnson Lin Journal: Environment International Published: Submission Contributions: Miss Atheesha Ganesh performed all experimental work and the writing of the article. Professor Johnson Lin performed the editing of the article and submission to the journal. The article forms part of Chapter Three and Four of the thesis. Conference Presentations Conference 1 Title: Detection of human pathogenic viruses in the Umgeni River of Durban, South Africa Authors: Atheesha Ganesh and Johnson Lin Conference: 2011 South African Society For Microbiology (SASM) Meeting, Southern Sun Cape Sun Hotel, Cape Town, South Africa, 6-9 November 2011 Contributions: Oral Presentation v Conference 2 Title: Detection of human pathogenic viruses in the Umgeni River of Durban, South Africa Authors: Atheesha Ganesh and Johnson Lin Conference: South Africa, 23rd congress of the South African Society of Biochemistry and Molecular Biology/ Federation of African Societies of Biochemistry and Molecular Biology (SASBMB-FASBMB congress), Champagne Sports Resort, Drakensberg, KZN, South Africa 29 January-1 February 2012. Contributions: Poster Presentation Conference 3 Title: Detection of human pathogenic viruses in the Umgeni River of Durban, South Africa Authors: Atheesha Ganesh and Johnson Lin Conference: First Biotechnology World Congress, Dubai Men’s College, Dubai, 14-15 February 2012 Contributions: Poster Presentation Conference 4 Title: Detecting the Presence of Virus-like Particles (VLPs) from the Umgeni River-South Africa Authors: Atheesha Ganesh and Johnson Lin Conference: School of Life Sciences and Engineering First Research Day, College of Agriculture, Engineering and Science, PMB Campus- UKZN, 29 October 2012. Contributions: Poster Presentation Conference 4 Title: Evaluation of the virus Population in the Umgeni River-South Africa Authors: Atheesha Ganesh and Johnson Lin Conference: 2nd International Water Research Conference, Singapore, 20-23rd January 2013 Contributions: Poster Presentation Signed: __________________________ Atheesha (Ganesh) Singh Declaration Publications FHDR 22/05/08 Approved vi TABLE OF CONTENTS CONTENTS PAGE PREFACE ii DECLARATION 1 – PLAGIARISM iii DECLARATION 2 – PUBLICATIONS iv ACKNOWLEDGEMENTS xi DEDICATION xiii ABSTRACT xiv LIST OF FIGURES xv LIST OF TABLES xxi CHAPTER ONE: Literature Review 1 1.1 Introduction 2 1.2 Current Environmental Water Situation in South Africa 2 1.3 Surface Water Pollution: Sources, Indicator Organisms and Detection Methods 4 1.3.1 Microbial Indicators of Water Quality and their Detection Methods 6 1.3.1.1 Total Coliforms and Faecal Coliforms 7 1.3.1.2 Faecal Streptococci and Enterococci 7 1.3.1.3 Vibrio cholerae 8 1.3.1.4 Bacteriophages 9 1.3.1.5 Somatic Coliphages 10 1.3.1.6 Male-Specific F-RNA Coliphages 10 1.3.2 Human Pathogenic Viruses as Potential Indicators of Water Quality 12 1.4 Viral Studies in Freshwater Environments 13 1.5 Human Pathogenic Viruses Present in Environmental Waters 14 1.6 Waterborne Human Pathogenic Viruses of Public Health Concern and their Associated Illnesses 17 1.6.1 Adenovirus (AdV) 19 1.6.2 Enterovirus (EV) 20 1.6.3 Hepatitis A Virus (HAV) 21 1.6.4 Norovirus (NoV) 23 vii 1.6.5 Rotavirus (RV) 25 1.6.6 Astrovirus (AstV) 26 1.7 Hepatitis B Virus 27 1.8 Methods for Isolating Viruses in Environmental Waters 27 1.8.1 Glass-Wool Adsorption-Elution Method 28 1.8.2 Ultrafiltration and Tangential Flow Filtration 29 1.8.3 Organic Flocculation 30 1.9 Methods for Detecting the Presence of Viruses in Environmental Waters 30 1.9.1 Cell-Culture Techniques 30 1.9.2 Electron Microscopy 32 1.9.2.1 Epifluorescence Microscopy (EFM) 32 1.9.2.2 Transmission Electron Microscopy (TEM) 33 1.9.3 Flow Cytometry (FCM) 33 1.9.4 Polymerase Chain Reaction (PCR) 34 1.9.5 Real-Time (RT) PCR 35 1.9.6 Pulsed-Field Gel Electrophoresis (PFGE) 36 1.9.7 Metagenomic Sequencing 36 1.9.8 Microfluidic Digital PCR 37 1.10 Scope of Present Study 37 1.11 Hypothesis 38 1.12 Research Objectives: 38 1.13 Experimental Design 38 CHAPTER TWO: Microbial and Physico-Chemical Quality of the Umgeni River in Durban, South Africa 40 2.1 Introduction 41 2.2.2 Determination of Physico-Chemical Parameters 45 2.2.2.1 Biological Oxygen Demand 46 2.2.2.2 Chemical Oxygen Demand 46 viii 2.2.3 Enumeration of Bacterial Indicator Microorganisms 46 2.3 Statistical Analysis 47 2.4 Results 48 2.4.1