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Detection of Human Rotavirus in Southern Ontario Source Waters

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Detection of Human Rotavirus in Southern Ontario Source Waters Detection of Human Rotavirus in Southern Ontario Source Waters A Thesis Presented to The University of Guelph By Bailey Helena Davis In partial fulfilment of requirements for the degree of Master of Science in Environmental Biology Guelph, Ontario, Canada © Bailey Helena Davis, December, 2012 ABSTRACT Detection of Human Rotavirus in Southern Ontario Source Waters Bailey Helena Davis Advisors: S. N. Liss University of Guelph, 2012 M. B. Habash As part of a larger quantitative microbial risk assessment (QMRA) study, the raw water intakes of 8 different drinking water treatment plants in Ontario were sampled for rotavirus. Group A rotavirus was detected and semi-quantified via RT-qPCR. Rotavirus was detected in 6 of 8 drinking water treatment plant raw water intakes at various sampling times during a 2 year period at estimated quantities of 0 – 513 viral genome copies/L water. As hypothesized, the virus counts showed a seasonal tendency with significant detection most likely to occur during the spring months and a correlation with turbidity measurements. To our knowledge this is the first study exploring the presence of rotavirus in Ontario source waters. With new proposed changes to the Health Canada guidelines regarding the viruses in drinking water, data on the presence of rotavirus in source waters is required for assessment of risk to public health. iii Acknowledgements Much gratitude is extended to my advisors, Steven Liss and Marc Habash. I would like to thank Steven for his support and patience as he saw me through to the end of this project even though he was hours away at Queens. Marc, I would like to thank you for all of your support and advice, especially with regards to method development. Without your input, I surely would have been lost. My lab mates were surely a key in this success. Patrick Soo, Sandra Tirado, Dr. Mehendran Basuvaraj, Mojtaba Kherzy, Derek Edwardson, and Robert Best – thank you for all of your support, encouragement, and laughter. I especially want to thank Patrick for all of his help in packing and shipping coolers weekly, for processing my last few samples while I was on leave, and most importantly, for his friendship. I want to thank my parents (especially my Mom, Wendy) and my husband, Michael, for all of their support, love, and encouragement through this endeavor. Many special thanks to my mother-in-law, Ellen, who cared for my daughter for months while I disappeared into the lab, I surely would not have been able to complete this without you. Above all, I want to express my gratitude to my beautiful daughter, Avery, born in the midst of this on June 16, 2011, for helping me to keep things in perspective and showing me what matters most in life. iv Table of Contents ABSTRACT ........................................................................................................................................ii Acknowledgements ............................................................................................................................ iii List of Tables ..................................................................................................................................... vi List of Figures .................................................................................................................................. viii List of Appendices ............................................................................................................................. ix List of Abbreviations........................................................................................................................... x Chapter 1: Introduction and Literature Review .................................................................................. 1 1.1 Rotavirus ............................................................................................................................ 1 1.2 Rotavirus and Public Health ............................................................................................... 2 1.2.1 Seasonality of Rotavirus Illness ................................................................................. 3 1.3 Persistence of Rotavirus in the environment ...................................................................... 5 1.4 Regulations and Policies on Viral Pathogens in Drinking Water .................................... 12 1.5 Methodologies for the Collection and Detection of Rotavirus ......................................... 16 1.5.1. Isolation and Concentration .................................................................................... 16 1.5.2 Detection and Enumeration ...................................................................................... 19 1.6 Quantitative Microbial Risk Assessment (QMRA) ......................................................... 22 1.7 Summary .......................................................................................................................... 23 1.8 Hypothesis ........................................................................................................................ 25 Chapter 2: Materials and Methods ............................................................................................. 26 2.1 MA104 cell line and Rotavirus WA ................................................................................. 26 2.1.1 Rotavirus Propagation .............................................................................................. 26 2.1.2 Harvesting Rotavirus ................................................................................................ 27 2.1.3 Semi-Purification of Rotavirus WA stock................................................................ 27 2.1.4 Rotavirus WA Stock Titration.................................................................................. 27 2.2 Drinking Water Treatment Plant Sampling ...................................................................... 29 2.2.1 Sampling Locations .................................................................................................. 29 2.2.2 Sampling Apparatus Construction ........................................................................... 29 2.2.3 Sampling Procedure ................................................................................................. 30 2.3 Sample Processing ........................................................................................................... 32 v 2.3.1 NanoCeram Filter Elution ........................................................................................ 32 2.3.2 Organic Flocculation ................................................................................................ 34 2.4 Rotavirus Detection .......................................................................................................... 35 2.4.1 Construction of a DNA plasmid standard ................................................................ 35 2.4.2 NanoDrop Spectrophotometer 2000 verification ..................................................... 36 2.4.3 RNA Extraction with MoBioPowerWater RNA Isolation Kit ................................. 36 2.4.4 cDNA synthesis by RT-PCR .................................................................................... 37 2.4.5 Real-time Polymerase Chain Reaction (qPCR)........................................................ 37 2.4.5. Check for PCR inhibition ......................................................................................... 38 2.4.6 Check for inhibition relief using BSA and DMSO .................................................. 38 2.4.7. Extraction Controls .................................................................................................. 39 2.5 Determination of virus recovery ...................................................................................... 40 2.6 Statistical Analysis ........................................................................................................... 41 Chapter 3: Results ...................................................................................................................... 42 Chapter 4: Discussion ................................................................................................................ 59 References ........................................................................................................................................ 70 Appendix 1 ....................................................................................................................................... 79 Appendix 2 ....................................................................................................................................... 82 vi List of Tables Table 1.1 Detection of rotavirus in source waters from a selection of publication 8 Table 1.2 Detection of rotavirus in sewage and wastewater from a selection of publications 10 Table 1.3 Different eluents used for virus elution from positively charged filters 18 Table 3.1 Results of preliminary test for PCR inhibition of environmental sample concentrates 44 Table 3.2 Results of preliminary testing of BSA and DMSO for relief of PCR inhibition from environmental sample concentrates 45 Table 3.3 RNA concentration of the extraction controls and RT-qPCR results back calculated to rotavirus genome copies per ng of RNA in the RT-PCR reaction 47 Table 3.4 Samples positive for rotavirus listed by plant and season 51 Table 3.5 Statistical calculation for Satterthwaite t-test comparing the virus counts
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