Environmental Surveillance of Human and Animal Pathogens in African River Water Using Metagenomics Alexandra Kossik A thesis submitted in partial fulfillment of the requirements for the degree of: Master of Public Health University of Washington 2020 Committee: J. Scott Meschke, Chair Alison Cullen Nicolette Zhou Program Authorized to Offer Degree: Environmental and Occupational Health Sciences School of Public Health ©Copyright 2020 Alexandra Kossik University of Washington Abstract Environmental Surveillance of Human and Animal Pathogens in African River Water Using Metagenomics Alexandra Kossik Chair of the Supervisory Committee: Professor J. Scott Meschke Department of Environmental and Occupational Health Sciences Diarrheal disease remains a persistent global health threat and is ranked as the second leading cause of death for children under the age of five, with approximately 525,000 child deaths worldwide annually. Children with HIV are eleven times more likely to die from a diarrheal pathogen, making studies that examine disease-causing organisms in HIV burdened areas like Sub-Saharan Africa particularly crucial. In Sub-Saharan African countries, such as Botswana, infectious agents of public health concern have been detected including norovirus, rotavirus, Salmonella Typhi, Mycobacterium, Vibrio cholera, Cryptosporidium, Giardia, and E. coli. As enteric viruses are responsible for a majority of acute waterborne diseases, their surveillance is critical to understand disease prevalence within a community. The specific aims for this project were (1) to analyze Chobe River water samples for both human and animal bacterial, protozoan, and viral pathogens using next-generation sequencing and (2) to determine the relevance and applicability of environmental surveillance and the analyzed Chobe River water samples to nationally reported health data, policy interests, and national priorities in order to determine potential public health and policy impact. A novel environmental surveillance method, originally developed for poliovirus detection called the bag-mediated filtration system (BMFS), enables in-field collection and filtration of large sample volumes. As the BMFS has been shown to be an effective way to sample, process, and analyze large volumes of wastewater and surface water in multiple countries for multiple pathogens, it was an ideal method for this study. For this study BMFS environmental samples were collected at ten transect points along the Chobe River in both Spring and Summer 2017, along with Fall 2019. The sampling locations encompassed areas of wastewater discharge, pit latrine discharge, and animal-impacts. This range of samples was meant to allow for a broad overview of what is impacting the surface water and to determine if there are fluctuations within the same sampling locations between seasons. BMFS samples, 6 liters each, were collected and filtered in the field at the respective sites along the Chobe River and transferred to a field laboratory in Kasane, Botswana for elution, secondary concentration, and nucleic acid extraction. Samples were then shipped to the University of Washington in Seattle for library preparation using the Nextera DNA Flex Library Prep Kit and were then sequenced on a NextSeq or a NovaSeq, depending on sample batch. Sequencing reads were analyzed for quality, assembled with SPAdes, aligned with Kraken, and pathogens of interest were filtered out using the Rosetta Stone Microbial Database. Pathogens of interest selected using this pipeline were further analyzed through BLAST searches and KMA pipeline analysis to allow for further interrogation and taxonomic annotation. The pathogens with the five highest number of sequence alignments based on the Kraken pipeline were Bacillus cereus, Clostridium botulinum, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus anthracis. This study demonstrates promising results for possible recovery of a wide range of viruses and bacteria that could be influencing both animals and humans residing in and along the Chobe River in Kasane, Botswana. Table of Contents Abstract ......................................................................................................................................................... 3 Acknowledgements ....................................................................................................................................... 7 List of Tables ................................................................................................................................................. 8 List of Figures ................................................................................................................................................ 9 Chapter 1: Introduction .............................................................................................................................. 10 1.1 Background ....................................................................................................................................... 10 1.2 Kasane, Botswana ............................................................................................................................. 10 1.3 Chobe Research Institute and CARACAL ........................................................................................... 11 1.4 Previous Research Efforts in The Chobe District ............................................................................... 12 1.5 Metagenomics Studies ...................................................................................................................... 13 1.6 Specific Aims ..................................................................................................................................... 14 Chapter 2: Environmental Surveillance ...................................................................................................... 16 2.1 Background and Significance of Environmental Surveillance ........................................................... 16 2.2 Typhoid Programs ............................................................................................................................. 16 2.3 Polio Programs .................................................................................................................................. 16 2.4 Campylobacter Programs .................................................................................................................. 17 2.5 Antimicrobial Resistance ................................................................................................................... 17 2.6 Risk Assessment and Risk Management Importance ....................................................................... 17 2.7 Botswana Statistics ........................................................................................................................... 19 Chapter 3: Methods and Sequencing Results ............................................................................................. 21 3.1 Sampling Locations ........................................................................................................................... 21 3.2 Sample Collection and Processing .................................................................................................... 21 3.2.1 Sample Elution ............................................................................................................................... 22 3.2.2 Removal and Processing of Viral and free DNA Portion of Samples .............................................. 23 3.2.3 Secondary Concentration............................................................................................................... 23 3.2.4 Sample Extraction .......................................................................................................................... 24 3.2.5 Sample Transport ........................................................................................................................... 24 3.2.6 Sequencing Round 1 ...................................................................................................................... 25 3.2.7 Sequencing Round 2 – DNA Sample Processing ............................................................................ 25 3.2.8 Sequencing Round 3 – RNA Sample Processing ............................................................................. 25 3.2.9 Nextera DNA Flex Library Prep Kit ................................................................................................. 29 3.2.10 Illumina NextSeq and NovaSeq Sequencers ................................................................................ 29 5 3.3 Data Analysis ..................................................................................................................................... 30 3.3.1 Sequencing Read Data ................................................................................................................... 31 3.3.2 Read Assembly ............................................................................................................................... 32 3.3.3 Assembly Assignment .................................................................................................................... 34 3.3.4 Assembly Pipeline .......................................................................................................................... 34 3.3.5 Future Laboratory Analysis ...........................................................................................................