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Identification of Viral Contamination on Lettuce from the Field to Post-Harvest Processing

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Identification of Viral Contamination on Lettuce from the Field to Post-Harvest Processing IDENTIFICATION OF VIRAL CONTAMINATION ON LETTUCE FROM THE FIELD TO POST-HARVEST PROCESSING By Samantha Lynn Wengert A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Fisheries and Wildlife - Master of Science 2015 ABSTRACT IDENTIFICATION OF VIRAL CONTAMINATION ON LETTUCE FROM THE FIELD TO POST-HARVEST PROCESSING By Samantha Lynn Wengert Viral foodborne outbreaks are a serious threat to public health and fresh produce is becoming increasingly recognized as a transmission vehicle. Potential pre- and post-harvest sources of contamination include irrigation and processing water, soil, manure, equipment, and human handling. Traditional detection methods limit studies on viruses in produce. New culture-independent metagenomic next generation sequencing (NGS) technologies present an opportunity for generating an improved understanding of the virus communities (virome) associated with foods. The goals of this study were to use NGS technology for the first time to identify the virome present in irrigation water and lettuce in the field and to investigate the efficacy of current post-harvest leafy green processing and disinfection practices during a contamination event. In this study, most viruses found in irrigation water and lettuce from the field environment could not be identified suggesting limited knowledge of the virome in these environments. Human enteric viruses such as rotavirus A and picobirnavirus were identified in field lettuce. On the processing side, the efficacy of a chlorine-based sanitizer against MS2 coliphage on fresh-cut romaine lettuce was assessed during simulated commercial production of fresh-cut lettuce. Flume washing lettuce in 25 ppm of free chlorine did not significantly reduce viral levels on romaine lettuce when compared to water without chlorine. Overall, this study suggests that metagenomic technology can be used as a potential tool for monitoring food safety. Viruses were present in field lettuce and resistant to current commercial chlorine disinfection techniques, posing a possible threat to public health. Copyright by SAMANTHA LYNN WENGERT 2015 ACKNOWLEDGEMENTS This work was supported by a grant from the United States Department of Agriculture, National Institute for Food and Agriculture Grant # 11281370 Proposal 2012-04389. I would like to thank my main advisor, Dr. Joan B. Rose, for all her support and encouragement throughout my time as a graduate student at MSU. I would also like to thank Tiong G. Aw, Rebecca Ives, and other Rose Lab members for their help and support in carrying out this research project. Thank you to my committee members Dr. Mohamad Faisal and Dr. Elliot Ryser for their guidance, with special recognition to Dr. Elliot Ryser who provided the small- scale leafy green processor for our research project. I would also like to acknowledge the Research Technology Support Facility and the High Performance Computing Center at Michigan State University for sequencing and computational support as well as Channah Rock and Kurt Nolte (The University of Arizona) for helping with the Yuma field sampling. Finally, I would like to thank my family for all their love and support. iv TABLE OF CONTENTS LIST OF TABLES ...................................................................................................................... vii LIST OF FIGURES ..................................................................................................................... ix KEY TO ABBREVIATIONS...................................................................................................... xi I. LITERATURE REVIEW ......................................................................................................... 1 1. Burden of Viral Foodborne Illness Associated with Fresh Produce on Public Health ...... 1 1.1 Foodborne Disease in the United States ........................................................................... 1 1.2 Foodborne Enteric Viruses and Public Health…………………………..……….………3 1.3 Significance of Fresh Produce as a Food Commodity and Vehicle of Pathogen Transmission ........................................................................................................................... 7 2. Methods for Studying Viruses in the Environment .............................................................. 8 3. Viruses in the Environment and Pre-Harvest Sources of Fresh Produce Contamination..............................................................................................................................12 3.1 Viral Types, Characteristics, and Role in Food Industry .................................................12 3.2 Irrigation Water as an Environmental and Pre-Harvest Source of Fresh Produce Contamination ........................................................................................................................15 4. Viral Metagenomics .................................................................................................................18 4.1 Viral Metagenomic Technology .......................................................................................18 4.2 Metagenomic Insights into the Virome and Food Safety .................................................22 5. Post-Harvest Leafy Green Processing and Viral Contamination .......................................25 5.1 Leafy Green Commercial Processing Practices ...............................................................25 5.2 Post-Harvest Fresh Produce Contamination and Viral Survival on Foods ......................27 II. RESEARCH GOALS AND OBJECTIVES ……………………...…………………….....31 III. METAGENOMIC IDENTIFICATION OF VIRUS COMMUNITIES ASSOCIATED WITH LETTUCE AND IRRIGATION WATER ................................................................... 32 1. Introduction ............................................................................................................................. 32 2. Research Goals and Objectives.............................................................................................. 35 3. Materials and Methods .......................................................................................................... 35 3.1 Virus Recovery from Lettuce.......................................................................................... 35 3.1.1 Bacterial Host and Bacteriophage Preparation ....................................................... 35 3.1.2 Lettuce Inoculation, Elution, and Plating of Bacteriophage ................................... 37 3.1.3 Plaque Concentration Assay and Percent Recovery Calculation ............................ 39 3.2 Virus Communities from Lettuce and Irrigation Water .................................................. 40 3.2.1 Irrigation Water and Lettuce Sample Collection ..................................................... 40 3.2.2 Virus Concentration and Purification ...................................................................... 42 3.2.3 Final Concentration, Purification, and Nucleic Acid Extraction ............................. 43 3.2.4 Random Amplification ............................................................................................. 43 3.2.5 Sequencing and Bioinformatics .............................................................................. 45 v 4. Results .................................................................................................................................... 46 4.1 Virus Recovery Efficiency from Lettuce ........................................................................ 46 4.2 Yuma Irrigation Water Quality ....................................................................................... 50 4.3 Irrigation Water Metagenomic Statistics ........................................................................ 52 4.4 Irrigation Water Virome ................................................................................................. 52 4.5 Yuma Lettuce Virome Statistics .......................................................................................69 4.6 Yuma Lettuce Virome ......................................................................................................71 5. Discussion..................................................................................................................................85 IV. VIRAL CROSS-CONTAMINATION OF LETTUCE DURING SMALL-SCALE LEAFY GREEN PROCESSING ............................................................................................... 92 1. Introduciton ............................................................................................................................. 92 2. Reseach Goals and Objectives ............................................................................................... 94 3. Materials and Methods ........................................................................................................... 95 3.1 Bacteriophage Inactivation and Free Chlorine Demand ................................................. 95 3.1.1 MS2 Inactivation during Sanitizer Exposure ........................................................... 95 3.1.2 Coliphage MS2 Chlorine Demand ........................................................................... 96 3.2 Bacteriophage Reduction during Small-Scale Leafy Green Processing With and Without a Chlorine-Based Sanitizer .................................................................................................... 96 3.2.1 Lettuce and Processing
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