Epidemiological studies on Non-O157 Shiga toxin-producing Escherichia coli DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anil Kenneth Persad DVM, MS Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2016 Dissertation Committee: Professor Jeffrey LeJeune (Advisor) Dr. Gireesh Rajashekara Dr. Qiuhong Wang Dr. Joshua Daniels Copyrighted by Anil Kenneth Persad 2016 Abstract Shiga toxin-producing Escherichia coli (STEC), a diverse group of bacteria with over 400 serogroups, is estimated to cause over two million cases of human disease globally each year. STEC O157:H7 has been the pathotype most associated with human disease; however the number of human diseases associated with non–O157 serogroups has been increasing. STEC can be part of the normal gastrointestinal flora of animals and shed asymptomatically. Humans are primarily infected via consumption of contaminated food or water, but can also be infected via direct-contact with animals and/or their environment and person to person transmission. In chapter 2, we investigated the extent to which E. coli transfers from deer feces to soils where crops are cultivated in Ohio. Two experiments were performed assessing persistence and dissemination of E. coli in deer feces. In the first experiment, the total coliform and E. coli counts were determined in soil and surface debris samples collected from a vegetable production field naturally contaminated with deer feces and compared with samples collected away from any visible signs of fecal contamination. Samples were similarly assessed 60 days post-redmediation. In the second experiment, deer feces were deposited on soils in vegetable production fields and dissemination of E. coli in surrounding soils was determined at multiple distances over several time intervals. For produce grown in Ohio we can conclude: remediation of fields contaminated by deer ii feces can minimize risk of transfer of microorganisms from feces to soil and employing no-harvest zones greater than 15cm around areas of fresh deer fecal contamination can minimize the risk of harvesting contaminated vegetables. In chapter 3, our aim was to ascertain the minimum number of colony picks from a MacConkey agar plate required to recover at least one stx-positive colony from ruminant fecal enrichment samples testing PCR-positive for stx. There are over 200 STEC serotypes associated with disease and unfortunately, to date, highly selective and specific methods for the isolation of many of these serotypes are not available. Two studies using stx PCR-positive fecal samples obtained from cattle and small ruminants were performed. Enriched fecal samples were streaked onto MacConkey agar and suspect E. coli colonies screened for stx genes. Overall, at least one stx-positive colony was recovered from 79% of the PCR-positive fecal samples. Based upon the proportion of stx- positive E. coli present, selecting more than 20 suspect colonies per sample did not significantly increase in the probability of recovering a stx-positive colony. In chapter 4, we investigated the prevalence of stx-positive colonies in healthy sheep and goats in Trinidad and Tobago. Based on PCR screening, goats had a higher stx prevalence than sheep. Upon culture, approximately two-thirds of all PCR-positive enrichments, regardless of species of origin, yielded colonies that only encoded stx1. The eae gene was detected in only three stx-positive isolates recovered from two different ovine enrichments. Possible risk factors for stx-positive carriage were also identified. From our results, we can conclude that stx-positive isolates shed by sheep and goats in Trinidad do not pose a significant risk to human health. iii Acknowledgments I am eternally grateful to my advisor Professor Jeffrey LeJeune for giving me the honor of being a member of his research team at The Ohio State University. I would like to sincerely thank him for all of his friendship, patience, guidance, support, and mentorship during both my Masters and PhD research. As a mentor he has not only fostered my development as a researcher, but also instilled in me the qualities of being a good supervisor, leader and mentor. I would like to thank my committee members Dr. Gireesh Rajashekara, Dr. Qiuhong Wang and Dr. Joshua Daniels for their guidance and support. To my parents, Seunarine Persad and Drupatee Persad, and sister Kavita Persad: I thank them for their love, support and encouragement, without them my academic and personal achievements would be impossible. I wish to thank all my fellow lab members a.k.a. “My Wooster family”: Michael Kauffman, Pamela Schlegel, Jennifer Schrock, Nicholas Anderson, Ken Schenge, Michele Williams, Gayeon Won, Ana Castillo and Dr. Zhanqiang Su for their support, advice, friendship and encouragement during my five years in Ohio. I would also like to acknowledge all my Wooster friends especially Anand Kumar, Huang-chi Huang, Chung-Ming Lin and Ruby-Pina Mimbela, I hope we can have many for “pizza parties” in the future! iv Finally I thank the major mentors in my life: Dr. Gustave Borde, for his guidance during my undergraduate studies and early Veterinary career; Dr. Jeffrey LeJeune for his mentorship during my graduate studies and introducing me to the sphere of Preharvest food safety; and my parents for being my life mentors. I thank you sincerely for all that you have taught me and for believing in me. v Vita 2002 – 2007 ..................................................Doctor of Veterinary Medicine, University of the West Indies, St. Augustine. 2007 – 2009 ...................................................Teaching Assistant–Food Animal Medicine, University of the West Indies, St. Augustine. 2009 – 2011 ...................................................Research Assistant/Instructor/ Veterinarian University of Trinidad and Tobago. 2011 – 2013 ..................................................Graduate Research Fellow M.S. Comparative and Veterinary Medicine The Ohio State University. 2013 – 2016 ...................................................Graduate Research Assistant, PhD Comparative and Veterinary Medicine The Ohio State University. vi Publications (1) Persad, Anil K., and Jeffrey T. Lejeune. "Animal Reservoirs of Shiga Toxin- Producing Escherichia coli." In Enterohemorrhagic Escherichia Coli and Other Shiga Toxin-Producing E. coli: American Society of Microbiology, 2015 OR Persad, A. K., & LeJeune, J. T. (2014). Animal reservoirs of Shiga toxin-producing Escherichia coli. Microbiology spectrum, 2(4). (2) Persad, A. K. (2013). Survival of Shiga Toxin-producing Escherichia coli and Salmonella serotypes in the feces of five animal species (Master of Science Thesis, The Ohio State University). (3) Adesiyun A.A., Fosgate G.T., Persad A., Campbell M., Seebaransingh R. & Stewart- Johnson A. (2010). Comparative study on responses of cattle and water buffalo (Bubalus bubalis) to experimental inoculation of Brucella abortus biovar 1 by the intraconjunctival route: a preliminary report. Trop. Anim. Health Prod. 42(8):1685- 94. (4) Persad, A., Charles, R., & Adesiyun, A. A. (2011). Frequency of toxoplasmosis in water buffalo (Bubalus bubalis) in Trinidad. Veterinary medicine international, 2011. Fields of Study Major Field: Comparative and Veterinary Medicine vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ..................................................................................................................................... vi Publications ....................................................................................................................... vii List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x Chapter 1: Literature Review: Shiga-toxin producing Escherichia coli ..............................1 Chapter 2: Escherichia coli in Ohio specialty-crop soils contaminated with deer feces. 133 Chapter 3: Isolation of non-O157 Shiga toxin – producing Escherichia coli from ruminant manure. .............................................................................................................156 Chapter 4: Shiga toxin (stx) encoding genes in sheep and goats reared in Trinidad and Tobago. ............................................................................................................................179 Bibliography: ...................................................................................................................214 Appendix A: Identification and subtyping of Salmonella isolates using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF) ...........270 viii List of Tables Table 1.1 Categorization of STEC Sero-pathotypes associated with illness in humans………………………………………………………………… 83 Table 2.1 Rainfall and temperature data from sampling the different sampling blocks………………………………………………………………….. 149 Table 3.1 Primers Sequences used in the PCR assays and the expected sizes of the products…………………………………………………………… 173 Table 4.1 Multivariate