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MOLECULAR AND BIOLOGIC CHARACTERIZATION OF TRYPANOSOMA CRUZI FROM THE UNITED STATES OF AMERICA by DAWN MARIE ROELLIG (Under the Direction of Michael J. Yabsley) ABSTRACT Trypanosoma cruzi, the causative agent of Chagas disease, is a flagellated protozoan parasite endemic to the Americas. There are an estimated 7.6 million people infected within Latin America, with 200,000 new cases occurring annually. In the United States, only six autochthonous human cases have been reported, but screening of US blood donors since January 2007 by the Chagas’ Disease Biovigilance Network has confirmed over 1,000 seropositive donations, some of which are believed to be autochthonously acquired infections. In addition to autochthonous human cases, naturally acquired infections have been reported in domestic dogs, captive exotic animals, and a wide range of wildlife species. Although T. cruzi is common in the sylvatic cycle (transmission between wildlife and vectors) and the peridomestic cycle (between animals that move between the wild and around homes), circulation within the domestic cycle (between hosts within or around homes) is less often documented in the US. In the sylvatic cycle, a number of wild mammals have been identified as reservoirs and the prevalence of T. cruzi in US wildlife based on serology, culture isolation, and/or PCR can be equally as high as in South America. Considerable work on T. cruzi has been conducted in Central and South America, but due to limited numbers of human cases in the US, little work has been conducted to characterize US isolates. Previous studies have determined prevalence in some host species through the use of serology, hemoculture, and/or PCR, identified transmission routes for a few reservoirs using non-native isolates, and molecularly characterized a limited number of isolates. With new human cases, increasing numbers of veterinary cases, and influx of potentially infected immigrants, understanding the ecology in the US is imperative. The goal of this dissertation was to elucidate characteristics of T. cruzi isolates from the US using molecular and in vivo techniques to better understand T. cruzi epizootiology in the region. INDEX WORDS: Trypanosoma cruzi, trypanosome, Chagas disease, vector-borne disease, zoonotic disease, experimental infection, United States, sylvatic cycle MOLECULAR AND BIOLOGIC CHARACTERIZATION OF TRYPANOSOMA CRUZI FROM THE UNITED STATES OF AMERICA by DAWN MARIE ROELLIG BA, Agnes Scott College, 2004 MS, Georgia Southern University, 2006 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2009 © 2009 Dawn Marie Roellig All Rights Reserved MOLECULAR AND BIOLOGIC CHARACTERIZATION OF TRYPANOSOMA CRUZI FROM THE UNITED STATES OF AMERICA by DAWN MARIE ROELLIG Major Professor: Michael J. Yabsley Committee: David S. Peterson Ray M. Kaplan David E. Stallknecht Daniel G. Mead Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2009 iv ACKNOWLEDGEMENTS I would like to extend a special thank you to Dr. Michael Yabsley for all his support, guidance, and patience through this project and my academic career. My entire committee, including Dr. David Peterson, Dr. Ray Kaplan, Dr. David Stallknect, Dr. Daniel Mead, and Dr. Michael Yabsley, has given me invaluable advice, encouragement, education, and critique for which I am grateful. I would also like to thank my mentors throughout my undergraduate and graduate education, Dr. Robert Massung, Dr. Dana Nayduch, and Dr. Lance Durden, all of whom were instrumental in introducing me to research, parasitology, and infectious diseases. I offer a sincere thanks to my parents, Ralph and Darlene Roellig, who instilled in me the courage to never give up, sound advice, and ice cream when I needed it most. Many thanks to all the faculty, staff, and students at the Southeastern Cooperative Wildlife Disease Study for all their assistance and support throughout the years. I especially would like to thank all those who have previously worked in the Yabsley lab that have helped with the various experiments and projects, including Letitia Saunders, Mason Savage, Wendy Fujita, Kate McMillan, Emily Rushton, Jessica Murdock, Barbara Shock, Emily Blizzard, Jess Gonynor, and Elizabeth Gleim. A gigantic thank you to all the friends I’ve made over the years in Athens, especially Lindsay Fann Killmaster and Sabrina McGraw who have been there for me through it all. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................... iv LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .......................................................................................................... xii CHAPTER 1 INTRODUCTION ............................................................................................1 References ...................................................................................................5 2 LITERATURE REVIEW ...............................................................................11 History, life cycle and epidemiology of Trypanosoma cruzi ....................11 Human cases of Trypanosoma cruzi in the United States.........................15 Trypanosoma cruzi vectors in the United States.......................................17 Trypanosoma cruzi in wildlife from the United States .............................17 Trypanosoma cruzi diagnostics.................................................................20 Trypanosoma cruzi molecular evolution and genotyping .........................20 Wildlife reservoirs and the genotype-strain dichotomy ............................24 References .................................................................................................28 3 MOLECULAR TYPING OF TRYPANOSOMA CRUZI ISOLATES, UNITED STATES ..........................................................................................................43 Abstract .....................................................................................................44 The Study ..................................................................................................45 vi Conclusion ................................................................................................46 Acknowledgments.....................................................................................48 Biographical Sketch ..................................................................................48 References .................................................................................................49 4 GENETIC VARIATION AND EXCHANGE IN TRYPANOSOMA CRUZI ISOLATES FROM THE UNITED STATES ..............................................55 Introduction ................................................................................................57 Materials and Methods ...............................................................................59 Results ........................................................................................................61 Discussion ..................................................................................................64 Acknowledgements ....................................................................................68 References ..................................................................................................68 5 GENETICALLY DIFFERENT ISOLATES OF TRYPANOSOMA CRUZI ELICIT DIFFERENT INFECTION DYNAMICS IN RACCOONS (PROCYON LOTOR) AND VIRGINIA OPOSSUMS (DIDELPHIS VIRGINIANA) ................................................................................................86 Abstract .....................................................................................................87 Introduction ...............................................................................................88 Materials and Methods ..............................................................................90 Results .......................................................................................................95 Discussion .................................................................................................99 Acknowledgements .................................................................................104 References ...............................................................................................105 vii 6 EXPERIMENTAL INFECTIONS OF TWO SOUTH AMERICAN RESERVOIRS WITH FOUR DISTINCT STRAINS OF TRYPANOSOMA CRUZI ..........................................................................................................119 Summary .................................................................................................120 Introduction .............................................................................................121 Materials and Methods ............................................................................123 Results .....................................................................................................126 Discussion ...............................................................................................129 Acknowledgements .................................................................................132 Financial Support ....................................................................................133
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