Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2009 Quantitative real-time polymerase chain reaction (QPCR) assay as a molecular tool to assess rickettsial replications in tick hosts Andrea S. Zanetti Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation Zanetti, Andrea S., "Quantitative real-time polymerase chain reaction (QPCR) assay as a molecular tool to assess rickettsial replications in tick hosts" (2009). LSU Master's Theses. 3859. https://digitalcommons.lsu.edu/gradschool_theses/3859 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR) ASSAY AS A MOLECULAR TOOL TO ASSESS RICKETTSIAL REPLICATION IN TICK HOSTS A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Interdepartmental Program in Veterinary Medical Sciences Through the Department of Pathobiological Sciences by Andre Serrano Zanetti D.V.M., Sao Paulo State University, 2006 May 2009 Acknowledgments It is a pleasure to thank the many people who made this thesis possible. It is difficult to overstate my gratitude to my M. S. supervisor, Dr. Kevin R. Macaluso. With his passion, his inspiration, and his great efforts to provide available resources, he helped to make the completion of this section of my academic life pleasant and straightforward. I would like to thank the many people who provided ideal conditions for the development of the research presented in this thesis: O. Strey (Texas A&M) for the Amblyomma ticks used to describe the natural kinetics of Rickettsia amblyommii during tick feeding; Marc J Boudreaux for teaching me how to run and prepare a qPCR reaction; Thaya Guedry for analyzing my sequences; Olga Borkhsenious and Felipe Sandoval for processing the samples for transmission electron microscopy (TEM) and DNA extraction, respectively, in the in vitro study; and my laboratory colleagues (Walairat Pornwiroon, Piyanate Sunyakumthorn and Natthida Petchampai) for helping me with the construction of the Ra17kDa/AaMIF plasmid, the maintenance of the cell lines and for providing the Ra17kDa/ISE6Crt plasmid, respectively. I am indebted to my many graduate student colleagues for providing a stimulating and fun environment in which to learn and grow. I am especially grateful to Katherine Reif, Soma Chowdhury, Paula Mischler, Sunita Seemanapalli, Rebecca Christofferson, Britton Grasperge and Niranjan Butchi. I am grateful to the secretaries of the Pathobiological Sciences Department and the librarians of the School of Veterinary Medicine of Louisiana State University for helping with the deadlines and for assisting me in many different ways. ii I would like to thank Dr. John Malone and Dr. James Miller for accepting to serve on my committee and for helpful career advice. I wish to thank my entire extended family for providing a loving environment for me. My siblings, Marcelo and Fernanda, and my grandmother, Elydia. Lastly, and most importantly, I wish to thank my parents, Jose Americo Zanetti and Marilene C. Serrano Zanetti. They bore me, raised me, supported me, taught me, and loved me. To them I dedicate this thesis. The research presented in this thesis was supported by the Louisiana Board of Regents Louisiana Educational Quality Support Fund (LEQSF), National Institutes of Health grant P20 RR0201595, and the National Institute of Allergy and Infectious Diseases grants AI60821 and AI070705. iii Table of Contents Acknowledgments........................................................................................................................... ii List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abstract .......................................................................................................................................... ix Chapter 1. General Introduction ..................................................................................................... 1 Chapter 2. Characterization of Rickettsial Infection in Amblyomma americanum Ticks (Acari: Ixodidae) by Quantitative Real-Time Polymerase Chain Reaction ........................ 18 2.1. Introduction .............................................................................................................. 18 2.2. Methods and Materials ............................................................................................. 19 2.2.1. Ticks ............................................................................................................ 19 2.2.2. Tick Feeding and Sample Collection .......................................................... 20 2.2.3. Tissue Recovery and DNA Isolation .......................................................... 22 2.2.4. Rickettsial Detection and Identification by PCR ........................................ 22 2.2.5. Construction of an Internal-Control Plasmid for Quantitative Real-Time PCR ....................................................................................................................... 24 2.2.6. Quantitative Real-Time PCR ...................................................................... 24 2.2.7. Statistical Analysis ...................................................................................... 26 2.3. Results ...................................................................................................................... 29 2.3.1. Tick Biology ............................................................................................... 29 2.3.2. Rickettsia Prevalence and Identification in Tick Samples .......................... 30 2.3.3. Analysis of Rickettsial Infection by qPCR ................................................. 30 2.3.4. Rickettsial Distribution and Growth in Tick Samples ................................ 32 2.4. Discussion................................................................................................................. 34 Chapter 3. Dynamics of Growth and Infectivity of Rickettsia amblyommii (WB-8-2), Rickettsia parkeri and Rickettsia montanensis in Vertebrate and Tick Cells: A Preliminary Study ............. 42 3.1. Introduction .............................................................................................................. 42 3.2. Methods and Materials ............................................................................................. 43 3.2.1. Cells Lines and Rickettsiae Strains ............................................................. 43 3.2.2. Cell-Rickettsiae Challenge.......................................................................... 44 3.2.3. Infectivity Determination by Diff Quicktm and Transmission Electron Microscopy ........................................................................................................... 46 3.2.4. High Through-Put DNA Extraction ............................................................ 46 2.2.5. Construction of an Internal-Control Plasmid for Quantitative Real-Time PCR ....................................................................................................................... 46 3.2.6. Quantitative Real-Time PCR ...................................................................... 48 iv 3.2.7. Statistical Analysis ...................................................................................... 51 3.3. Results ...................................................................................................................... 51 3.3.1. Rickettsial Infectivity in Host Cells ............................................................ 51 3.3.2. Rickettsial Growth in Atypical Host Cells .................................................. 55 3.3.3. Ultrastructural Comparison of Infected Cell Lines ..................................... 63 3.4. Discussion................................................................................................................. 63 Chapter 4. Conclusions ................................................................................................................. 73 References ..................................................................................................................................... 75 Vita ................................................................................................................................................ 85 v List of Tables 1. Officially named Spotted Fever Group Rickettsia (SFGR), their tick vectors, life cycle and geographical distribution in the United States ................................................................................ 2 2. Bacteria isolated or identified from Amblyomma americanum ................................................ 10 3. Primers used for PCR amplification ........................................................................................