Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2016 Rickettsia felis, Transmission Mechanisms of an Emerging Flea-borne Rickettsiosis Lisa Diane Brown Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation Brown, Lisa Diane, "Rickettsia felis, Transmission Mechanisms of an Emerging Flea-borne Rickettsiosis" (2016). LSU Doctoral Dissertations. 2215. https://digitalcommons.lsu.edu/gradschool_dissertations/2215 This Dissertation 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 Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. RICKETTSIA FELIS, TRANSMISSION MECHANISMS OF AN EMERGING FLEA-BORNE RICKETTSIOSIS A Dissertation 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 Doctor of Philosophy in The Interdepartmental Program in Biomedical and Veterinary Medical Sciences Through the Department of Pathobiological Sciences by Lisa Diane Brown B.S., University of Texas at Tyler, 2009 M.S., University of Louisiana at Monroe, 2012 May 2016 ACKNOWLEDGEMENTS This dissertation project could not have been accomplished alone, and numerous individuals helped me throughout this time- and labor-intensive process. First, I would like to thank my husband, Sam Holcomb, for his unwavering love and support. I fully acknowledge that pursuit of this degree was a challenge for both of us, but it would not have been possible without the encouragement of my husband to continue. I would also like to thank my parents, Tom and Karen, who supported me during this project even though they really had no clue as to what I’ve been doing these past three-and-a-half years. I would like to thank Dr. Natthida Petchampai, Dr. Kaikhushroo H. Banajee, Emma K. Harris, and Victoria I. Verhoeve for their invaluable assistance in the laboratory, including numerous demonstrations of molecular techniques. I would like to thank Dr. Kevin R. Macaluso for taking a chance on a naïve field biologist with virtually no skills in molecular biology. He provided me with the opportunity to pursue my interest in vector-borne diseases, and I am eternally grateful to have worked on such an interesting and rewarding project. I would also like to thank Dr. Rebecca C. Christofferson for her expertise as well as helpful suggestions and advice on my first manuscript. I would like to thank the other members of my graduate committee: Dr. Lane D. Foil, Dr. Christopher Mores, and Dr. John Battista for their guidance and support. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii LIST OF TABLES ...............................................................................................................v LIST OF FIGURES ........................................................................................................... vi ABSTRACT ...................................................................................................................... vii CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ....................................1 1.1. Introduction .....................................................................................................1 1.2. Transmission biology of Rickettsia felis .........................................................2 1.3. Epidemiology of Rickettsia felis ...................................................................10 1.4. Genetic diversity of Rickettsia felis ..............................................................13 1.5. Prospective research for Rickettsia felis .......................................................18 1.6. Conclusions ...................................................................................................21 1.7. Reference List ...............................................................................................22 CHAPTER 2. COFEEDING INTRA- AND INTERSPECIFIC TRANSMISSION OF AN EMERGING INSECT-BORNE RICKETTSIAL PATHOGEN ................................37 2.1 Introduction ...................................................................................................37 2.2 Materials and Methods..................................................................................42 2.2.1. Species and strains of bacteria, fleas, and mice ..........................42 2.2.2. Ethics Statement..........................................................................42 2.2.3. Cat flea bloodmeal treatments in the artificial dog unit ..............43 2.2.4. Rickettsial horizontal transmission bioassays on C3H/HeJ mice .............................................................................43 2.2.5. Sustained rickettsial horizontal transmission bioassay ...............46 2.2.6. Detection of Rickettsia in fleas and mice ...................................46 2.2.7. Histopathology and immunohistochemistry ...............................48 2.2.8. Statistical analyses and model of cofeeding transmission ..........48 2.3 Results ...........................................................................................................52 2.3.1. Horizontal transmission of R. felis occurs between cofeeding cat fleas .......................................................................................52 2.3.2. Interspecific transmission of R. felis occurs between cofeeding fleas ............................................................................54 2.3.3. Transcriptionally active R. felis was detected in mouse skin between cofeeding fleas ......................................................55 2.3.4. Cofeeding transmission of R. felis is sustainable amongst cat flea populations .....................................................................56 2.3.5. Cofeeding transmission is sufficient to cause secondary transmission events after introduction of an infected flea(s), and can lead to persistence of the pathogen ................................57 2.4 Discussion .....................................................................................................61 2.5 Reference List ...............................................................................................67 iii CHAPTER 3. TRANSMISSION MECHANISMS OF AN EMERGING INSECT- BORNE RICKETTSIAL PATHOGEN .............................................................................74 3.1 Introduction ...................................................................................................74 3.2 Materials and Methods..................................................................................78 3.2.1. Source of fleas and cultivation of Rickettsia-infected fleas ........78 3.2.2. Experimental design ...................................................................79 3.2.2.1. Kinetics of cofeeding transmission bioassays.............79 3.2.2.2. Mechanism of early-phase rickettsial transmission ................................................................79 3.2.3. Detection of Rickettsia in fleas ...................................................83 3.2.4. Statistical analysis .......................................................................85 3.3 Results ...........................................................................................................85 3.3.1. Cofeeding transmission of R. felis to naïve fleas is dependent upon the exposure time to produce infectious fleas and the association time with infected fleas ............................................85 3.3.2. Cat flea salivary glands are not the anatomical source of R. felis for early-phase transmission ...........................................88 3.3.3. Cat fleas release R. felis from contaminated mouthparts during probing events .................................................................89 3.3.4. Early-phase transmission of R. felis is due to a mechanical mechanism ..................................................................................91 3.4 Discussion .....................................................................................................93 3.5 Reference List .............................................................................................100 CHAPTER 4. DISCUSSION OF RESULTS AND FUTURE DIRECTIONS ...............105 4.1. Discussion of Results and Future Directions ................................................105 4.2 Reference List ................................................................................................110 APPENDIX A: COMMONLY USED ABBREVIATIONS ...........................................113 APPENDIX B: PERMISSION ........................................................................................115 VITA ................................................................................................................................116 iv LIST OF TABLES Table 1.1. Geographic distribution of R. felis in wild-caught arthropods since 2009 review ................................................................................................................5 Table 1.2. Geographic distribution of RFLO in wild-caught arthropods ..........................16 Table 2.1. Parameter values and