Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2011 Characterization of Spiroplasma mirum and its role in transmissible spongiform encephalopathies Hilari Maree French Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation French, Hilari Maree, "Characterization of Spiroplasma mirum and its role in transmissible spongiform encephalopathies" (2011). LSU Doctoral Dissertations. 3012. https://digitalcommons.lsu.edu/gradschool_dissertations/3012 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]. CHARACTERIZATION OF SPIROPLASMA MIRUM AND ITS ROLE IN TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES 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 Veterinary Medical Sciences through the Department of Pathobiological Sciences by Hilari French B.S., Louisiana Tech University, 2006 D.V.M, Louisiana State University, 2009 August 2011 ACKNOWLEDGEMENTS I would first like to thank my major professor and mentor Dr. Philip Elzer. Without his patience, understanding, and friendship, I would not have been successful in completing this degree program. Our “master” and “grasshopper” relationship kept me on course and will continue to guide me in my future endeavors. I will be forever grateful to him and his family for all they have done for me. I would also like to thank the members of the spiroplasma working group that have spent countless hours brainstorming and assisting me with all my experiments. Sue Hagius and Charles Mitch Boudreaux have been instrumental in helping me with laboratory techniques as well as finalizing projects and publications. I have also benefited from the molecular work and advice provided by Christie Landry and Dr. Ron Thune. I am also grateful to Joel Walker and Tony Bridges who assisted me with all of my animal experiments. There are many others that have assisted me through my degree program. Mrs. Cheryl Crowder processed all of my histological sections. Mr. Rob Poston taught me how to work with embryonated eggs. Dr. Sara Lyle conducted pregnancy checks on all my animals so that I could time my experiments appropriately. My committee: Drs Elzer, Thune, Enright, Hawke, and Godke - were all very helpful and always gave me the time I needed for advice and training. I must also thank the LSU School of Veterinary Medicine for my previous veterinary training as well as the Pathobiological Sciences department for my financial support. The LSU AgCenter and Veterinary Science department provided my laboratory space and animal housing. Lastly I would like to thank my family. My mother Nina French has been my rock of support every day since I was born, and I will be forever in debt to her for her selfless love and attention. My father Dennis French has been my role model and the driving force in my life, and I will always strive to follow his example on the path which I have chosen to follow. My best friend and confidant Sarah “Chicken” Drake has kept me grounded and help me see the “big picture” so that I can enjoy life as I progress in my profession. Without all of their combined love and support, I would not be the person I am today nor would I have achieved this latest goal in my career. ii TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………………………….. ii LIST OF TABLES………………………………………………………………………….. v LIST OF FIGURES………………………………………………………………………… vii ABSTRACT………………………………………………………………………………... x CHAPTER 1: INTRODUCTION/ REVIEW OF LITERATURE…………………………. 1 Introduction…………………………………………………………... 1 Spiroplasma citri................................................................................... 5 Spiroplasma melliferum………………………………………………. 6 Spiroplasma mirum…………………………………………………... 6 Transmissible Spongiform Encephalopathies………………………... 11 Shortcomings to the Prion Theory……………………………………. 13 Discrepancies Seen in Spiroplasma mirum/TSE Publications……….. 15 CHAPTER 2: A COMPARISON OF CULTURE MEDIA FOR SPIROPLASMA SPP………………….............................................................................................................. 18 Introduction ………………………………………………………….. 18 Materials and Methods ...…………………………………………….. 22 Results ……………………………………………………………….. 26 Discussion ……………………………………………………………. 38 Conclusion……………………………………………………………. 40 CHAPTER 3: EFFECTS OF DISINFECTANTS AND ANTIBIOTICS ON SPIROPLASMA SPP………………………………………………………………………... 41 Introduction ………………………………………………………….. 41 Materials and Methods ...…………………………………………….. 42 Results ……………………………………………………………….. 47 Discussion ……………………………………………………………. 57 Conclusion……………………………………………………………. 62 CHAPTER 4: CLINICAL SIGNS AND PATHOLOGY OF SPIROPLASMA MIRUM SUCKLING MOUSE CATARACT AGENT IN SMALL RUMINANTS………………... 64 Introduction ………………………………………………………….. 64 Materials and Methods ...…………………………………………….. 67 Results ……………………………………………………………….. 71 Discussion ……………………………………………………………. 75 Conclusion……………………………………………………………. 78 iii CONCLUSION……………………………………………………………………………... 79 REFERENCES……………………………………………………………………………... 82 VITA………………………………………………………………………………………... 96 iv LIST OF TABLES 1.1 Spiroplasma media……………………………………………………………………. 4 1.2 Summary of differences seen in separate species of Spiroplasmas…………………... 4 1.3 Summary of Spiroplasma mirum experimentally infected animals…………………... 9 2.1 Spiroplasma media……………………………………………………………………. 19 2.2 ATCC SP4 recipe……………………………………………………………………... 23 2.3 Summary of Spiroplasma growth…………………………………………………….. 28 2.4 Dark field counts for diluted (1x10-2) SMCA and GT48 cultures ……………...…... 30 2.5 Dark field counts for diluted (1x10-3) SMCA and GT48 cultures ……………...…... 30 2.6 Dark field counts for diluted (1x10-4) SMCA and GT48 cultures ……………...…... 30 2.7 Dark field counts for diluted (1x10-5) SMCA and GT48 cultures ……………...…... 30 2.8 Dark field counts for diluted (1x10-6) SMCA and GT48 cultures ……………...…... 32 2.9 Dark field counts for diluted (1x10-7) SMCA and GT48 cultures ……………...…... 32 2.10 Dark field counts for diluted (1x10-8) SMCA and GT48 cultures ……………...…... 32 2.11 Dark field counts for diluted (1x10-9) SMCA and GT48 cultures ……………...…... 32 2.12 Summary of CFUs found 21 days post-inoculation from diluted 1 day old cultures on 37 modified SP4 plates with 1.6% Noble agar…………………………………………… 3.1 Efficacy of decontamination procedures for inactivating prions……………………... 43 3.2 M1D media……………………………………………………………………………. 44 3.3 Summary of disinfectants tested for spiroplasma susceptibility……………………... 45 3.4 Summary of antibiotics used to test spiroplasma susceptibility………………………. 46 3.5 Concentration of ethanol (70-0.02%) that inhibited spiroplasma growth…………... 47 3.6 Concentration of methanol (100-0.02%) that inhibited spiroplasma growth……….. 47 v 3.7 Concentration of methanol (100-0.02%) that inhibited spiroplasma growth……….. 47 3.8 Concentration of formalin (20-0.002%) that inhibited spiroplasma growth………….. 48 3.9 Concentration of gluteraldehyde (100-0.02%) that inhibited spiroplasma growth…. 48 3.10 Concentration of sodium hypochlorite (5.25-0.001%) that inhibited spiroplasma growth…………………………………………………………………………………. 48 3.11 Concentration of povidone-iodine (100-0.02%) that inhibited spiroplasma growth…. 49 3.12 Concentration of hydrogen peroxide (3-0.001%) that inhibited spiroplasma growth… 49 3.13 Concentration of acetone (100-0.02%) that inhibited spiroplasma growth…….…….. 50 3.14 Concentration of chlorhexidine (2-0.001%) that inhibited spiroplasma growth..….…. 50 3.15 Concentration of sodium hydroxide (1 mol) (100-0.02%) that inhibited spiroplasma growth…………………………………………………………………………………. 50 3.16 Concentration of SDS (10-0.002%) that inhibited spiroplasma growth…………..….. 51 3.17 Concentration of Roccal-D Plus (100-0.02%) that inhibited spiroplasma growth…… 51 3.18 Spiroplasma growth in the presence of ultraviolet irradiation at various times………. 51 3.19 Spiroplasma growth following autoclaving (121º, 15psi)…………………………….. 52 3.20 Spiroplasma growth in the presence of heat (45º C) at various times………………… 52 3.21 Spiroplasma growth in the presence of heat (50º C) at various times………………… 52 3.22 Spiroplasma growth in the presence of heat (56º C) at various times………………… 52 3.23 Minimal inhibitory concentration and time required of disinfectants for spiroplasma…... 54 3.24 Minimal inhibitory concentration of disinfectant for spiroplasma after 60 minutes….. 55 3.25 Spiroplasma susceptibility to antibiotics……………………………………………… 56 3.26 Spiroplasma growth in the presence of normal goat complement……………………. 57 3.27 Spiroplasma growth in the presence of hyperimmunized goat complement…………. 57 3.28 Comparison of disinfectant efficacy against Spiroplasma and prions………………... 63 vi 4.1 Summary of Spiroplasma mirum experimentally infected animals…………………... 65 4.2 Summary of experimental animals……………………………………………………. 71 4.3 Summary of ruminant experiments…………………………………………………… 76 vii LIST OF FIGURES 1.1 Spiroplasma-like inclusions……………………………………………………………. 15 2.1 Egg Candling…………………………………………………………………………... 22 2.2 Egg inoculation…………………………………………………………………………