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Exploration of Laboratory Techniques Relating to Cryptosporidium Parvum Propagation, Life Cycle Observation, and Host Immune Responses to Infection

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Exploration of Laboratory Techniques Relating to Cryptosporidium Parvum Propagation, Life Cycle Observation, and Host Immune Responses to Infection EXPLORATION OF LABORATORY TECHNIQUES RELATING TO CRYPTOSPORIDIUM PARVUM PROPAGATION, LIFE CYCLE OBSERVATION, AND HOST IMMUNE RESPONSES TO INFECTION A Thesis Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Cheryl Marie Brown In Partial Fulfillment for the Degree of MASTER OF SCIENCE Major Department: Veterinary and Microbiological Sciences February 2014 Fargo, North Dakota North Dakota State University Graduate School Title EXPLORATION OF LABORATORY TECHNIQUES RELATING TO CRYPTOSPORIDIUM PARVUM PROPAGATION, LIFE CYCLE OBSERVATION, AND HOST IMMUNE RESPONSES TO INFECTION By Cheryl Marie Brown The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of MASTER OF SCIENCE SUPERVISORY COMMITTEE: Dr. Jane Schuh Chair Dr. John McEvoy Dr. Carrie Hammer Approved: 4-8-14 Dr. Charlene Wolf-Hall Date Department Chair ii ABSTRACT Cryptosporidium causes cryptosporidiosis, a self-limiting diarrheal disease in healthy people, but causes serious health issues for immunocompromised individuals. Cryptosporidiosis has been observed in humans since the early 1970s and continues to cause public health concerns. Cryptosporidium has a complicated life cycle making laboratory study challenging. This project explores several ways of studying Cryptosporidium parvum, with a goal of applying existing techniques to further understand this life cycle. Utilization of a neonatal mouse model demonstrated laser microdissection as a tool for studying host immune response to infeciton. A cell culture technique developed on FrameSlides™ enables laser microdissection of individual infected cells for further analysis. Finally, the hypothesis that the availability of cells to infect drives the switch from asexual to sexual parasite reproduction was tested by time-series infection. The results suggest this isn’t accurate. These experiments open the door to several avenues of Cryptosporidium study and the host response to cryptosporidiosis. iii ACKNOWLEDGMENTS I would like to thank Dr. Jane Schuh, my advisor for allowing me into your lab and for the ongoing patient support. I also thank Dr. John McEvoy, you opened your lab to me and taught me so much about Crypstosporidium. Your help is much appreciated. Thank you to Catherine Giddings, your smiling face, countless flasks of MDCK cells, and patient explanations were priceless. Thank you Scott Hoselton for the help with microscopy and baby mice. Thank you, Dr. Amali Samarasinghe: your cheerleading, encouragement and nagging would have done it, if only you were about 1,000 miles closer! I truly appreciate all of your help and advice and moral support. iv DEDICATION I would have loved to have had one more chance to thank Dr. David Berryhill face- to-face for his ongoing support and encouragement and pep talks when things got stinky. Doc, you may be gone, but you are not forgotten! I still half expect to bump into you on one of your wanders when I come around corners in Van Es! This thesis is dedicated to my loving family and to my darling Monkeys. It is finished. We will never speak of this again. v TABLE OF CONTENTS ABSTRACT ............................................................................................................................ iii ACKNOWLEDGMENTS ....................................................................................................... iv DEDICATION .........................................................................................................................v LIST OF TABLES .................................................................................................................. xi LIST OF FIGURES ............................................................................................................... xii LITERATURE REVIEW ......................................................................................................... 1 Thesis outline ................................................................................................................. 1 Introduction ................................................................................................................... 1 Cryptosporidium classification and taxonomy ............................................................ 3 The Cryptosporidium life cycle .................................................................................... 5 Ingestion, excystation, and attachment ........................................................................ 6 Host cell invasion ......................................................................................................... 11 Asexual reproduction: Type I merogony ..................................................................... 11 Asexual reproduction: Type II merogony .................................................................... 12 Sexual reproduction: Gametogony .............................................................................. 13 Advantageous characteristics ...................................................................................... 13 Oocyst structure ........................................................................................................... 14 Life cycle terminology .................................................................................................. 16 Transmission: Route of infection ................................................................................ 17 Cryptosporidiosis ......................................................................................................... 17 Course of disease in the immunocompetent host........................................................ 18 Course of disease in the immunocompromised host................................................... 19 Pathology of cryptosporidiosis .................................................................................... 19 Host immune response ............................................................................................... 20 vi Current treatment and chemotherapy options ............................................................ 21 Potential treatments for cryptosporidiosis ................................................................. 22 Epidemiology: Surveillance and outbreaks ................................................................ 23 Milwaukee outbreak ...................................................................................... 24 England outbreak: Rabbit genotype ............................................................ 25 Outbreak among firefighters in Indiana and Michigan ................................ 25 Water treatments to remove Cryptosporidium oocysts ............................................. 26 Research challenges .................................................................................................... 26 Available Cryptosporidium isolates ........................................................................... 27 Culturing techniques and limitations ......................................................................... 27 Understanding and identifying life stages .................................................................. 29 Genetic analysis .......................................................................................................... 30 Objectives of the project ............................................................................................. 30 References .................................................................................................................... 31 CHAPTER 1: UTILIZATION OF A FETAL MOUSE MODEL OF CRYPTOSPORIDIOSIS FOR IMMUNE STUDIES ................................................................................................... 52 Introduction ................................................................................................................ 52 Materials and methods ............................................................................................... 53 Animals and institutional use compliance .................................................... 53 Infection of neonatal mice with C. parvum .................................................. 53 Collection and preparation of tissue samples from neonatal mice ............... 53 Lectin-VVL staining of tissue sections .......................................................... 54 Immunohistochemical staining of tissue sections ........................................ 56 Results ........................................................................................................................ 58 H&E staining ................................................................................................. 58 Lectin-VVL staining ...................................................................................... 59 vii Immunohistochemical staining .................................................................... 60 Discussion ................................................................................................................... 60 Infection of neonatal mice and staining of tissues........................................ 60 Future directions ............................................................................................ 61 Acknowledgements ..................................................................................................... 62 References ..................................................................................................................
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