AN ABSTRACT OF THE DISSERTATION OF Justin L. Sanders for the degree of Doctor of Philosophy in Microbiology presented on September 3, 2013. Title: Pseudoloma neurophilia: Progression of Infection and Transmission Characteristics of a Microsporidian Parasite in a Model Vertebrate, Danio rerio Abstract approved:________________________________________________________ Michael L. Kent The microsporidian parasite, Pseudoloma neurophilia, is the most commonly diagnosed infectious disease in laboratory populations of the zebrafish, Danio rerio. Infections by P. neurophilia are generally subclinical, however, they can become acute either incidentally or due to experimental immune suppression. Non-protocol induced variation can confound results in laboratory experiments using such fish. As a result, there has been growing interest in sensitive diagnostic assays for P. neurophilia and demand for P. neurophilia specific-pathogen free zebrafish lines among the zebrafish research community. The high prevalence of P. neurophilia in zebrafish provides the opportunity to investigate the progression of infection and transmission characteristics of a microsporidian parasite in a well-developed model vertebrate host species. I developed a real-time PCR-based assay combined with the use of sonication to improve spore disruption which has a sensitivity that is 10-100 times more sensitive than a previously published conventional PCR-based assay. The microsporidium infects ovaries and eggs, thus, I developed a sampling method for the testing of water from spawning fish and demonstrated the utility of testing spawn water, eggs, and sperm for the non-lethal detection of P. neurophilia in adult fish. The presence of P. neurophilia in spawn water and eggs from infected adults provided the initial evidence of vertical transmission of P. neurophilia in zebrafish. Intraovum transmission of P. neurophilia was directly observed by histological screening of larval fish spawned from infected adults and by analyzing eggs using a transmitted light dissecting microscope. The prevalence of intraovum transmission was determined by screening surface-decontaminated eggs from 27 paired spawns using the real-time PCR based assay. Intraovum transmission was detected in 4 of the 27 spawns and the prevalence of intraovum P. neurophilia in the eggs from these spawns was determined to be approximately 1%. Parasite DNA was also detected in the spawning water from 11 of the 27 spawns, highlighting the potential for extraovum transmission of P. neurophilia. I investigated the early stages of P. neurophilia infection in experimentally infected larval zebrafish. This was accomplished using a combination of standard hematoxylin and eosin stain, the Luna stain, and an in situ hybridization probe specific to the small-subunit ribosomal DNA gene of P. neurophilia, which I developed. At 12 hours post exposure, P. neurophilia was mainly visualized as intact spores in the intestinal lumen, and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx, and within the hepatocytes of the liver. Proliferative (presporogonic) stages were visualized in these tissues and in the pancreas and kidney at 36 and 48 hours post exposure and in the spinal cord, eye, and skeletal muscle beginning at 72 hours post exposure. The first spore stages of P. neurophilia were observed at 96 hours post exposure in the pharyngeal epithelium, liver, spinal cord and skeletal muscle. The parasite was observed in the brain of larval fish at 120 hours post- exposure. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hours post exposure suggests that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tubule, and that autoinfection does not occur at any detectable frequency during the initial stages of infection. ©Copyright by Justin L. Sanders September 3, 2013 All Rights Reserved Pseudoloma neurophilia: Progression of Infection and Transmission Characteristics of a Microsporidian Parasite in a Model Vertebrate, Danio rerio by Justin L. Sanders A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented September 3, 2013 Commencement June 2014 Doctor of Philosophy dissertation of Justin L. Sanders presented on September 3, 2013. APPROVED: Major Professor, representing Microbiology Chair of the Department of Microbiology Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Justin L. Sanders, Author ACKNOWLEDGEMENTS There have been numerous people who have helped me in some way on this journey to whom I owe a huge debt of gratitude. First and foremost, I thank Dr. Michael Kent. His mentorship and guidance, as well as his infectious enthusiasm and optimism have made this work possible and he has provided me with a multitude of great opportunities. His encouragement to always "keep paddling" has proven invaluable in research, writing, surfing, and life. I also must thank my thesis committee members, Drs. Luiz Bermudez, Kathy O'Reilly, Michelle Steinauer, and Robert Tanguay, for their guidance, advice, and time. I must thank my former employers and mentors who encouraged me to embark on this journey, especially Sandi Fredrickson, Dr. Patty McVay, and Jerry Hurst, and my friends and colleagues at the Humboldt County Public Health Branch. This project would have been far more difficult if not for my training and experience as a public health microbiologist. To that I owe the excellent training staff at the California State Viral and Rickettsial Disease/Microbial Disease laboratories, and the staff at the numerous public health laboratories in which I had the great opportunity to train. I want to thank the faculty and staff of the Department of Microbiology here at OSU for all of their help and time. I also want to thank Carrie Barton and Carrie Buchner of the Tanguay Laboratory for maintaining and providing the Pseudoloma-free fish which were used in the majority of the experiments for this project. Thanks also to the staff at the histology laboratory here at OSU, Kay Fisher, Misty Corbus and Renee Norred, for their excellent histotechnical assistance and the hundreds of high-quality slides which were prepared during the course of this project. A big thank you to the members of the Kent Laboratory, especially Virginia Watral, for putting up with me during these years and Dr. Trace Peterson for his fish pathology expertise, friendship, and hours of comic relief. I also want to thank the many friends and colleagues I have made during my time here whose encouragement, collaborations, and discussions have helped me immensely throughout this project and will continue long afterward: Drs. Ann Cali, Peter Takvorian, Louis Weiss, Kevin Lafferty, Liz Didier, Nicolas Corradi and so many others. I am also grateful to Mark Francis and the folks at Aquaneering for sponsoring the fish disease section of the zebrafish husbandry session at the Aquaculture America meeting of the World Aquaculture Society. It has been very rewarding for me to give talks at this meeting and I have really enjoyed watching it grow every year. Major funding for this project came from the National Institutes of Health (NIH NCRR 5R24RR017386-02 and NIH NCRR P40 RR12546-03S1). A travel award from the College of Sciences assisted my travel to Tarrytown, NY to present at the International Workshop for Opportunistic Protists in 2012. Finally, I am also enormously grateful for my family, without whose constant love and support, this would not have been possible: My parents, Jim and Jeanne, my sister, Julie Gagner, who also provided excellent review and editing of this dissertation, and especially Tara, Gavin, and Maeve: this is for you. CONTRIBUTION OF AUTHORS Virginia Watral assisted with data collection and experimental setup for the vertical transmission studies. Kerri Clarkson assisted with spawning fish and egg collection for the vertical transmission studies. Doctor Tracy Peterson provided assistance with the histological descriptions in the parasite progression study. TABLE OF CONTENTS Page Chapter 1. Introduction ...................................................................................................... 1 General Characteristics of the Microsporidia ..........................................................1 Impacts of Microsporidiosis ....................................................................................1 Summary and Thesis Overview ...............................................................................4 References ................................................................................................................5 Chapter 2. Microsporidiosis in zebrafish research facilities ...............................................9 Abstract ..................................................................................................................10 Introduction ............................................................................................................11 Current methods of detection .................................................................................13 Transmission ..........................................................................................................15