ABSTRACT SCHREEG, MEGAN ELIZABETH. Cytauxzoon felis in a Post-Genomic Era: Taxonomy, Diagnosis, Treatment, and Prevention. (Under the direction of Dr. Adam Birkenheuer, Chair, and Dr. Michael Levy, Vice Chair.) Cytauxzoonosis is an emerging disease affecting felines throughout the Western hemisphere. Cytauxzoonosis is caused by the tick-transmitted parasite Cytauxzoon felis, an organism for which little is known. Since being discovered 40 years ago in Missouri, C. felis has spread across 1/3 of the United States, and is expected to continue spreading given the continental distribution of competent feline and tick hosts. C. felis is highly pathogenic to domestic cats. Disease progresses rapidly and even with the best treatment mortality remains high. No vaccine exists, so disease prevention relies on indoor confinement and acaricide prophylaxis. Given the rapid dispersal of this virulent parasite that lacks effective treatment or prevention options, further investigation is warranted. However, the inability to culture the parasite in vitro, ethical concerns with in vivo studies, and lack of funding for feline diseases has limited C. felis research. To counteract this, we have sequenced the parasite’s mitochondrial and chromosomal genomes. Using these resources, we have identified useful genetic targets that resolve the taxonomy of C. felis and improve the treatment, diagnosis and prevention of cytauxzoonosis. The taxonomic placement of Cytauxzoon felis within Piroplasmida remains unsolved due to discrepancies between morphological and molecular data. We have clarified phylogeny of C. felis and other Piroplasmida using mitochondrial genome sequences and structures. Mitochondrial genome analysis supported the placement of C. felis within the Theileria clade, and indicated that T. equi, B. conradae, and B. microti organisms are genetically distinct lineages. Characterization of additional mitochondrial genomes and subsequent reclassification of Piroplasmida genera are merited. Mortality of infected cats treated with atovaquone and azithromycin (A&A) is 40%. Atovaquone targets an electron transport protein encoded by mitochondrial cytb. Mutations in the cytb gene are associated with atovaquone resistance in related parasites. We hypothesized that C. felis cytb genotype would be associated with response to A&A treatment. After cytb-genotyping 69 samples, we identified a C. felis cytb genotype (cytb1) associated with increased survival of cats treated with A&A. Given this association, we hypothesized that cytb1 could aid in the prognosis of cytauxzoonosis. By developing a quantitative PCR panel that identifies cytb1-specific SNPs with high resolution melt (HRM) analysis, we distinguished C. felis cytb1 from all other C. felis cytb genotypes with 100% sensitivity and 98.2% specificity. This assay is cost-effective and can be completed in less than 3 hours, which is important given the high cost of A&A and rapid disease course. Diagnosis of C. felis is challenging during early infection when parasitemia is low and clinical signs remain vague. Mitochondrial genes are more sensitive molecular diagnostic targets for parasite detection than 18S in related parasites. We demonstrated that mitochondrial cox3 copy number is increased relative to 18S in blood and tissue samples from cats with acute cytauxzoonosis, and that cox3 is more sensitive for identifying early C. felis infection than 18S. This assay will aid in early detection of Cytauxzoon felis infection. Sequencing the C. felis genome has allowed for identification of 33 vaccine candidates. We have manufactured two DNA vaccines: an expression library vaccine including all candidates and a vaccine consisting of the recently described cf76. We describe the immunization approach, serological response to vaccination, and subsequent clinical outcome following challenge with C. felis. All vaccinated cats became infected and developed disease, rendering both vaccines inadequate methods for prevention of cytauxzoonosis. However, the expression library vaccine showed evidence of aiding in disease control, providing a baseline for development of future subunit vaccines against cytauxzoonosis. In conclusion, the mitochondrial and chromosomal genomes have clarified the taxonomy of Cytauxzoon felis and further advanced our understanding of the treatment, diagnosis, and prevention of cytauxzoonosis. © Copyright 2015 by Megan Elizabeth Schreeg All Rights Reserved Cytauxzoon felis in a Post-Genomic Era: Taxonomy, Diagnosis, Treatment, and Prevention by Megan Elizabeth Schreeg A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Comparative Biomedical Sciences Raleigh, North Carolina 2015 APPROVED BY: _______________________ _______________________ Adam Birkenheuer Michael Levy Chair of Advisory Committee Vice Chair of Advisory Committee ___________________ ___________________ ___________________ David Bird Luke Borst Jeffrey Yoder Committee Member Committee Member Committee Member BIOGRAPHY Megan was born and raised in Kokomo, Indiana, where she was constantly surrounded by cats and consequently was inspired to become a veterinarian. Megan graduated summa cum laude with a Bachelor of Arts Degree in Biology and minors in Biochemistry and Latin from Hanover College in Hanover, Indiana in 2010. Megan is currently in the Combined DVM/PhD Program at North Carolina State University College of Veterinary Medicine. Megan has interests in feline medicine, immunology, comparative anatomy and Photo credit to Wendy Savage pathology, comparative molecular genomics, and parasitology. Following completion from the DVM/PhD program, Megan currently intends to apply for a residency in either anatomic or clinical pathology, and also hopes to pursue board certification as a veterinary parasitologist. Megan also has a passion for teaching, and her ultimate goal is to become a professor at a school of veterinary medicine. ii ACKNOWLEDGEMENTS A researcher is only as good as her support system, and I’ve been blessed enough to have a great team of lab mates, friends, and family helping me through every step of completing this work. First, I would like to thank my advisor Adam, who has mentored me not only in research, but also in life. None of this work would be possible without your passion for companion animal medicine, love for learning, or aptitude for training developing clinician scientists. The countless hours you have spent helping me both inside the lab and out have meant so much to me, and taught me what it truly means to be a mentor. You have taken a naive student and molded her into a slightly less naïve researcher and human being, and for that I cannot thank you enough! Next I would like to thank Henry, our laboratory technician, for teaching me the ropes of working at the bench, for helping me with my work and always patiently answering my often stupid questions, and for being a great friend. Together you and Adam have made me feel like part of a little “lab family,” which meant the world to a girl who left everything she knew behind and moved across the country to pursue an education. There are endless other mentors and supporters I have had in the world of science. First, a huge thank you to Dr. Sam Jones for accepting me in the DVM/PhD program—you believed in my potential when I didn’t, and have offered unending support throughout the program! A special thank you to my undergraduate advisor, Dr. Walter Bruyninckx, who showed me that a career as a researcher, veterinarian, teacher, and life-long learner was possible. An additional thank you to Dr. Leah Cohn, who I consider not only a key collaborator, but also a phenomenal clinician, a friend, and one of the most kind-hearted iii people I know. Thanks to Dr. Brian Wiegmann for patiently teaching me about molecular systematics! A huge thanks as well to my committee members: Dr. Levy, Dr. Bird, Dr. Borst, and Dr. Yoder—your support and collaboration have been critical for me on this journey. Last but certainly not least, I also would like to thank the rest of my office and lab members, including Kaye, Candace, Mitsu, Jingjing, Jaime, the folks of the VBDDL, and all the others that I am forgetting. You are like family to me and make coming to work a joy! A huge thanks as well to all of my friends both near and far, who have been with me through high school, college, and beyond. I would be lost without the Great 8 and my Hanover ladies, who have been some of my biggest cheerleaders in the pursuit of this degree. A special thanks to the newly minted Dr. Susan Grayden Shapiro, my DVM/PhD partner in crime—I wouldn’t have had the opportunity to do any of this work if you hadn’t told me about the amazing veterinary school at NC State, and I can’t wait to see how our destinies continue to cross in the future. My family deserves infinite praise for endlessly supporting and loving me. To Mom and Dad—you imparted a love for problem-solving, nature, and animals in me from a very young age. You always nurtured my passions through the years, whether that meant getting me yet another cat, helping me tend to my science fair project, or pitching batting practice to me. You taught me the meaning of hard work and persistence, and instilled in me that anything was possible if I put my mind to it. I couldn’t ask for better parents or role models, and I’m forever grateful and proud to be your daughter. To my brothers Danny and Keagan—whether you know it or not, you both have inspired me in your own way, and I wouldn’t trade you for any other siblings on the planet! iv More than any other person, I need to thank my husband Jacob. You are my anchor in all things I do! You moved across the country to come with me on this journey, and for some crazy reason along the way decided that I was worthy to be your wife. You have supported me every step of the way, whether that mean helping me study, coming into the lab with me, listening to me blather about my latest silly discovery, or taking care of our house and our animal family—Murray, Pip, and Rue—while I focused on school.