DISSERTATION A STUDY OF FACTORS ASSOCIATED WITH GIARDIA AND CRYPTOSPORIDIUM INFECTIONS IN HUMANS, DOGS AND CATS IN THE USA Submitted by Hanaa Thigeel Department of Clinical Sciences In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2016 Doctoral Committee: Advisor: Michael Lappin Co-Advisor: Francisco Olea-Popelka David Twedt Doreene Hyatt Copyright by Hanaa Thigeel 2016 All Rights Reserved ABSTRACT A STUDY OF FACTORS ASSOCIATED WITH GIARDIA AND CRYPTOSPORIDIUM INFECTIONS IN HUMANS, DOGS AND CATS IN THE USA Giardia spp. and Cryptosporidium spp. are two of the leading causal agents of parasitic diarrhea in humans, dogs and cats. The two pathogens contain both host-adapted and zoonotic strains and dogs and cats can harbor both strains. There is critical need to understand factors potentially associated with the risk and prevalence of infection due to Giardia spp. and Cryptosporidium spp. in dogs, cats and humans. This will ultimately aid in disease management and control. Furthermore, molecular characterization of the human, dog or cat isolates may identify zoonotic genotypes and may provide further information concerning the transmission routes between humans, dogs and cats. In Chapter 1, a review of literature regarding Giardia duodenalis and Cryptosporidium spp. in humans and companion animals (dogs and cats) was conducted. The review involves a brief description of the two pathogens’ current taxonomy, epidemiology, and diagnostic methods. Chapter 2 presents a retrospective study designed to analyze results from dog and cat polymerase chain reaction (PCR) panels from the commercial laboratory, ANTECH Diagnostics. The main purpose of this study was to evaluate associations between the probability of testing positive to Giardia spp. and Cryptosporidium spp. and risk factors such as animal’s age, sex, region, and season. The results of this study showed that age (younger animals) was significantly associated with the risk of PCR positive results for Giardia spp. and Cryptosporidium spp. in ii both dogs and cats. Region was significantly associated with Cryptosporidium spp. in both dogs and cats, whereas season was only associated with Giardia spp. in dogs. Chapter 3 describes the validation and optimization a previously published 60 kDa glycoprotein (gp60) gene-based PCR assay. The objective of this study was to use the assay to subtype C. parvum and C. hominis isolated from human fecal samples. The analytical sensitivity of this PCR assay was determined by assaying serial dilutions of C. parvum oocysts and C. hominis DNA. The analytic specificity was determined by assaying Cryptosporidium and non- Cryptosporidium spp. DNA. The gp60 PCR assay consistently detected DNA of C. parvum if oocysts were present at 104/mL. The assay was detected the DNA of C. hominis in the lowest concentration. In Chapter 4, a prospective study was conducted to assess the risk of factors potentially associated with Giardia duodenalis and Cryptosporidium spp. infections and estimate the prevalence of these two pathogens in senior veterinary students and their pet dogs and cats. A structured questionnaire was developed to assess a baseline exposure of the students to large and small animals. In addition, a single voluntary sample was requested from students and their dogs or cats that live within the household. Giardia duodenalis and Cryptosporidium spp. were detected by the PCR and immunofluorescence (IFA) assays in students and their dogs and cats. As a result of the recruitment, 51 surveys, 42 human fecal samples, 31 dog fecal samples, and 17 cat fecal samples were collected. Clinical rotation, track preference, gender, pet ownership and farm exposure were factors selected to be evaluated for the risk of both pathogens in senior veterinary students. As a result of this evaluation, none of these factors selected was statistically associated with the risk of infection due to G. duodenalis or Cryptosporidium spp. All Giardia iii isolated from dogs were host-adapted assemblages. However, a zoonotic Cryptosporidium genotype (C. parvum subtype family IIa) was identified in one human sample. The analysis conducted in this dissertation provided an evaluation of potential risk factors associated with giardiasis and cryptosporidiosis in pet dogs and cats. The results of this research enhanced the understanding of the disease prevalence of Giardia spp. and Cryptosporidium spp. among senior veterinary students and their dogs and cats. The survey collected valuable and novel information on the students’ characteristics, student health status, their pets’ health status and activities that may have led to an increased risk of infection during their clinical rotations or intense handling of small or large animals. The analysis of the survey provided an evaluation of potential risk factors associated with the risk of infection in senior veterinary students. Molecular analysis of isolates of human, dog and cat origin helped in differentiating between G. duodenalis assemblages and Cryptosporidium spp. genotypes. Future directions may include an evaluation for associations of positive test results with clinical findings and further studies determining the likelihood dogs or cats are carrying zoonotic Giardia spp. or Cryptosporidium spp.. National research is recommended to be conducted to identify risk factors in veterinary students from different states in the United States. Additionally, a larger study should be performed to determine the baseline exposure of veterinary school faculty, specifically, those who work on large animal rotations and collect fecal samples from their pet dogs and cats to for genotyping to detail whether zoonotic infections with these two protozoans occur. iv ACKNOWLEDGMENTS I must offer my sincere thanks and appreciation to my advisors, Dr. Michael Lappin and Dr. Francisco Olea–Popelka, for their support and encouragement to complete my research and pursue my interests. Dr. Lappin has been supportive and has provided valuable guidance throughout my training. I highly appreciate and give special thanks to Dr. Olea–Popelka for his patience and support both academically and morally over this journey. Without my advisors, I would never have reached this point of my study. Thanks to my committee members, Dr. Doreene Hyatt and Dr. David Twedt, for supporting me in the first phase of this program, particularly, in my preliminary examination. Special thanks to Dr. Valeria Scorza for her patience, support, and laboratory training. Beyond providing guidance as a Research Scientist, Dr. Scorza became a valuable friend who supported me personally, through good and bad times over the last three years of my study. Special thanks go to Dr. Lappin’s laboratory personnel in the Center for Companion Animal Studies for allowing me to use the laboratories and for their guidance and teaching. Thanks to Ms. Jennifer Hawley for taking care of the student financial compensation which was part of my study. Special thanks to the Animal Population Health Institute, which I am a member of, for the financial support in the first two years of this Ph.D. program. Thank you to Dr. Lyndsey Linke, a Research Scientist and the APHI Laboratory Coordinator, for being a good, supportive friend and for her guidance in the laboratory diagnostic part of this project. Thanks to ANTECH® Diagnostics, particularly Dr. David Aucoin for facilitating and providing the dataset that was used in chapter 2 of this dissertation. v My thanks and appreciation to Dr. Paula Cowen for her support both morally and financially. I thank her for being an aspiration to me. She taught me to never give up and always start again. I thank her family for being my best friends and family. Thank you to Dr. John Belfrage, and the boys, Brice, Seth and Sean Belfrage. Last but not least, I would like to thank my friends in the USA and outside the country. Special thanks to Ms. Loretta Scott in Greeley, Colorado for her continuous encouragement to pursue my degree. Special thanks go to my friends: Huda Tawir in Fort Collins, Colorado; Estabraq Abdulhadi in Detroit, Michigan and Hanaa Salih in Plymouth, England for their emotional support, much like a sister. My heartfelt thanks go to my family in Iraq: my wonderful, loving husband Bahaa Abosaooda and my two brothers: Ali and Raeid Thigeel for their support and love. A portion of this project was funded by the Research Council of the College of Veterinary Medicine and Biomedical Sciences (CRC) and the Young Investigator Grant program in the Center for Companion Animal Studies at Colorado State University. vi DEDICATION This dissertation is dedicated to my mother. May God rest her soul in peace vii TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGMENTS ...............................................................................................................v DEDICATION .............................................................................................................................. vii CHAPTER 1: GIARDIA DUODENALIS AND CRYPTOSPORIDIUM SPP.: A REVIEW ..........1 1.1 Giardia duodenalis ...................................................................................................................1 1.1.1Taxonomy .......................................................................................................................1 1.1.2