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Gibson Daniel 201908 Msc.Pdf Determination of common causes of morbidity and mortality in captive psittacine birds submitted to the Ontario Veterinary College Teaching Hospital and development of targeted diagnostic tests by Daniel Gibson A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Pathobiology Guelph, Ontario, Canada © Daniel Gibson, July, 2019 ABSTRACT Determination of common causes of morbidity and mortality in captive psittacine birds submitted to the Ontario Veterinary College Teaching Hospital and development of targeted Daniel Gibson Advisors: University of Guelph, 2019 Dr. Leonardo Susta Dr. Nicole Nemeth Veterinary medical literature focused on diagnostic pathology of psittacine birds (e.g., parrots, macaws, cockatoos) is based largely on data derived from case reports with an overall lack of systematic approaches with the exception of a few zoonotic or economically important diseases. In an effort to increase our understanding of disease prevalence and presentation, and improve diagnostic approaches, we conducted a retrospective study of pathological data in psittacine birds submitted to the Ontario Veterinary College from 1998-2017 (n = 1,850). Using this database, we identified cohorts of birds with which to test and validate new diagnostic methods. First, we developed a multiplex real-time PCR assay for the detection of three DNA viruses in psittacine birds. Second, we used cases of round cell neoplasia to assess the utility of immunohistochemical markers, commonly used in mammalian species, for the diagnosis of lymphoma in psittacine birds. ACKNOWLEDGMENTS I would like to thank my advisors Drs. Leonardo Susta and Nicole Nemeth for encouraging me and supporting me on this adventure. You both have communicated to me many times how much you appreciate me, and this is one of the things that keeps me going, so thank you very much. I will always remember how much time and effort you have put into me both for personal and professional development. Also, to my other committee members Drs. Hugues Beaufrère, and Csaba Varga, thank you very much for all the guidance, your perspectives were very valuable in helping form and disseminate the information in this thesis, and specifically Csaba, for the endless advice on statistics. I would like to thank all the Animal Health Laboratory staff for tirelessly working to produce good histological material and accurate diagnostic tests. Specifically, I would like to thank Dr. Beverly McEwen for help in extracting data from the databases, all the pathologists and graduate students, who have made my time at pathobiology very enjoyable and much easier than it could have been. I would like to acknowledge Kai Moore for her original work on the database on which I could build the current projects. I would like to thank all my lab mates, both past and present for helping me get oriented and answering my many menial questions on sometimes a daily basis, specifically, Alex Leacy, Dr. John Pham, as well as all the summer students who have helped to make my time in the lab enjoyable. iii STATEMENT OF WORK PERFORMED All the work described in the present thesis was performed by myself (Daniel Gibson) in collaboration with and under the guidance of my advisory committee (Drs. Leonardo Susta, Nicole Nemeth, Hugues Beaufrère, and Csaba Varga) with the following exceptions: Chapter 3: Immunohistochemistry for CD3, MUM-1, and all other markers described in Chapter 5 were performed by the staff of the Animal Health Laboratory, with the exception of Pax5 and KUL01, for which all laboratory work was performed by myself (Daniel Gibson). Chapter 4: Dr. Davor Ojkic, Animal Health Laboratory, designed the primer/probe sets and oversaw the PCR testing. The Animal Health Laboratory histology technicians cut the paraffin scrolls, and Anna Marom, Animal Health Laboratory, conducted RNA extraction and performed PCR. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGMENTS ............................................................................................................. iii STATEMENT OF WORK PERFORMED ................................................................................... iv TABLE OF CONTENTS ................................................................................................................ v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ........................................................................................................................ x LIST OF ABBREVIATIONS ....................................................................................................... xii LIST OF APPENDICES .............................................................................................................. xiii Chapter 1. Literature review ....................................................................................................... 1 1.1 Definition of retrospective case studies, and utility to investigate diseases in psittacine birds… ......................................................................................................................................... 1 1.1.1 Objectives of retrospective studies ........................................................................... 2 1.1.2 Inherent selection bias in retrospective studies ......................................................... 7 1.1.3 Gaps of knowledge: Current state of psittacine research .......................................... 8 1.2 Characterization of round cell neoplasia in psittacines and methods of diagnosis .......... 8 1.2.1 Prevalence ................................................................................................................. 9 1.2.2 Etiology ................................................................................................................... 10 1.2.3 Pathology and presentation ..................................................................................... 10 1.2.4 Prognosis ................................................................................................................. 11 1.2.5 Immunohistochemistry ........................................................................................... 11 1.2.6 Tissue microarray methods to assess immunohistochemical cross-reactivity ........ 13 1.3 Real-time PCR for DNA viruses in psittacines .............................................................. 14 1.3.1 Psittacid herpesvirus-1 ............................................................................................ 15 1.3.2 Beak-and-feather disease virus ............................................................................... 17 1.3.3 Aves polyomavirus-1 .............................................................................................. 21 1.3.4 Overall diagnostic methods for psittacine DNA viruses......................................... 23 1.4 Proposal .......................................................................................................................... 25 1.4.1 Gaps of knowledge ................................................................................................. 25 1.4.2 Objectives ............................................................................................................... 25 v 1.4.3 Methods................................................................................................................... 26 1.5 Tables ............................................................................................................................. 28 Chapter 2. Captive psittacine birds in Ontario, Canada: A 19-year retrospective study of the causes of morbidity and mortality................................................................................................. 34 2.1 Summary ........................................................................................................................ 35 2.2 Introduction .................................................................................................................... 36 2.3 Materials and methods ................................................................................................... 37 2.3.1 Study population and data extraction ...................................................................... 37 2.3.2 Coding of final diagnoses by severity, etiology, and organ system ....................... 38 2.3.3 Database formatting ................................................................................................ 39 2.3.4 Statistical analysis ................................................................................................... 39 2.4 Results ............................................................................................................................ 41 2.4.1 Demographic data ................................................................................................... 41 2.4.2 Overview of final diagnoses and disease processes ................................................ 41 2.4.3 Infectious disease processes .................................................................................... 42 2.4.4 Non-infectious disease processes ............................................................................ 45 2.4.5 Exploratory statistics ..............................................................................................
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