COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Director of Copyright Services sydney.edu.au/copyright Feline Sino-nasal and Sino-orbital Aspergillosis Vanessa R.D. Barrs BVSc(hons) MVetClinStud GradCertEd FANZCVSc (Feline Medicine) A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Veterinary Science The University of Sydney 2015 Acknowledgements I would like to thank, foremost, my supervisors, Professor Katherine Belov, Professor Rosanne Taylor and Emeritus Professor Paul Canfield for their support, encouragement and guidance. Kathy has been an inspirational mentor and I cannot thank her enough for welcoming me into her lab and for strategic advice and friendship. There are many other people to thank. Within the Faculty of Veterinary Science, special thanks to Dr. Trish Martin for mycological expertise, ideas and support since 2006 when we first isolated unusual Aspergillus species from pet cats, A/Professor Mark Krockenberger and Dr. Nathan Saul who facilitated molecular mycology in the Faculty and Dr. Jessica Talbot for recent contributions to our fungal diagnostics team, Dr. Beata Ujvari for sharing the highs and lows of developing an Aspergillus-specific ELISA with me, Dr. Navneet Dhand and Dr. Bethany Wilson for statistical advice and support and the team at the University Veterinary Teaching Hospital, Sydney who have been involved in so many ways with this research through interactions with affected cats and their owners. Thanks to Dr. Mariano Makara for his enthusiastic collaboration in advanced imaging analysis, to Helen Laurendet and Kathy Hughes for technical assistance, to Marianna Koureas for taking the time to track me down to take calls about cats with aspergillosis and to all the nursing and veterinary staff whose hearts were broken when many of our patients died despite months of caring for cats with the devastating sino-orbital form of disease. I would also like to thank my many external collaborators who agreed to work with me on the projects contained within this thesis. Very heartfelt thanks go to Dr. Catriona Halliday and Sue Sleiman from the NSW Centre for Infectious Diseases and Microbiology Laboratory Services for introducing me to molecular mycology. Also, thanks to Dr. Ailsa Hocking, who demystified fungal mating tests and, in turn, helped discover the teleomorphic phase of Aspergillus felis. I am enormously grateful to Professor Rob Samson, who on the basis of an email and a few preliminary PCR ii results, agreed to host my visit to the CBS-KNAW Fungal Biodiversity Centre at Utrecht. Thanks to Dr. Jos Houbraken and Ms. Tineke van Doorn at CBS for helping me to up- skill in molecular mycology and for sharing their knowledge and expertise and to Dr. Janos Varga for assisting with phylogenetic analyses. Thanks also to Dr. Sarah Kidd Head, National Mycology Reference Centre Microbiology & Infectious Diseases, for her expertise in antifungal susceptibility testing, to Dr. Iain Peters for technical advice in interpretation of ELISA data and to Professor Michael Day for hosting my visit to the University of Bristol that will result in future research publications. Contributions of clinical case material often go unrecognized, yet these have been pivotal in demonstrating that upper respiratory aspergillosis is a global emerging disease, that A. felis is not confined to Australia and that it is a human pathogen. I would also like to make special mention of my excellent clinical collaborators overseas; Professor Lynelle Johnson at the University of California, Davis, Dr. Frederic Billen from the University of Liege, Dr Peter Chapman, from the Veterinary Specialty and Emergency Centre in Levittown, Philadelphia, Professor Malcolm Richardson from the Mycology Reference Centre, Manchester UK and Dr Dolores Pinheiro from Hospital de Sao Joao, Porto, Portugal for contributing Aspergillus isolates and sera. Within Australia many other veterinary colleagues have donated clinical material and deserve special thanks – Drs. Erin Bell, Linda Abraham, Sue Bennett, Rob La Buc, Reuben Fliegner, Eloise Koelmeyer, Marcus Gunew and Anne Thompson. I am also grateful to the various funding bodies that have made this work possible; the Australian Government Endeavour Research Fellowship, the Faculty of Veterinary Sciences’ bequest funds, and especially the Australian Small Animal Veterinary Association’s funding body (the Australian Companion Animal Health Foundation), for their continued support of my research. Finally, my thanks and love to my life partner Jules for all of your support – I could not have done it without you. iii Table of Contents Acknowledgements ii Table of contents iv Declaration viii Author contributions ix Abbreviations xv Abstract xvi CHAPTER 1. INTRODUCTION 1 CHAPTER 2. LITERATURE REVIEW 4 2.1 Respiratory Aspergillosis: a one medicine approach 4 2.2 Nomenclature and taxonomy 5 2.2.1 The Aspergillus fumigatus complex 5 2.2.2 Fungal species identification – a polyphasic approach 9 2.2.2.1 Macro- and micro- morphology 12 2.2.2.2 Mating tests 13 2.2.2.3 Fungal Growth temperature regimes 13 2.2.2.4 Metabolomics - Extrolite profiles 14 2.2.2.5 Phylogenetic methods 16 2.3 Aetiological agents of aspergillosis in humans, dogs and cats 17 2.3.1 Humans 17 2.3.2 Dogs 18 2.3.3 Cats 19 2.4. Aspergillosis - parallels between humans, dogs and cats with regard to epidemiology, pathogenesis and clinical disease 19 2.4.1 Humans 20 2.4.1.1 Chronic granulomatous fungal rhinosinusitis 22 2.4.1.2 Chronic erosive non-invasive fungal rhinosinusitis 23 iv 2.4.2 URT aspergillosis in dogs and cats 23 2.4.2.1 Pathogenesis 23 2.4.2.1.1 Innate anti-fungal immunity 24 2.4.2.1.2 Adaptive anti-fungal immunity 26 2.4.2.1.3 Fungal virulence factors and evasion of the immune system 28 2.4.2.1.4 Immunology of canine SNA 30 2.4.2.1.5 Immunology of feline SNA 32 2.4.2.2 Epidemiology 32 2.4.2.3 Clinical presentation in dogs and cats 34 2.5 Diagnosis of aspergillosis 34 2.5.1 Serology and antigen detection in tissues and/or body fluids 35 2.5.1.1 Galactomannan 35 2.5.1.1.1 Galactomannan detection – humans 36 2.5.1.1.2 Galactomannan detection – dogs 37 2.5.1.1.3 Galactomannan detection – cats 38 2.5.1.2 Serum 1-3-B-D-glucan detection 39 2.5.1.3 Serum antibody detection - humans 39 2.5.1.4 Serum antibody detection – dogs and cats 40 2.5.2 Molecular methods for diagnosis of aspergillosis 41 2.5.2.1Molecular methods using formalin fixed paraffin embedded tissues (FFPET) – humans 42 2.5.2.1.1 Molecular methods using FFPET- dogs and cats 43 2.5.2.2 Real time quantitative PCR using clinical specimens 44 2.5.3 Diagnostic imaging 45 2.5.3.1 Computed tomographic findings in aspergillosis 46 2.5.3.1 Computed tomographic findings in aspergillosis 46 2.5.4 Histology and Immunohistochemistry 49 2.5.4.1 Canine SNA 50 2.5.4.2 Feline URT aspergillosis 51 v 2.6 Treatment and Prognosis 51 2.6.1 Treatment of canine SNA 51 2.6.2 Treatment of feline URT aspergillosis 52 2.7 Aims 54 CHAPTER 3. CHARACTERISATION OF CLINICAL DISEASE AND TREATMENT OUTCOMES IN CATS WITH UPPER RESPIRATORY ASPERGILLOSIS 3.1 Background 56 3.2 Main article 57 3.3 Conclusions 64 CHAPTER 4. IDENTIFICATION OF THE AETIOLOGICAL AGENTS OF FELINE ASPERGILLOSIS AND DISCOVERY OF A NOVEL SPECIES, ASPERGILLUS FELIS. 4.1 Background 66 4.2 Main article 67 4.3 Conclusions 78 CHAPTER 5. COMPUTED TOMOGRAPHIC FEATURES OF FELINE SINO-NASAL AND SINO-ORBITAL ASPERGILLOSIS. 5.1 Background 80 5.2 Main article 81 5.3 Conclusions 89 CHAPTER 6. DETECTION OF ANTI-ASPERGILLUS SPECIFIC ANTIBODIES BY AGAR GEL IMMUNODIFFUSION AND IMMUNOGLOBULIN-G ELISA 6.1 Background 91 6.2 Main article 93 6.3 Conclusions 98 vi CHAPTER 7. GENERAL DISCUSSION 7.1 Background 100 7.2 Summary of results 100 7.3 Main article 103 7.4 Further discussion 126 7.5 Future directions 130 CHAPTER 8. REFERENCES 133 CHAPTER 9. APPENDICES 150 Appendix 1 Barrs VR. Feline sino-orbital aspergillosis: an emerging clinical syndrome. American College of Veterinary Internal Medicine 27th Annual Congress Proceedings, 30 May- June 2nd 2009 Barrs VR. Feline upper respiratory tract aspergillosis. The European College of Veterinary Internal Medicine – Companion Animals 22nd ECVIM-CA Congress September 2012. Appendix 2 159 Barrs VR, Beatty JA 2010. Chapter 2: Feline Upper Respiratory Aspergillosis: An Emerging Clinical Syndrome. In: Consultations in Feline Internal Medicine Volume 6. Ed: August JR, Elsevier Saunders Ed August, pp 36-50.