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Feline Upper Respiratory Disease Complex: the Detection

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Feline Upper Respiratory Disease Complex: the Detection Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2014 Feline Upper Respiratory Disease Complex: The detection and epidemiology of respiratory pathogens in Midwestern feline shelter populations Uri Donnett Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Epidemiology Commons, and the Veterinary Medicine Commons Recommended Citation Donnett, Uri, "Feline Upper Respiratory Disease Complex: The detection and epidemiology of respiratory pathogens in Midwestern feline shelter populations" (2014). Graduate Theses and Dissertations. 13814. https://lib.dr.iastate.edu/etd/13814 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Feline Upper Respiratory Disease Complex: The detection and epidemiology of respiratory pathogens in Midwestern feline shelter populations by Uri Baruch Donnett A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Veterinary Preventive Medicine Program of Study Committee: Jessie D. Trujillo, Major Professor Annette M. O’Connor Brett A. Sponseller Claudia J. Baldwin Iowa State University Ames, Iowa 2014 Copyright © Uri Baruch Donnett, 2014. All rights reserved. ii TABLE OF CONTENTS Page LIST OF FIGURES ................................................................................................... v LIST OF TABLES ..................................................................................................... vi ABSTRACT………………………………. .............................................................. viii CHAPTER 1 GENERAL INTRODUCTION ....................................................... 1 Background ..................................................................................................... 1 Goals and objectives ............................................................................................ 2 Thesis Organization ............................................................................................. 3 References ......................................................................................................... 4 CHAPTER 2 LITERATURE REVIEW ............................................................... 5 Feline Upper Respiratory Disease Pathogens ................................................... 5 Feline Herpesvirus ................................................................................... 5 Feline Calicivirus .................................................................................... 7 Chlamydophila felis ................................................................................. 9 Bordetella bronchiseptica ........................................................................ 10 Mycoplasma felis ..................................................................................... 12 Evidence for predisposing factors ........................................................................ 13 Age ......................................................................................................... 13 Sex and sterilization status ...................................................................... 14 Source ...................................................................................................... 14 Breed ........................................................................................................ 15 Vaccination history .................................................................................. 15 Time in shelter.......................................................................................... 15 Hygiene ................................................................................................... 16 Presence of dogs ...................................................................................... 16 Season ...................................................................................................... 17 Review of the Prevalence of FURD and FURD Associated Pathogens in Shelter Cats ......................................................................................................... 17 Review of prevalence studies in literature .............................................. 17 Data extraction and quality assessment .................................................. 18 Data Analysis ........................................................................................... 20 Results ...................................................................................................... 21 Literature search .......................................................................... 21 FURD in shelters ......................................................................... 22 Pathogens in shelters ................................................................... 22 Critical review of the prevalence of FURD ................................. 23 Critical review of the prevalence of target pathogens ................. 29 iii Discussion ................................................................................................ 31 References ............................................................................................................ 34 CHAPTER 3 IDENTIFICATION OF DIVERSE AND UNIQUE MYCOPLASMA SPECIES IN THE FELINE UPPER RESPIRATORY TRACT UTILIZING THE PAN-MYCO qPCR ASSAY ................................................................................. 43 Abstract ......................................................................................................... 43 Introduction ......................................................................................................... 44 Materials and Methods ......................................................................................... 48 Mycoplasma Strains and Colony Forming Unit Determination .................... 48 Clinical Feline Samples ................................................................................. 48 Nucleic Acid Extraction ................................................................................. 49 Pan-Myco SYBR qPCR assay ........................................................................ 49 Assay Limits of Detection and PCR Processing of Clinical Sample ............. 50 Mycoplasma species detected by the Pan-Myco SYBR qPCR ....................... 52 Results ......................................................................................................... 52 Validation and Limits of Detection of Pan-Myco SYBR qPCR for Stock Mycoplasma Species ...................................................................................... 53 Mycoplasma Species Detected in Clinical Samples ...................................... 54 Discussion ......................................................................................................... 59 References ......................................................................................................... 64 CHAPTER 4 VALIDATION OF A PORTABLE MOLECULAR DIAGNOSTIC DEVICE FOR DETECTION OF PATHOGENS ASSOCIATED WITH FELINE UPPER RESPIRATORY DISEASE IN CATS ........................................................................ 69 Abstract ......................................................................................................... 69 Introduction ........................................................................................................ 70 Materials and Methods ......................................................................................... 73 Viruses and Bacteria ...................................................................................... 73 Nucleic Acid Extraction ................................................................................. 74 Real-time PCR Reference Assays................................................................... 75 Internal Laboratory Validation of Selected Reference qPCR Assays ............ 76 Insulated Isothermal PCR Reagents .............................................................. 76 Limits of detection and exclusivity testing ..................................................... 78 Absolute quantitation of DNA targets ............................................................ 79 Clinical samples and selection methods ........................................................ 80 Statistical Analysis ......................................................................................... 81 Results ......................................................................................................... 81 Stage 1: Assay Selection ................................................................................ 81 Stage 2: Validation of qPCR Reference Assays ............................................. 81 Stage 2: 100% Limits of Detection for Point of Need Reagents vs. Reference Assays ............................................................................................ 84 Stage 2: Exclusivity of Reference and Point of Need Reagents ..................... 87 Stage 3: Selection of Clinical Samples .......................................................... 87 iv Stage 3: Measures of Agreement Utilizing Clinical Sample Sets .................
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