Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2014 Investigation of swine dysentery associated with "Brachyspira hampsonii" strain EB107 and comparison of diagnostic methods Bailey Lauren Wilberts Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Animal Diseases Commons, Pathology Commons, and the Veterinary Medicine Commons Recommended Citation Wilberts, Bailey Lauren, "Investigation of swine dysentery associated with "Brachyspira hampsonii" strain EB107 and comparison of diagnostic methods" (2014). Graduate Theses and Dissertations. 14266. https://lib.dr.iastate.edu/etd/14266 This Dissertation 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]. Investigation of swine dysentery associated with “Brachyspira hampsonii” strain EB107 and comparison of diagnostic methods by Bailey L. Wilberts A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Veterinary Pathology Program of Study Committee: Eric R. Burrough, Co-Major Professor Michael J. Yaeger, Co-Major Professor Darin M. Madson Jesse M. Hostetter Paul J. Plummer Iowa State University Ames, Iowa 2014 Copyright © Bailey L. Wilberts, 2014. All rights reserved. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ....................................................................................... v ABSTRACT………………………………. .............................................................. vi CHAPTER 1 GENERAL INTRODUCTION .................................................... 1 Statement of Problem ......................................................................................... 1 Specific Aims ..................................................................................................... 1 Dissertation Organization .......................................................................................... 2 Literature Review....................................................................................................... 3 Historical background .......................................................................................... 3 Diagnostic assays ........................................................................................... 8 Identification of a novel etiologic agent of swine dysentery ......................... 10 Fluorescent in situ hybridization for bacterial identification ......................... 12 Background ........................................................................................... 12 Technique .............................................................................................. 13 Challenges ............................................................................................. 16 Selected applications ............................................................................ 17 Pathogenesis of swine dysentery ................................................................... 18 Differential mucin expression of the colonic mucus bilayer in health and disease ................................................................................................ 21 References ......................................................................................................... 27 CHAPTER 2 COMPARISON OF LESION SEVERITY, DISTRIBUTION, AND COLONIC MUCIN EXPRESSION IN PIGS WITH ACUTE SWINE DYSENTERY FOLLOWING ORAL INOCULATION WITH “BRACHYSPIRA HAMPSONII” OR BRACHYSPIRA HYODYSENTERIAE ... 44 Abstract ......................................................................................................... 44 Introduction ......................................................................................................... 45 Materials and Methods ......................................................................................... 47 Animals ......................................................................................................... 47 Bacterial strains, growth conditions, and preparation of inocula ................... 47 Animal inoculation......................................................................................... 48 Molecular identification ................................................................................. 49 Animal observations and necropsy ................................................................ 49 Histopathology ............................................................................................... 50 Immunohistochemistry .................................................................................. 51 Histochemistry ............................................................................................... 52 Image analysis ................................................................................................ 52 iii Fluorescent in situ hybridization .................................................................... 53 Statistical analyses ......................................................................................... 53 Results ......................................................................................................... 53 Animal observations ...................................................................................... 53 Microbial culture ............................................................................................ 54 Gross pathology ............................................................................................. 54 Histopathology ............................................................................................... 57 Immunohistochemistry .................................................................................. 58 Histochemistry ............................................................................................... 60 Fluorescent in situ hybridization .................................................................... 62 Molecular identification ................................................................................. 62 Discussion ......................................................................................................... 62 Acknowledgements .............................................................................................. 67 References ......................................................................................................... 68 CHAPTER 3 COMPARISON OF CULTURE, PCR, AND FLUORESCENT IN SITU HYBRIDIZATION FOR DETECTION OF BRACHYSPIRA HYODYSENTERIAE AND “BRACHYSPIRA HAMPSONII” IN PIG FECES . 72 Abstract ......................................................................................................... 72 Introduction ......................................................................................................... 73 Materials and Methods ......................................................................................... 74 Bacterial strains and growth conditions ......................................................... 74 Preparation of fecal dilutions and standard plate counts ............................... 75 Fluorescent in situ hybridization .................................................................... 75 Real-time polymerase chain reaction primers, probes, and protocol ............. 77 Preparation of samples from experimentally infected pigs ............................ 77 Results ......................................................................................................... 78 Culture ......................................................................................................... 78 Fluorescent in situ hybridization .................................................................... 79 Real-time polymerase chain reaction ............................................................. 80 Fluorescent in situ hybridization: Samples from experimentally infected pigs ........................................................................................................ 80 Discussion ......................................................................................................... 82 Sources and Manufacturers .................................................................................. 84 References ......................................................................................................... 85 CHAPTER 4 INVESTIGATION OF THE IMPACT OF INCREASED DIETARY INSOLUBLE FIBER THROUGH THE FEEDING OF DRIED DISTILLERS GRAINS WITH SOLUBLES ON THE INCIDENCE AND SEVERITY OF BRACHYSPIRA-ASSOCIATED COLITIS IN PIGS ............... 87 Abstract ......................................................................................................... 87 Introduction ......................................................................................................... 88 Materials and Methods ......................................................................................... 90 Animals ......................................................................................................... 90 iv Diet ......................................................................................................... 91 Inoculum .......................................................................................................