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Effect of Genetic and Proteomic Variation in Field Isolates of Mycoplasma Hyopneumoniae on Virulence, Diagnosis and Prevention O Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2008 Effect of genetic and proteomic variation in field isolates of Mycoplasma hyopneumoniae on virulence, diagnosis and prevention of disease in the pig Erin Linn Strait Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, Animal Sciences Commons, Large or Food Animal and Equine Medicine Commons, Microbiology Commons, and the Veterinary Pathology and Pathobiology Commons Recommended Citation Strait, Erin Linn, "Effect of genetic and proteomic variation in field isolates of Mycoplasma hyopneumoniae on virulence, diagnosis and prevention of disease in the pig" (2008). Retrospective Theses and Dissertations. 15640. https://lib.dr.iastate.edu/rtd/15640 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 Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Effect of genetic and proteomic variation in field isolates of Mycoplasma hyopneumoniae on virulence, diagnosis and prevention of disease in the pig by Erin Linn Strait A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Immunobiology Program of Study Committee: Eileen L. Thacker, Major Professor Patrick J. Halbur Douglas E. Jones F. Chris Minion Brad J. Thacker Iowa State University Ames, Iowa 2008 Copyright © Erin Linn Strait, 2008. All rights reserved 3307067 3307067 2008 ii TABLE OF CONTENTS LIST OF FIGURES vi LIST OF TABLES vii ABSTRACT ix CHAPTER 1. GENERAL INTRODUCTION 1 Introduction 1 Dissertation Organization 1 Literature Review 2 Mollicutes 2 Mycoplasmas and Acholeplasmas of swine 3 Mycoplasma hyopneumoniae 3 Disease characteristics 3 Epidemiology 4 Field isolate diversity 5 Virulence factors 6 Immune response 7 Vaccines 8 Diagnostics 9 Culture 9 Serology 9 Polymerase chain reaction 10 Summary with Hypothesis and Goals 11 References 12 CHAPTER 2. COMPARISON OF MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES 22 Abstract 22 Introduction 22 Materials and Methods 24 Mycoplasma hyopneumoniae field isolates 24 Study design 24 Sample collection 25 Serology 25 iii BALF analysis 25 Growth curves 26 Statistics 26 Results 26 Challenge inoculum titers 26 Percent pneumonia 27 Bacterial isolation 27 Quantitative real-time PCR 29 IL-1ß and IL-18 ELISAs 29 IgA and IgG ELISAs 29 Serology 29 Growth curves 29 Discussion 31 Acknowledgements 32 References 33 CHAPTER 3. IMPACT OF GENETIC DIVERSITY AMONG MYCOPLASMA HYOPNEUMONIAE FIELD ISOLATES ON PCR ASSAY DEVELOPMENT 35 Abstract 35 Introduction 35 Materials and Methods 36 Mycoplasmas 36 PCR assays 36 M. hyopneumoniae mhp165 real-time PCR 37 M. hyopneumoniae mhp183 real-time PCR 43 mhp165 and mhp183 multiplex real-time PCR assay 43 DNA sequencing and analysis 43 Results 45 Mycoplasma PCR assay panel 45 Real-time PCR assays 45 DNA sequence results 45 Detection level 46 Discussion 47 Acknowledgements 49 iv References 50 CHAPTER 4. COMPARISON OF MYCOPLASMA HYOPNEUMONIAE- SPECIFIC ELISAS AND THE EFFECT OF FIELD ISOLATE VARIABILITY 53 Abstract 53 Introduction 54 Materials and Methods 55 Animals, housing and experimental design 55 Sample collection 55 Macroscopic pneumonia 56 ELISA assays 56 PCR from nasal swabs, bronchial swabs and bronchial alveolar lavage fluid 57 Western blot assay 57 Statistics 57 Results 57 Macroscopic pneumonia 57 Bacteriological findings 58 Oxoid, IDEXX and 232 Tween 20 ELISAs 58 97MP0001 Tween 20 ELISA 62 PCR 63 Western blot 63 Discussion 64 Acknowledgements 68 References 68 CHAPTER 5. EFFICACY OF A MYCOPLASMA HYOPNEUMONIAE BACTERIN IN PIGS CHALLENGED WITH TWO CONTEMPORARY PATHOGENIC ISOLATES OF M. HYOPNEUMONIAE PROVIDES AN INNOVATIVE APPROACH TO EVALUATING M. HYOPNEUMONIAE VACCINES 71 Summary 71 Introduction 72 Materials and Methods 73 Study animals 73 v Experimental design 73 Serum antibody tests 74 Necropsy 74 Data analysis 75 Results 76 Death of animals during the study 76 Serology 76 Macroscopic lung lesions 76 M. hyopneumoniae real-time PCR 76 IgA and IgG antibodies 78 Bacterial isolation from bronchial swabs 79 Discussion 81 Implications 83 Acknowledgements 83 References 83 CHAPTER 6. GENERAL CONCLUSIONS 87 General Discussion 87 Recommendations for Future Research 89 ACKNOWLEDGEMENTS 90 vi LIST OF FIGURES Figure 2.1. Box-plot comparing the percent of macroscopic pneumonia in pigs challenged with one of four field isolates of M. hyopneumoniae, strain 232, or unchallenged controls 27 Figure 2.2. Growth-curves for four M. hyopneumoniae field isolates and strain 232 generated from real-time PCR on culture samples 30 Figure 3.1. Sequence alignment of the mhp165 gene real-time PCR target region for isolates of M. hyopneumoniae compared to the consensus sequence from M. hyopneumoniae strain 232 (AE017332) 42 Figure 3.2. Sequence alignment of the mhp183 gene real-time PCR target region for isolates of M. hyopneumoniae compared to the consensus sequence from M. hyopneumoniae strain 232 (AE017332) 44 Figure 3.3. DNA sequence analysis of the Kurth et al. inner PCR product of strains 00MP1502 and 00MP1301 47 Figure 4.1. 232 Tween 20 OD comparison of group means in M. hyopneumoniae-infected groups versus days-post-challenge 61 Figure 4.2. 97MP0001 Tween 20 OD comparison of group means in M. hyopneumoniae-infected groups versus days-post-challenge 63 Figure 4.3. Western blot results detecting proteins from antigen prepared from M. hyopneumoniae isolate 97MP0001 64 vii LIST OF TABLES Table 2.1. Summary of pneumonia and bronchial alveolar lavage fluid analysis 28 Table 2.2. A comparison of Tween 20, IDEXX and Oxoid ELISA results from sera collected at necropsy (28 DPC) from pigs challenged with one of four field isolates of M. hyopneumoniae or strain 232 30 Table 3.1. Bacteria used in this study 38 Table 3.2. Summary of results for PCR assays tested against M. hyopneumoniae isolates 41 Table 3.3. List of primer and probe sequences used in this study 42 Table 3.4. Analysis of clinical samples by real-time PCR 46 Table 4.1. Summary of the percentage of pneumonia, culture, and PCR results or pigs challenged with M. hyopneumoniae field isolates 59 Table 4.2. Summary of pneumonia and culture results for pigs challenged with M. flocculare, M. hyorhinis and M. hyosynoviae 60 Table 4.3. ELISA results from pigs challenged with M. hyopneumoniae field isolates 62 Table 5.1. Frequency of pigs positive for M hyo Tween 20 and DAKO serum antibody titers in pigs vaccinated twice with an M hyo bacterin, or saline, then challenged with M hyo, and necropsied 28 DPC (95MP1509 challenge) or 30DPC (00MP1301 challenge) 77 Table 5.2. M hyo Tween 20 serum antibody titers in pigs vaccinated twice with an M hyo bacterin, or saline, then challenged with M hyo, and necropsied 28 DPC (95MP1509 challenge) or 30DPC (00MP1301 challenge) 78 Table 5.3. Parametric analysis of M hyo percent lung lesions in pigs vaccinated twice with an M hyo bacterin, or saline, then challenged with M hyo, and necropsied 28 DPC (95MP1509 challenge) or 30DPC (00MP1301 challenge) 79 viii Table 5.4. Frequency distribution of percentage lung lesions at necropsy in pigs vaccinated twice with an M hyo bacterin, or saline, then challenged with M hyo, and necropsied 28 DPC (95MP1509 challenge) or 30DPC (00MP1301 challenge) 80 Table 5.5. M hyo real-time polymerase chain reaction levels in bronchial alveolar lavage fluids (BALF) at necropsy in pigs vaccinated twice with an M hyo bacterin, or saline, then challenged with M hyo, and necropsied 28 DPC (95MP1509 challenge) or 30DPC (00MP1301 challenge) 80 Table 5.6. M hyo-specific IgA and IgG antibodies in bronchial alveolar lavage fluids (BALF) at necropsy in pigs vaccinated twice with an M hyo bacterin, or saline, then challenged with M hyo, and necropsied 28 DPC (95MP1509 challenge) or 30DPC (00MP1301 challenge) 81 ix ABSTRACT The purpose of these studies was to investigate the implications of variability in virulence, genetics and antigenic profiles among field isolates of Mycoplasma hyopneumoniae. Increased virulence was compared with in vitro and in vivo growth characteristics of the organism and with immune parameters in the pig, although none was found to be significantly correlated. The impact of proteomic and genetic variation among field isolates on diagnostic assays was examined by both in vivo and in vitro studies. Three enzyme-linked immunosorbent assays (ELISAs) used in the United States were compared and shown to not detect all isolates of M. hyopneumoniae equally. Similarly, polymerase chain reaction (PCR) assays were tested against a panel of M. hyopneumoniae field isolates and it was shown that some isolates are not detected by all published PCR assays. Therefore, two new real-time PCR assays were developed that appear to be both sensitive and specific for M. hyopneumoniae. The ability of a commercial vaccine to protect against field isolates of M. hyopneumoniae was evaluated in a challenge study using two recent field isolates. One isolate produced nearly twice as many macroscopic lung lesions as the other isolate, but vaccinated pigs had less pneumonia compared to non-vaccinated pigs in both challenge groups. Together these results demonstrate that differences exist among isolates of M. hyopneumoniae in the
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