THE EFFECTS OF THE ABORTIFACIENT PARASITE, NEOSPORA CANINUM ON BOVINE FOETUSES IN EARLY AND LATE GESTATION Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Patrick Sylvester Craig July 2014 i AUTHORS’S DECLARATION Apart from the help and advice acknowledged, this thesis represents the unaided work of the author ……………………………………………… Patrick Sylvester Craig July 2014 This research was carried out in the Department of Infection Biology and School of Veterinary Science, University of Liverpool ii TABLE OF CONTENTS AUTHORS’S DECLARATION .................................................................................. ii TABLE OF CONTENTS ............................................................................................ iii DEDICATION ........................................................................................................... vii ACKNOWLEDGEMENTS ...................................................................................... viii ABSTRACT ................................................................................................................. x LIST OF FIGURES ................................................................................................... xii LIST OF TABLES ..................................................................................................... xv LIST OF ABBREVIATIONS ................................................................................... xvi CHAPTER ONE: INTRODUCTION .......................................................................... 1 1.1 Neospora caninum .............................................................................................. 1 1.2 History and economic impact of N. caninum ..................................................... 2 1.3 Life cycle of N. caninum .................................................................................... 4 1.4 Structure and biology of N. caninum .................................................................. 5 1.5 Epidemiology of neosporosis and N. caninum infection .................................... 7 1.6 Transmission of N. caninum in cattle ................................................................. 9 1.7 Pathogenesis of N. caninum infection in cattle ................................................ 11 1.8 Host cell invasion and parasite replication ....................................................... 12 1.9 Host immune response to N. caninum .............................................................. 14 1.10 In vitro cultivation of N. caninum .................................................................. 15 1.11 Pathological changes in N. caninum infected cattle ....................................... 18 1.12 Diagnosis of bovine neosporosis .................................................................... 20 1.13 Prevention and control of N. caninum in cattle .............................................. 23 1.14 Development of the bovine foetal immune response and its relevance for N. caninum infection ................................................................................................... 25 1.15 Pro- and anti-apoptotic activities of intracellular protozoan parasites ........... 29 1.16 Parasite manipulation of host cells ................................................................. 32 1.18 Parasite egress from host cells ........................................................................ 33 1.19 Host cell death ................................................................................................ 34 1.20 The apoptosis-necrosis continuum ................................................................. 34 1.21 Aims and objectives of the study ................................................................... 36 CHAPTER TWO: Histological and immunohistological analysis of bovine foetuses infected with N. caninum in early and late gestation ........................................ 38 2.1 ABSTRACT ......................................................................................................... 39 iii 2.2 INTRODUCTION ............................................................................................ 40 2.3 Aim of the study ............................................................................................... 42 2.4 MATERIALS AND METHODS ..................................................................... 43 2.4.1 Animals and foetal tissues, histological examination ....................................... 43 2.4.2 Histology and immunohistology for the characterisation of pathological changes and the identification of the composition of haemolymphatic tissues 44 2.4.3 Lendrum’s Carbol Chromotrope stain for the demonstration of eosinophils ... 46 2.4.4 Immunohistological staining ............................................................................. 46 2.4.5 Staining for myeloid/histiocyte antigen/calprotectin (method: PAP mouse) .... 48 2.4.6 Staining for N. caninum antigen (method: PAP rabbit) .................................... 49 2.4.7 Staining for CD3 antigen (method: ABC) ........................................................ 50 2.4.8 Quantitative data analysis ................................................................................. 51 2.5 RESULTS ......................................................................................................... 52 2.5.1 Histological and immunohistological findings in foetuses from dams infected in early gestation (day 70 dg) and control foetuses (91±1 dg) ............................. 52 2.5.1.1 Non-haemolymphatic tissues ......................................................................... 52 2.5.1.2 Haemolymphatic tissues ................................................................................ 60 2.5.1.3 Thymus ........................................................................................................... 60 2.5.1.4 Spleen ............................................................................................................. 61 2.5.1.5 Bone marrow .................................................................................................. 62 2.5.2 Histological and immunohistological findings in foetuses from dams infected in late gestation (day 210) and control foetuses (231±1 dg) ................................. 66 2.5.2.1 Non-haemolymphatic tissues ......................................................................... 66 2.5.2.2 Haemolymphatic tissues ................................................................................ 73 2.5.2.3 Thymus ........................................................................................................... 73 2.5.2.4 Spleen ............................................................................................................. 74 2.5.2.5 Mesenteric lymph nodes ................................................................................ 75 2.5.2.6 Bone marrow .................................................................................................. 75 2.5.3 Histological and immunohistological findings in a foetus and a new-born calf from naturally infected dams after recrudescence of N. caninum in mid to late gestation ............................................................................................................ 79 2.5.3.1 Non-haemolymphatic tissues of naturally infected foetus ............................. 80 2.5.3.2 Non-haemolymphatic tissues of new-born calf ............................................. 86 2.5.3.3 Haemolymphatic tissues ................................................................................ 88 2.5.3.4 Thymus ........................................................................................................... 88 2.5.3.5 Spleen ............................................................................................................. 89 2.5.3.6 Mesenteric lymph node .................................................................................. 90 2.5.3.7 Bone marrow .................................................................................................. 91 iv 2.6 DISCUSSION .................................................................................................. 96 CHAPTER THREE: Investigation of parasite-induced apoptosis in hepatocyte and cardiomyocyte cell lines ................................................................................. 114 3.1 ABSTRACT ................................................................................................... 115 3.2 INTRODUCTION .......................................................................................... 116 3.3 Aims of the study ........................................................................................... 118 3.4 MATERIALS AND METHODS ................................................................... 119 3.4.1 Culturing and maintenance of Vero cells ........................................................ 119 3.4.2 Culturing and maintenance of N. caninum parasites ....................................... 119 3.4.3 Isolation and culturing of primary bovine hepatocytes ................................... 120 3.4.4 Culturing and maintenance of hepatocyte cell lines ....................................... 122 3.4.5 N. caninum in vitro invasion-egression study ................................................. 123 3.4.6 Culturing and maintenance of murine cardiomyocyte cell line .....................