Analysis of the Vaccine-Induced Immune Response Against the Bovine Parasites Ostertagia Ostertagi and Cooperia Oncophora
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Laboratory of Parasitology Department of Virology, Parasitology and Immunology Faculty of Veterinary Medicine Ghent University Analysis of the vaccine-induced immune response against the bovine parasites Ostertagia ostertagi and Cooperia oncophora. Frederik Van Meulder Dissertation submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Science, 2019 Promotor Prof. Dr. Peter Geldhof Table of Contents LIST OF ABBREVIATIONS 5 1 LITERATURE REVIEW: 9 1.1 NEMATODE HELMINTH INFECTIONS IN CATTLE 9 1.1.1 LIFE CYCLE 11 1.1.2 PATHOPHYSIOLOGY - PATHOLOGY OF O. OSTERTAGI AND C. ONCOPHORA 14 1.1.3 ECONOMIC IMPACT OF HELMINTH INFECTIONS 16 1.2 TREATMENT OF HELMINTHS WITH CHEMICAL ANTHELMINTHICS 17 1.3 ALTERNATIVE TO ANTHELMINTHIC DRUGS: VACCINES 18 1.3.1 VACCINES AGAINST OSTERTAGIA OSTERTAGI 19 1.3.2 VACCINES AGAINST COOPERIA ONCOPHORA 21 1.3.3 RECOMBINANT VACCINES 22 1.4 ANTI-PARASITE IMMUNE RESPONSES 27 1.4.1 CELLULAR RESPONSE IN GASTROINTESTINAL TRACT 27 1.4.2 EOSINOPHILS 30 1.4.3 MUCOSAL MAST CELLS AND GLOBULAR LEUKOCYTES 31 1.4.4 GOBLET CELLS AND MUCINS 31 1.4.5 HUMORAL RESPONSE 32 1.4.6 –OMICS RESULTS OF IMMUNE RESPONSES 34 1.5 OBJECTIVES: 41 2 GRANULE-EXOCYTOSIS OF GRANULYSIN AND GRANZYME B AS A POTENTIAL KEY MECHANISM IN VACCINE-INDUCED IMMUNITY IN CATTLE AGAINST THE NEMATODE OSTERTAGIA OSTERTAGI 45 2.1 ABSTRACT 45 2.2 INTRODUCTION: 45 2.3 MATERIALS AND METHODS: 48 2.3.1 PREPARATIONS OF ASP ANTIGENS 48 2.3.2 VACCINATION TRIAL 48 2.3.3 ENZYME-LINKED IMMUNOSORBENT ASSAYS 48 2.3.4 TISSUE COLLECTION, RNA EXTRACTION AND CDNA PREPARATION 49 2.3.5 MICROARRAY ANALYSIS 49 2.3.6 QUANTITATIVE REAL-TIME PCR 50 2.3.7 HISTOCHEMISTRY 51 2.3.8 CELL COUNTS 52 2.3.9 SDS-PAGE AND WESTERN BLOTTING OF MUCUS SAMPLES 52 2.3.10 MASS SPECTROMETRIC PROTEIN IDENTIFICATION 53 2.3.11 STATISTICAL ANALYSES 53 2.4 RESULTS: 54 2.4.1 VACCINATION TRIAL 54 2.4.2 HUMORAL AND CELLULAR IMMUNE RESPONSES 54 2.4.3 MICRO-ARRAY ANALYSIS ON ABOMASAL MUCOSA 55 2.4.4 CORRELATION STUDY 61 2.4.5 LOCALISATION AND CELLULAR SOURCE OF GRANULYSIN IN THE ABOMASUM 63 2.4.6 WESTERN BLOT AND SDS-PAGE ON MUCUS SAMPLES 65 2.5 DISCUSSION 66 2.6 ACKNOWLEDGMENTS 70 3 3 EFFECT OF GRANULYSIN (GNLY) ON THE GROWTH AND FECUNDITY OF THE MODEL NEMATODE CAENORHABDITIS ELEGANS 75 3.1 ABSTRACT 75 3.2 INTRODUCTION 75 3.3 MATERIALS AND METHODS 78 3.3.1 GNLY PEPTIDE 78 3.3.2 ESCHERICHIA COLI TEST OF BOVINE GNLY PEPTIDE 79 3.3.3 C. ELEGANS CULTURES 79 3.3.4 STATISTICS 81 3.4 RESULTS 82 3.4.1 EFFECTS ON E. COLI 82 3.4.2 EFFECTS OF THE BOGNLY PEPTIDE ON C. ELEGANS 82 3.5 DISCUSSION 85 4 ANALYSIS OF THE PROTECTIVE IMMUNE RESPONSE FOLLOWING INTRAMUSCULAR VACCINATION OF CALVES AGAINST THE INTESTINAL PARASITE COOPERIA ONCOPHORA 91 4.1 ABSTRACT 91 4.2 INTRODUCTION 91 4.3 MATERIALS AND METHODS 93 4.3.1 PREPARATION OF COOPERIA ONCOPHORA PROTEIN MATERIAL 93 4.3.2 ANIMAL EXPERIMENTS AND SAMPLE COLLECTION 93 4.3.3 ANTIBODY RESPONSES 94 4.3.4 HISTOLOGY AND CELL COUNTS 95 4.3.5 IN VITRO RE-STIMULATION ASSAYS AND FLOW CYTOMETRY 95 4.3.6 RNA EXTRACTION, CDNA PRODUCTION AND QUANTITATIVE REAL-TIME PCR 97 4.3.7 RNA-SEQ ANALYSIS 97 4.3.8 STATISTICAL AND CORRELATION ANALYSES 98 4.4 RESULTS 99 4.4.1 SYSTEMIC AND INTESTINAL TISSUE-ASSOCIATED ANTIBODY RESPONSES 99 4.4.2 CELLULAR IMMUNE RESPONSES 100 4.4.3 INTESTINAL TRANSCRIPTOME ANALYSIS 101 4.4.4 CORRELATION ANALYSIS OF IMMUNOLOGICAL AND PARASITOLOGICAL PARAMETERS 107 4.5 DISCUSSION 109 4.6 ACKNOWLEDGEMENTS 111 5 GENERAL DISCUSSION 115 6 REFERENCES 125 7 APPENDIX 145 7.1 APPENDIX OF CHAPTER 2 145 7.2 APPENDIX OF CHAPTER 4 153 8 SUMMARY – SAMENVATTING 167 8.1 SUMMARY 167 8.2 SAMENVATTING 171 DANKWOORD 177 CURRICULUM VITAE 185 4 List of abbreviations ALB Albendazole (ALB, from BZs) APCs Antigen Presenting Cells ASPs Activation-associated secreted proteins BZs Benzimidazoles CD Cluster of Differentiation DC Dendritic Cell ddASPs double-domain Activation-associated secreted proteins EPG Eggs Per Gram ES Excretion/Secretion ESP Excretory/Secretory products EST Expressed Sequence Tags FEC Fecal Egg Count FGS First Grazing Season GI Gasto-intestinal GL Globule Leukocytes GCNT Glycosyltransferases GNLY Granulysin IPA Ingenuity Pathway Analysis IFN Interferon IHF Immunohistofluorescence Ig Immunoglobulin IL Interleukin IVM Ivermectin (IVM from MLs) I/T Imidothiazoles and tetrahydropyrimidines LEV Levamisole (LEV from I/Ts) MHC Major Histocompatibility Complex MLN Mesenteric Lymph Nodes MLs macrocyclic lactones MDR Multi-drug resistance MUC Mucin OPA Ostertagia polyprotein allergen PAMP Pathogen-Associated Molecular Pattern PRR Pattern-Recognition Receptor PGE Parasitic Gastroenteritis RNA-Seq RNA sequencing SAGE Serial Analysis of Gene Expression SIM Small Intestine mucosa TCR T-cell receptor TFF Trefoil Factors TGF Transforming Growth Factor Th T helper cell Treg Regulatory T-cell 5 1 Introduction Introduction ___________________________________________________________________________ 1 Literature review: 1.1 Nematode helminth infections in cattle Ostertagia ostertagi and Cooperia oncophora are the most important gastro-intestinal (GI) parasitic roundworms of grazing cattle (Bos taurus) [1-3]. More specifically, they are both members of the nematode order Strongylida and family Trichostrongylidae. Nematodes are bilaterally symmetrical, the digestive system is fully equipped with mouth, oesophagus, intestine and anus. There are mostly separated sexes, and the life cycle of parasites is direct with one host, or indirect with one invertebrate intermediary host. More than 10.000 different nematode species are described, but presumably more than 100.000 exist. Most of the described nematodes are free living, such as the famous transparent laboratory model organism Caenorhabditis elegans [4], which belongs to the order Rhabditida. A large number of nematodes exist as parasites, such as the order Strongylida, to which many veterinary parasites belong [5]. A taxonomic overview of nematodes in which both of these closely related orders are shown is presented in Figure 1 [6]. Strongylid nematodes comprise nine families, but the family of Trichostrongylidae contains many gastrointestinal (GI) parasites of ruminants. They are filiform worms with a simple mouth with or without a rudimentary developed bursa copulatrix. Beside O. ostertagi and C. oncophora, Trichostrongylidae also contains the important sheep and goat parasites Haemonchus contortus, and Teladorsagia circumcincta and different species of Trichostrongylus spp. infecting the abomasum or small intestine. Most Trichostrongylid parasites have an important economic impact on livestock production [7, 8]. 9 Chapter 1 ________________________________________________________________________ Figure 1 Molecular evolutionary framework for the phylum Nematoda. Right: nematode orders; Left: tropic ecology. The order Strongylida (to which O. ostertagi and C. oncophora belong) and the order Rhabditida (to which C. elegans belongs) are closely related to each other. An MP (maximum parsimony) analysis of SSU (Small Subunit) ribosomal DNA sequences from 53 nematode taxa is shown. Nearly complete SSU sequences were aligned and processed for MP analysis. Twenty- four shortest trees (requiring 3,583 nucleotide changes) were found; the tree presented is the strict consensus of these. Branch lengths are drawn to be proportional to the number of changes inferred. The trophic ecologies of the taxa are represented by coloured icons. (based on Blaxter et. al, 1998) 10 Introduction ___________________________________________________________________________ 1.1.1 Life cycle 1.1.1.1 Ostertagia ostertagi There are three phases of the pre-parasitic part of the life cycle of Ostertagia ostertagi (see Figure 2) [1, 7-9]. Inside the egg, an L1 larvae develops, which leaves the egg after approximately 24h. The first two larval stages (L1 + L2), which retain in the feces, feed from the coliform bacteria. The third stage (L3), the infective stage which will spread out of the feces, keeps the cuticle of the L2 stage. Therefore, this larvae cannot take up food and it lives from its glycogen reserve. The temperature is especially important for the development of embryonated eggs to infectious larvae as higher temperatures accelerate the speed of development. During a normal summer in countries with a moderate climate like Belgium, infectious L3 larvae development takes between 7 to 14 days. In the early spring, development takes several weeks. Before the parasites can be taken up by the hosts, they have to migrate from the feces to the grass of the pasture. There are both active and passive movement processes, and although larvae of parasites are motile, they can only move for a short distance and a moisture film is necessary for the active movement. Passive movements are dependent of feces consistency, rain that decomposes feces, animals touching and spreading the feces with their feet and feces spread by insects, earthworms, etc. Infectious larvae leave the feces in a period that can take, especially in dry weather, several months. Within their protective L2 cuticle they are quite well protected. The time in which they will survive is difficult to predict, as it is dependent of temperature fluctuations, humidity and soil condition [10]. The majority of larvae die within a year. Low temperatures improve the survival of infectious larvae because they are less active and can thus survive longer on their food reservoir. Cattle infect themselves by taking up infectious larvae from the grass. 11 Chapter 1 ________________________________________________________________________ Once ingested by the cow, the L3 larvae lose their sheath (L2 cuticle) in the rumen. The larvae subsequently move to the gastric glands of the abomasum, more specifically the fundus glands where the third shake off takes place (L3 to L4 on day 4 after infection). Also the fourth shake off (L4 to L5) takes place in the fundus gland, starting 10 days after infection, and after approximately 16 days of infection the L5 larvae appear from the fundus gland and become adults.