Association of Markers in the Vitamin D Receptor with Mhc Class Ii Expression and Marek's Disease Resistance
Total Page：16
File Type：pdf, Size：1020Kb
ASSOCIATION OF MARKERS IN THE VITAMIN D RECEPTOR WITH MHC CLASS II EXPRESSION AND MAREK'S DISEASE RESISTANCE Dana Praslickova Department of Animal Science McGill University Montreal Canada December, 2007 A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of Doctor of Philosophy © Dana Praslickova, 2007 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-50980-7 Our file Notre reference ISBN: 978-0-494-50980-7 NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada MOTTO "...for it is not the rock that is the most solid and it is not steel that is the most firm. In fact, it is an ordinary Joe whose endurance is greatest." J. C. Hronsky: Jozef Mak. Published in Czechoslovakia by Tatran, 1965, p. 271 This is dedicated to my beloved daughter Zuzana for being supportive and patient throughout all these years... Contents TABLE OF CONTENTS Page TABLE OF CONTENTS i ABSTRACT v RESUME vii ACKNOWLEDGEMENTS ix LIST OF TABLES xi LIST OF FIGURES xii ABBREVIATIONS xiv STATEMENT OF ORIGINALITY xvii CONTRIBUTION OF CO-AUTORS TO MANUSCRIPTS FOR PUBLICATION xix CHAPTER 1 - INTRODUCTION 1 1.1 GENERAL INTRODUCTION 1 1.1.1 Hypothesis 2 1.1.2 Objective 2 1.1.3 Experimental model 3 1.2 OVERVIEW OF THESIS CONTENT 3 1.3 REFERENCES 4 CHAPTER 2 - LITERATURE REVIEW 5 2.1 GENERAL DESCRIPTOPN OF MAREK'S DISEASE 5 2.1.1 Marek's disease virus 5 2.1.2 Phatogenesis 7 2.1.3 Immune response of the organism 10 2.1.4 Diagnosis of Marek's disease 12 2.2 CONTROL STRATEGY 13 2.2.1 Vaccination 13 2.2.2 Genetic resistance 15 2.2.3 Major histocompactibility complex genes 16 2.2.4 Non-major histocompactibility complex genes 16 2.3 GENES USED IN OUR STUDY 17 2.3.1 Growth hormone 17 2.3.2 Growth hormone receptor 18 2.3.3 Macrophage inflammatory protein 3a 19 2.3.4 Vitamin D 20 2.4 REFERENCES 23 CONNECTING STATEMENT I 35 CHAPTER 3 - SEQUENCE VARIATIONS IN GENES ENCODING ENZYMES INVOLVED IN THE VITAMIN D METABOLISM AND ASSOCIATION WITH SUBCLASSES OF PERIPHERAL BLOOD MONONUCLEAR CELLS IN CHICKENS 36 3.1 ABSTRACT 37 3.2 INTRODUCTION 38 3.3 MATERIALS AND METHODS 39 3.3.1 Strains of chickens and data collection 39 3.3.2 Flow cytometry 39 3.3.3 Genetic analysis 40 3.3.4 Statistics and graphics 41 3.4 RESULTS 41 3.4.1 Determination of blocks of co-segregating SNP 41 3.4.2 Association of single genes with the cell differentiation antigens on peripheral blood mononuclear cells 42 3.4.3 Gene interaction 43 3.4.4 Correlation with production traits 43 3.5 DISCUSSION 44 3.6 ACKNOWLEDGEMENTS 47 3.7 REFERENCES 60 CONNECTING STATEMENT II 63 CHAPTER 4 - EFFECT OF MARKER ASSISTED SELECTION ON INDICATORS OF MAREK'S DISEASE IN A VACCINATED COMMERCIAL WHITE LEGHORN STRAIN 64 4.1 ABSTRACT 65 4.2 INTRODUCTION 66 4.3 MATERIALS AND METHODS 68 4.3.1 Strains of chickens and selection strategy 68 4.3.2 Markers selection 68 4.3.3 Challenge 69 4.3.4 Apramycin treatment 69 4.3.5 Viral titers in feather tip extracts 69 4.3.6 Statistical analysis 70 4.4 RESULTS 70 4.4.1 Efficacy of vaccination 70 4.4.2 Effect of selection trial on viral titers 71 4.4.3 Survival analysis 71 4.4.4 Necropsy analysis 72 4.4.5 Effect on body weight, spleen weight and bursal weight 73 4.5 DISCUSSION 73 4.6 REFERENCES 86 CONNECTING STATEMENT III 89 CHAPTER 5 - ASSOCIATION OF A MARKER IN THE VITAMIN D RECEPTOR GENE WITH MAREK'S DISEASE RESISTANCE IN POULTRY 90 5.1 ABSTRACT 91 5.2 INTRODUCTION 92 5.3 MATERIALS AND METHODS 93 5.3.1 Strains of chickens and challenge test 93 5.3.2 DNA extraction and viral titration 93 5.3.3 Genetic analysis of the VDR gene 94 Contents 5.3.4 Statistical analysis 94 5.4 RESULTS 95 5.4.1 Association with viral proliferation 95 5.4.2 Association with MD lesions, mortality and weight of the bursa 95 5.5 DISCUSSION 96 5.6 REFERENCES 106 CHAPTER 6 - GENERAL CONCLUSION 109 APPENDIX 113 Abstract ABSTRACT Vaccination, biosecurity and selection for genetic resistance are used world-wide in the poultry industry against the threat of Marek's disease (MD). Unfortunately there are new outbreaks of MD that cause serious economic problems. Scientists are therefore searching for new and more effective ways to improve existing controls of the disease. Knowledge of the chicken genome and progress in the study of the molecular biology of the MD virus are providing new approaches to MD control. A particularly useful strategy is the identification of genes that affect viral and tumor susceptibility. Genetic markers in the growth hormone receptor (GHR), the growth hormone (GH) and the chemokine MIP- 3 a that are associated with MD resistance have previously been identified in our laboratory. In this thesis we identified additional candidate genes by analyzing genes of the vitamin D metabolism; conducted a large scale challenge experiment with Marek's disease virus and tested genes encoding enzymes involved in vitamin D metabolism for association with disease resistance. The first manuscript describes the analysis of three genes of the vitamin D metabolism for sequence variability and their association with the proportion of peripheral blood mononuclear cells (PBMC) that display the surface antigens LYB, MHC II, CD3, CD4, CD8, TCR1 (Ty8) and TCR2 (Tap1). We identified a genetic marker in the vitamin D receptor (VDR) gene that affected the frequency of the MHC class II expressing leukocytes (P=0.0007), and a marker in the vitamin D binding protein gene that (DBP SIP 15) affected the expression of TCR1. The goal of the second study was to conduct selection in a commercial strain of White Leghorns for markers in the GH, GHR and MIP-3a genes that had previously been associated with MD resistance and to compare the resistance of the selected and non- selected commercial cross. We conducted two challenge experiments three months apart with 100 chickens from the selected and 100 chickens from a non-selected population in each challenge. To maintain similarity in poultry management, we followed a commercial vaccination procedure. A database was compiled that comprised measurements of the viral titers in extracts of feather tips on a weekly basis up to 8 weeks post infection, a record of mortality and a necropsy analysis of all chickens, including those that died Abstract during the experiment. The outcome in challenge 1 differed from challenge 2. In challenge 1 the selected population had a two-fold lower viral load than the non-selected control population (P= 10") while in challenge 2 the situation was reversed (P=T0"). A comparison of the effect of the challenge on the two populations shows that the titers in the non-selected population in the two challenges were similar, while the titers in the selected population differed by a factor of four. Hence the challenges were reproducible for the non-selected population but not for the selected population. The same conclusion was reached when other indicators of MD, such as mortality, frequency of proliferative lesions, loss of body weight or atrophy of the bursa were measured. The source of the different behaviors is unknown, but it raises the possibility that immune compromising factors such as stress, nutritional status, maternal antibodies or infections may compromise the response to MD infection in a manner that is dependent on the genetic background. In the third manuscript we used the database we had created to analyze the influence of three markers in the chicken VDR gene on MD resistance. We found that the marker that had been found to be associated with MHC class II was also associated with a reduced viral titer (P=0.002).