NUCLEAR, PLASTID AND MITOCHONDRIAL GENES FOR DNA IDENTIFICATION, BARCODING AND PHYLOGENETICS OF APICOMPLEXAN PARASITES A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by JOSEPH DAIRO OGEDENGBE In partial fulfillment of requirements for the degree of Doctor of Philosophy May, 2011 ©J. D. Ogedengbe,2011 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-82844-1 Our file Notre reference ISBN: 978-0-494-82844-1 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives 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 Plnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, 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 this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada•*• ABSTRACT NUCLEAR, PLASTID AND MITOCHONDRIAL GENES FOR DNA IDENTIFICATION, BARCODING AND PHYLOGENETICS OF APICOMPLEXAN PARASITES Joseph Dairo Ogedengbe Advisor: University of Guelph, 2011 Dr. J.R. Barta The phylum Apicomplexa consists of parasitic organisms that are of major health and economic importance to man and other animals especially domestic ones. The systematics of these parasites has received some attention in recent years but there are still many areas of study that need elucidation. Morphological data has been shown to be insufficient in answering most phylogenetic questions especially within the Emerioriniid coccidia which is the main focus of this study. The need to corroborate and in some instances modify classical systematics with molecular data is a major thrust of phylogenetic studies. Most studies have however focused on limited number of genomes and genes and have led to new challenges in the molecular phylogenetics of the phylum Apicomplexa. In this study, nuclear as well as organellar genomes were explored both as molecular identification tools as well as markers for molecular phylogenetic studies. It is concluded that mitochondrial genes such as cytochrome oxidase c I is a useful diagnostic (barcoding) and corroborative molecular tool and that organellar (both plastid and mitochondrial) genes in conjunction with nuclear genes in a combined, robust phylogenetic analysis coupled with extensive taxon sampling offer a useful approach to understanding phylogenetic relationships within the Apicomplexa. ACKNOWLEDGEMENTS I am indebted and grateful to have worked with my advisor, Dr John R. Barta for his insights, encouragement and constant support from the conception of this work to its completion. I would also like to thank my graduate committee members; Drs Robert H. Hanner; Patrick Boerlin; and Bruce Hunter for their support, interest and suggestions to this Thesis. I would also like to thank Dr. Katarzyna B. Miska for accepting the task of being my external examiner. I wish to express my sincere thanks to Julie Cobean for her kind provision and untiring efforts in making available pure isolates of most of the eimeriid coccidia used for this study. The contributions of Dustin Curts to DNA isolation from field isolates of coccidia are acknowledged. I thank the National Veterinary Research Institute, Vom Nigeria, NSERC National Science and Engineering Research Council of Canada (NSERC), and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) for funding this study. I would last but not the least thank all members of my family especially my wife, Mosunmola and children for bearing with my long absence and their encouraging words when I needed it the most. 1 TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF TABLES iv LIST OF FIGURES v LIST OF APPENDICES vi LIST OF ABBREVIATIONS vii Declaration of Work 1 Chapter I: Introduction and Literature Review 2 1.1. INTRODUCTION 2 1.2. THE PHYLUM APICOMPLEXA 4 1.3. METHODS OF IDENTIFICATION 9 1.4. MOLECULAR PHYLOGENETICS 11 1.4.1. Molecular Phylogeny of the Apicomplexa (Levine, 1970) 16 1.5. MOLECULAR MARKERS AND GENOMES USED IN APICOMPLEXAN PHYLOGENY 31 1.5.1. Nuclear small subunit gene (18S rRNA gene) 31 1.5.2. Mitochondrial genome 33 1.5.3. Plastid genome 36 1.6. MULTIGENOME AND MULTIGENE ANALYSES IN THE APICOMPLEXA 38 1.7. THE OBJECTIVES THIS STUDY WAS TO: 41 Chapter II: Molecular identification ofEimeria species infecting market-age meat chickens in commercial flocks in Ontario 42 2.1. ABSTRACT 42 2.2. INTRODUCTION 42 2.3. MATERIALS AND METHODS 44 2.3.1. Samples 44 2.3.2. Sample Processing and Oocyst Preparation 44 2.3.3. Multiplex PCR Identification of Coccidia 45 2.3.4. Statistical Analysis 46 2.4. RESULTS 46 2.4.1. Histological Observations 46 2.4.2. Prevalence of Infections with Eimeria spp 46 2.4.3. Multiplex PCR Identifications 47 2.4. DISCUSSION 47 Chapter III: Phylogenetic Position of the Adeleorinid Coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) Inferred using 18S rDNA sequences 54 3.1. ABSTRACT 54 3.2. INTRODUCTION 55 3.3. MATERIALS AND METHODS 57 3.3.1. Parasite Material 57 3.3.2. DNA extraction and rDNA PCR Amplification 58 3.3.3. Phylogenetic analyses 60 3.4. RESULTS 62 3.4.1. Parasite Sequences: 62 3.4.2. Phylogenetic Analyses: 62 3.5. DISCUSSION 67 4.1. ABSTRACT 78 ii 4.2. INTRODUCTION 79 4.3.1. Oocysts and DNA extraction 82 4.3.2. PCR reaction parameters 83 4.3.3. Sequence Alignment and Phylogenetic Analysis 84 4.3.3. Species delimitation using mt COI or nu 18S rDNA sequences 87 4.4. RESULTS 88 4.5. DISCUSSION 92 4.5.1. DNA Barcoding for Parasite Species Identification 93 4.5.2. Use of mt COI partial sequences for molecular phylogenetics 96 CHAPTER V: Molecular Phylogenetics of Eimeriid coccidia (Eimeridae, Emeriorina, Apicomplexa, Alveolata): A Multi-gene and Multi-genome approach 101 5.1. ABSTRACT 101 5.2. INTRODUCTION 102 5.3. MATERIALS AND METHODS 104 5.3.1. Sources of Parasites and Parasite DNA 104 5.3.2. DNA Extraction: 105 5.3.3. PCR 105 5.3.4. Phylogenetic Analysis 106 5.3.4.1 Sequencing and Sequence Alignments 106 5.3.4.2. Data Analysis 110 5.4. RESULTS Ill 5.4.1.18S rDNA sequence analysis Ill 5.4.2. Cytochrome c oxidase subunit I sequence analysis 120 5.4.3. Plastid gene analysis 123 5.4.4. Multiple gene and genome consensus tree 126 5.5. DISCUSSION 128 6.0. GENERAL DISCUSSION AND CONCLUSIONS 133 7.0. REFERENCES 141 8.0. APPENDICES 172 111 LIST OF TABLES TABLE 1.1. TAXONOMIC CLASSIFICATION OF TAXA IN THE PHYLUM APICOMPLEXA 8 TABLE 2.1. DETECTION OF EIMERIA SPECIES IN MARKET-AGE MEAT BIRDS FROM COMMERCIAL BROILER FLOCKS 51 TABLE 3.1. ORGANISMS ANALYZED PHYLOGENETICALLY USING NUCLEAR 18SRDNA SEQUENCES 73 TABLE 4.1. COMPARISON OF PAIRWISE DIFFERENCES (MEAN±STANDARD ERROR) OF 18S NUCLEAR RDNA GENE SEQUENCES AND MITOCHONDRIAL CYTOCHROME OXIDASE C SUBUNIT 1 (COI) SEQUENCES 99 TABLE 4.2. COMPARISON OF THE GENETIC VARIATION WITHIN SPECIES AND BETWEEN CLOSEST SPECIES OF SEVEN ELMERIA SPECIES OF CHICKENS 100 TABLE 5.1. PLASTID AND MITOCHONDRIAL GENE PRIMER SETS 106 IV LIST OF FIGURES Figure 1.1: Representation of the probable evolutionary relationships among major groups within the Alveolata, based principally on 18S rDNA sequences 17 Figure 1.2: General layout of nuclear genes (18S rDNA; large subunit, 28S; 5.8S and 5S) ribosomal gene in eukaryotes 32 Figure 1.3: Mitochondrial genome structure of Eimeria tenella and Plasmodium falciparum 34 Figure 1.4: Gene organization oftheplastid genome of Eimeria tenella 37 Figure 2.1: Histological appearance of the cecal epithelium of a commercial broiler that had visible macroscopic cecal lesions and tested positive for Eimeria tenella using multiplex PCR 52 Figure 2.2: RAPD-SCAR-based multiplex PCR products obtained when using single Eimeria species as templates 52 Figure 2.3: Typical agarose gel showing separated multiplex PCR products with positive amplification products from individual DNA samples from intestinal contents containing oocysts 53 Figure 3.1: Bayesian phylogenetic analysis using 85 18S rDNA sequences from members of the Apicomplexa including the adeleorinid coccidia 65 Figure 3.2: Ingroup Bayesian phylogenetic analysis using 78 18S rDNA sequences from adeleorinid coccidia 66 Figure 4.1: Phylogenetic