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Genomic Comparison of Mycoplasma Species Isolated from Commercial Chickens in South Africa by Amanda Beylefeld Submitted in Fulf

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Genomic Comparison of Mycoplasma Species Isolated from Commercial Chickens in South Africa by Amanda Beylefeld Submitted in Fulf Genomic comparison of mycoplasma species isolated from commercial chickens in South Africa by Amanda Beylefeld submitted in fulfilment in accordance with the requirements for the degree Doctor of Philosophy (PhD) in Production Animal Studies in the Faculty of Veterinary Sciences, University of Pretoria October 2018 Supervisor: Prof Celia Abolnik DECLARATION I, Amanda Beylefeld, student number 25285573 hereby declare that this dissertation, “Genomic comparison of mycoplasma species isolated from commercial chickens in South Africa”, is submitted in accordance with the requirements for the Doctor of Philosophy (PhD) degree at University of Pretoria, is my own original work and has not previously been submitted to any other institution of higher learning. All sources cited or quoted in this research paper are indicated and acknowledged with a comprehensive list of references. Amanda Beylefeld 31 October 2018 i ETHICS STATEMENT The author, whose name appears on the title page of this thesis, has obtained, for the research described in this work, the applicable research ethics approval. The author declares that she has observed the ethical standards required in terms of the University of Pretoria’s Code of ethics for researchers and the Policy guidelines for responsible research. ii ACKNOWLEDGEMENTS First and foremost, I would like to give my praise and thanks to my father God, who put this opportunity on my path and walked this road with me. I would also like to give a very special thanks to Prof. Celia Abolnik for her outstanding leadership, invaluable advice, guidance, support and patience and for granting me this amazing opportunity. I would also like to take the time to express my sincere gratitude to the following people, without whom this thesis would not be possible: Mr. Johan Gouws and Ms. Pamela Wambulawaye for their help with the mycoplasma isolations and identification Dr. Buks Wandrag for his help, support and invaluable talks about the poultry industry The whole poultry group for their support, friendship and encouragement, in no particular order Thandeka Phiri, Dr. Dauda Bwala, Dr. Adeyemi Modupeore, Dr Agnes Oladokun and the late Dr Ponman Solomon The Technology Innovation Agency-Tshwane Animal Health Innovation Cluster Initiative, the Welfare Sector Education and Training Authority, University of Pretoria and the National Research Foundation for their financial support. Then also my parents, Arnold and Sharon van Tonder for all their love and support and for giving me all the opportunities to become the successful young adult I am today And my grandparents for their love, support and financial aid throughout my studies Then to my amazing husband, Jaco Beylefeld for all his love, encouragement, support, never-ending patience and invaluable help with the command line programs (or Linux headaches, as would like to call it) This has been a great journey, without many ups and downs, but I enjoyed every minute of it and I am very grateful for all the love, support and patience provided by all my friends and family iii ABSTRACT Mycoplasma gallisepticum and Mycoplasma synoviae are listed by the World Organisation for Animal Health (OIE) as notifiable avian diseases agents and are considered as economically important species affecting the South African poultry industry. A decreased number of cases identified by culture with growth inhibition of M. gallisepticum and M. synoviae and an increase in unidentified mycoplasma species were observed at the University of Pretoria diagnostic laboratory. Samples were isolated from chickens displaying typical signs associated with pathogenic mycoplasma infection, thus necessitating a closer look at the lesser known mycoplasma species found in poultry. The aim of this study was to use second generation sequencing to identify and compare mycoplasma species isolated from commercial chickens in South Africa. Mycoplasma samples were isolated, sequenced and de novo assembled for identification by 16S rRNA phylogeny. A total of 124 samples were received between 2003 and 2015, 44 of these samples contained multiple species resulting in 179 isolates identified as M. gallisepticum (24.58%), M. synoviae (9.50%), M. gallinaceum (24.02%), M. gallinarum (24.58%), M. pullorum (13.97%) and M. iners (2.79%) and one Acholeplasma laidlawii (0.56%). Antimicrobial resistance (AMR) to chlortetracycline, enrofloxacin, tylosin and tiamulin and the genes involved in AMR were studied. Three M. gallinaceum samples showed possible multidrug resistance and novel point mutations associated with AMR in M. gallinaceum and M. gallinarum were identified. The first complete genome of M. pullorum was assembled, annotated and published. Draft genome assemblies for axenic strains were constructed and candidate genes that can be tested for novel diagnostic and vaccine targets were identified. Key terms: Mycoplasma, poultry, antimicrobial resistance, 16S rRNA, Mycoplasma gallisepticum, M. synoviae, Mycoplasma pullorum, Mycoplasma gallinaceum, sequencing, whole genome sequencing iv LIST OF ABBREVIATIONS AIC - Akaike information criteria AMR - Antimicrobial resistance ATCC - American Type Culture Collection BIC - Bayesian information criteria CDS - coding sequences CI - Consistency index COG - Clusters of Orthologous Group CRD - Chronic respiratory disease crmA - Cytokine response modifier A gene cysP - cysteine proteases gene DIVA - Differentiating infected from vaccinated animals DOE-JGI - Department of Energy Joint Genome Institute DNA - Deoxyribonucleic acid dNTP - deoxynucleoside triphosphate DVTD - Department of Veterinary Tropical Diseases ELISA - Enzyme-linked immunosorbent assays gapA - Glyceraldehyde-3-phosphate dehydrogenase A gene GTS - Gene-targeted sequencing gyrA - DNA gyrase subunit A gene gyrB - DNA gyrase subunit B gene HGT - Horizontal gene transfer HI - Haemagglutination inhibition IBV - Infectious bronchitis virus IgG - immunoglobulin G IGSR - Intergenic spacer region IMG - Integrated Microbial Genomes and Microbiomes LCB - Locally collinear block MAFFT - Multiple Alignment using Fast Fourier Transform MDR - Multi-drug resistant MG - Mycoplasma gallisepticum MGAP - Microbial Genome Annotation Pipeline MIC - Minimum inhibitory concentration MS Mycoplasma synoviae NAD - Nicotinamide adenine dinucleotide NCBI - National Centre for Biotechnology Information v ncRNAs - non-coding RNAs NDV - Newcastle disease virus NNI - Nearest neighbour interchange OIE - World Organisation for Animal Health osmC - osmotically inducible protein C gene OLC - Overlap-layout-consensus ORF - Open reading frames ohr - organic hydroperoxide resistance gene parC - Topoisomerase IV subunit A gene parE - Topoisomerase IV subunit B gene PCR - Polymerase chain reaction PGAP - Prokaryotic Genome Annotation Pipeline PGM - Personal genome machine PTS - phosphotransferase system PvpA - Phase-variable putative adhesin qPCR - Real-time PCR QRDR - Quinolone resistance-determining resistance regions RAPD - random amplification of polymorphic DNA RAST - Rapid Annotation using Subsystem Technology RFLP - Restriction fragment length polymorphism RI - Retention index rplD - ribosomal protein L4 gene rplV - ribosomal protein L22 gene rRNA - Ribosomal ribonucleic acid RSA - Rapid serum agglutination rt-PCR - reverse transcription PCR SGS - Second-generation sequencing SMRT - Single molecule real time SNP - Single-nucleotide polymorphisms SOLiD - Sequencing by oligonucleotide ligation and detection SPR - Subtree pruning and regrafting TBR - Tree bisection and reconnection tRNA - transfer RNA UP - University of Pretoria VlhA - Variable lipoprotein haemagglutinin WGS - Whole genome sequencing w/v - Weight per volume vi TABLE OF CONTENTS DECLARATION ........................................................................................................................ I ETHICS STATEMENT............................................................................................................. II ACKNOWLEDGEMENTS ...................................................................................................... III ABSTRACT ............................................................................................................................ IV LIST OF ABBREVIATIONS .................................................................................................... V TABLE OF CONTENTS ........................................................................................................ VII LIST OF FIGURES .................................................................................................................. X LIST OF TABLES ..................................................................................................................XV CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW .............................................. 1 1.1. INTRODUCTION ....................................................................................................... 1 1.2. MYCOPLASMA ......................................................................................................... 2 1.2.1. General characteristics ..................................................................................................2 1.2.2. Poultry mycoplasmas ....................................................................................................3 1.2.3. Role of poultry mycoplasmas in disease .........................................................................4 1.2.4. Diagnosis of poultry mycoplasma ...................................................................................6
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