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Development and Evaluation of Sequence Typing Assays for Investigating The Development and Evaluation of Sequence Typing Assays for investigating the Epidemiology of Mycoplasma synoviae Outbreaks in Poultry DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mohamed El-Gazzar Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2014 Dissertation Committee: Richard D. Slemons, Advisor Päivi J. Rajala-Schultz Daral J. Jackwood Copyrighted by Mohamed Medhat El-Gazzar 2014 Abstract Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the two most pathogenic avian mycoplasma species. In birds Mycoplasma can be transmitted both horizontally and vertically, and understanding the epidemiology of the infections is an integral part of controlling the disease. Several molecular assays can be used for mycoplasma genotyping. Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) are two finger printing assays commonly used for this purpose. But sequence typing assays are the preferred method for avian mycoplasma genotyping. Sequence typing is preferred because it can be performed directly on clinical samples without the need to isolate the microorganism in a pure culture. Multiple genes have been identified for sequence typing of MG, while the partial vlhA gene sequence is the only target being used for MS sequence typing. Relying on a single gene sequence creates ambiguity in the genetic relatedness between MS strains. In an effort to enlarge the repatoir of MS sequence typing assays, we explore expantion of the currently described vlhA assay segment and we investigate one new genomic target for sequence typing of MS. We also report the development of a new MLST assay for MS. ii The first chapter is a literature review summarizing the main features of class Mollicutes which is the class to which avian mycoplasma belong. In addition we also outline the characteristics of avian mycoplasma diseases and their diagnosis, control and molecular epidemiology. The second chapter of this document describes the attempt to expand the targeted area of the vlhA gene used for MS sequence typing in an effort to maximize its genotypic potential. The expansion was successful and the target segment was extended from 420 bp to 900 bp. The gene conversion recombination process was discovered to occur immediately downstream of the original 420 bp. Hence the sequence information generated beyond 420 bp was found not to be useful for MS sequence typing purposes. This study identified the maximum potential of vlhA gene for MS sequence typing. The third chapter describes an attempt to amplify another DNA segment that can be used for MS sequence typing by sequencing. Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR) system in prokaryotes. This system serves as an acquired immune system of prokaryotes against invading phage infections. Amplification and sequencing of the CRISPR segment was successful in a few MS samples, and CRISPR sequences showed variations between MS samples. However, amplification of the CRISPR segment was not successful in the rest of the tested MS samples using conventional PCR techniques at hand. Whole genome sequencing can be one of the ways to harness the discriminatory power and the genotypic potential of this region. While for the time being the whole genome sequencing is not one of the used techniques in the iii regular investigation of mycoplasma outbreaks in the poultry industry, but probably will be in the future. The fourth chapter describes the development Multilocus Sequence Typing (MLST) assay for MS. Twenty four housekeeping genes were studied and seven internal gene segments were finally selected. The amplification and sequencing of sevenn gene segments was successful in all 58 tested MS strains, field isolates and clinical samples. Out of the tested 58 samples MLST generated 30 sequence types. The vlhA sequence typing assay also generated 30 sequence types for the same population. In spite of having the same apparent discriminatory power, MLST provided links between the related outbreaks that is more congruent with the available epidemiological information. Therefore using both MLST and vlhA in tandem is recommended for a more refined understanding of the epidemiological picture of mycoplasma outbreaks. In conclusion, the maximum potential of vlhA sequence typing assay was determined and a new MS sequence typing assay (MLST) was developed. As a result a tenfold increase in the size of genomic sequence used for MS sequence typing was achieved. The author recommends to use both MLST and vlhA assays in tandem for more accurate and refined epidemiological analysis of MS outbreaks. iv Acknowledgments I‘m privileged to be in this position by the help and the support of many individuals along the journey who encouraged me to pursuit my dreams when they seemed impossible and assured me when I was in doubt. For that I am forever indebted to them and forever grateful. I first want to pay tribute to both of my parents who are no longer with us in this life. I want to let them know that the love of science and knowledge they instilled in me and their insistence that I should always follow my passions has paid dividends. It goes without saying that my advisors and committee members are the pillar without which I would have not been able to rise to this new peak in my career. However, I‘m beholden to Dr. Richard Slemons for accepting to be my advisor and for his sincere guidance. I would like also to thank Dr. Päivi Rajala-Schultz and Dr. Daral Jackwood for their valuable contributions to the success of this program. Among my committee member I want to especially thank to Dr. Mo Saif for his priceless input and continuous encouragement. I want to acknowledge Dr. Ziv Raviv who afforded me the opportunity to come to The Ohio State University, and put me on the right track. Especial thanks Dr. William J. v A. Saville our department chair who made my graduation possible by his unflinching support through uncertain times. I also want to take the opportunity to recognize other individual who had profound effect on my life. Thanks to Dr. Alaa Abdo of Cairo University for his guidance that immensely helped me to push forward and reach for my goals. I thank him not only for his mentorship but also for being the role model that I aspired to follow on both the professional and personal levels. I would like to thank Dr. Nicolas Dorko of Aviagen who opened for me the horizon of better poultry education and pointed me in the right direction. With that I‘m ready to close one chapter of my life and excited to start a new one. vi Vita 1995................................................................Ismail El-Kabbany High School 2000................................................................B.S. Veterinary Medicine, Cairo University 2000 to 2006 ..................................................Technical Service Veterinarian, Cairo Poultry Group 2006 to 2008 ..................................................Avian Range Product Specialist, Ceva Santé Animale – Egypt Ltd. 2009................................................................Master of Avian Medicine, The University of Georgia 2010 to present ..............................................Graduate Research Associate, Department of Veterinary Preventive Medicine, The Ohio State University Publications 1. M. El Gazzar, V. A. Laibinis, and N. Ferguson-Noel. Characterization of a ts- 11–like Mycoplasma gallisepticum isolate from commercial broiler chickens. Avian Dis. 55:569–574. 2011. vii 2. Kyung-Il Kang, Mohamed El-Gazzar, Holly S. Sellers, Fernanda Dorea, Susan M. Williams, Taejoong Kim, Stephen Collett and Egbert Mundt. Investigation into the aetiology of runting and stunting syndrome in chickens. Avian Pathol. 41:41–50. 2012. 3. Mohamed M. El-Gazzar, Amy N. Wetzel, and Ziv Raviv. The genotyping potential of the Mycoplasma synoviae vlhA gene. Avian Dis. 56:711–719. 2012. Fields of Study Major Field: Veterinary Preventive Medicine viii Table of Contents Abstract ................................................................................................................... ii Acknowledgments................................................................................................... v Vita ........................................................................................................................ vii Table of Contents ................................................................................................... ix List of Tables ....................................................................................................... xiv List of Figures ....................................................................................................... xv Introduction ............................................................................................................. 1 Chapter 1: Litruature Review ................................................................................. 5 1.1 History and evolution of Class Mollicutes ............................................... 5 1.1.1 History and significance ........................................................... 5 1.1.2 Evolution and phylogeny .......................................................... 7 1.2 Biology of Class Mollicutes ................................................................... 10 1.2.1 Lack of cell wall.....................................................................
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