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Comparative Genomics in Brucella Suis: from Intra-Specific and Inter-Specific Distinctive Features to Diagnostic Molecular Markers

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Comparative Genomics in Brucella Suis: from Intra-Specific and Inter-Specific Distinctive Features to Diagnostic Molecular Markers UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Comparative genomics in Brucella suis: from intra-specific and inter-specific distinctive features to diagnostic molecular markers Doutoramento em Biologia Especialidade de Microbiologia Ana Cristina Ribeiro Alves Ferreira Inácio Tese orientada por: Professor Doutor Rogério Paulo de Andrade Tenreiro Doutora Maria Inácia Aleixo Vacas de Carvalho Corrêa de Sá Documento especialmente elaborado para a obtenção do grau de doutor 2017 vii ii UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Comparative genomics in Brucella suis: from intra-specific and inter-specific distinctive features to diagnostic molecular markers Doutoramento em Biologia Especialidade de Microbiologia Ana Cristina Ribeiro Alves Ferreira Inácio Tese orientada por: Professor Doutor Rogério Paulo de Andrade Tenreiro Doutora Maria Inácia Aleixo Vacas de Carvalho Corrêa de Sá Júri: Presidente: ● Doutor José Manuel Gonçalves Barroso Vogais: ● Doutor João Paulo dos Santos Gomes ● Doutor Albano Gonçalo Beja-Pereira ● Doutora Maria Inácia Vacas de Carvalho Corrêa de Sá ● Doutora Lélia Mariana Marcão Chambel ● Doutor Ricardo Pedro Moreira Dias Documento especialmente elaborado para a obtenção do grau de doutor 2017 iii iv It is not the strongest of the species that survive, nor the most intelligent, but the one most adaptable to change. Wrongly attributed to Charles Darwin. Leon Megginson, 1964. v vi Acknowledgments First of all I would like to thank my supervisors, Professor Rogério Tenreiro and Doctor Maria Inácia Corrêa de Sá, for giving me the opportunity to work on this thesis, as well as for their friendly and good-humored mentoring, constant availability and guidance. I am very grateful for their support throughout my studies and doubts. Professor Rogério, thank you for receiving me so well at BioISI Research group M&B, and for always trying to meet the best conditions so that I could achieve my goals. Doctor Maria Inácia, I would like to express my admiration and gratitude for you and for all the opportunities of valorization and scientific training you have given me during these last 20 years. Thank you for your interest and most of all, thank you for all the advices (often personal) and words of encouragement. A special acknowledge to Doctor Ricardo Dias from the BioISI Research group M&B for open the doors for me on the whole-genome sequencing field, and for all the support and guidance in the comparative genomic studies. Thank you for sharing knowledge and suggestions, constant availability, constructive criticism and for your friendship, having always respected the way I work. Without your support this journey would have been much more difficult! Thanks to all the members of the M&B group (the “bugworkers family”) who in one way or another contributed for the success of this PhD work. Thanks for making me feel as a bugworker member, for all the good moments and fruitful scientific (or not) discussions. A special thanks to Doctor Lélia Chambel for being always available to assist me with BioNumerics and for all the precious advices and friendship. I also want to thank the colleagues that help me out with the Sanger sequencing in the beginning of this work. I would like to thank my superiors, Doctor Miguel Fevereiro and Doctor Ana Botelho, and all my colleagues from the Instituto Nacional de Investigação Agrária e Veterinária (INIAV, IP) that, directly or indirectly, contributed to the accomplishment of this work. In particular, special thanks to my colleagues from the brucellosis sector, Isabel Travassos Dias and Regina Cardoso, and to my dear friends Helga Waap, Cristina Ferreira and Alice Geraldes, for their friendship, support in the most difficult moments and for always believe that I could do this. I’m very grateful to my friends Lúcia, Teresa and Marta for making part of my life, and for always believing in my capability to go further. I wish to thank my parents, in-laws, brothers, sisters-in-law and nephews, for being always around and for encouraging me to go on. A special thanks to my parents and in-laws for all the support. Without you it would be much more difficult. Finally, I want to deeply thank my dearest husband, Daniel, for always being by my side, for the patience and above of all, for all the love. You will be in my heart forever! To my favorite persons vii in the world, my children, Rodrigo and Maria, I just want to say that I love you. This thesis is dedicated to you! Support from Biosystems and Integrative Sciences Institute (BioISI, FCT/UID/Multi/04046/2013), FCT/MCTES/PIDDAC (Portugal) and FCT project PTDC/CVT/104050/2008 is also acknowledged. viii Table of Contents Abstract............................................................................................................................................... xiii Resumo ................................................................................................................................................ xv Table Index ......................................................................................................................................... xix Figure Index ........................................................................................................................................ xxi Supplementary Material Index ......................................................................................................... xxiii List of abbreviations…………..……………………………………………..……………………………………………..…………. xxv Research papers integrated into this thesis ..................................................................................... xxvii Submitted research papers .............................................................................................................. xxvii Chapter 1. General Introduction .................................................................................................. 1 1.1. The Genus Brucella: a taxonomic and phylogenetic overview ............................................... 3 1.2. The Brucella species: approaches on diversity and epidemiology ......................................... 6 1.3. Brucella suis biovar 2 and brucellosis ..................................................................................... 9 Occurrence of B. suis biovar 2 infection in Portugal ............................................................. 10 1.4. From the outside-in: the cell envelope and virulence of brucellae ...................................... 11 1.5. Comparative genomics as a tool to understand evolution in brucellae ............................... 17 1.6. Objectives, research strategy and thesis organization ......................................................... 21 Thesis organization ............................................................................................................... 23 References ................................................................................................................................... 24 Chapter 2. Isolation and molecular characterization of Brucella suis strains from swine and wild boars in Portugal ...................................................................................................................... 33 Subchapter 2.1. Development and evaluation of a selective medium for Brucella suis ................ 35 1.Introduction .............................................................................................................................. 37 2.Material and Methods .............................................................................................................. 38 2.1.Brucella strains .................................................................................................................... 38 2.2.Culture media ...................................................................................................................... 38 2.3.Culture conditions ............................................................................................................... 38 2.4.Evaluation of the relative diagnostic performance of LNIV-M in the primary isolation of B. suis with respect to FAR and MTM ...................................................................................... 38 3.Results and Discussion .............................................................................................................. 39 4.Conclusion ................................................................................................................................. 41 5.References ................................................................................................................................ 42 Subchapter 2.2. Genetic diversity of Brucella suis biovar 2 strains circulating in Europe.............. 43 1.Introduction .............................................................................................................................. 45 2.Material and Methods .............................................................................................................. 46 2.1.B. suis isolates and genomic DNA bank ............................................................................ 46 2.2.Suis ladder multiplex PCR and PCR-RFLP analysis ............................................................ 46 2.3.MLVA-16 assay and data analysis ..................................................................................... 46 3.Results and Discussion .............................................................................................................
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