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MOLECULAR DETECTION and CHARACTERIZATION of HEMOPLASMAS and Bartonella Spp

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MOLECULAR DETECTION and CHARACTERIZATION of HEMOPLASMAS and Bartonella Spp UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL MOLECULAR DETECTION AND CHARACTERIZATION OF HEMOPLASMAS AND Bartonella spp. IN SMALL MAMMALS, LARGE RUMINANTS AND ASSOCIATED ECTOPARASITES Luiz Ricardo Gonçalves Biólogo 2020 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL MOLECULAR DETECTION AND CHARACTERIZATION OF HEMOPLASMAS AND Bartonella spp. IN SMALL MAMMALS, LARGE RUMINANTS AND ASSOCIATED ECTOPARASITES Discente: Luiz Ricardo Gonçalves Orientador: Prof. Dr. Marcos Rogério André Coorientadora: Profa. Dra. Rosangela Zacarias Machado Tese apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Doutor em Microbiologia Agropecuária 2020 DADOS CURRICULARES DO AUTOR Luiz Ricardo Gonçalves – nascido na cidade de Jaboticabal, São Paulo, em 4 de novembro de 1985. Formado em Ciências Biológicas (Bacharelado) pela Universidade Estadual Paulista “Júlio de Mesquita Filho” – Faculdade de Ciências Agrárias e Veterinárias – FCAV/UNESP, Jaboticabal, São Paulo, no ano de 2013. Durante a graduação, foi bolsista CNPQ e FAPESP (2010-2010: CNPQ - Apoio Técnico/ Bolsa: 370406/2010–9; 2010–2011: FAPESP - 2010/12873–0; 2012–2014: CNPQ - Apoio Técnico/ Bolsa: 350104/2012–3), trabalhando com pesquisas na área de Parasitologia sob a orientação dos Professores: Profa. Dra. Rosangela Z. Machado e Prof. Dr. Marcos R. André. Em junho de 2016, concluiu o Curso de Mestrado em Microbiologia Agropecuária na FCAV/UNESP, com bolsa da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Em agosto de 2016, ingressou no Curso de Doutorado no mesmo programa de pós- graduação e instituição sob a orientação dos Professores: Prof. Dr. Marcos R. André e Profa. Dra. Rosangela Z. Machado. No segundo semestre de 2016, ministrou as aulas de protozoologia na disciplina de Parasitologia para o Curso de Ciências Biológicas da FCAV/UNESP, sob a supervisão do Prof. Dr. Marcos R. André. Entre novembro de 2018 e abril de 2019, realizou Estágio de Doutorado Sanduíche na Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment – The Hebrew University of Jerusalem – Rehovot, Israel, sob orientação do Prof. Dr. Shimon Harrus, com bolsa da CAPES. Durante o primeiro semestre de 2020, atuou como docente na disciplina de Parasitologia Geral e Humana para o curso de Ciências Biológicas da FCAV/UNESP sob a supervisão do Prof. Dr. Marcos R. André e Profa. Dra. Rosangela Z. Machado. Epígrafe Retribui-se mal a um mestre continuando a ser apenas um aluno. “Ecce homo” Friedrich Nietzsche DEDICATÓRIA Aos meus queridos pais, Luzia C. Gonçalves e Luiz C. Gonçalves, pelo amor, carinho e apoio em todas as decisões da minha vida. Aos meus irmãos, Kátia, Maiko e Luana e minhas sobrinhas, Aghata e Yasmin, pelo carinho e apoio incondicional. À minha namorada Rafaela, pelo amor, parceria, compreensão e companherismo diário. AGRADECIMENTOS A Deus, por tudo aquilo que tenho experienciado. À UNESP e ao Programa de Pós-graduação em Microbiologia Agropecuária pela oportunidade e pelo crescimento acadêmico e científico que me proporcionaram durante minha formação doutoral. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelas bolsas de estudos. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo Auxílio-pesquisa (Processo 2018/02753-0) concedido para a realização deste trabalho. Ao Professor Heitor Miraglia Herrera, pelo respeito, confiança e por todo suporte durante as coletas. Esse auxílio foi imprescindível para o desenvolvemento deste projeto. Ao Professor Shimon Harrus, pela oportunidade, confiança e ensinamentos durante o estágio de Doutorado Sanduíche. Sempre serei grato. Aos meus parceiros de coleta de amostras, Wesley, Filipe, Grasiela, Wanessa, Gabriel, William, João, Thiago e Inalda. Ao Dr. Ricardo Gutiérrez, pelos ensinamentos compartilhados e amizade durante meu estágio de Doutorado Sanduíche. À Professora Darci Moraes Barros-Battesti, pelo auxílio na identificação dos ectoparasitas amostrados. À Dra. Keyla C. M. de Sousa, pelo companherismo e todo auxílio em Israel. Aos funcionários do Deparatamento de Patologia, Reprodução e Saúde Única, em especial à Mabel Mastro Custódio. Aos amigos que passaram ou que ainda estão no Laboratório de Imunoparasitologia: Arvelino, Carlos, Otávio, Paulo, Márcia, Carla, Thiago, Natalia, Victória, Ana Cláudia, Matheus, Lucas, Paulo, Inalda, Simone, Kayo, Renan, Jyan, Priscila, Maria, Lucas, Lívia, Laryssa, Ana Júlia, Jhessye, Leidiane, Jaqueline e Ana Carolina. Venho aprendendo com vocês a cada dia. AGRADECIMENTOS ESPECIAIS Minha imensa gratidão ao Professor Dr. Marcos R. André e à Professora Dra. Rosangela Z. Machado. Orientadores e motivadores incansáveis que, com entusiasmo, sabedoria e competência, sempre me apoiaram, mesmo nos momentos turbulentos. Sempre os terei como um exemplo a ser seguido. i CONTENTS Page CERTIFICADO DA COMISSÃO DE ÉTICA NO USO DE ANIMAL.................... iv RESUMO.............................................................................................................. v ABSTRACT.......................................................................................................... vii LIST OF ABBREVIATIONS................................................................................. ix CHAPTER 1 – General consideration............................................................... 1 1. INTRODUCTION.............................................................................................. 1 2. LITERATURE REVIEW................................................................................... 3 2.1. INFECTION BY HEMOTROPIC MYCOPLASMAS (HEMOPLASMAS).... 3 2.1.1. Taxonomy........................................................................................... 3 2.1.2. Etiologic agents................................................................................... 3 2.1.3. Transmission....................................................................................... 4 2.1.4. Diagnosis............................................................................................ 5 2.1.5. Epizootiology of hemoplasmas in rodents, marsupials and associated ectoparasites...................................................................................... 6 2.1.6. Epizootiology of hemoplasmas in bovids............................................ 8 2.1.7. Human infection with hemoplasmas species...................................... 9 2.2. INFECTION BY Bartonella spp................................................................. 11 2.2.1. Taxonomy........................................................................................... 11 2.2.2. Etiologic agents.................................................................................. 12 2.2.3. Transmission...................................................................................... 14 2.2.4. Diagnosis............................................................................................ 16 2.2.5. Epizootiology of Bartonella spp. in rodents, marsupials and associated ectoparasites...................................................................................... 17 2.2.6. Epizootiology of Bartonella spp. in bovids.......................................... 23 2.2.7. Human infection with Bartonella species............................................ 26 3. GENERAL OBJECTIVES................................................................................ 30 4. REFERENCES……………………………………………………………………… 31 CHAPTER 2 – Genetic diversity, and lack of molecular evidence for hemoplasma cross-species transmission between wild and synanthropic mammals from Central-Western Brazil............................................................. 51 Abstract................................................................................................................ 51 1. Introduction..................................................................................................... 51 2. MATERIALS AND METHODS......................................................................... 53 2.1. Study sites, mammal trapping and sample collection................................. 53 2.2. DNA extraction and molecular detection of hemoplasmas......................... 55 2.3. Phylogenetic analysis………………………………………………………….. 56 2.4. Identification and genetic relationship of identified hemoplasmas genotypes……………………………………………………………………………….. 57 3. RESULTS……………………………………………………………………………. 57 3.1. Ectoparasites and hemoplasma occurrence and BLAST analysis………... 57 3.2. Phylogenetic and genotype analyses………………………………………… 58 4. DISCUSSION………………………………………………………………………... 63 5. CONCLUSION………………………………………………………………………. 66 REFERENCES...................................................................................................... 66 ii CHAPTER 3 – Low occurrence of Bartonella in synanthropic mammals and associated ectoparasites in peri-urban areas from Central-Western and Southern Brazil............................................................................................ 75 ABSTRACT…………………………………………………………………………….. 75 1. INTRODUCTION……………………………………………………………………. 75 2. MATERIALS AND METHODS……………………………………………………. 77 2.1. Sampling sites, mammals capture and biological samples collection……. 77 2.2. Bartonella isolation……………………………………………………………..
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