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WANDERLEY MORENO QUINTEIRO FILHO Efeitos Do Estresse Por Calor WANDERLEY MORENO QUINTEIRO FILHO Efeitos do estresse por calor sobre a imunidade e a migração de Salmonela enteritidis em frangos de corte Tese apresentada ao Programa da Pós- Graduação em Patologia Experimental e Comparada da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo para obtenção do título de Doutor em Ciências Departamento: Patologia Área de Concentração: Patologia Experimental e Comparada Orientador: Prof. Dr. João Palermo-Neto São Paulo 2013 Autorizo a reprodução parcial ou total desta obra, para fins acadêmicos, desde que citada a fonte. DADOS INTERNACIONAIS DE CATALOGAÇÃO-NA-PUBLICAÇÃO (Biblioteca Virginie Buff D’Ápice da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo) T.2756 Quinteiro Filho, Wanderley Moreno FMVZ Efeitos do estresse por calor sobre a imunidade e a migração de Salmonela enteritidis em frangos de corte / Wanderley Moreno Quinteiro Filho. -- 2013. 168 f. : il. Tese (Doutorado) - Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Patologia, São Paulo, 2013. Programa de Pós-Graduação: Patologia Experimental e Comparada . Área de concentração: Patologia Experimental e Comparada . Orientador: Prof. Dr. João Palermo-Neto. 1. Estresse por calor. 2. INF-γ. 3. Citocinas. 4. Macrófagos. 5. Corticosterona. I. Título. FOLHA DE AVALIAÇÃO Nome: WANDERLEY MORENO QUINTEIRO FILHO Titulo: Efeitos do estresse por calor sobre a imunidade e a migração de Salmonela enteritidis em frangos de corte Tese apresentada ao Programa da Pós-Graduação em Patologia Experimental e Comparada da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo para obtenção do título de Doutor em Ciências Data: ____/____/____ Banca examinadora Prof. Dr.:___________________________________________________________________ Instituição:________________________________Julgamento:________________________ Prof. Dr.:___________________________________________________________________ Instituição:________________________________Julgamento:________________________ Prof. Dr.:___________________________________________________________________ Instituição:________________________________Julgamento:________________________ Prof. Dr.:___________________________________________________________________ Instituição:________________________________Julgamento:________________________ Prof. Dr.:___________________________________________________________________ Instituição:________________________________Julgamento:________________________ Ao Prof. Dr. João Palermo Neto, um exemplo de profissional e pessoa. Durante essa longa jornada que inclui a IC, o Mestrado e, agora, o Doutorado foram mais de 9 anos de muitos ensinamentos. A constante busca pelo conhecimento e pelo aprendizado fazem do Prof. João um cientista e um professor de muito respeito e credibilidade. Um exemplo a ser seguido. Seus ensinamentos perdurarão por toda minha vida profissional, por esse motivo dedico esta obra a você, que me orientou, me fez entender, aprender e fazer ciência. Muito Obrigado! À Karen Fanti, minha companheira de hoje e sempre. Sem você, nada disso seria possível. Obrigado pela paciência e apoio incondicional. Palavras não são sufientes para descrever o quanto você é importante para mim. TE AMO “O verdadeiro amor nunca se desgasta. Quanto mais se dá mais se tem” Antoine de Saint-Exupéry Às minhas irmãs, Wanice e Gabi. Obrigado pelo apoio, carinho e compreensão. Obrigado por sempre estarem dispostas a ajudar! E, finalmente, dedico esta tese aos meus pais, Eunice e Wanderley, que me viram crescer, guiaram-me e educaram-me. Acreditaram nessa longa jornada. Não me questionaram. Apoiaram-me. Doaram- se por inteiros e renunciaram aos seus sonhos, para que, muitas vezes, pudéssemos realizar os nossos. Obrigado por ensinarem o que é caráter e respeito. Amo vocês!!! “A dúvida é o principio da sabedoria” Aristóteles AGRADECIMENTOS À Fundação de Amparo à Pesquisa do Estado de São Paulo (Projeto Tematico - nº 2009/51886-3; Bolsas - n° 2009/52487-5 e n° 2011/23391-0) e ao CNPq (Projeto Universal - nº 470776/2009-9) pelo suporte financeiro, que permitiu a execução deste estudo. À Faculdade de Medicina Veterinária e Zootecnia e ao Departamento de Patologia, pela formação e estruturas. Ao Prof. João Palermo Neto por acreditar em mim e por me orientar em mais esta jornada. Palavras são poucas para expressar minha gratidão nesses anos de trabalho. Ao Prof. Antonio Piantino Ferreira, pela parceria e por estar sempre pronto a ajudar. Obrigado por abrir as portas do LABOR a todo o grupo de Neuroimunomodulação. Ao Prof. Dr. Shayan Sharif, que gentilmente me aceitou em seu Laboratorio (Laboratory of Immunology/OVC-University of Guelph) e proporciou uma grande experiência no Canada. Aproveito para agradecer toda sua equipe, sempre muito atenciosos e pacientes. À Dra. Claudete Astolfi-Ferreira pelo apoio nas metodologias microbiológicas desta tese e de inúmeros outros trabalhos. À Profa. Dra. Cris Massoco pelas discussões e conversas. Sempre muito gentil, fez o final do doutorado ser mais tranquilo. Ao Prof. Dr. Frederico Pinto e ao Dr. Daniel Cohn (Natô) pelos conselhos e pelas discussões enriquecedoras sobre a ciência e sobre a vida. A Prof. Dr. Silvana Lima Gorniák, chefe do Programa de Patologia Experimental e Comparada, pela ótima convivência e pelo apoio nesses anos. Às secretarias do VPT, em especial à Cris (Programa de Patologia Experimental e Comparada), por toda a ajuda em todas as etapas da pós-graduação. Aos técnicos do Laboratório de Farmacologia e Toxicologia do VPT: das antigas, Priscila, Ricardo, Magali e os atuais, Dra. Nicolle, Vagner e Herculano pelo auxilio na realização dos experimentos. Aos técnicos do Laboratório de Histopatologia, Luciano (Buga) e Claúdio, pelo auxilio no processamento e preparo das laminas histopatológicas. A todos os funcionários do VPT, peças essenciais para o bom funcionamento deste Departamento, que direta ou indiretamente contribuíram com esta dissertação. A todos os funcionários da biblioteca (FMVZ-USP), em especial à Helena, Elza e Neusa, sempre dispostas a ajudar. Aos funcionários da Manutenção, em especial o Cabelo, Damião e Suli, parceiros em solucionar os problemas técnicos das nossas instalações. A equipe do Laboratório de Ornitopatologia pela convivência harmôniosa. Ao Grupo de Neuroimunomodulação (VPT/FMVZ/USP) por todo o crescimento científico. Ao Rodrigo por estar sempre presente e disposto a nos auxiliar com os processos burocráticos e, principalmente, por toda a amizade construída nesses anos. Dr. Thiago Aloia e ao Atilio pela ajuda nas técnicas de biologia molecualr e imunohistoquímica. Aos amigos e colaboradores desta tese: Atilio, Andre Gomes, Bruno Honda, Tieme, Dra. Milena, Dra. Viviane, Dr. Alison e.Dr. Thiago Ao meu primo Dr. Fabio Ribeiro da Silva, sempre atencioso e disposto a ajudar. A toda minha Família, tios, avós, primos, sogros, cunhados e, em especial aos meus pais e irmãs. Muito Obrigado por me ajudarem a realizar esse sonho. Ao Meu Avô Gabriel Silva (in memory). Realmente muito obrigado! As figuras ilustres da pós-graduação do VPT: Adriana, Aline, Bruno, Camila, Carol Costola, Daniel Sanches, Daniel, Denise, Domenica, Dudu, Eduardo, Emily, Fernando, Gabi, Glaucie, João Gimenes,Luciana Allegretti, Luciana Cunha, Martinha, Monica, Poliana, Renato Moraes, Samantha, Tereza, Thaisa, Thalita, Thiago Marinho,Vinicius entre outros. Muito Obrigado! RESUMO QUINTEIRO-FILHO, W. M. Efeitos do estresse por calor sobre a imunidade e a migração de Salmonela enteritidis em frangos de corte. [Effects of heat stress on immunity and Salmonella enteritidis invasion in broiler chickens]. 2013. 168 f. Tese (Doutorado em Ciências) – Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, 2013. O estresse é uma realidade na produção avícola mundial. Sabe-se que ambientes estressores prejudicam o bem-estar, os parâmetros produtivos e a imunidade de frangos de corte. Sabe-se, também, que o estresse por calor diminui a atividade de macrófagos em frangos de corte e, que existem, inúmeros estressores ambientais que insidem sobre a produção animal e podem aumentar a susceptibilidade às doenças. A Salmonella spp. é uma das maiores zoonoses do mundo, causando mais de 1 bilhão de casos de infecção. Nesse sentido, o presente trabalho analisa os efeitos do estresse por calor (31±1°C) sobre os índices zootécnicos, a imunidade, a invasão bacteriana e a integridade intestinal em frangos de corte infectados com Salmonella enteritidis; os dados obtidos foram discutidos dentro de uma perspectiva neuroimune. Os frangos foram divididos em quatro grupos: 1) Controle (C); 2) Estresse por Calor a 31±1 °C (HS31°C); 3) Controle infectados com Salmonella enteritidis (Controle Positivo [PC]) e; 4) Estresse por calor a 31±1 °C e infectados com Salmonella (PHS31°C). Nossos resultados mostraram que o estresse por calor em uma situação de infecção experimental por Salmonella enteritidis (grupo PHS31°C) 1) diminuiu os índices zootécnicos; especificamente, diminuiu o ganho de peso, consumo de alimento e a conversão alimentar; 2) diminuiu os níveis plasmáticos de INF-γ e IgA; 3) diminuiu a expressão de, IL-6 e IL-12 em baço e diminui IL1- β, IL-10 e TGF-β em tonsila cecal; 4) diminuiu a expressão de AvBD-4 e AvBD-6 em tonsila cecal e; 5) diminuiu a expressão de TLR-2 em baço e tonsila cecal. Observamos, também, 6) aumento dos níveis séricos de corticosterona nos animais dos grupos HS31°C e PHS31°C e; 7) piora no quadro de enterite produzida
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