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Múcio Flávio Barbosa Ribeirohttp://lattes.cnpq.br/0037573905724394Deborah A. Negrão- CorrêaAntônio Marcos GuimarãesArildo Pinto da CunhaJoaquim Hernán Patarroyo SalcedoRicardo Nascimento Araújohttp://lattes.cnpq.br/4016084792475146Bruna Torres Silvestre da Silva2021-01-20T23:04:36Z2021-01-20T23:04:36Z2017-03-23http://hdl.handle.net/1843/34806Anaplasmose bovina é uma doença infecciosa causada pela riquétsia intraeritrocitária A. marginale. Atualmente não há uma vacina inativada comercialmente disponível. Vacinas inativadas produzidas em células IDE8 falharam devido a ausência da expressão da MSP1a nas riquetsias cultivadas neste sistema. Camundongos imunizados com rMSP1a, utilizando nanotubos de carbono (MWNT) como molécula carreadora, apresentaram resposta celular e humoral significativa, indicando que esta tecnologia possa auxiliar no desenvolvimento de vacina inativada. Desta forma, o presente estudo avaliou o efeito da imunização de bezerros com MWNT funcionalizado com rMSP1a, associado com a vacina inativada de A. marginale produzida in vitro, na indução da resposta imunológica, no controle da riquetsemia e no desenvolvimento da doença clínica após desafio experimental com amostra heteróloga A. marginale UFMG1. A rMSP1a foi expressa em Escherichia coli BL21, purificada e ligada covalentemente à MWNTs. Extrato solúvel de A. marginale (AmUFMG2) foi produzido em cultivo in vitro da riquétsia em células IDE8. No Capítulo I foi avaliado a resposta humoral e celular dos bezerros imunizados nos diferentes protocolos e possíveis efeitos tóxicos desta imunização. Para isso, vinte e quatro bezerros machos foram divididos em quatro grupos e imunizados (via SC) com três doses, 21-21 dias, no esquema: G1 (controle), G2 (AmUFMG2), G3 (MWNT+rMSP1a) e G4 (AmUFMG2 e MWNT+rMSP1a). Amostras de sangue foram coletadas para a contagem global de leucócitos, avaliação da resposta imune e análise bioquímica. A imunização com MWNT+rMSP1a induziu aumento na contagem de leucócitos totais, no número de células NK circulantes, na população de linfócitos T CD4+, CD8+, linfócitos T ativados, células B e níveis elevados de anticorpos IgG, IgG1 e IgG2. Além disso, os MWNTs não induziram alterações no perfil bioquímico, indicando que MWNT+rMSP1a foi capaz de induzir resposta celular e humoral significativa, sem gerar toxicidade. No Capítulo II foram avaliados as alterações imunológicas, os parâmetros clínicos e parasitológicos de bezerros imunizados e experimentalmente desafiados com a amostra heteróloga A. marginale UFMG1. Quinze bezerros machos foram divididos em três grupos e imunizados (via SC) com três doses, 21-21 dias, no esquema: G1 (controle), G2 (MWNT+rMSP1a) e G3 (AmUFMG2 e MWNT+rMSP1a). Após as imunizações, os animais foram desafiados com 4x107 eritrócitos infectados com A. marginale UFMG1. Parâmetros clínicos, tais como temperatura retal, volume globular, riquetsemia e escore clínico foram avaliados. Amostras de sangue foram coletadas para a análise da resposta celular e humoral. Animais do G3 apresentaram redução significativa dos níveis de riquetsemia, escore clínico moderado, melhor capacidade de recuperação do volume globular e forte resposta de IgG2. Estes resultados sugerem que a combinação de antígenos presentes no imunógeno recebido pelo grupo G3 foi capaz de proteger os animais do escore grave, permitindo que novas abordagens sejam realizadas utilizando a vacina inativada produzida in vitro associada com rMSP1a.Bovine anaplasmosis is an infectious disease caused by intracytocytic rickettsia A. marginale. There is no commercially available inactivated vaccine. Inactivated vaccine produced in IDE8 cells failed due to absence of the MSP1a expression in the rickettsia cultured in this system. Mice immunized with rMSP1a, linked to carbon nanotubes (MWNT), showed significant humoral and cellular responses, indicating that this technology may help in the development of inactivated vaccine. Therefore, the general objective was to evaluate the effect of the immunization of calves with MWNT+rMSP1a, associated with inactivated vaccine of A. marginale produced in vitro, in the induction of immune response, control of rickettsemia and in the development of clinical disease after experimental challenge with heterologous sample A. marginale UFMG1. rMSP1a was expressed in Escherichia coli BL21, purified and covalently linked to MWNTs. Soluble extract of A. marginale (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. In the Chapter I was evaluated the humoral and cellular response of the calves immunized with the different protocols and possible toxic effects of this immunization. Twenty four holstein calves were divided into four groups and immunized subcutaneously with three doses, 21-21 days, in the following scheme: G1 (control), G2 (AmUFMG2), G3 (MWNT+rMSP1a) and G4 (AmUFMG2 and MWNT+rMSP1a). Blood and plasma samples were collected for total leukocyte counts, evaluation of the immune response and biochemical profile. Immunization with MWNT+rMSP1a induced an increase in the total leukocyte counts, in the number of circulating NK cells, in the T lymphocytes CD4+, CD8+ T, activated T lymphocytes, B cells and high levels of IgG, IgG1 and IgG2 antibodies. Furthermore, MWNTs did not induce changes in the biochemical profile, indicating that the immunization of calves with MWNT+rMSP1a was able to induce significant cellular and humoral response, without generate toxicity. In the Chapter II were evaluated the immunological alterations, clinical and parasitological parameters of calves immunized and experimentally challenged with the heterologous sample A. marginale UFMG1. Fifteen holstein calves were divided into three groups and immunized (via SC) with three doses, 21-21 days, in the following scheme: G1 (control), G2 (MWNT+rMSP1a) and G3 (AmUFMG2 and MWNT+rMSP1a). After immunizations, calves were challenged with 4x107 infected erythrocytes with A. marginale UFMG1. Clinical parameters, such as rectal temperature, packed cell volume, rickettsemia and clinical score were evaluated. Blood and plasma samples were collected for the cellular and humoral response analysis. Calves from G3 showed significant reduction of the levels of rickettsemia, moderate clinical score, better recovery capacity of the packed cell volume and high levels of IgG2. These results suggest that the antigens combination received by the G3 group was able to protect animals from severe score, allowing further approaches to be performed using the inactivated vaccine produced in vitro associated with rMSP1a.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em ParasitologiaUFMGBrasilICB - INSTITUTO DE CIÊNCIAS BIOLOGICAShttp://creativecommons.org/licenses/by-nc-nd/3.0/pt/info:eu-repo/semantics/openAccessParasitologiaAnaplasma marginaleNanotubos de carbonoAnaplasma marginaleVacina inativadaMSP1aNanotubos de carbonoAvaliação da vacina de Anaplasma marginale utilizando rMSP1a funcionalizada a nanotubos de carbono, associado ao antígeno inativado produzido in vitro: Parâmetros imunológicos, clínicos e a proteção induzida em bezerros, após desafio experimentalinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTese Final_Bruna Silvestre 2017.pdfTese Final_Bruna Silvestre 2017.pdfapplication/pdf8477269https://repositorio.ufmg.br/bitstream/1843/34806/1/Tese%20Final_Bruna%20Silvestre%202017.pdff6c92a75e506c1dafa2bacfcbb4cdad6MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufmg.br/bitstream/1843/34806/2/license_rdfcfd6801dba008cb6adbd9838b81582abMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82119https://repositorio.ufmg.br/bitstream/1843/34806/3/license.txt34badce4be7e31e3adb4575ae96af679MD531843/348062021-01-20 20:04:36.204oai:repositorio.ufmg.br:1843/34806TElDRU7Dh0EgREUgRElTVFJJQlVJw4fDg08gTsODTy1FWENMVVNJVkEgRE8gUkVQT1NJVMOTUklPIElOU1RJVFVDSU9OQUwgREEgVUZNRwoKQ29tIGEgYXByZXNlbnRhw6fDo28gZGVzdGEgbGljZW7Dp2EsIHZvY8OqIChvIGF1dG9yIChlcykgb3UgbyB0aXR1bGFyIGRvcyBkaXJlaXRvcyBkZSBhdXRvcikgY29uY2VkZSBhbyBSZXBvc2l0w7NyaW8gSW5zdGl0dWNpb25hbCBkYSBVRk1HIChSSS1VRk1HKSBvIGRpcmVpdG8gbsOjbyBleGNsdXNpdm8gZSBpcnJldm9nw6F2ZWwgZGUgcmVwcm9kdXppciBlL291IGRpc3RyaWJ1aXIgYSBzdWEgcHVibGljYcOnw6NvIChpbmNsdWluZG8gbyByZXN1bW8pIHBvciB0b2RvIG8gbXVuZG8gbm8gZm9ybWF0byBpbXByZXNzbyBlIGVsZXRyw7RuaWNvIGUgZW0gcXVhbHF1ZXIgbWVpbywgaW5jbHVpbmRvIG9zIGZvcm1hdG9zIMOhdWRpbyBvdSB2w61kZW8uCgpWb2PDqiBkZWNsYXJhIHF1ZSBjb25oZWNlIGEgcG9sw610aWNhIGRlIGNvcHlyaWdodCBkYSBlZGl0b3JhIGRvIHNldSBkb2N1bWVudG8gZSBxdWUgY29uaGVjZSBlIGFjZWl0YSBhcyBEaXJldHJpemVzIGRvIFJJLVVGTUcuCgpWb2PDqiBjb25jb3JkYSBxdWUgbyBSZXBvc2l0w7NyaW8gSW5zdGl0dWNpb25hbCBkYSBVRk1HIHBvZGUsIHNlbSBhbHRlcmFyIG8gY29udGXDumRvLCB0cmFuc3BvciBhIHN1YSBwdWJsaWNhw6fDo28gcGFyYSBxdWFscXVlciBtZWlvIG91IGZvcm1hdG8gcGFyYSBmaW5zIGRlIHByZXNlcnZhw6fDo28uCgpWb2PDqiB0YW1iw6ltIGNvbmNvcmRhIHF1ZSBvIFJlcG9zaXTDs3JpbyBJbnN0aXR1Y2lvbmFsIGRhIFVGTUcgcG9kZSBtYW50ZXIgbWFpcyBkZSB1bWEgY8OzcGlhIGRlIHN1YSBwdWJsaWNhw6fDo28gcGFyYSBmaW5zIGRlIHNlZ3VyYW7Dp2EsIGJhY2stdXAgZSBwcmVzZXJ2YcOnw6NvLgoKVm9jw6ogZGVjbGFyYSBxdWUgYSBzdWEgcHVibGljYcOnw6NvIMOpIG9yaWdpbmFsIGUgcXVlIHZvY8OqIHRlbSBvIHBvZGVyIGRlIGNvbmNlZGVyIG9zIGRpcmVpdG9zIGNvbnRpZG9zIG5lc3RhIGxpY2Vuw6dhLiBWb2PDqiB0YW1iw6ltIGRlY2xhcmEgcXVlIG8gZGVww7NzaXRvIGRlIHN1YSBwdWJsaWNhw6fDo28gbsOjbywgcXVlIHNlamEgZGUgc2V1IGNvbmhlY2ltZW50bywgaW5mcmluZ2UgZGlyZWl0b3MgYXV0b3JhaXMgZGUgbmluZ3XDqW0uCgpDYXNvIGEgc3VhIHB1YmxpY2HDp8OjbyBjb250ZW5oYSBtYXRlcmlhbCBxdWUgdm9jw6ogbsOjbyBwb3NzdWkgYSB0aXR1bGFyaWRhZGUgZG9zIGRpcmVpdG9zIGF1dG9yYWlzLCB2b2PDqiBkZWNsYXJhIHF1ZSBvYnRldmUgYSBwZXJtaXNzw6NvIGlycmVzdHJpdGEgZG8gZGV0ZW50b3IgZG9zIGRpcmVpdG9zIGF1dG9yYWlzIHBhcmEgY29uY2VkZXIgYW8gUmVwb3NpdMOzcmlvIEluc3RpdHVjaW9uYWwgZGEgVUZNRyBvcyBkaXJlaXRvcyBhcHJlc2VudGFkb3MgbmVzdGEgbGljZW7Dp2EsIGUgcXVlIGVzc2UgbWF0ZXJpYWwgZGUgcHJvcHJpZWRhZGUgZGUgdGVyY2Vpcm9zIGVzdMOhIGNsYXJhbWVudGUgaWRlbnRpZmljYWRvIGUgcmVjb25oZWNpZG8gbm8gdGV4dG8gb3Ugbm8gY29udGXDumRvIGRhIHB1YmxpY2HDp8OjbyBvcmEgZGVwb3NpdGFkYS4KCkNBU08gQSBQVUJMSUNBw4fDg08gT1JBIERFUE9TSVRBREEgVEVOSEEgU0lETyBSRVNVTFRBRE8gREUgVU0gUEFUUk9Dw41OSU8gT1UgQVBPSU8gREUgVU1BIEFHw4pOQ0lBIERFIEZPTUVOVE8gT1UgT1VUUk8gT1JHQU5JU01PLCBWT0PDiiBERUNMQVJBIFFVRSBSRVNQRUlUT1UgVE9ET1MgRSBRVUFJU1FVRVIgRElSRUlUT1MgREUgUkVWSVPDg08gQ09NTyBUQU1Cw4lNIEFTIERFTUFJUyBPQlJJR0HDh8OVRVMgRVhJR0lEQVMgUE9SIENPTlRSQVRPIE9VIEFDT1JETy4KCk8gUmVwb3NpdMOzcmlvIEluc3RpdHVjaW9uYWwgZGEgVUZNRyBzZSBjb21wcm9tZXRlIGEgaWRlbnRpZmljYXIgY2xhcmFtZW50ZSBvIHNldSBub21lKHMpIG91IG8ocykgbm9tZXMocykgZG8ocykgZGV0ZW50b3IoZXMpIGRvcyBkaXJlaXRvcyBhdXRvcmFpcyBkYSBwdWJsaWNhw6fDo28sIGUgbsOjbyBmYXLDoSBxdWFscXVlciBhbHRlcmHDp8OjbywgYWzDqW0gZGFxdWVsYXMgY29uY2VkaWRhcyBwb3IgZXN0YSBsaWNlbsOnYS4KCg==Repositório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2021-01-20T23:04:36Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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