Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal

Detalhes bibliográficos
Ano de defesa: 2016
Autor(a) principal: Kowalski, Ananda Paula lattes
Orientador(a): Vargas, Agueda Palmira Castagna de lattes
Banca de defesa: Scherer, Charles Ferando Capinos lattes, Frandoloso, Frandoloso lattes
Tipo de documento: Dissertação
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Federal de Santa Maria
Centro de Ciências Rurais
Programa de Pós-Graduação: Programa de Pós-Graduação em Medicina Veterinária
Departamento: Medicina Veterinária
País: Brasil
Palavras-chave em Português:
CIB
Palavras-chave em Inglês:
IBK
Área do conhecimento CNPq:
Link de acesso: http://repositorio.ufsm.br/handle/1/11282
Resumo: Infectious bovine keratoconjunctivitis (IBK) is the main ocular disease of cattle. Highly contagious, it is responsible for significant economic losses of livestock worldwide. Moraxella bovis is recognized as the primary agent of the disease in cattle. However, the occurrence of other similar species, including M. ovis and M. bovoculi, recently described, have been common in outbreaks of the disease and are suspected to be causally related to the limited success of preventive measures, especially the use of bacterins containing only strains of M. bovis. This dissertation describes the antigenic characterization of M. bovis, M. bovoculi and M. ovis strains and a commercial vaccine by cross-reactivity using flow cytometry analysis and identification of immunodominant conserved antigens by Western blotting. Antisera against strains of the three species were obtained from immunization of New Zealand rabbits and challenged before a panel of strains isolated from cattle and sheep clinically affected by the disease between 1983 and 2013 as well as reference strains of each species. Field strains of M. bovoculi (Mbv2 and Mbv3) recognized satisfactorily all heterologous strains. However, Mbv3 (M. bovoculi), Mov2 (M. ovis) and Mov3 (M. ovis) strains stood out as the most intense recognition strains of their respective species and suggest that species-specific antigens play an important role in the host immune response. The association of reactivity percentage with the antigenic profiles, evidenced by Western blotting analysis, indicates that the immune response induced by Moraxella spp. appears to be mediated by multiple surface antigens of which many are shared between the three species. Among 32 different proteins identified, 22 (68.7%) were recognized by at least one antiserum in the protein extracts of all strains analyzed by Western blotting. Our study suggests (1) the selection and combination of M. bovis, M. bovoculi and M. ovis strains to be used in the composition of antigenic unit vaccines as IBK control strategy and (2) the use of flow cytometry as the most appropriate methodology for this selection.
id UFSM-20_7b66a5037945cb2bb50ecafdcb4603dd
oai_identifier_str oai:repositorio.ufsm.br:1/11282
network_acronym_str UFSM-20
network_name_str Manancial - Repositório Digital da UFSM
repository_id_str
spelling 2017-07-31T16:06:09Z2017-07-31T16:06:09Z2016-09-13http://repositorio.ufsm.br/handle/1/11282Infectious bovine keratoconjunctivitis (IBK) is the main ocular disease of cattle. Highly contagious, it is responsible for significant economic losses of livestock worldwide. Moraxella bovis is recognized as the primary agent of the disease in cattle. However, the occurrence of other similar species, including M. ovis and M. bovoculi, recently described, have been common in outbreaks of the disease and are suspected to be causally related to the limited success of preventive measures, especially the use of bacterins containing only strains of M. bovis. This dissertation describes the antigenic characterization of M. bovis, M. bovoculi and M. ovis strains and a commercial vaccine by cross-reactivity using flow cytometry analysis and identification of immunodominant conserved antigens by Western blotting. Antisera against strains of the three species were obtained from immunization of New Zealand rabbits and challenged before a panel of strains isolated from cattle and sheep clinically affected by the disease between 1983 and 2013 as well as reference strains of each species. Field strains of M. bovoculi (Mbv2 and Mbv3) recognized satisfactorily all heterologous strains. However, Mbv3 (M. bovoculi), Mov2 (M. ovis) and Mov3 (M. ovis) strains stood out as the most intense recognition strains of their respective species and suggest that species-specific antigens play an important role in the host immune response. The association of reactivity percentage with the antigenic profiles, evidenced by Western blotting analysis, indicates that the immune response induced by Moraxella spp. appears to be mediated by multiple surface antigens of which many are shared between the three species. Among 32 different proteins identified, 22 (68.7%) were recognized by at least one antiserum in the protein extracts of all strains analyzed by Western blotting. Our study suggests (1) the selection and combination of M. bovis, M. bovoculi and M. ovis strains to be used in the composition of antigenic unit vaccines as IBK control strategy and (2) the use of flow cytometry as the most appropriate methodology for this selection.Ceratoconjuntivite infecciosa bovina (CIB) é a principal doença ocular de bovinos. Altamente contagiosa, é responsável por significativas perdas econômicas para a pecuária no mundo inteiro. Moraxella bovis é, reconhecidamente, o agente primário da enfermidade em bovinos. Contudo, a ocorrência de outras espécies do gênero, incluindo M. ovis e M. bovoculi, recentemente descrita, têm sido comuns em surtos da doença e são suspeitas de serem causalmente associadas ao sucesso limitado de medidas preventivas, sobretudo do uso de bacterinas contendo apenas cepas de M. bovis. Esta dissertação descreve a caracterização antigênica de cepas de M. bovis, M. bovoculi e M. ovis e de uma vacina comercial através da análise de reatividade cruzada por citometria de fluxo e da identificação de antígenos imunodominantes e conservados através de Western blotting. Antissoros contra cepas das três espécies foram obtidos a partir da imunização de coelhos Nova Zelândia e desafiados frente a um painel de cepas isoladas de bovinos e ovinos clinicamente acometidos pela doença entre 1983 e 2013 assim como cepas de referência de cada espécie. Cepas de campo de M. bovoculi (Mbv2 e Mbv3) reconheceram satisfatoriamente todas as cepas heterólogas. Contudo, as cepas Mbv3 (M. bovoculi), Mov2 (M. ovis) e Mov3 (M. ovis) destacaram-se pela maior intensidade de reconhecimento de cepas de suas respectivas espécies e sugerem que antígenos espécie-específicos desempenham importante papel na resposta imune do hospedeiro. A associação dos percentuais de reatividade aos perfis antigênicos evidenciados através da análise por western blotting indica que a resposta imune induzida por Moraxella spp. parece ser mediada por múltiplos antígenos de superfície dos quais, diversos são compartilhados entre as três espécies. Entre 32 diferentes proteínas detectadas, 22 (68,7%) foram reconhecidas por pelo menos um antissoro nos extratos protéicos de todas as cepas analisadas por western blotting. Nosso estudo sugere a seleção e combinação de cepas de M. bovis, M. bovoculi e M. ovis circulantes para composição da unidade antigênica de vacinas como estratégia de controle de IBK e a utilização de citometria de fluxo como metodologia mais adequada para esta seleção.porUniversidade Federal de Santa MariaCentro de Ciências RuraisPrograma de Pós-Graduação em Medicina VeterináriaUFSMBrasilMedicina VeterináriaAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessPinkeyeCitometria de fluxoReatividade cruzadaMoraxella sp.CIBPinkeyeFlow cytometryCross-reactivityMoraxella spIBKCNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIACaracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinalAntigenic characterization of Moraxella bovis, Moraxella bovoculi and Moraxella ovis strains with potential use in vaccinesinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisVargas, Agueda Palmira Castagna dehttp://lattes.cnpq.br/1383126157031968Scherer, Charles Ferando Capinoshttp://lattes.cnpq.br/1396090596936396Frandoloso, Frandolosohttp://lattes.cnpq.br/2502891354017410http://lattes.cnpq.br/0800999372009907Kowalski, Ananda Paula50050000000760086704c10-1997-4b84-868c-5547143be87b7ec33c06-2c43-4eb6-978d-d6b58e3b1b1d30897278-c5e7-4146-b3be-7083f7c6fef2d5663dc0-bd95-4390-8ce4-ebb64c664094reponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMORIGINALKowalski, Ananda Paula.pdfKowalski, Ananda Paula.pdfDissertação de Mestradoapplication/pdf1211677http://repositorio.ufsm.br/bitstream/1/11282/1/Kowalski%2c%20Ananda%20Paula.pdfb6361dc13ab236d9002b5afbfa0c57cdMD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8804http://repositorio.ufsm.br/bitstream/1/11282/2/license_rdfc1efe8e24d7281448e873be30ea326ffMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-81956http://repositorio.ufsm.br/bitstream/1/11282/3/license.txt2f0571ecee68693bd5cd3f17c1e075dfMD53TEXTKowalski, Ananda Paula.pdf.txtKowalski, Ananda Paula.pdf.txtExtracted texttext/plain106538http://repositorio.ufsm.br/bitstream/1/11282/4/Kowalski%2c%20Ananda%20Paula.pdf.txt48b704ed7cfb3665c089dceedb663a3bMD54THUMBNAILKowalski, Ananda Paula.pdf.jpgKowalski, Ananda Paula.pdf.jpgIM Thumbnailimage/jpeg4315http://repositorio.ufsm.br/bitstream/1/11282/5/Kowalski%2c%20Ananda%20Paula.pdf.jpg8b4d5314f410f7f6a5575434150f3b41MD551/112822022-06-10 15:30:18.742oai:repositorio.ufsm.br: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ório Institucionalhttp://repositorio.ufsm.br/PUBhttp://repositorio.ufsm.br/oai/requestopendoar:39132022-06-10T18:30:18Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false
dc.title.por.fl_str_mv Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
dc.title.alternative.eng.fl_str_mv Antigenic characterization of Moraxella bovis, Moraxella bovoculi and Moraxella ovis strains with potential use in vaccines
title Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
spellingShingle Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
Kowalski, Ananda Paula
Pinkeye
Citometria de fluxo
Reatividade cruzada
Moraxella sp.
CIB
Pinkeye
Flow cytometry
Cross-reactivity
Moraxella sp
IBK
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA
title_short Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
title_full Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
title_fullStr Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
title_full_unstemmed Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
title_sort Caracterização antigênica de cepas de Moraxella bovis, Moraxella bovoculi e Moraxella ovis com potencial uso vacinal
author Kowalski, Ananda Paula
author_facet Kowalski, Ananda Paula
author_role author
dc.contributor.advisor1.fl_str_mv Vargas, Agueda Palmira Castagna de
dc.contributor.advisor1Lattes.fl_str_mv http://lattes.cnpq.br/1383126157031968
dc.contributor.referee1.fl_str_mv Scherer, Charles Ferando Capinos
dc.contributor.referee1Lattes.fl_str_mv http://lattes.cnpq.br/1396090596936396
dc.contributor.referee2.fl_str_mv Frandoloso, Frandoloso
dc.contributor.referee2Lattes.fl_str_mv http://lattes.cnpq.br/2502891354017410
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/0800999372009907
dc.contributor.author.fl_str_mv Kowalski, Ananda Paula
contributor_str_mv Vargas, Agueda Palmira Castagna de
Scherer, Charles Ferando Capinos
Frandoloso, Frandoloso
dc.subject.por.fl_str_mv Pinkeye
Citometria de fluxo
Reatividade cruzada
Moraxella sp.
CIB
topic Pinkeye
Citometria de fluxo
Reatividade cruzada
Moraxella sp.
CIB
Pinkeye
Flow cytometry
Cross-reactivity
Moraxella sp
IBK
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA
dc.subject.eng.fl_str_mv Pinkeye
Flow cytometry
Cross-reactivity
Moraxella sp
IBK
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA
description Infectious bovine keratoconjunctivitis (IBK) is the main ocular disease of cattle. Highly contagious, it is responsible for significant economic losses of livestock worldwide. Moraxella bovis is recognized as the primary agent of the disease in cattle. However, the occurrence of other similar species, including M. ovis and M. bovoculi, recently described, have been common in outbreaks of the disease and are suspected to be causally related to the limited success of preventive measures, especially the use of bacterins containing only strains of M. bovis. This dissertation describes the antigenic characterization of M. bovis, M. bovoculi and M. ovis strains and a commercial vaccine by cross-reactivity using flow cytometry analysis and identification of immunodominant conserved antigens by Western blotting. Antisera against strains of the three species were obtained from immunization of New Zealand rabbits and challenged before a panel of strains isolated from cattle and sheep clinically affected by the disease between 1983 and 2013 as well as reference strains of each species. Field strains of M. bovoculi (Mbv2 and Mbv3) recognized satisfactorily all heterologous strains. However, Mbv3 (M. bovoculi), Mov2 (M. ovis) and Mov3 (M. ovis) strains stood out as the most intense recognition strains of their respective species and suggest that species-specific antigens play an important role in the host immune response. The association of reactivity percentage with the antigenic profiles, evidenced by Western blotting analysis, indicates that the immune response induced by Moraxella spp. appears to be mediated by multiple surface antigens of which many are shared between the three species. Among 32 different proteins identified, 22 (68.7%) were recognized by at least one antiserum in the protein extracts of all strains analyzed by Western blotting. Our study suggests (1) the selection and combination of M. bovis, M. bovoculi and M. ovis strains to be used in the composition of antigenic unit vaccines as IBK control strategy and (2) the use of flow cytometry as the most appropriate methodology for this selection.
publishDate 2016
dc.date.issued.fl_str_mv 2016-09-13
dc.date.accessioned.fl_str_mv 2017-07-31T16:06:09Z
dc.date.available.fl_str_mv 2017-07-31T16:06:09Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://repositorio.ufsm.br/handle/1/11282
url http://repositorio.ufsm.br/handle/1/11282
dc.language.iso.fl_str_mv por
language por
dc.relation.cnpq.fl_str_mv 500500000007
dc.relation.confidence.fl_str_mv 600
dc.relation.authority.fl_str_mv 86704c10-1997-4b84-868c-5547143be87b
7ec33c06-2c43-4eb6-978d-d6b58e3b1b1d
30897278-c5e7-4146-b3be-7083f7c6fef2
d5663dc0-bd95-4390-8ce4-ebb64c664094
dc.rights.driver.fl_str_mv Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Universidade Federal de Santa Maria
Centro de Ciências Rurais
dc.publisher.program.fl_str_mv Programa de Pós-Graduação em Medicina Veterinária
dc.publisher.initials.fl_str_mv UFSM
dc.publisher.country.fl_str_mv Brasil
dc.publisher.department.fl_str_mv Medicina Veterinária
publisher.none.fl_str_mv Universidade Federal de Santa Maria
Centro de Ciências Rurais
dc.source.none.fl_str_mv reponame:Manancial - Repositório Digital da UFSM
instname:Universidade Federal de Santa Maria (UFSM)
instacron:UFSM
instname_str Universidade Federal de Santa Maria (UFSM)
instacron_str UFSM
institution UFSM
reponame_str Manancial - Repositório Digital da UFSM
collection Manancial - Repositório Digital da UFSM
bitstream.url.fl_str_mv http://repositorio.ufsm.br/bitstream/1/11282/1/Kowalski%2c%20Ananda%20Paula.pdf
http://repositorio.ufsm.br/bitstream/1/11282/2/license_rdf
http://repositorio.ufsm.br/bitstream/1/11282/3/license.txt
http://repositorio.ufsm.br/bitstream/1/11282/4/Kowalski%2c%20Ananda%20Paula.pdf.txt
http://repositorio.ufsm.br/bitstream/1/11282/5/Kowalski%2c%20Ananda%20Paula.pdf.jpg
bitstream.checksum.fl_str_mv b6361dc13ab236d9002b5afbfa0c57cd
c1efe8e24d7281448e873be30ea326ff
2f0571ecee68693bd5cd3f17c1e075df
48b704ed7cfb3665c089dceedb663a3b
8b4d5314f410f7f6a5575434150f3b41
bitstream.checksumAlgorithm.fl_str_mv MD5
MD5
MD5
MD5
MD5
repository.name.fl_str_mv Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)
repository.mail.fl_str_mv
_version_ 1794524352624984064