Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples
Autor(a) principal: | |
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Data de Publicação: | 2015 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Brazilian Journal of Veterinary Medicine |
Texto Completo: | https://rbmv.org/BJVM/article/view/383 |
Resumo: | ABSTRACT. Mesquita S.M.C., Mansur F.J., Nascimento E.R., Barreto M.L. & Kimura L.M.S. [Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples.] Padroniza- ção e aplicação de ELISA indireto para diagnóstico de Mycoplasma bovis em amostras de soro sanguíneo bovino. Revista Brasileira de Medicina Veterinária, 37(2):101-107, 2015. Universidade Federal Fluminense, Faculdade de Veteriná- ria, Rua Vital Brazil Filho, 64, Vital Brazil, Niterói, RJ 24230-340, Brasil. E-mail: samira.veterinaria@gmail.com International researchers presented results indicating frequent involvement of Mycoplasma spp. as a causative agent of mastitis in cattle, associating its presence with significant economic losses to farmers. Mycoplasma bovis is the species most reported and relevant, because it causes more severe disease. The level of antibodies against M. bovis remains high for several months and can be detected by ELISA. The aim of this work was to develop an indirect ELISA with whole cell antigen of M. bovis (strain Donetta PG 45) with subsequent application in bovine blood serum samples for detection of antibodies against M. bovis. The immunization of cows A and B by inoculating an immunogen against M. bovis to obtain hyperimmune blood serum was the first stage of this work, then the stage of standardization of ELISA was proceeded. The concentration of 2 mg of antigen/mL for coating the microtiter plates was decided by statistical analyses. The optical density value 0,2 was determined as the limit of reactivity discrimination of samples (the cut-off point). The hyperimmune blood serum sample of the cow A (collected 30 days after immunization) was chosen as the positive control and, the fetal calf serum was chosen as negative control of the assay. In addition, the ideal optimal dilutions found for blood serum samples was 1:400 and for conjugate was 1:10.000 and the substrate used was the ortho-phenylenediamine. The amount of 321 samples of cow blood serum from 15 farms was obtained for the application of the standardized indirect ELISA. It was diagnosed animals that have been exposed to M. bovis and the prevalence rate found in blood serum samples was 3,1% (10/321) and 46,7% (7/15) was the prevalence rate found between the corresponding farms studied. |
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Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samplesPadronização e aplicação de ELISA indireto para diagnóstico de Mycoplasma bovis em amostras de soro sanguíneo bovinoMycoplasma bovisimunógenoELISAsoro sanguíneoMycoplasma bovisimmunogenELISAblood serumABSTRACT. Mesquita S.M.C., Mansur F.J., Nascimento E.R., Barreto M.L. & Kimura L.M.S. [Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples.] Padroniza- ção e aplicação de ELISA indireto para diagnóstico de Mycoplasma bovis em amostras de soro sanguíneo bovino. Revista Brasileira de Medicina Veterinária, 37(2):101-107, 2015. Universidade Federal Fluminense, Faculdade de Veteriná- ria, Rua Vital Brazil Filho, 64, Vital Brazil, Niterói, RJ 24230-340, Brasil. E-mail: samira.veterinaria@gmail.com International researchers presented results indicating frequent involvement of Mycoplasma spp. as a causative agent of mastitis in cattle, associating its presence with significant economic losses to farmers. Mycoplasma bovis is the species most reported and relevant, because it causes more severe disease. The level of antibodies against M. bovis remains high for several months and can be detected by ELISA. The aim of this work was to develop an indirect ELISA with whole cell antigen of M. bovis (strain Donetta PG 45) with subsequent application in bovine blood serum samples for detection of antibodies against M. bovis. The immunization of cows A and B by inoculating an immunogen against M. bovis to obtain hyperimmune blood serum was the first stage of this work, then the stage of standardization of ELISA was proceeded. The concentration of 2 mg of antigen/mL for coating the microtiter plates was decided by statistical analyses. The optical density value 0,2 was determined as the limit of reactivity discrimination of samples (the cut-off point). The hyperimmune blood serum sample of the cow A (collected 30 days after immunization) was chosen as the positive control and, the fetal calf serum was chosen as negative control of the assay. In addition, the ideal optimal dilutions found for blood serum samples was 1:400 and for conjugate was 1:10.000 and the substrate used was the ortho-phenylenediamine. The amount of 321 samples of cow blood serum from 15 farms was obtained for the application of the standardized indirect ELISA. It was diagnosed animals that have been exposed to M. bovis and the prevalence rate found in blood serum samples was 3,1% (10/321) and 46,7% (7/15) was the prevalence rate found between the corresponding farms studied.Pesquisadores internacionais apresentaram resultados indicativos do envolvimento frequente de Mycoplasma spp. como agente causador de mastite em bovinos, associando sua presença com perdas econômicas significativas para os criadores. Mycoplasma bovis é a espécie mais relatada e relevante, pois causa doença mais grave. Os níveis de anticorpos contra M. bovis permanecem elevados por muitos meses e podem ser detectados pelo ELISA. O objetivo do presente trabalho foi a padronização de ELISA indireto com antígeno celular total de M. bovis (cepa Donetta PG 45) com subsequente aplicação em amostras de soro sanguíneo de vacas para detecção de anticorpos anti- M. bovis. A imunização das vacas A e B, para obtenção de soro sanguíneo com imunógeno contra M. bovis foi a primeira etapa do trabalho. Em seguida, procedeu-se à etapa de padronização do ELISA indireto e após serem realizadas análises estatísticas, optou- -se por utilizar a concentração de 2 mg de antígeno/ mL para sensibilização das placas de microtitula- ção. O valor de densidade ótica 0,2 foi estabelecido como limite de discriminação de reatividade das amostras (ponto de corte). Foi escolhido como controle positivo dos ensaios a amostra de soro sanguí- neo da vaca A (coletado 30 dias após a imunização) e, como controle negativo dos ensaios foi escolhido o soro fetal bovino estéril. Além disso, as diluições consideradas ideais para as amostras de soro sanguíneo e para o conjugado utilizado foram respectivamente 1:400 e 1:10.000 e o substrato utilizado foi o Orto- fenilenodiamina. Para a aplicação do ELISA indireto foram obtidas 321 amostras de soro sanguíneo de vacas de 15 fazendas. Na aplicação do ELISA indireto padronizado, foram diagnosticados animais que já foram expostos a M. bovis e as prevalências encontradas foram 3,1% (10/321) nas amostras de soro sanguíneo e 46,7% (7/15) entre as respectivas fazendas estudadas.Sociedade de Medicina Veterinária do Estado do Rio de Janeiro.2015-06-28info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionpeer reviewedAvaliado pelos paresapplication/pdfhttps://rbmv.org/BJVM/article/view/383Brazilian Journal of Veterinary Medicine; Vol. 37 No. 2 (2015); 101-107Revista Brasileira de Medicina Veterinária; v. 37 n. 2 (2015); 101-1072527-21790100-2430reponame:Brazilian Journal of Veterinary Medicineinstname:Sociedade de Medicina Veterinária do Estado do Rio de Janeiro (SOMVERJ)instacron:SBMVporhttps://rbmv.org/BJVM/article/view/383/275de Mesquita, Samira Moraes CunhaMansur, Felipe Jansendo Nascimento, Elmiro RosendoBarreto, Maria LúciaKimura, Leda Maria Silvainfo:eu-repo/semantics/openAccess2020-12-23T17:33:12Zoai:ojs.rbmv.org:article/383Revistahttps://rbmv.org/BJVMONGhttps://rbmv.org/BJVM/oaicontato.rbmv@gmail.com2527-21790100-2430opendoar:2020-12-23T17:33:12Brazilian Journal of Veterinary Medicine - Sociedade de Medicina Veterinária do Estado do Rio de Janeiro (SOMVERJ)false |
dc.title.none.fl_str_mv |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples Padronização e aplicação de ELISA indireto para diagnóstico de Mycoplasma bovis em amostras de soro sanguíneo bovino |
title |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples |
spellingShingle |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples de Mesquita, Samira Moraes Cunha Mycoplasma bovis imunógeno ELISA soro sanguíneo Mycoplasma bovis immunogen ELISA blood serum |
title_short |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples |
title_full |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples |
title_fullStr |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples |
title_full_unstemmed |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples |
title_sort |
Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples |
author |
de Mesquita, Samira Moraes Cunha |
author_facet |
de Mesquita, Samira Moraes Cunha Mansur, Felipe Jansen do Nascimento, Elmiro Rosendo Barreto, Maria Lúcia Kimura, Leda Maria Silva |
author_role |
author |
author2 |
Mansur, Felipe Jansen do Nascimento, Elmiro Rosendo Barreto, Maria Lúcia Kimura, Leda Maria Silva |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
de Mesquita, Samira Moraes Cunha Mansur, Felipe Jansen do Nascimento, Elmiro Rosendo Barreto, Maria Lúcia Kimura, Leda Maria Silva |
dc.subject.por.fl_str_mv |
Mycoplasma bovis imunógeno ELISA soro sanguíneo Mycoplasma bovis immunogen ELISA blood serum |
topic |
Mycoplasma bovis imunógeno ELISA soro sanguíneo Mycoplasma bovis immunogen ELISA blood serum |
description |
ABSTRACT. Mesquita S.M.C., Mansur F.J., Nascimento E.R., Barreto M.L. & Kimura L.M.S. [Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples.] Padroniza- ção e aplicação de ELISA indireto para diagnóstico de Mycoplasma bovis em amostras de soro sanguíneo bovino. Revista Brasileira de Medicina Veterinária, 37(2):101-107, 2015. Universidade Federal Fluminense, Faculdade de Veteriná- ria, Rua Vital Brazil Filho, 64, Vital Brazil, Niterói, RJ 24230-340, Brasil. E-mail: samira.veterinaria@gmail.com International researchers presented results indicating frequent involvement of Mycoplasma spp. as a causative agent of mastitis in cattle, associating its presence with significant economic losses to farmers. Mycoplasma bovis is the species most reported and relevant, because it causes more severe disease. The level of antibodies against M. bovis remains high for several months and can be detected by ELISA. The aim of this work was to develop an indirect ELISA with whole cell antigen of M. bovis (strain Donetta PG 45) with subsequent application in bovine blood serum samples for detection of antibodies against M. bovis. The immunization of cows A and B by inoculating an immunogen against M. bovis to obtain hyperimmune blood serum was the first stage of this work, then the stage of standardization of ELISA was proceeded. The concentration of 2 mg of antigen/mL for coating the microtiter plates was decided by statistical analyses. The optical density value 0,2 was determined as the limit of reactivity discrimination of samples (the cut-off point). The hyperimmune blood serum sample of the cow A (collected 30 days after immunization) was chosen as the positive control and, the fetal calf serum was chosen as negative control of the assay. In addition, the ideal optimal dilutions found for blood serum samples was 1:400 and for conjugate was 1:10.000 and the substrate used was the ortho-phenylenediamine. The amount of 321 samples of cow blood serum from 15 farms was obtained for the application of the standardized indirect ELISA. It was diagnosed animals that have been exposed to M. bovis and the prevalence rate found in blood serum samples was 3,1% (10/321) and 46,7% (7/15) was the prevalence rate found between the corresponding farms studied. |
publishDate |
2015 |
dc.date.none.fl_str_mv |
2015-06-28 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion peer reviewed Avaliado pelos pares |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://rbmv.org/BJVM/article/view/383 |
url |
https://rbmv.org/BJVM/article/view/383 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.none.fl_str_mv |
https://rbmv.org/BJVM/article/view/383/275 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Sociedade de Medicina Veterinária do Estado do Rio de Janeiro. |
publisher.none.fl_str_mv |
Sociedade de Medicina Veterinária do Estado do Rio de Janeiro. |
dc.source.none.fl_str_mv |
Brazilian Journal of Veterinary Medicine; Vol. 37 No. 2 (2015); 101-107 Revista Brasileira de Medicina Veterinária; v. 37 n. 2 (2015); 101-107 2527-2179 0100-2430 reponame:Brazilian Journal of Veterinary Medicine instname:Sociedade de Medicina Veterinária do Estado do Rio de Janeiro (SOMVERJ) instacron:SBMV |
instname_str |
Sociedade de Medicina Veterinária do Estado do Rio de Janeiro (SOMVERJ) |
instacron_str |
SBMV |
institution |
SBMV |
reponame_str |
Brazilian Journal of Veterinary Medicine |
collection |
Brazilian Journal of Veterinary Medicine |
repository.name.fl_str_mv |
Brazilian Journal of Veterinary Medicine - Sociedade de Medicina Veterinária do Estado do Rio de Janeiro (SOMVERJ) |
repository.mail.fl_str_mv |
contato.rbmv@gmail.com |
_version_ |
1798313107599130624 |