RESUMO
Contagious agalactia (CA) is a serious disease notifiable to the World Organisation for Animal Health (OIE) causing severe economic losses to sheep and goat producers worldwide. Mycoplasma agalactiae, considered as its main etiological agent, inflicts a variety of symptoms in infected animals, including keratoconjunctivitis, mastitis, arthritis, ankylosis, abortions, stillbirths and granular vulvovaginitis. Despite its significance, developing a successful vaccine remains elusive, mostly due to the lack of knowledge about M. agalactiae's pathogenicity factors and pathogenic mechanisms, including its "core" antigens. The aim of this study was to identify, characterize and express antigenic proteins of M. agalactiae as potential vaccine candidates. Predicted proteins of type strain PG2 were analyzed using bioinformatic algorithms to assess their cellular localization and to identify their linear and conformational epitopes for B cells. Out of a total of 156 predicted membrane proteins, three were shortlisted as potential antigenic surface proteins, namely [MAG_1560 (WP_011949336.1), MAG_6130 (WP_011949770.1) and P40 (WP_011949418.1)]. These proteins were expressed in recombinant Escherichia coli strains. Purified proteins were evaluated for their antigenicity using Western blot and ELISA using sera of M. agalactiae-naturally infected and non-infected sheep and goats. All 3 proteins were specifically recognized by the tested sera of M. agalactiae-infected animals. Also, specific rabbit antisera raised against each of these 3 proteins confirm their membrane localization using TritonX-114 phase partioning, Western and colony immunoblotting. In conclusion, our study successfully identified P40 (as proof of concept and validation) and two novel antigenic M. agalactiae proteins as potential candidates for developing effective CA vaccines.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Mycoplasma agalactiae/química , Testes Sorológicos/métodos , Animais , Antígenos de Bactérias/genética , Epitopos de Linfócito B/imunologia , Feminino , Genoma Bacteriano , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Mycoplasma agalactiae/genética , Mycoplasma agalactiae/imunologia , CoelhosRESUMO
Mycoplasma agalactiae é o principal microrganismo causador da agalaxia contagiosa (AC), doença caracterizada por mastite seguida de agalaxia, poliartrite e ceratoconjuntivite. Em 2001, M. agalactiae foi isolado e identificado no Brasil, determinando grandes prejuízos econômicos. Considerando a necessidade de caracterização de amostras brasileiras de M. agalactiae e da implantação de medidas eficazes de controle, esse trabalho foi realizado em três etapas. O primeiro artigo descreve o perfil bioquímico e protéico de isolados de M. agalactiae de pequenos ruminantes através do cultivo em meio Hayflick modificado, testes bioquímicos e eletroforese em gel de poliacrilamida (SDS-PAGE) e a imunogenicidade destas proteínas por Western Blot (WB). As amostras apresentaram similaridade no perfil protéico com bandas variando de 30 a 135 kDa no SDS-PAGE, além da presença de uma proteína imunodominante de 48 kDa no WB. Para dar continuidade a identificação do agente envolvido na AC no Brasil, o segundo artigo descreve a análise de dez sequências de M. agalactiae isolados de caprinos e ovinos. Um fragmento de DNA de 360 bp do gene 16S rRNA amplificado por PCR foi sequenciado. A análise revelou alto grau de similaridade, entre as dez sequências e uma similaridade maior que 99% com amostras de referência de M. agalactiae. No terceiro artigo, a eficiência de três vacinas inativadas, preparadas com amostra local de M. agalactiae e adsorvidas com três adjuvantes, foi avaliada em caprinos e ovinos. A resposta sorológica dos animais vacinados foi analisada através de um ELISA indireto. As três vacinas induziram produção de anticorpos, podendo ser utilizadas como uma alternativa para reduzir as perdas econômicas e prevenir a agalaxia contagiosa. Estes resultados confirmam a presença e disseminação do M. agalactiae no país e fortalece a possibilidade de controle da doença pela adoção da vacinação dos rebanhos.
Mycoplasma agalactiae is the main causative organism of contagious agalactia (CA), a disease characterized by mastitis followed by agalactia, polyarthritis, and keratoconjunctivitis. In 2001, M. agalactiae was isolated and identified in Brazil, causing great economic losses. Considering the need for characterization of Brazilian strains of M. agalactiae and implementation of effective control, this work was performed in three steps. The first article describes the biochemical and protein profile of isolates of M. agalactiae in small ruminants through the culture in modified Hayflick medium, biochemical tests, polyacrylamide gel electrophoresis (SDS-PAGE) and immunogenicity of these proteins by Western blot (WB). The samples had similar protein in the profile bands ranging from 30-135 kDa in SDS-PAGE, in addition the presence of a protein of 48 kDa immunodominant WB was shown. To continue identifying the agent involved in the AC in Brazil, the second article describes the analysis of ten sequences of M. agalactiae isolated from goats and sheeps. A DNA fragment of 360 bp of the 16S rRNA gene was amplified by PCR and sequenced. The analysis revealed a high degree of similarity among all the sequences. The study revealed greater than 99% similarity with the reference samples of M. agalactiae. In the third article, efficacy of three inactivated vaccines prepared with a local of M. agalactiae sample and adsorbed with three adjuvants was evaluated in goats and sheep. The antibody response in vaccinated animals was analyzed using an indirect ELISA. The three vaccines induced antibody production, and can be an alternative to reduce economic losses and prevent contagious agalactia. These results confirms the presence and spread of M. agalactiae in the country and enhances the possibility of controlling the disease through the adoption of livestock vaccination.