Exoantigens from Fusarium verticillioides strains were analysed by polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and Western blot and were used for production of polyclonal antibodies. Rabbits were immunized with exoantigens from strains 97K, 162A and 113F. Polyclonal antibodies against F. verticillioides 97K exoantigen showed the highest titre in ELISA (1:12.800) and were used in further studies. The crossreactivity of anti-97K antibodies with 14 species from 7 other fungal genera was low, ranging from 4.5 Á/14.7%; the highest reactivities (51.2 Á/76.0%) were observed in Fusarium genera. The detection limit of exoantigen by indirect competitive ELISA was 0.27 mg/ml. Western blot analysis showed two immunodominant antigens (67 kDa and 113 kDa) common to all the F. verticillioides strains analysed. The results suggest that polyclonal antibodies raised against exoantigens from F. verticillioides 97K are promising for Fusarium sp. immunodetection.
ResumoFumonisinas são toxinas produzidas por Fusarium verticillioides Sacc. Niremberg (F. moniliforme Sheldon), patógeno primário de milho, que está implicado em inúmeras doenças animais, com ênfase em leucoencefalomalácia em eqüinos, edema pulmonar em suínos e carcinomas hepáticos e renais em ratos e camundongos. Em seres humanos, os estudos epidemiológicos têm associado o consumo de milho altamente contaminado com fumonisinas ao câncer esofágico em populações da China e África do Sul. Além disso, a contaminação de milho por Fusarium spp. causa perdas econômicas consideráveis em todo o mundo. Estruturalmente, a fumonisina B 1 (FB 1 ) assemelha-se às bases esfingóides, sendo a toxicidade e carcinogenicidade associada à capacidade de inibir a ceramida sintetase, uma enzima chave na via de biossíntese dos esfingolipídeos, causando acúmulo de esfinganina e depleção de esfingolipídeos complexos importantes nas funções celulares. Devido à ocorrência global, as fumonisinas têm sido alvo de intensas investigações na tentativa de desenvolver biomarcadores capazes de avaliar a exposição à FB 1 e, estabelecer os níveis de contaminação que não causem danos à saúde humana e animal. Esta revisão apresenta os principais aspectos referentes à toxicidade de fumonisinas em animais e sistemas in vitro, aliado ao mecanismo de ação bioquímica e aos biomarcadores visando avaliação da exposição. Palavras-chave: Fumonisinas, esfingolipídios, Fusarium verticillioides, biomarcadores, milho. AbstractFumonisins, mycotoxins produced by Fusarium verticillioides Sacc. Niremberg (F. moniliforme Sheldon), the prevalent seed borne fungus of corn (Zea mays), have been associated with several animal diseases, such as equine leukoencephalomalacia, porcine pulmonary edema and hepatic and renal carcinomas in rats and mice. Epidemiological studies have linked consumption of fumonisin-contaminated corn with esophageal cancer in human populations in China and South Africa. In addition, fungal growth and further corn contamination by fumonisins cause great economic losses throughout the world. The
Fusarium verticillioides is a primary corn pathogen and one of the main producers of fumonisins, a group of mycotoxins that cause several diseases in animals and probably also affect humans. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on polyclonal antibodies was developed to detect the exoantigen of this fungus in corn and its correlation with traditional methods for mould detection was evaluated. Forty freshly harvested corn samples were analysed for F. verticillioides exoantigens, as well as for total mould count, ergosterol and fumonisin levels in order to evaluate the relationship between these parameters. In addition, F. verticillioides was grown in brain heart infusion (BHI) broth in order to evaluate the correlation between biomass and exoantigen concentration. There was no significant correlation between exoantigen concentration and total mould count, Fusarium sp. count and fumonisin levels. The correlation coefficient between exoantigens and ergosterol content was 0.52 and between biomass and F. verticillioides exoantigens in BHI broth was 0.84. These results suggest that this ic-ELISA has potential for Fusarium sp. detection in corn samples.
ResumoO monitoramento constante da contaminação fúngica é imprescindível para assegurar a qualidade e segurança dos alimentos, reduzindo as perdas econômicas, assim como os riscos à saúde humana e animal. Os métodos tradicionais de identificação e detecção de fungos (cultivo em diversos meios, exame microscópico e análises bioquímicas) geralmente consomem muito tempo e exigem pessoal com experiência. Os imunoensaios, particularmente os ensaios imunoenzimáticos, constituem uma alternativa promissora aos métodos tradicionais devido à alta sensibilidade, especificidade, reprodutibilidade e potencial como método rápido de controle de qualidade. Dentre os ensaios imunoenzimáticos, aqueles baseados em exoantígenos são os mais empregados na resolução de problemas taxonômicos, detecção e identificação de fungos toxigênicos. Nesta revisão serão abordados conceitos básicos de imunoensaios, métodos de detecção de fungos, assim como diversos ensaios imunoenzimáticos para a detecção de fungos toxigênicos em alimentos. Palavras-chave: Imunoensaios, fungos toxigênicos, detecção de fungos, imunodetecção AbstractConstant monitoring of mould contamination is essential in order to assure the food quality and safety and reduce the economic losses, as well as to minimize the potential hazards to human and animal health. The traditional methods for mould identification and detection (culture in several media, microscopic examination and chemical analysis) are usually time-consuming and require trained staff. Immunoassays, particularly enzyme-linked immunosorbent assay (ELISA) could be a promising alternative to the traditional methods due to high sensitivity, specificity, reproducibility and potential for use in rapid quality control. Among ELISAs, those based on exoantigens are the most employed in the resolution of taxonomic problems, detection and identification of toxigenic fungi. This review discusses the basic principles of immunoassays, methods of mould detection and the several ELISAs developed for toxigenic fungi detection in food.
Resumo: O Cerrado é reconhecido como a savana mais rica do mundo em biodiversidade, assim o seu estudo no ensino se reveste de grande importância no sentido de fortalecer a formação dos alunos para conhecer e praticar a preservação ambiental deste bioma. O objetivo deste trabalho foi verificar o processo de aprendizagem sobre o bioma cerrado pela interação dos alunos em uma aula diferenciada, utilizando os cinco sentidos (tato, olfato, paladar, audição e visão). O método fundamenta-se fato de tentar reaproximar as pessoas da natureza temporariamente, despertando para o uso dos sentidos na exploração de “trilhas sensitivas”. Os resultados demonstraram que o uso da trilha sensitiva mostrou-se satisfatório no aumento do interesse dos alunos e no desenvolvimento do aprendizado sobre o cerrado. Abstract The Brazilian’s Cerrado is recognized as the richest in biodiversity savanna in the world, with the presence of several ecosystems. Therefore, their study in education has great importance in strengthening the training of students to learn and practice environmental conservation of this biome. The objective of this study was to assess the process of learning about the cerrado by the interaction of students in a differentiated class, using the five senses (touch, smell, taste, hearing and vision). The methodology is based on the fact of trying to reconnecting people of temporary nature without the use of vision, awakening to the use of other senses in the exploration of "sensory trails." The results demonstrated that the use of sensory trail was satisfactory in increasing student interest and learning about the development of the cerrado. Key words: Environmental education. Sensory trail. Science. Teaching-learning process.
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