Ex vivo and in vitro poultry intestinal models to evaluate antimycotoxins additives / Uso de modelos ex vivo e in vitro de intestino de aves para avaliar aditivos antimicotoxinas

In vitro tests are performed to evaluate the efficacy of antimycotoxins additives (AMAs); nevertheless, such assays show a low correlation with in vivo trials, which are also required to determine AMAs' efficacy. In search of an alternative method, the current study investigated the use of an ex vivo technique. Six AMAs (AMA1 to AMA6) had their ability to reduce intestinal absorption of aflatoxin B1 (AFB1) evaluated. Jejunal explants were obtained from broilers and subjected to two treatments per AMA in Ussing chambers: T1 (control) - 2.8 mg/L AFB1, and T2 - 2.8 mg/L AFB1 + 0.5% AMA. AMAs were also tested in vitro to assess adsorption of AFB1 in artificial intestinal fluid. In the ex vivo studies, AMA1 to AMA6 decreased intestinal absorption of AFB1 by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively. As for the in vitro results, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. The evaluated ex vivo model proved useful in the assessment of AMAs. No correlation was reported between ex vivo and in vitro findings. Further studies are needed to elucidate the correlation between ex vivo and in vivo results seeking to reduce animal testing.

Testes in vitro são realizados para avaliar a eficácia de aditivos antimicotoxinas (AAMs); entretanto, tais experimentos apresentam uma baixa correlação com ensaios in vivo, que também são exigidos para determinar a eficácia de AAMs. Em busca de um método alternativo, o presente estudo investigou o uso de uma técnica ex vivo. A capacidade de seis AAMs (AAM1 a AAM6) de reduzir a absorção intestinal de aflatoxina B1 (AFB1) foi avaliada. Explantes jejunais foram coletados de frangos de corte e submetidos a dois tratamentos por AAM em câmaras de Ussing: T1 (controle) - 2,8 mg/L AFB1, e T2 - 2.8 mg/L AFB1 + 0,5% AAM. Os AAMs também foram testados in vitro para verificar a adsorção de AFB1 em fluido intestinal artificial. Nos ensaios ex vivo, AAM1 ao AAM6 diminuíram a absorção intestinal de AFB1 em 67,11%, 73,82%, 80,70%, 85,86%, 86,28% e 82,32%, respectivamente. Quanto aos achados in vitro, AAM1 ao AAM6 apresentaram adsorção de 99,72%, 99,37%, 99,67%, 99,53%, 99,04% e 99,15%, respectivamente. O modelo ex vivo avaliado mostrou-se eficiente na avaliação de AAMs. Não houve correlação entre os resultados ex vivo e in vitro. Estudos adicionais são necessários para definir a correlação entre achados ex vivo e in vivo na tentativa de reduzir os testes em animais.

Effect of aflatoxin B1 on digestibility and blood parameters in horses / Digestibilidade e efeitos hematológicos da aflatoxina B 1 em equinos

This study aimed to evaluate the effect of aflatoxin B1 (AFB1) on the digestibility of nutrients and the hematological profile of horses. The assay lasted 40 days, with 12 days for adaptation and 28 days for experimentation. In the experimental stage, the horses were distributed in a completely randomized design, including three treatment groups with four animals in each group. The treatments used included 0 µg/kg (control), 50 µg/kg, and 100 µg/kg of AFB1 added to a concentrate in the basal diet. The basal diet contained mycotoxins from naturally contaminated feed. A digestibility essay was carried out at the end of the experimental period through partial feces collection, with LIPE® as an indicator. Blood samples were collected once a week during the assay for hematological and biochemical evaluations. The results of the hematological and biochemical parameters were submitted to analysis of variance (ANOVA) and compared by the Tukey test at 5% significance. The aflatoxins in the diet increased the leukocyte count, especially that of mature neutrophils. Creatine kinase and alkaline phosphatase (P < 0.05) activities were higher in horses fed more toxic diets. The digestibility of nutrients was unaffected by the level of mycotoxins in the diet (P > 0.05).

Objetivou-se com esta pesquisa avaliar o efeito da Aflatoxina B1 (AFB1) na digestibilidade dos nutrientes e no perfil hematológico de equinos. O ensaio durou 40 dias, sendo 12 dias para adaptação e 28 dias para experimentação. No período experimental, os cavalos foram distribuídos em delineamento inteiramente casualizado, com três tratamentos, com quatro animais cada. Os tratamentos utilizados foram 0 µg/kg (controle), 50 µg/kg e 100 µg/kg de AFB1 adicionados ao concentrado da dieta basal. A dieta basal continha alimentos naturalmente contaminados com micotoxinas. Um ensaio de digestibilidade foi realizado no final do período experimental, pelo método de coleta parcial de fezes utilizando o LIPE® como indicador. Amostras de sangue foram coletadas uma vez por semana, durante o ensaio para avaliações hematológicas e bioquímicas. Os resultados dos parâmetros hematológicos, bioquímicos e do ensaio de digestibilidade foram submetidos à análise de variância (ANOVA) e comparados pelo teste de Tukey a 5% de significância. As aflatoxinas da dieta aumentaram a contagem de leucócitos, principalmente os neutrófilos maduros. A creatina quinase e a fosfatase alcalina (P<0,05) apresentaram maior atividade nos equinos alimentados com dietas com maior toxicidade. A digestibilidade dos nutrientes não foi alterada pelos níveis de micotoxinas presentes nas dietas (P>0,05).

Saccharomyces cerevisiae strain associated to a fish culture additive: antifungal activity and AFB1 adsorption potential / Saccharomyces cerevisiae associada a um aditivo de piscicultura: atividade antifúngica e potencial de adsorção de AFB1

Saccharomyces cerevisae is widely applied as a probiotic in aquaculture activities, due to its ability to decontaminate the mycotoxin Aflatoxin B1 (AFB1). With this in mind, the aim of the present study was to evaluate the antifungal and anti-aflatoxigenic activities of inactivated Saccharomyces cerevisiae mixed with a commercial product (CP) of animal feed to assess its influence on the Aspergillus flavus and A. parasiticus fungi growth and on the AFB1 production. In addition, AFB1 adsorption potential of S. cerevisae and commercial product was also investigated. Different concentrations of commercial product alone and in the presence of inactivated yeast were analyzed by Aspergillus growth inhibition test, aflatoxin production by Aspergillus species and AFB1 adsorption capacity. AFB1 detection and quantification were carried out by High Performance Liquid Chromatography. The inactivated yeast and commercial product combination was effective in reducing A. flavus and A. parasiticus growth. A. flavus produced less AFB1 after the inactivated yeast treatment, whereas A. parasiticus produced significantly less AFB1 under a combination of inactivated yeast and 50% CP. Regarding AFB1 adsorption, 100% CP displayed the highest adsorption capacity at 10 ng mL-1 AFB1. At 25 ng mL-1 AFB1, only the treatment comprising inactivated yeast associated to 50% CP led to AFB1 adsorption, albeit

Saccharomyces cerevisae é uma levedura largamente utilizada como probiótico na aquicultura e que tem se destacado devido a capacidade para a descontaminação de AFB1. O objetivo desse trabalho foi avaliar a atividade anti-fúngica e anti-aflatoxigênica da Saccharomyces cerevisae inativada e misturada a um produto comercial, observando sua influência no crescimento dos fungos Aspergillus flavus e A. parasiticus e na produção destes da Aflatoxina B1 (AFB1), investigar o potencial de adsorção da levedura e do produto comercial sobre a AFB1. Foram realizadas analises de preparação de leveduras inativadas e das soluções com o produto comercial, Ensaio do efeito inibitório de espécies de Aspergillus in vitro, Teste in vitro sobre a produção de aflatoxinas, avaliação in vitro da capacidade de adsorção de AFB1, condições cromatográficas para detecção e quantificação de AFB1 por cromatografia Líquida de Alta Eficiência. A associação do aditivo comercial EPICIN – G2® e a levedura S. cerevisiae inativada foi eficaz na diminuição do cresci-mento fúngico dos A. flavus e A parasiticus. Quanto à produção de micotoxina, o A. flavus obteve menor produção com o tratamento composto por leveduras inativadas, enquanto, o A. parasiticus teve diminuição significativa na produção de AFB1 com a associação da levedura a uma concentração de 50% do produto. No referente a adsorção, em concentra-ção de 10ng/mL, o tratamento que demonstrou melhor capacidade de adsorver AFB1 foi o 100% Produto (100%). Já na concentração 25 ng/mL, somente o tratamento que associava levedura e 50% do produto, apresentou uma pequena capacidade de adsorção.

Saccharomyces cerevisiae strain associated to a fish culture additive: antifungal activity and AFB1 adsorption potential / Saccharomyces cerevisiae associada a um aditivo de piscicultura: atividade antifúngica e potencial de adsorção de AFB1

Saccharomyces cerevisae is widely applied as a probiotic in aquaculture activities, due to its ability to decontaminate the mycotoxin Aflatoxin B1 (AFB1). With this in mind, the aim of the present study was to evaluate the antifungal and anti-aflatoxigenic activities of inactivated Saccharomyces cerevisiae mixed with a commercial product (CP) of animal feed to assess its influence on the Aspergillus flavus and A. parasiticus fungi growth and on the AFB1 production. In addition, AFB1 adsorption potential of S. cerevisae and commercial product was also investigated. Different concentrations of commercial product alone and in the presence of inactivated yeast were analyzed by Aspergillus growth inhibition test, aflatoxin production by Aspergillus species and AFB1 adsorption capacity. AFB1 detection and quantification were carried out by High Performance Liquid Chromatography. The inactivated yeast and commercial product combination was effective in reducing A. flavus and A. parasiticus growth. A. flavus produced less AFB1 after the inactivated yeast treatment, whereas A. parasiticus produced significantly less AFB1 under a combination of inactivated yeast and 50% CP. Regarding AFB1 adsorption, 100% CP displayed the highest adsorption capacity at 10 ng mL-1 AFB1. At 25 ng mL-1 AFB1, only the treatment comprising inactivated yeast associated to 50% CP led to AFB1 adsorption, albeit

Saccharomyces cerevisae é uma levedura largamente utilizada como probiótico na aquicultura e que tem se destacado devido a capacidade para a descontaminação de AFB1. O objetivo desse trabalho foi avaliar a atividade anti-fúngica e anti-aflatoxigênica da Saccharomyces cerevisae inativada e misturada a um produto comercial, observando sua influência no crescimento dos fungos Aspergillus flavus e A. parasiticus e na produção destes da Aflatoxina B1 (AFB1), investigar o potencial de adsorção da levedura e do produto comercial sobre a AFB1. Foram realizadas analises de preparação de leveduras inativadas e das soluções com o produto comercial, Ensaio do efeito inibitório de espécies de Aspergillus in vitro, Teste in vitro sobre a produção de aflatoxinas, avaliação in vitro da capacidade de adsorção de AFB1, condições cromatográficas para detecção e quantificação de AFB1 por cromatografia Líquida de Alta Eficiência. A associação do aditivo comercial EPICIN G2® e a levedura S. cerevisiae inativada foi eficaz na diminuição do cresci-mento fúngico dos A. flavus e A parasiticus. Quanto à produção de micotoxina, o A. flavus obteve menor produção com o tratamento composto por leveduras inativadas, enquanto, o A. parasiticus teve diminuição significativa na produção de AFB1 com a associação da levedura a uma concentração de 50% do produto. No referente a adsorção, em concentra-ção de 10ng/mL, o tratamento que demonstrou melhor capacidade de adsorver AFB1 foi o 100% Produto (100%). Já na concentração 25 ng/mL, somente o tratamento que associava levedura e 50% do produto, apresentou uma pequena capacidade de adsorção.(AU)

Biopanning the mimotopes of aflatoxin B1 and their immunogenicity / Panning de mimotopo aflatoxina B1 e sua imunogenicidade

Utilizando um anticorpo monoclonal contra a aflatoxina B1 (AFB1) como ligante, foi identificado um mimotopo específico de aflatoxina B1 após se realizarem quatro ciclos de seleção biológica de 7-peptídeos aleatórios em biblioteca de fago exibida. O mimotopo é denominado P10, e sua sequência de aminoácidos é YRRHEKD. O soro imunológico de ratos Balb/c imunizados com P10 foi especificamente ligado à aflatoxina B1-albumina, indicando que o anticorpo era específico ao AFB1. Esses resultados sugerem que é possível desenvolver a vacina baseada em mimotopo associado à toxina.(AU)

Biopanning the mimotopes of aflatoxin B1 and their immunogenicity / Panning de mimotopo aflatoxina B1 e sua imunogenicidade

Utilizando um anticorpo monoclonal contra a aflatoxina B1 (AFB1) como ligante, foi identificado um mimotopo específico de aflatoxina B1 após se realizarem quatro ciclos de seleção biológica de 7-peptídeos aleatórios em biblioteca de fago exibida. O mimotopo é denominado P10, e sua sequência de aminoácidos é YRRHEKD. O soro imunológico de ratos Balb/c imunizados com P10 foi especificamente ligado à aflatoxina B1-albumina, indicando que o anticorpo era específico ao AFB1. Esses resultados sugerem que é possível desenvolver a vacina baseada em mimotopo associado à toxina.(AU)

Saccharomyces cerevisiae as a probiotic agent and a possible aflatoxin B1 adsorbent in simulated fish intestinal tract conditions / Saccharomyces cerevisiae como agente probiótico e possível adsorvente de aflatoxina B1 em condições simuladas do trato intestinal de peixes

The aim of this study was to evaluate in vitro the probiotic potential and absorption of Saccharomyces cerevisiae for the aflatoxin B1 in simulated fish intestinal tract conditions. Three yeast strains were used, two from brewery: S. cerevisiae RC1 and S. cerevisiae RC3 and one from a fish farming environment: S. cerevisiae A8L2. The selected yeasts were subjected to the following in vitro tests: homologous inhibition, self-aggregation, co-aggregation, antibacterial activity, gastrointestinal conditions tolerance and adsorption of AFB1. All S. cerevisiae strains showed good capability of self-aggregation and co-aggregation with pathogenic bacteria. All yeast strains were able to survive the gastrointestinal conditions. In acidic conditions, the factors (strain vs. time) had interaction (P=0.0317), resulting in significant variation among the strains tested in the time periods analyzed. It was observed that there was also interaction (P=0.0062) in intestinal conditions, with an increased number of cells in the 12-hour period for all strains tested. In the adsorption test, the A8L2 strain was statistically more effective (P<0.005) for both AFB1 concentrations evaluated in this study (10 and 25ng/mL). Thus, it was observed that the strains of S. cerevisiae have potential probiotic and adsorbent of AFB1.(AU)

Objetivou-se, com esta pesquisa, avaliar in vitro o potencial probiótico e adsorvente de Saccharomyces cerevisiae para aflatoxina B1 em condições simuladas do trato intestinal de peixes. Foram utilizadas três cepas de leveduras, sendo duas provenientes de cervejaria: S. cerevisiae RC1 e S. cerevisiae RC3, e uma de ambiente de piscicultura: S. cerevisiae A8L2. As leveduras selecionadas foram submetidas aos seguintes testes in vitro: inibição homóloga, autoagregação, coagregação, atividade antibacteriana, viabilidade às condições gastrointestinais e adsorção de AFB1. Todas as estirpes de S. cerevisiae mostraram boa capacidade de autoagregação e coagregação com bactérias patogênicas. Todas as estirpes de levedura foram capazes de sobreviver às condições gastrointestinais. Em condições ácidas, os fatores (cepa x tempo) tiveram interação (P=0,0317), resultando em variações significativas entre as cepas testadas nos períodos de tempo analisados. Observou-se que também houve interação (P=0,0062) em condições intestinais, havendo um aumento do número de células no período de 12h para todas as cepas avaliadas. No ensaio de adsorção, a estirpe A8L2 foi a mais eficaz estatisticamente (P<0,005), para as duas concentrações de AFB1 avaliadas neste estudo (10 e 25ng. mL-1). Dessa forma, conclui-se que as cepas de Saccharomyces cerevisiae possuem potencial probiótico e adsorvente de AFB1.(AU)

Saccharomyces cerevisiae as a probiotic agent and a possible aflatoxin B1 adsorbent in simulated fish intestinal tract conditions / Saccharomyces cerevisiae como agente probiótico e possível adsorvente de aflatoxina B1 em condições simuladas do trato intestinal de peixes

The aim of this study was to evaluate in vitro the probiotic potential and absorption of Saccharomyces cerevisiae for the aflatoxin B1 in simulated fish intestinal tract conditions. Three yeast strains were used, two from brewery: S. cerevisiae RC1 and S. cerevisiae RC3 and one from a fish farming environment: S. cerevisiae A8L2. The selected yeasts were subjected to the following in vitro tests: homologous inhibition, self-aggregation, co-aggregation, antibacterial activity, gastrointestinal conditions tolerance and adsorption of AFB1. All S. cerevisiae strains showed good capability of self-aggregation and co-aggregation with pathogenic bacteria. All yeast strains were able to survive the gastrointestinal conditions. In acidic conditions, the factors (strain vs. time) had interaction (P=0.0317), resulting in significant variation among the strains tested in the time periods analyzed. It was observed that there was also interaction (P=0.0062) in intestinal conditions, with an increased number of cells in the 12-hour period for all strains tested. In the adsorption test, the A8L2 strain was statistically more effective (P<0.005) for both AFB1 concentrations evaluated in this study (10 and 25ng/mL). Thus, it was observed that the strains of S. cerevisiae have potential probiotic and adsorbent of AFB1.(AU)

Objetivou-se, com esta pesquisa, avaliar in vitro o potencial probiótico e adsorvente de Saccharomyces cerevisiae para aflatoxina B1 em condições simuladas do trato intestinal de peixes. Foram utilizadas três cepas de leveduras, sendo duas provenientes de cervejaria: S. cerevisiae RC1 e S. cerevisiae RC3, e uma de ambiente de piscicultura: S. cerevisiae A8L2. As leveduras selecionadas foram submetidas aos seguintes testes in vitro: inibição homóloga, autoagregação, coagregação, atividade antibacteriana, viabilidade às condições gastrointestinais e adsorção de AFB1. Todas as estirpes de S. cerevisiae mostraram boa capacidade de autoagregação e coagregação com bactérias patogênicas. Todas as estirpes de levedura foram capazes de sobreviver às condições gastrointestinais. Em condições ácidas, os fatores (cepa x tempo) tiveram interação (P=0,0317), resultando em variações significativas entre as cepas testadas nos períodos de tempo analisados. Observou-se que também houve interação (P=0,0062) em condições intestinais, havendo um aumento do número de células no período de 12h para todas as cepas avaliadas. No ensaio de adsorção, a estirpe A8L2 foi a mais eficaz estatisticamente (P<0,005), para as duas concentrações de AFB1 avaliadas neste estudo (10 e 25ng. mL-1). Dessa forma, conclui-se que as cepas de Saccharomyces cerevisiae possuem potencial probiótico e adsorvente de AFB1.(AU)

Effect of aflatoxin B1 on blood serum oestradiol-17β and progesterone concentrations during the luteal phase and the synchronized oestrus of goats

The effect of prolonged aflatoxin B1 (AFB1) administration on blood serum oestradiol-17β and progesterone concentrations in goats during the luteal phase and the synchronized oestrus was investigated. Thirty-six Greek indigenous primiparous goats were used, during the oestrus period; 12 goats received, per os, 50 μg (treated group T50) and 12 goats received 100 μg (treated group T100) AFB1/day/head, respectively, for approximately 1.5 month, while 12 goats served as controls (C). On day 36 of the experiment, each goat was injected, i.m, 0.5 ml prostaglandin F2α (PGF2α). Blood samples were collected from each goat twice a week, before PGF2α injection, as well as every 4 hours from the onset to the end of the synchronized oestrus. Oestradiol-17β and progesterone concentrations in blood serum were determined using radioimmunoassay. During the whole luteal(s) phase(s), linear regression analysis revealed a significant negative dependence (P 0.05). In conclusion, prolonged AFB1 administration at doses of 100 or even of 50 μg/day/head changes the hormonal pattern in blood during the luteal phase and the synchronized oestrus of goats, being in oestrus period.

Effect of aflatoxin B1 on blood serum oestradiol-17β and progesterone concentrations during the luteal phase and the synchronized oestrus of goats

The effect of prolonged aflatoxin B1 (AFB1) administration on blood serum oestradiol-17β and progesterone concentrations in goats during the luteal phase and the synchronized oestrus was investigated. Thirty-six Greek indigenous primiparous goats were used, during the oestrus period; 12 goats received, per os, 50 μg (treated group T50) and 12 goats received 100 μg (treated group T100) AFB1/day/head, respectively, for approximately 1.5 month, while 12 goats served as controls (C). On day 36 of the experiment, each goat was injected, i.m, 0.5 ml prostaglandin F2α (PGF2α). Blood samples were collected from each goat twice a week, before PGF2α injection, as well as every 4 hours from the onset to the end of the synchronized oestrus. Oestradiol-17β and progesterone concentrations in blood serum were determined using radioimmunoassay. During the whole luteal(s) phase(s), linear regression analysis revealed a significant negative dependence (P < 0.05) of oestradiol-17β and a significant positive dependence (P < 0.05) of progesterone over group (C = 0, T50 = 50, T100 = 100), in a dose dependent manner. During the synchronized oestrus, multiple linear regression analysis revealed a significant negative dependence (P < 0.05) of oestradiol-17β, as well as a significant positive dependence (P < 0.05) of progesterone over group (C = 0, T50 = 50, T100 = 100) and over time (hours, from the onset to the end of the synchronized oestrus). No significant differences were noticed among the three groups, regarding the body weight of the goats from the onset to the end of AFB1 administration, the occurrence or the duration of the synchronized oestrus presented by the goats (P > 0.05). In conclusion, prolonged AFB1 administration at doses of 100 or even of 50 μg/day/head changes the hormonal pattern in blood during the luteal phase and the synchronized oestrus of goats, being in oestrus period.(AU)

Avaliação in vitro da adsorção de aflatoxina B1 por produtos comerciais utilizados na alimentação animal / In vitro evaluation of aflatoxin B1 adsorption by commercial products used in animal feed

The aim of this study was to evaluate the aflatoxin B1 (AFB1) adsorption capacity, in vitro, by commercial products used in animal feed. Many studies are being conducted for the decontamination of aflatoxins in feed. The commercial products destined to fish feed that are available as probiotics and are formed by strains of bacteria and yeasts used in most mycotoxins adsorption assays. Three commercial products were studied: A, consisting of Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium and Lactobacillus acidophilus; B, consisting of dry yeast of Saccharomyces cerevisiae from brewery; and C, consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Five suspensions of the maximum dose recommended by the manufacturer of each product (0; 25; 50; 75 and 100%) were tested against AFB1 (1000 ng.mL-1) in microtubes to determine the adsorption capacity. To simulate the pH of the stomach and intestine of the Nile tilapia (Oreochromis niloticus), phosphate buffered saline solutions (PBS) at pH 1.5 and 7.5, respectively, were formulated. Microtubes were introduced into a centrifuge with mechanical agitation at 37ºC for 1 h and then centrifuged for 10 min at 14.000 rpm; the supernatants were quantified by high-performance liquid chromatography. The commercial products in the maximum concentration were capable of adsorbing AFB1 in amounts from 45.01 to 129.59; from 123.90 to 215.59; and from 209.98 to 370.73 (ng.mL-1), respectively. It was concluded that all commercial products, which are added to animal feed, adsorbed AFB1 under simulated gastrointestinal pH conditions and are potential candidates for AFB1 adsorption for future in vivo studies.

Objetivou-se avaliar a capacidade de adsorção in vitro de aflatoxina B1 (AFB1) por produtos comerciais utilizados na alimentação animal. Muitas pesquisas estão sendo realizadas para a descontaminação de AFB1 em alimentos. Os produtos comerciais utilizados frequentemente na alimentação de peixes, disponíveis na forma de probióticos, são formados por cepas de bactérias e leveduras utilizadas na maioria dos ensaios de adsorção de micotoxinas. Foram utilizados três produtos comerciais: A, composto por Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium e Lactobacillus acidophilus; B, por leveduras secas de Saccharomyces cerevisiae provenientes de cervejaria; e C, por Bacillus subtilis, Bacillus licheniformis e Bacillus pumilus. Cinco suspensões da dose máxima recomendada pelo fabricante de cada produto (0; 25; 50; 75 e 100%) foram testadas contra AFB1 (1000 ng.mL-1) em microtubos para determinação da capacidade de adsorção. Para simular o pH do estômago e do intestino de tilápias do Nilo (Oreochromis niloticus) foram formuladas soluções tampão fosfato salino (PBS), com pH 1,5 e 7,5; respectivamente. Os microtubos foram introduzidos em uma centrífuga com agitação mecânica, a 37ºC por 1 h e depois centrifugados por 10 min a 14.000 rpm; os sobrenadantes foram quantificados por cromatografia líquida de alta eficiência. Os produtos comerciais, nas concentrações máximas, foram capazes de adsorver AFB1 em quantidades de 45,01 a 129,59; 123,90 a 215,59 e 209,98 a 370,73 ng.mL-1, respectivamente. Concluiu-se que todos os produtos comerciais analisados adsorvem AFB1 em condições simuladas de pH gastrointestinal e são candidatos potenciais para adsorção de AFB1 para futuros ensaios in vivo.

Avaliação in vitro da adsorção de aflatoxina B1 por produtos comerciais utilizados na alimentação animal / In vitro evaluation of aflatoxin B1 adsorption by commercial products used in animal feed

Objetivou-se avaliar a capacidade de adsorção in vitro de aflatoxina B1 (AFB1) por produtos comerciais utilizados na alimentação animal. Muitas pesquisas estão sendo realizadas para a descontaminação de AFB1 em alimentos. Os produtos comerciais utilizados frequentemente na alimentação de peixes, disponíveis na forma de probióticos, são formados por cepas de bactérias e leveduras utilizadas na maioria dos ensaios de adsorção de micotoxinas. Foram utilizados três produtos comerciais: A, composto por Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium e Lactobacillus acidophilus; B, por leveduras secas de Saccharomyces cerevisiae provenientes de cervejaria; e C, por Bacillus subtilis, Bacillus licheniformis e Bacillus pumilus. Cinco suspensões da dose máxima recomendada pelo fabricante de cada produto (0; 25; 50; 75 e 100%) foram testadas contra AFB1 (1000 ng.mL-1) em microtubos para determinação da capacidade de adsorção. Para simular o pH do estômago e do intestino de tilápias do Nilo (Oreochromis niloticus) foram formuladas soluções tampão fosfato salino (PBS), com pH 1,5 e 7,5; respectivamente. Os microtubos foram introduzidos em uma centrífuga com agitação mecânica, a 37ºC por 1 h e depois centrifugados por 10 min a 14.000 rpm; os sobrenadantes foram quantificados por cromatografia líquida de alta eficiência. Os produtos comerciais, nas concentrações máximas, foram capazes de adsorver AFB1 em quantidades de 45,01 a 129,59; 123,90 a 215,59 e 209,98 a 370,73 ng.mL-1, respectivamente. Concluiu-se que todos os produtos comerciais analisados adsorvem AFB1 em condições simuladas de pH gastrointestinal e são candidatos potenciais para adsorção de AFB1 para futuros ensaios in vivo.(AU)

The aim of this study was to evaluate the aflatoxin B1 (AFB1) adsorption capacity, in vitro, by commercial products used in animal feed. Many studies are being conducted for the decontamination of aflatoxins in feed. The commercial products destined to fish feed that are available as probiotics and are formed by strains of bacteria and yeasts used in most mycotoxins adsorption assays. Three commercial products were studied: A, consisting of Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium and Lactobacillus acidophilus; B, consisting of dry yeast of Saccharomyces cerevisiae from brewery; and C, consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Five suspensions of the maximum dose recommended by the manufacturer of each product (0; 25; 50; 75 and 100%) were tested against AFB1 (1000 ng.mL-1) in microtubes to determine the adsorption capacity. To simulate the pH of the stomach and intestine of the Nile tilapia (Oreochromis niloticus), phosphate buffered saline solutions (PBS) at pH 1.5 and 7.5, respectively, were formulated. Microtubes were introduced into a centrifuge with mechanical agitation at 37ºC for 1 h and then centrifuged for 10 min at 14.000 rpm; the supernatants were quantified by high-performance liquid chromatography. The commercial products in the maximum concentration were capable of adsorbing AFB1 in amounts from 45.01 to 129.59; from 123.90 to 215.59; and from 209.98 to 370.73 (ng.mL-1), respectively. It was concluded that all commercial products, which are added to animal feed, adsorbed AFB1 under simulated gastrointestinal pH conditions and are potential candidates for AFB1 adsorption for future in vivo studies.(AU)

Avaliação in vitro da adsorção de aflatoxina B1 por produtos comerciais utilizados na alimentação animal / In vitro evaluation of aflatoxin B1 adsorption by commercial products used in animal feed

The aim of this study was to evaluate the aflatoxin B1 (AFB1) adsorption capacity, in vitro, by commercial products used in animal feed. Many studies are being conducted for the decontamination of aflatoxins in feed. The commercial products destined to fish feed that are available as probiotics and are formed by strains of bacteria and yeasts used in most mycotoxins adsorption assays. Three commercial products were studied: A, consisting of Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium and Lactobacillus acidophilus; B, consisting of dry yeast of Saccharomyces cerevisiae from brewery; and C, consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Five suspensions of the maximum dose recommended by the manufacturer of each product (0; 25; 50; 75 and 100%) were tested against AFB1 (1000 ng.mL-1) in microtubes to determine the adsorption capacity. To simulate the pH of the stomach and intestine of the Nile tilapia (Oreochromis niloticus), phosphate buffered saline solutions (PBS) at pH 1.5 and 7.5, respectively, were formulated. Microtubes were introduced into a centrifuge with mechanical agitation at 37ºC for 1 h and then centrifuged for 10 min at 14.000 rpm; the supernatants were quantified by high-performance liquid chromatography. The commercial products in the maximum concentration were capable of adsorbing AFB1 in amounts from 45.01 to 129.59; from 123.90 to 215.59; and from 209.98 to 370.73 (ng.mL-1), respectively. It was concluded that all commercial products, which are added to animal feed, adsorbed AFB1 under simulated gastrointestinal pH conditions and are potential candidates for AFB1 adsorption for future in vivo studies.(AU)

Objetivou-se avaliar a capacidade de adsorção in vitro de aflatoxina B1 (AFB1) por produtos comerciais utilizados na alimentação animal. Muitas pesquisas estão sendo realizadas para a descontaminação de AFB1 em alimentos. Os produtos comerciais utilizados frequentemente na alimentação de peixes, disponíveis na forma de probióticos, são formados por cepas de bactérias e leveduras utilizadas na maioria dos ensaios de adsorção de micotoxinas. Foram utilizados três produtos comerciais: A, composto por Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium e Lactobacillus acidophilus; B, por leveduras secas de Saccharomyces cerevisiae provenientes de cervejaria; e C, por Bacillus subtilis, Bacillus licheniformis e Bacillus pumilus. Cinco suspensões da dose máxima recomendada pelo fabricante de cada produto (0; 25; 50; 75 e 100%) foram testadas contra AFB1 (1000 ng.mL-1) em microtubos para determinação da capacidade de adsorção. Para simular o pH do estômago e do intestino de tilápias do Nilo (Oreochromis niloticus) foram formuladas soluções tampão fosfato salino (PBS), com pH 1,5 e 7,5; respectivamente. Os microtubos foram introduzidos em uma centrífuga com agitação mecânica, a 37ºC por 1 h e depois centrifugados por 10 min a 14.000 rpm; os sobrenadantes foram quantificados por cromatografia líquida de alta eficiência. Os produtos comerciais, nas concentrações máximas, foram capazes de adsorver AFB1 em quantidades de 45,01 a 129,59; 123,90 a 215,59 e 209,98 a 370,73 ng.mL-1, respectivamente. Concluiu-se que todos os produtos comerciais analisados adsorvem AFB1 em condições simuladas de pH gastrointestinal e são candidatos potenciais para adsorção de AFB1 para futuros ensaios in vivo.(AU)

In vivo amelioration of aflatoxin B1 in broiler chicks by magnetic carbon nanocomposite

In this study an Iron oxide/carbon nanocomposite from maize straw was prepared and was characterized by XRD, SEM, EDX, FTIR, TG/DTA and Surface area analyzer. The adsorbent was fed to different groups of poultry birds along with aflatoxin B1. Different physiological and blood parameters were monitored in order to study the efficacy of the prepared adsorbent for binding of aflatoxin B1 in the gastrointestinal tract of chickens. It was found that adsorbent at dose of 0.3%/ kg feed was highly effective in detoxifying aflatoxin B1 in gastrointestinal tract of poultry birdswith no harmful effects. The high doses given to groups E and F; 0.4% and 0.5% respectively showed slight variation in tested parameters from group A. No negative symptoms associated with the use of activated carbon as previously reported were observed for the adsorbent under study.(AU)

In vivo amelioration of aflatoxin B1 in broiler chicks by magnetic carbon nanocomposite

In this study an Iron oxide/carbon nanocomposite from maize straw was prepared and was characterized by XRD, SEM, EDX, FTIR, TG/DTA and Surface area analyzer. The adsorbent was fed to different groups of poultry birds along with aflatoxin B1. Different physiological and blood parameters were monitored in order to study the efficacy of the prepared adsorbent for binding of aflatoxin B1 in the gastrointestinal tract of chickens. It was found that adsorbent at dose of 0.3%/ kg feed was highly effective in detoxifying aflatoxin B1 in gastrointestinal tract of poultry birdswith no harmful effects. The high doses given to groups E and F; 0.4% and 0.5% respectively showed slight variation in tested parameters from group A. No negative symptoms associated with the use of activated carbon as previously reported were observed for the adsorbent under study.(AU)

Acute aflatoxicosis in swines in Northeastern Brazil / Aflatoxicose aguda em suínos no Nordeste do Brasil

Background: Aflatoxins are hepatotoxic mycotoxins derived from the secondary metabolism of toxigenic fungi belonging to the genus Aspergillus, especially A. flavus and A. parasiticus. Aflatoxin B1 is the most important metabolite, because of its deleterious effect mainly to the liver, especially for its carcinogenic, mutagenic and haemorrhagic properties, and usually is detected in higher concentrations in contaminated substrates. This paper reports the epidemiological, clinical, pathological and toxicological aspects of an outbreak of acute aflatoxicosis in pigs raised in Northeastern Brazil. Case: The cases occurred in a complete cycle farm, in the city of Mossoró, Rio Grande do Norte, Northeastern Brazil, in pigs ingesting low quality (moldy) corn being produced and processed on the farm. Sixty (73.1%) out of 82 two to five-months old pigs were clinicaly affected and 54 (65.8%) died. All animals showed fever, weight loss, tachycardia, tachypnea, lethargy, muscle tremors, muscle weakness and diarrhea. The clinical course ranged from five to 48 hours. Adult pigs were not affected. At necropsies, generalized jaundice, ascites, hydropericardium, petechial hemorrhages in the mesentery, subcutaneous edema and mesocolon were observed. The liver was yellow-orange with reddish diffuse multifocal areas, enlarged and extremely friable. Histologically there was swelling of hepatocytes [...]

Acute aflatoxicosis in swines in Northeastern Brazil / Aflatoxicose aguda em suínos no Nordeste do Brasil

Background: Aflatoxins are hepatotoxic mycotoxins derived from the secondary metabolism of toxigenic fungi belonging to the genus Aspergillus, especially A. flavus and A. parasiticus. Aflatoxin B1 is the most important metabolite, because of its deleterious effect mainly to the liver, especially for its carcinogenic, mutagenic and haemorrhagic properties, and usually is detected in higher concentrations in contaminated substrates. This paper reports the epidemiological, clinical, pathological and toxicological aspects of an outbreak of acute aflatoxicosis in pigs raised in Northeastern Brazil. Case: The cases occurred in a complete cycle farm, in the city of Mossoró, Rio Grande do Norte, Northeastern Brazil, in pigs ingesting low quality (moldy) corn being produced and processed on the farm. Sixty (73.1%) out of 82 two to five-months old pigs were clinicaly affected and 54 (65.8%) died. All animals showed fever, weight loss, tachycardia, tachypnea, lethargy, muscle tremors, muscle weakness and diarrhea. The clinical course ranged from five to 48 hours. Adult pigs were not affected. At necropsies, generalized jaundice, ascites, hydropericardium, petechial hemorrhages in the mesentery, subcutaneous edema and mesocolon were observed. The liver was yellow-orange with reddish diffuse multifocal areas, enlarged and extremely friable. Histologically there was swelling of hepatocytes [...](AU)

In vitro ability of beer fermentation residue and yeast-based products to bind aflatoxin B1

This study aimed to verify the in vitro ability of beer fermentation residue (BFR) containing Saccharomyces cerevisiae cells and five commercial products that differed in the viability and integrity of S. cerevisiae cells to remove aflatoxin B1 (AFB1) from a citrate-phosphate buffer solution (CPBS). BFR was collected at a microbrewery and prepared by drying and milling. The commercial yeast-based products were as follows: inactive intact yeast cells from beer alcoholic fermentation, inactive intact yeast cells from sugarcane alcoholic fermentation, hydrolyzed yeast cells, yeast cell walls and active yeast cells. Adsorption assays were performed in CPBS spiked with 1.0 μg AFB1/mL at pH 3.0 and 6.0 for a contact time of 60 min at room temperature. Analysis of AFB1 in the samples was performed by high performance liquid chromatography. AFB1 adsorption by the products ranged from 45.5% to 69.4% at pH 3.0 and from 24.0% to 63.8% at pH 6.0. The higher percentages (p < 0.05) of AFB1 binding at both pH values were achieved with products containing hydrolyzed yeast cells or yeast cell walls rather than intact cells. The AFB1 binding percentages of BFR were 55.0 ± 5.0% at pH 3.0 and 49.2 ± 4.5% at pH 6.0, which was not significantly different (p > 0.05) from commercial products containing inactive intact yeast cells. The results of this trial indicate that the yeast-based products tested, especially the BFR, have potential applications in animal feeds as a suitable biological method for reducing the adverse effects of aflatoxins.(AU)

Estudo sobre a ocorrência de fungos e aflatoxina B1 na dieta de bovinos leiteiros em São Paulo / Study on the occurrence of fungi and aflatoxina B1 in the diet of dairy cattle in São Paulo, Brazil

A qualidade da dieta ofertada às vacas em lactação é uma preocupação dos agentes de saúde devido à possibilidade da detecção de micotoxinas prejudiciais a saúde humana e animal. Os objetivos do trabalho foram avaliar o perfil da micobiota, determinar a atividade de água (Aa) e a ocorrência natural de aflatoxina B1 (AFB1) em dietas ofertadas a vacas em lactação de fazendas leiteiras no estado de São Paulo, Brasil. As amostragens das dietas foram realizadas diretamente dos cochos de lote de 15 vacas, em dois dias consecutivos com intervalos de 24h e a cada 15 dias, perfazendo um período de 45 dias de amostragens por fazenda. A purificação e determinação de AFB1 foram realizadas em colunas de imunoafinidade e Cromatografia Líquida de Alta Eficiência (CLAE). O estudo da micobiota presente nas amostras das dietas (288) revelou que as leveduras foram predominantes em todas as dietas (83,97 a 99,98%). Foram isolados 15 gêneros de fungos filamentosos, com os gêneros Aspergillus spp (20,09%), Fusarium spp (14,16%) e Penicillium spp (11,48%) os mais prevalentes. As contagens de Unidades Formadoras de Colônias por grama de alimento (UFC. g-1) variaram de 102 a 1011. A atividade de água das amostras variou entre 0,91 a 0,98. Foi detectada a presença de AFB1 em 31,44% das amostras com teores entre 1,68 a 194,51μg.kg-1. Medidas de boas práticas de produção, estocagem e utilização devem ser tomadas para diminuir a ocorrência de AFB1 nas dietas ofertadas às vacas em lactação.(AU)

The quality of the diet offered to lactating cows is a concern to health officials the possibility of detecting mycotoxins harmful to human and animal health. The objectives were to evaluate the profile of mycoflora, determine the water activity (Aw) and the natural occurrence of aflatoxin B1 (AFB1) in diets offered to lactating cows from dairy farms in the state of São Paulo, Brazil. Samples of the diets were taken directly from the troughs batch of 15 cows, on two consecutive days at intervals of 24 hours and every 15 days with a period of 45 sampling days per farm. Purification and determination of AFB1 were performed on immunoaffinity columns and High Performance Liquid Chromatography (HPLC). The study of mycobiota present in samples of diets (288) revealed that yeast cells were predominant in all diets (83.97 to 99.98%). 15 genera were isolated from filamentous fungi, with Aspergillus spp (20.09%), Fusarium spp. (14.16%) and Penicillium spp. (11.48%) the most prevalent. The counts of colony forming units per gram of food (UFC.g-1) ranged from 102 a1011. The water activity of the samples ranged from 0.91 to 0.98. We have detected the presence of AFB1 in 31.44% of samples with levels between 1.68 a 194.51μg.kg-1. Measures of good production, storage and use should be taken to reduce the occurrence of aflatoxin B1 in the diet offered to lactating cow.(AU)

In-vitro cytotoxicity of aflatoxin B1 to broiler lymphocytes of broiler chickens

The aim of the present work was to study the in-vitro cytotoxic effects of different concentrations of aflatoxin B1 (AFB1) on broiler lymphocytes. Lymphocyte-rich mononuclear cells were separated by Ficoll-Histopaque density and cultured in 96-wellplates containing the evaluated AFB1 concentrations in 5% CO2 atmosphere at 39°C. Thereafter, MTT, PicoGreen, and reactive oxygen species assays were performed. Cell viability decreased in the presence of 10 µg/mL AFB1 at 48 h (p 0.05) and of 10 and 20 µg/mL AFB1 at 72 h (p 0.01 and p 0.001, respectively) when compared to the control (0 µg/mL). However, a dose-dependent increase in the cell-free DNA at 24 h was observed at 1, 10 and 20 µg/mL (p 0.001). ROS formation significantly increased at 24 h at all concentrations (p 0.001). The in-vitro results demonstrate that AFB1 is cytotoxic and causes biomolecular oxidative damage in broiler lymphocytes.(AU)