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.

Minerais séricos, características morfométricas ósseas e deposição de minerais ósseos de frangos de corte alimentados com dieta com inclusão de bentonita / Serum minerals, bone morphometric characteristics and deposition of bone minerals from broilers fed diet containing bentonite

O objetivo deste trabalho foi avaliar a concentração sérica de cálcio, cloretos, ferro, fósforo e magnésio, as características morfométricas ósseas e a deposição de cálcio e fósforo nas tíbias de frangos de corte recebendo dieta com zero, 0,25 ou 0,50% de bentonita. Um ensaio foi conduzido com 288 frangos de corte de 14 a 21 dias de idade, submetidos a três dietas experimentais: sem inclusão (0,0); com inclusão de 0,25 e com inclusão de 0,50% do adsorvente bentonita. Não foram observadas diferenças (P>0,05) no desempenho das aves, nos níveis séricos de cálcio, cloretos, ferro e magnésio, no entanto os níveis de fósforo foram reduzidos (P<0,05) nas aves que ingeriram dieta com 0,50% de bentonita. Em relação às tíbias, observou-se redução (P<0,05) na matéria mineral (g e %) e no teor de cálcio com a inclusão de 0,50% de bentonita. Houve redução (P<0,05) nos níveis de fósforo das tíbias com a inclusão de 0,25 e 0,50% de bentonita. Conclui-se que a inclusão de até 0,50% do adsorvente de micotoxinas bentonita na dieta de frangos de corte não altera o desempenho zootécnico das aves. A inclusão de 0,25% de bentonita, na dieta de frangos de corte, não altera a concentração dos minerais séricos e a deposição de minerais nas tíbias, entretanto a inclusão de 0,5% reduz os níveis séricos de fósforo, o teor de matéria mineral e a concentração de cálcio e fósforo ósseos, sem afetar as características morfométricas ósseas.(AU)

The aim of this study was to evaluate performance, serum concentration of calcium, chloride, iron, magnesium, phosphorus, and bone characteristics, ash, calcium, and phosphorus in tibias of broilers receiving diet with zero, 0.25 or 0.50% of bentonite. No differences were found on performance of poultry, on serum mineral calcium, chloride, iron, magnesium, however phosphorus levels of broilers fed on diets containing 0.5% bentonite was reduced. With respect to tibia, reduction was observed on mineral matter (g and %) and calcium levels with inclusion of 0.50% bentonite, and reduction on phosphorus levels with inclusion of 0.25 or 0.50% of bentonite on diet. We conclude that the inclusion of up to 0.50% of mycotoxin adsorbent bentonite in diet of broiler does not change broiler performance. The inclusion of 0.25% of bentonite in diet of broiler does not change serum mineral concentration and mineral deposition; however, the inclusion of 0.5% decrease serum levels of phosphorus, the content of bone mineral matter, with not effects on bone morphometric characteristics.(AU)

Minerais séricos, características morfométricas ósseas e deposição de minerais ósseos de frangos de corte alimentados com dieta com inclusão de bentonita / Serum minerals, bone morphometric characteristics and deposition of bone minerals from broilers fed diet containing bentonite

O objetivo deste trabalho foi avaliar a concentração sérica de cálcio, cloretos, ferro, fósforo e magnésio, as características morfométricas ósseas e a deposição de cálcio e fósforo nas tíbias de frangos de corte recebendo dieta com zero, 0,25 ou 0,50% de bentonita. Um ensaio foi conduzido com 288 frangos de corte de 14 a 21 dias de idade, submetidos a três dietas experimentais: sem inclusão (0,0); com inclusão de 0,25 e com inclusão de 0,50% do adsorvente bentonita. Não foram observadas diferenças (P>0,05) no desempenho das aves, nos níveis séricos de cálcio, cloretos, ferro e magnésio, no entanto os níveis de fósforo foram reduzidos (P<0,05) nas aves que ingeriram dieta com 0,50% de bentonita. Em relação às tíbias, observou-se redução (P<0,05) na matéria mineral (g e %) e no teor de cálcio com a inclusão de 0,50% de bentonita. Houve redução (P<0,05) nos níveis de fósforo das tíbias com a inclusão de 0,25 e 0,50% de bentonita. Conclui-se que a inclusão de até 0,50% do adsorvente de micotoxinas bentonita na dieta de frangos de corte não altera o desempenho zootécnico das aves. A inclusão de 0,25% de bentonita, na dieta de frangos de corte, não altera a concentração dos minerais séricos e a deposição de minerais nas tíbias, entretanto a inclusão de 0,5% reduz os níveis séricos de fósforo, o teor de matéria mineral e a concentração de cálcio e fósforo ósseos, sem afetar as características morfométricas ósseas.(AU)

The aim of this study was to evaluate performance, serum concentration of calcium, chloride, iron, magnesium, phosphorus, and bone characteristics, ash, calcium, and phosphorus in tibias of broilers receiving diet with zero, 0.25 or 0.50% of bentonite. No differences were found on performance of poultry, on serum mineral calcium, chloride, iron, magnesium, however phosphorus levels of broilers fed on diets containing 0.5% bentonite was reduced. With respect to tibia, reduction was observed on mineral matter (g and %) and calcium levels with inclusion of 0.50% bentonite, and reduction on phosphorus levels with inclusion of 0.25 or 0.50% of bentonite on diet. We conclude that the inclusion of up to 0.50% of mycotoxin adsorbent bentonite in diet of broiler does not change broiler performance. The inclusion of 0.25% of bentonite in diet of broiler does not change serum mineral concentration and mineral deposition; however, the inclusion of 0.5% decrease serum levels of phosphorus, the content of bone mineral matter, with not effects on bone morphometric characteristics.(AU)

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...(AU)

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...

Kinetics of epsilon antitoxin antibodies in different strategies for active immunization of lambs against enterotoxaemia / Cinética dos anticorpos antitoxina epsilon em diferentes estratégias de imunização ativa em cordeiros contra a enterotoxemia

Enterotoxaemia, a common disease that affects domestic small ruminants, is mainly caused by the epsilon toxin of Clostridium perfringens type D. The present study tested four distinct immunization protocols to evaluate humoral response in lambs, a progeny of non-vaccinated sheep during gestation. Twenty-four lambs were randomly allocated into four groups according to age (7, 15, 30 and 45 days), receiving the first dose of epsilon toxoid commercial vaccine against clostridiosis with booster after 30 days post vaccination. Indirect ELISA was performed after the first vaccine dose and booster to evaluate the immune response of the lambs. Results showed that for the four protocols tested all lambs presented serum title considered protective (≥0.2UI/ml epsilon antitoxin antibodies) and also showed that the anticipation of primovaccination of lambs against enterotoxaemia conferred serum title considered protective allowing the optimization of mass vaccination of lambs.(AU)

Enterotoxemia, uma das mais comuns enfermidades que acomete os pequenos ruminantes domésticos, é causada principalmente pela toxina épsilon de Clostridium perfringens tipo D. O presente estudo avaliou a resposta humoral conferida por quatro protocolos distintos de primovacinação na progênie de ovelhas não vacinadas durante a gestação. Vinte e quatro cordeiros foram aleatoriamente divididos em quatro grupos de acordo com a idade (dias) que receberam a primeira dose da vacina comercial contra clostridiose contendo toxóide epsilon na sua formulação. Todos os cordeiros foram vacinados aos 7, 15, 30 ou 45 dias de idade e receberam um reforço da dose 30 dias após a vacinação. A avaliação sorológica dos cordeiros pelo teste de ELISA indireto foi realizada por ocasião da administração da primeira dose da vacina. Os resultados elucidaram não haver comprometimento da resposta imune de cordeiros vacinados tanto aos 7, 15, 30 ou 45 dias de idade associada ao reforço da dose 30 dias após, demonstrando assim que a antecipação da primeira vacinação conferiu proteção aos cordeiros contra a enterotoxemia, permitindo otimizar o planejamento da vacinação em massa dos cordeiros.(AU)

Modified toxin-binding inhibition (ToBI) test for quantification of epsilon antitoxin in serum from immunized sheep

This study evaluated the use of the TOBI test as a substitute for the in vivo technique of the mouse serum neutralization test to manage quality control of vaccines against clostridiosis. Serum samples with known concentrations of anti-epsilon antibodies were obtained from 3 sheep immunized with vaccine against C. perfringens type D epsilon toxoid. The main results obtained indicate correlation coefficients above 97%, corroborating literature data. The inter and intraplate coefficients of variation were low (less than 1.3%). In conclusion, the in vitro TOBI test is suitable to evaluate the potency of clostridial vaccines. However, the TOBI test must be first validated before it can replace the in vivo serum neutralization methodology used in mice.

Anti-snake venom: use and adverse reaction in a snake bite study clinic in Bangladesh

Snakebites can present local or systemic envenomation, while neurotoxicity and respiratory paralysis are the main cause of death. The mainstay of management is anti-snake venom (ASV), which is highly effective, but liable to cause severe adverse reactions including anaphylaxis. The types of adverse reaction to polyvalent anti-snake venom have not been previously studied in Bangladesh. In this prospective observational study carried out between 1999 and 2001, in the Snake Bite Study Clinic of Chittagong Medical College Hospital, 35 neurotoxic-snake-bite patients who had received polyvalent anti-snake venom were included while the ones sensitized to different antitoxins and suffering from atopy were excluded. The common neurotoxic features were ptosis (100%), external ophthalmoplegia (94.2%), dysphagia (77.1%), dysphonia (68.5%) and broken neck sign (80%). The percentage of anti-snake venom reaction cases was 88.57%; pyrogenic reaction was 80.64%; and anaphylaxis was 64.51%. The common features of anaphylaxis were urticaria (80%); vomiting and wheezing (40%); and angioedema (10%). The anti-snake venom reaction was treated mainly with adrenaline for anaphylaxis and paracetamol suppository in pyrogenic reactions. The average recovery time was 4.5 hours. Due to the danger of reactions the anti-snake venom should not be withheld from a snakebite victim when indicated and appropriate guidelines should be followed for its administration.(AU)