AN IN OVO TOXICOLOGICAL ASSESSMENT OF INDIVIDUAL AND COMBINED FUSARIUM MYCOTOXINS IN THE CHICKEN EMBRYO

The increasing occurrence of Fusarium fungi and associated mycotoxins in cereal grains is a significant issue for global agriculture. The mycotoxin deoxynivalenol (DON) is the most prevalent feed contaminant worldwide and causes a variety of adverse effects in animals. While the individual toxicity of DON is a concern, exposure to multiple mycotoxins in feed is more common, necessitating information on the outcome of mixture exposures. However, characterizing the toxicity of DON and other mycotoxins has been difficult due to highly varied responses in long-term animal feeding trials. In addition, resources required for whole animal testing are only amplified in combinatorial mycotoxin studies given the large sample sizes and number of treatment groups required. The chicken embryo has been widely and successfully utilized as a non-animal alternative to evaluate the toxicity of environmental pollutants and could be used as a screening tool to evaluate mycotoxin mechanisms of action and mixture toxicity. The overall objective of this thesis research is to characterize the effects of DON (administered in ovo) alone and in combination with a commonly co-occurring mycotoxins, zearalenone (ZEA), to the late-term chicken embryo in order to determine whether an in ovo approach for conducting exposures to Fusarium mycotoxins could be used as a predictive tool for assessing the toxicity of Fusarium mycotoxins alone and in combination. The overall hypothesis is that responses of the late-term chicken embryo to single doses of Fusarium mycotoxins, alone or in combination, are similar to those reported in whole animal feeding trials with poultry. In the first experiment, the effect of in ovo administration of DON was evaluated in terms of embryotoxicity, growth and development, pathological changes to tissue, and biochemical/molecular indicators of oxidative and immune stress. A single injection of purified DON was administered to the late-term chicken embryo (embryonic day 14, ED14) at five doses ranging from 0.0 – 5.0 μg DON/g egg weight. Eggs were opened on ED20 and embryos were evaluated for survivability and growth parameters. Tissues were sampled for subsequent analysis. At the highest dose, DON decreased embryo survivability and increased the absolute and relative weight of both liver and spleen. Hepatic bile stagnation and concurrent splenic inflammation were frequently detected among groups receiving 5.0 and 1.0 μg DON/g egg weight but were observed less often in the latter. A dose-dependent increase in granulopoiesis and lipid peroxidation (as measured by TBARS assay) were observed in the liver; however, mRNA expression of genes related to immune and oxidative stress were mostly unchanged. These results suggest that the chicken embryo responds to in ovo DON exposure with effects on immunity and oxidative stress that are supported by previous in vivo and in vitro findings. The in ovo approach developed and validated in the first experiment was then carried forward to a second experiment with the aim of characterizing the combined toxicity of DON and another mycotoxin, ZEA to the chicken embryo. ZEA was chosen for this experiment because the combination of DON and ZEA is considered to be the most prevalent mycotoxin mixture in North America and worldwide. Treatments included an untreated control group (CON), a vehicle-injected control group (20% DMSO), 0.5 and 2.5 μg DON/g egg weight, 0.5 and 2.5 μg ZEA/g egg weight, and a low and high combination treatment at 0.5 μg DON + 0.5 μg ZEA/g egg weight and 2.5 μg DON + 2.5 μg ZEA/g egg weight, respectively. The results demonstrated that interactive effects of DON and ZEA differed across endpoints and tended to vary from antagonistic at low doses to non-interactive or possibly potentiated at high doses. At low doses, DON and ZEA had antagonistic effects on liver weight as well as liver lipid peroxidation. At high doses, effects of DON and ZEA were mostly independent and effects of DON, specifically, were in line with our previous observations. At a combined, high dose of DON and ZEA there was evidence of possible potentiation with respect to embryo survivability, hepatic bile stagnation and splenic inflammation, and hepatic granulopoiesis. These results suggest the chicken in ovo model is useful for studying combinatorial mycotoxin toxicity; however, further research regarding ZEA-induced toxicity would improve response interpretation. Overall, the results presented in this thesis indicate that in ovo responses to Fusarium mycotoxins, alone and in combination, are supported by previous in vitro and in vivo findings. While in ovo mycotoxin exposures cannot replace in vivo experimentation, there is potential for the in ovo model to inform whole animal studies by identifying and prioritizing emerging mycotoxins and high-risk mycotoxin combinations for further in vivo assessment. In the future, the in ovo model could be used in a more practical application as a rapid-screening tool to assess the toxicity of mycotoxin grain extracts or to evaluate the efficacy of new mycotoxin mitigation techniques

Regulation of genes associated with avian B cell receptors involved in innate and adaptive immunity

Epigenetics is a group of mechanisms associated with gene expression control. One of these mechanisms is methylation of cytosines which are part of a cytosine-guanine (CpG) dinucleotide. When this happens in the promoter region of a gene, it will control unwanted expression. For this process to take place, a methyl group donor is needed. One of the principal nutritional compounds that fit this role is folic acid (FA). Our aim in this research was to examine whether exposure to FA would change the methylation profile and expression of toll like receptor (TLR) 2, immunoglobulin (Ig) β and major histocompatibility class (MHC) II β chain in chicken B cells. The initial experiment used the chicken B cell derived DT-40 cell line. Cell cultures were incubated with 0, 1.72 or 3.96 mM of FA for 4 or 8 hours, with the 8 hour group treated with 0, 1 or 10 µg/ml of Lipopolysaccharide (LPS). A positive association was found between FA concentration and percent of Igβ promoter methylation and a negative association between FA concentration and percent of MHCII β chain promoter methylation after 4 hours of incubation. The expression of those two genes was affected as well. Both were downregulated after 8 hours compared to the 4 hours group. Incubation with 1.72 mM FA for 4 hours upregulated TLR2b and 3.96 mM FA for 4 hours upregulated MHCII β chain expression. FA at 3.96 mM for 8 hours downregulated TLR2b and upregulated Igβ expression. Treatment with LPS downregulated TLR2b expression. We concluded that FA indeed has an immunomodulatory effect on chicken B cells in a way that may possibly affect their ability to both recognize antigen through the TLR and B cell receptor (BCR) pathways and their ability to present antigen in an MHCII context. A second series of experiments examined the effect of FA on embryonic B cells extracted from the bursa of Fabricius (BoF) at days (ED) 15, 18 and 21 of embryonic development. A novel protocol was developed to obtain optimal growth and survival of the cells. The amount of chicken serum in the growth media was doubled and time of ex vivo culture was determined to ensure that the cells survive for the duration of the experiment. We found that our modified growth media maintained cells for the 4 hour duration of the experiment. When compared to cells that were not incubated (0 hours), cells harvested at ED15 and incubated for 4 hours had a lower population of IgMlow/med cells, increased population of IgMhigh cells and upregulated Igβ expression. When the cells were harvested at ED21 and incubated for 4 hours TLR2b, MHCII β chain as well as the expression of the reduced folate carrier (RFC), a FA transporter were downregulated compared to 0 hours. Exposure to FA did not have an effect on cell viability. For cells harvested at ED15 both 1.72 and 3.96 mM FA for 4 hours caused a downregulation of Igβ and the 3.96 mM treatment reduced the population of IgMmed cells when compared to these values after 4 hour incubation with no FA (0 mM FA). At ED18 both FA treatments reduced the IgMmed population and the 1.72 mM FA treatment reduced RFC expression compared to the 0 mM FA group. At ED21, 3.96 mM FA increased the IgMhigh population. The methylation status of the promoter region of all examined genes was not affected by either removal of the cells from the BoF or treatment with FA. In this experiment, we demonstrated the immune modulating capabilities of FA in the developing B cell, and provided a protocol for maintaining the viability of these cells ex vivo for longer periods then previously published

Dietary mycotoxins: an overview on toxicokinetics, toxicodynamics, toxicity, epidemiology, detection, and their mitigation with special emphasis on aflatoxicosis in humans and animals

Mycotoxins are secondary metabolites of filamentous fungi and ubiquitous dietary contaminants. Aflatoxins, a group of mycotoxins with high prevalence and toxicity, have raised a high level of public health concern, the most prevalent and toxic being aflatoxin B1 (AFB1). Many aspects appertaining to AFB1 poisoning are not well understood. Yet this information is necessary to devise appropriate surveillance and mitigation strategies against human and animal aflatoxicosis. This review provides an in-depth update of work carried out on mycotoxin poisoning, particularly aflatoxicosis in humans and animals, to identify gaps in knowledge. Hypotheses explaining the functional significance of mycotoxins in fungal biology and their dietary epidemiological data are presented and briefly discussed. The toxicology of aflatoxins and the challenges of their mitigation are discussed in depth. It was concluded that the identification of potential mycotoxin-hazard-prone food items and quantification of the associated risk of cancer ailments in humans is a prime priority. There is a dearth of reliable sampling methodologies for estimating AFB1 in animal feed. Data update on AFB1 in animal feed and its implication in animal production, mitigation strategies, and elucidation of risk factors to this hazard is required. To reduce the burden of aflatoxins, surveillance employing predictive technology, and biocontrol strategies seem promising approaches

Marine Polysaccharides Volume 1

The field of marine polysaccharides is constantly evolving, due to progress in the discovery and production of new marine polysaccharides. Seaweed remains the most abundant source of polysaccharides, but recent advances in biotechnology have allowed the production of large quantities of polysaccharides from a variety of micro-algae, by controlling growth conditions and tailoring the production of bioactive compounds in a bioreactor. Of particular interest are polysaccharides produced by micro-organisms from extreme marine environments, due to their recognized different biochemistry. Extracellular polysaccharides (EPSs) with unique properties produced by a number of micro-algae are known. The first volume is a collection of papers concerning the identification and characterization of novel marine polysaccharides. It is divided into three chapters; the first two are dedicated to polysaccharides from different marine sources (algae, micro-algae, animals), while the third one gathers information on the isolation, characterization and bioactivity of new EPSs

Desenvolvimento da mucosa intestinal e imunidade de frangos de cote alimentados com dietas de baixa e alta concentração de fibre e betaína

Orientadora: Prof. Dra. Ana Vitória Fischer da SilvaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Fisiologia. Defesa : Curitiba, 27/03/2018Inclui referências: p. 95-117Área de concentração: Fisiologia Animal Comparativa e dos Animais DomésticosResumo: Um total de 250 frangos de corte Cobb 500 com 1 dia de foram alocados em 16 gaiolas com 15 frangos cada. Os tratamentos consistiam de duas formulações com baixa ou alta concentração de fibra e 4 níveis de adição de betaína (0, 1, 3 e 5 kg/ton) em um delineamento fatorial 2x4. No alojamento, 10 aves foram pesadas e eutanaziadas para determinação de padrão experimental no momento anterior ao fornecimento das rações. Aos 4, 9 e 14 dias, 10 aves por tratamento foram retiradas, pesadas e eutanaziadas para avaliações de: peso do intestino, saco vitelínico, pâncreas e fígado em relação ao peso da ave; duodeno foi coletado para determinação de altura de vilosidade, profundida de cripta, numero de celulas em replicação e numero de celulas caliciformes. O conteúdo intestinal foi coletado para determinação de osmolalidade e concentração de ácido siálico. Sangue foi coletado para determinação da população de leucócitos e osmolalidade plasmática e amostras de jejuno para determinação de expressão gênica de proteinas de junção "Tight" (claudina 1, claudina 5 e ocludina) e interleucina 2. Dados foram analisados por ANOVA em desenho completamente ao acaso considerando inclusão de betaína, concentração de fibra e idade como fatores principais. Quando efeitos foram considerados significantes (P<0,05), médias foram separadas usando o teste de significância de Tukey. O peso do saco vitelínico reduziu (P<0,05) entre a eclosão e 4 dias e entre 4 e 14 dias, o número de celulas caliciformes e de enterócitos em mitose foram menores em aves aos 14 dias (P<0,05), enquanto o tamanho da vilosidade aumentou (P<0,05) durante todo o período analizado e a profundidade de cripta aumentou (P<0,05) entre eclosão e 4 dias e entre 4 e 14 dias, suportando o desenvolvimento do trato gastrointestinal nesse período inicial. Aves aos 4 e 9 dias de idade apresentaram maior (P<0,05) número de vilos fusionados em comparação com aves à eclosão e 14 e aves aos 4 dias apresentaram maior (P<0.05) osmolalidade do conteúdo intestinal comparado com aves aos 9 e 14 dias. A secreção de ácido siálico foi maior (P<0,05) aos 4 dias em comparação com aves aos 9 dias, posteriormente reduzindo aos 14 dias e a expressão de interleucina 2 apresentou uma tendência (P<0,06) de aumentar entre a eclosão e 4 dias e reduzindo (P<0,05) aos 9 dias, caracterizando um efeito inflamatório temporário. As aves que receberam betaina apresentaram menor (P<0,05) osmolalidade do conteúdo intestinal e menores números de celulas caliciformes e de celulas em mitose. Vilos de aves que receberam betaína apresentaram maior (P<0,05) tamanho e menor (P<0,05) espessura. A expressão de genes de Claudina 5 e ocludina reduziu (P<0,05) em aves alimetnadas com betaína independente da idade. Aves que receberam ração com maior concentração de fibra apresentaram aumento (P0,05) na altura da vilosidade e menor (P<0,05) número de celulas caliciformes independente da idade. O fornecimento de fibra e betaína reduziu o efeito inflamatório inicial gerado pela presença de ração no trato gastrointestinal e estimulou o desenvolvimento e adaptação do trato gastrointestinal entre a eclosão e 14 dias de vida. Palavras-chave: Betaína, Fibra, Frango de corte, Intestino.Abstract: A total of 250 broilers Cobb 500 were allocated at 16 cages with 15 birds each at day of hatch. Treatments were represented by two feed formulation with low or high fibre concentration and 4 levels of betaine inclusion (0, 1, 3 or 5kg/tonne) in a factorial design 2x4. At house, 10 broilers that were not distributed in the cages were weight and euthanized by cervical dislocation for standard reference before the feed supply. At 4, 9 and 14 days of age, 10 birds per treatment were selected weight and euthanized. Intestine, pancreas, liver and yolk sac weight was determined as a proportion of the animal weight, duodenum samples were collected for villus height and weidth, crypt dept and number of enterocyte cells in replication and caliciform cells at the centre of the villus. Intestinal content was collected for determination of the osmolality and sialic acid concentration and blood samples collected for determination of the leukocyte population and plasmatic osmolality. Jejunal samples were collected for gene expression of "Tight" junction proteins (claudin 1 and 5, and occludin) and interleukine 2. Data were subjected to least squares ANOVA for a completely randomized design in a three-way interaction considering age, non-starch polysaccharide content of the diet and betaine inclusion. Each animal served as the experimental unit. When the effects were found to be significant (P<0.05), treatment means were separated using Tukey's Significant Difference test. The weight of the yolk sac reduced (P<0.05) between hatch and 4 days and between 4 and 14 days, number of caliciform cells and cells in mitosis at the villus were lower (P<0.05) at 14 days of age while villus height increased (P<0.05) during all period and crypt dept increased (P<0,05) between hatch 4 and 14 days, supporting the development of the gastrointestinal tract after hatch. At 4 and 9 days of age birds had more (P<0.05) fusied villus than at hatch and 14 days and at 4 days had higher (P<0.05) digesta osmolality compared with 9 and 14 days. Sialic acid secretion was higher (P<0.05) at 4 days compared to 9 days and further reduced (P<0.05) at 14 days and the gene expression of interleukine 2 had a trend (P<0,06) to increase between hatch and 4 days, reducing (P<0.05) at 9 days supporting that animals at these ages passed through a proinflamatory status. Birds fed diets with betaine had lower (P<0,05) digesta osmolality, number of caliciform cells and cells in mitosis. Villus of birds fed diets included with betaine were bigger (P<0.05) and had smaller (P<0.05) weidth. Gene expression for Claudin 5 and Occludin reduced (P<0,05) in birds fed betaine independent of the age. Broilers fed high fibre diet presented higher (P<0,05) villus and lower (P<0,05) number of caliciform cells independent of the age. Formulation of diets with high fibre and/or betaine reduce the inflammatory status generated by the presence of feed in the intestinal tract of broilers and stimulates the gut development and adaptation of broilers between hatch and 14 days of age. Keywords: Betaine, Broiler, Fibre, Intestine