Le but de ce travail a été de mieux comprendre les défenses mises en œuvre par l’hôte infecté par le champignon opportuniste humain Aspergillus fumigatus (Af). 1) Un modèle d’infection a été redéveloppé chez l’organisme modèle Drosophila melanogaster. Seules les mouches mutantes pour le gène MyD88 de la voie immunitaire Toll succombent à l’injection d’une poignée de conidies, sans toutefois qu’Af dissémine dans l’hôte. Ce travail a révélé que ce n’est pas la réponse immunitaire qui joue un rôle prépondérant dans la défense de l’hôte, mais sa capacité de résilience à l’exposition à des mycotoxines sécrétées par Af. 2) Un crible génétique d’envergure a été établi pour identifier des lignées transgéniques mutantes ARNi sensibles à l’infection par Af. 6.471 lignées ont été criblées et 241 gènes-candidats identifiés, dont peu fonctionnent dans la réponse immunitaire. Ainsi, ce travail a contribué à identifier de nombreux gènes impliqués dans la résilience de l’hôte à Af et ses mycotoxines. / The overarching goal of this work is to better understand host defenses against the human opportunistic fungus Aspergillus fumigatus (Af). 1) An infection model has been reestablished in the genetic model organism Drosophila melanogaster. Only flies mutant for the immune response Toll pathway gene MyD88 succumb to the injection of a handful of conidia even though Af is unable to disseminate throughout its host. This work revealed that it is not the immune response that plays a cardinal role in host defense but its resilience capacity to the exposure to some mycotoxins secreted by Af. 2) A large-scale genetic screen has been implemented to identify transgenic RNAi mutant lines susceptible to Af infection in survival experiments. 6,471 lines have been screened and 241 candidate genes identified, few of which are known to act in the immune response. Thus, this work has contributed to identifying numerous genes involved in host resilience to Af and to some of its mycotoxins.

Text in English / Fungal species and their mycotoxins are the most predorminant contaminants of dried agricultural products in sub-Saharan Africa (SSA) and the main species of fungi that can synthesize mycotoxins are Aspergillus, Fusarium and Penicillium. In Africa, aflatoxin is labelled as a great threat to human and animal health due to its high contamination levels reported of aflatoxins in foods. The aim of this study was to survey fungi and aflatoxin contamination of small-scale processed foodstuffs sold at informal retail outlets in the Johannesburg metropolis, South Africa. A total of 270 food samples (10 starch and legume based foods, 11 meat and fish based foods, 22 spices and local condiments, 14 dried fruits and vegetables) were collected from retailers; and analysed four (4) times in different seasons of spring, summer, autumn and winter. Out of the 270 samples analysed, only 27.8% were contaminated with fungal. Of all the six categories of foods analysed, roots and tubers (60.0%), nuts and seeds (40.0%), dried vegetables (37.1%), and the Meat and Insect foods (33.3%) respectively, had the most contaminated samples with fungal respectively. The least contaminated food groups were the fish foods (10.0%) and spices and local condiments (16.7%) respectively. Twenty percent of the 270 dried food analysed were contaminated by Aspergillus species out of which 61.1% of the contaminated samples had fungal counts above 103 cfu/g. Aspergillus niger was the most predominant Aspergillus species identified in all the categories of food samples analysed. Fruits and vegetables (24.4%) and the nuts and seeds (20.0%) food groups had the highest number of samples contaminated with aflatoxin. Peanut flour and Cardamom had the most incidence of aflatoxin. AFB1, AFB2 & AFG1 were the most prominent aflatoxin types recovered from the food samples. Almost all the food samples in which aflatoxin were identified had aflatoxin values above 10μg/ml. / Life and Consumer Sciences / M.Sc. (Life Science)

Cette étude visait à évaluer l’impact d’une exposition à des doses faible et élevée d’un mélange de mycotoxines émergentes du groupe des enniatines sur le microbiote intestinal à l’aide d’un modèle murin humanisé Sprague Dawley. L'hypothèse était que l'utilisation de ce modèle humanisé permettrait de caractériser la dysbiose induite par les enniatines. L'étude comprenait trois parties. Dans la phase initiale, une déplétion du microbiote a été induite chez 24 rats Sprague Dawley par un traitement par un cocktail d'antibiotiques mélangé à du sirop de cassis pendant 14 jours. Par la suite, les rats ont été répartis de façon aléatoire en deux groupes, recevant chacun des matières fécales fraîches par transferts fécaux bi-hebdomadaires de deux donneurs humains adultes (un mâle et une femelle) en bonne santé, pendant 3 semaines. Dans la phase finale, les 24 rongeurs humanisés ont été divisés en trois groupes en fonction de leur dose d’exposition orale aux enniatines pendant quatre semaines: un groupe contrôle (0 μg/kg de poids corporel d’enniatine), un groupe recevant 2 μg/kg de poids corporel d’enniatines par jour, correspondant au niveau d’exposition moyen aux enniatines dans la population humaine, et un groupe recevant 200 μg/kg de poids corporel d’enniatines par jour. Les doses d’enniatines étaient administrées mélangées à du syrop de cassis. Des échantillons fécaux ont été collectés au début, et chaque semaine tout au long de cette phase. La structure et la diversité du microbiote ont été évaluées à l’aide du kit d’extraction d’ADN de matières fécales QIAamp DNA stool micro kit et de la méthode du séquençage du gène codant pour l’ARNr 16S. Les résultats de la phase de déplétion ont montré des différences significatives avant et après le traitement antibiotique, confirmant l'épuisement de la flore intestinale initiale des rats par le cocktail d'antibiotiques. De plus, nous avons réussi à générer un modèle de rat humanisé à partir du microbiote intestinal humain. L'établissement réussi d'un modèle animal humanisé a été confirmé par le transfert de divers phylums bactériens, notamment Bacteroidaceae, Bacteroidales, Bacteroides, Barnesiella, Bifidobacterium, Desulfovibriionales, Eggerthella, Enterococcaceae, Hungatella, Lachnospiraceae, Lactobacillaceae, Limosilactobacillus, Mycobacteriales, Peptostreptococcaceae, Romboutsia et Streptococcus. Dans la phase finale de l'étude, l'abondance relative de certains genres bactériens a été modifiée de manière dose-dépendante par l’exposition aux enniatines, bien que l'impact global des mycotoxines ait été limité. Cet impact global limité peut être dû au fort effet homogénéisant du sirop de cassis sur le microbiote dans tous les groupes expérimentaux, masquant potentiellement les effets des enniatines sur la structure du microbiote. Ces résultats indiquent que l’exposition aux enniatines, même aux doses moyennes observées dans la population humaine pourraient modifier significativement la composition du microbiote intestinal humain. / This study aimed to evaluate the impact of exposure to low and high doses of a mixture of emerging mycotoxins from the enniatin group on the gut microbiota using a humanized Sprague Dawley mouse model. The hypothesis was that using this humanized model would allow for the characterization of dysbiosis induced by enniatins. The study consisted of three parts. In the initial phase, microbiota depletion was induced in 24 Sprague Dawley rats through a treatment with an antibiotic cocktail mixed with blackcurrant syrup for 14 days. Subsequently, the rats were randomly divided into two groups, each receiving fresh fecal matter through bi-weekly fecal transplants from two healthy adult human donors (one male and one female) for three weeks. In the final phase, the 24 humanized rodents were divided into three groups based on their oral exposure dose to enniatins over four weeks: a control group (0 μg/kg body weight of enniatin), a group receiving 2 μg/kg body weight of enniatins mixed with blackcurrant syrup daily , and a group with 200 μg/kg body weight of enniatin daily. The daily 2 μg/kg body weight of enniatins represents the mean chronic exposure calculated for humans in Europe. Fecal samples were collected at the beginning and every week throughout this phase. The structure and diversity of the microbiota were evaluated using the QIAamp DNA Stool Mini Kit and 16S rRNA sequencing method. The results indicated significant differences before and after antibiotic treatment, confirming the initial depletion of the gut flora by the antibiotic cocktail. Additionally, we successfully generated a humanized rat model from human gut microbiota. The successful establishment of a humanized animal model was confirmed by the transfer of various bacterial genus, including Bacteroidaceae, Bacteroidales, Bacteroides, Barnesiella, Bifidobacterium, Desulfovibriionales, Eggerthella, Enterococcaceae, Hungatella, Lachnospiraceae, Lactobacillaceae, Limosilactobacillus, Mycobacteriales, Peptostreptococcaceae, Romboutsia, and Streptococcus. In the final phase of the study, the relative abundance of certain bacterial genera was dose-dependently modified by exposure to enniatins, although the global impact of the mycotoxins was limited. This limited global impact may be due to the strong homogenizing effect of the blackcurrant syrup on the microbiota across all experimental groups, potentially masking the effects of enniatins on the microbiota structure. Altogether, these results indicate that exposure to enniatins, even at the mean level observed in the human population, could significantly modify the composition of the human intestinal microbiota.

This study assessed the mycological and aflatoxin contamination of peanuts collected from Kinshasa, DRC and Pretoria, South Africa. Forty peanut samples were collected randomly at informal markets in the two cities and analysed for mycoflora and aflatoxins (B1, B2, G1 and G2) using standard methods. The results indicated that 95% and 100% of peanut samples collected from Kinshasa and Pretoria, respectively were contaminated with aflatoxigenic fungi with Kinshasa’s samples being the most contaminated (up to 49, 000 CFU/g). Seventy percent (70 %) of Kinshasa-samples and 35% of Pretoria-samples exceeded the maximum allowable limit of aflatoxin B1 set by JECFA (5 ppb). Statistical evidence showed a significant positive correlation between mycoflora and aflatoxin level for Kinshasa-samples (r = 0.4743, p < 0.005) while Pretoria-samples showed no correlation. The study reveals that high level of contamination in Kinshasa-samples could be due to the tropical nature of the climate and poor storage conditions as compared to Pretoria which is sub-tropical and sanitary regulations are enforced. / Life & Consumer Sciences / M. Sc. (Life Sciences)

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life & Consumer Sciences / M. Sc. (Life Sciences)

No description available.

This study assessed the mycological and aflatoxin contamination of peanuts collected from Kinshasa, DRC and Pretoria, South Africa. Forty peanut samples were collected randomly at informal markets in the two cities and analysed for mycoflora and aflatoxins (B1, B2, G1 and G2) using standard methods. The results indicated that 95% and 100% of peanut samples collected from Kinshasa and Pretoria, respectively were contaminated with aflatoxigenic fungi with Kinshasa’s samples being the most contaminated (up to 49, 000 CFU/g). Seventy percent (70 %) of Kinshasa-samples and 35% of Pretoria-samples exceeded the maximum allowable limit of aflatoxin B1 set by JECFA (5 ppb). Statistical evidence showed a significant positive correlation between mycoflora and aflatoxin level for Kinshasa-samples (r = 0.4743, p < 0.005) while Pretoria-samples showed no correlation. The study reveals that high level of contamination in Kinshasa-samples could be due to the tropical nature of the climate and poor storage conditions as compared to Pretoria which is sub-tropical and sanitary regulations are enforced. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Maize is the principal staple food/feed crop exposed to mycotoxins, and the co-occurrence of multiple mycotoxins and their metabolites has been well documented. Dietary (co)-exposure to mycotoxins is associated with human and animal health concerns as well as economic losses. The present thesis aims to apply a holistic approach for the risk assessment of mycotoxin mixtures in food and feed, i.e. from fungal production and occurrence to harmonised risk characterisation. This was done in three folds. Firstly, available environmental, ecological, and agronomic factors that may affect the relative abundance of co-occurring mycotoxins in the contaminated crops were collected from peer-reviewed literature, with focus on maize (Chapter I). Secondly, (co-)occurrence data on mycotoxins in core cereals was extracted from available articles in the scientific literature and analysed to estimate potential pattern of co-exposure in humans and animals (Chapter II). Finally, Chapter III investigates the applicability of the EFSA guidance to multiple mycotoxins through a scenario of possible co-exposure in humans and animals, using maize as a case study. In particular, a human and animal risk assessment to mycotoxin mixture in maize was conducted using a modelled component-based approach for selected mixture of mycotoxins, that, according to our data, co-occur in maize based feed and food products. / Maize is the principal staple food/feed crop exposed to mycotoxins, and the co-occurrence of multiple mycotoxins and their metabolites has been well documented. Dietary (co)-exposure to mycotoxins is associated with human and animal health concerns as well as economic losses. The present thesis aims to apply a holistic approach for the risk assessment of mycotoxin mixtures in food and feed, i.e. from fungal production and occurrence to harmonised risk characterisation. This was done in three folds. Firstly, available environmental, ecological, and agronomic factors that may affect the relative abundance of co-occurring mycotoxins in the contaminated crops were collected from peer-reviewed literature, with focus on maize (Chapter I). Secondly, (co-)occurrence data on mycotoxins in core cereals was extracted from available articles in the scientific literature and analysed to estimate potential pattern of co-exposure in humans and animals (Chapter II). Finally, Chapter III investigates the applicability of the EFSA guidance to multiple mycotoxins through a scenario of possible co-exposure in humans and animals, using maize as a case study. In particular, a human and animal risk assessment to mycotoxin mixture in maize was conducted using a modelled component-based approach for selected mixture of mycotoxins, that, according to our data, co-occur in maize based feed and food products.

Les mycotoxines sont des métabolites fongiques toxiques qui contaminent une variété d’aliments destinés à l’alimentation humaine et animale, notamment les céréales. Les porcs et les humains sont très sensibles aux mycotoxines. La mycotoxine zéaralénone (ZEN) se retrouve dans les aliments pour animaux et dans l'alimentation humaine principalement la suite d’une contamination fongique des céréales par Fusarium spp. la ZEA peut se fixer au 17-β-estradiol récepteur en raison de sa flexibilité, explique ses effets délétères sur les fonctions reproductives. Différentes stratégies de décontamination ont été développées pour réduire l’exposition des animaux d’élevage aux mycotoxines, parmi lesquelles des méthodes de conversion enzymatique de ZEN par certains micro-organismes. Cependant, les données préliminaires indiquent que certains de ces produits de biotransformation peuvent être reconvertis en ZEN. Des données récentes indiquent que Bacillus spp. S62-W peut phosphoryler ZEN sur ses 14e ou 16e carbones, la convertissant respectivement en ZEN-14-phosphate (ZEN-14-P) ou en ZEN-16-phosphate (ZEN-16-P). Cependant, la toxicité résiduelle et la stabilité de ZEN-14-P et de ZEN-16-P par rapport à ZEN sont encore inconnues. Cette étude visait à étudier la cytotoxicité, le stress oxydatif, l'activité pro-inflammatoire et l'activité œstrogénique des ZEN phosphorylés en utilisant des cellules épithéliales intestinales et endométriales porcines et humaines, ainsi que des explants d’utérus de cochettes prépubères. Nous avons également analysé les voies métaboliques de biotransformation des ZEN phosphorylés par les cellules porcines et humaines. Nos résultats ont démontré que l’exposition à ZEN-14-P ou à ZEN-16-P diminue significativement la viabilité des cellules intestinales porcines IPEC-J2 et des cellules endométriales humaines d’Ishikawa. ZEN, les produits de phosphorylation ont induit de manière significative un stress oxydatif dans les cellules IPEC-J2 à 10 µM. De même, 5 μM ou 25 μM de ZEN-14-P et ZEN-16-P ont activé de manière significative l'expression de cytokines pro-inflammatoires TNF-α, IL-1β, IL-6 et IL-8 dans les cellules IPEC-J2 différenciées. Pour ce qui est des effets œstrogéniques, de manière similaire à ZEN, l'activité intracellulaire de la phosphatase alcaline a été augmentée de façon significative et dose dépendante par ZEN-14-P et ZEN-16-P, dans les cellules d'Ishikawa. Ces produits de phosphorylation ont également, à l’image de ZEN, activé de manière significative l'expression de gènes sensibles aux œstrogènes ALPP, ALPG, PGR, ESR1, ESR2 et GPER1 dans les cellules d'Ishikawa. Enfin, l’exposition à ZEN ou aux ZEN phosphorylés a induit la prolifération des glandes endométriales dans les explants utérins de cochettes prépubères. L'analyse LC-MS/MS des surnageants de culture cellulaire de IPEC-J2 et Ishikawa indique que, bien que les ZEN phosphorylés aient été partiellement hydrolysés en ZEN, leurs métabolites de phase I et de phase II sont différents de ceux de ZEN. Globalement, ces résultats indiquent que la phosphorylation ne réduit pas la toxicité de ZEN pour les lignées cellulaires porcine et humaine. De plus, les voies métaboliques impliquées dans la biotransformation de ZEN et celle des produits de phosphorylation sont différentes, ce qui suggère une toxicité intrinsèque des ZEN phosphorylés. / Pigs and humans are highly susceptible to mycotoxins, which are toxic fungal metabolites that contaminate a variety of food and feedstuffs. Among these, Zearalenone (ZEN) is found in the feed and food supply from Fusarium fungal contamination in cereals. ZEN is functionally similar to 17-β estradiol, and therefore exerts deleterious effects on reproductive functions. Different decontamination strategies are developed to reduce the exposure of farm animals to mycotoxins. At many of these, there are methods of biotransformation of ZEN by the enzymatic activity of certain microorganisms. Bacillus sp. S62 reportedly phosphorylates ZEN on its 14th or 16th carbons, yielding, ZEN-14-P and ZEN-16-P. However, the residual toxicity and stability of ZEN-14-P and ZEN-16-P compared to ZEN are still unknown. This study aimed to investigate the cytotoxicity, oxidative stress, pro-inflammatory activity, and estrogenic activity of phosphorylated ZENs, as well as its metabolic fate when incubated either with intestinal or reproductive cells. Our results showed that ZEN-14-P and ZEN-16-P did not decrease the cytotoxic effect of ZEN in porcine intestinal IPEC-J2 cells and human endometrial Ishikawa cells. ZEN and both phosphorylated ZENs significantly induced oxidative stress in IPEC-J2 cells at 10 µM. Likewise, 5 μM or 25 μM ZEN-14-P and ZEN-16-P altered the expression of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 in differentiated IPEC-J2 cells. In addition, intracellular activity of alkaline phosphatase increased by ZEN, ZEN-14-P and ZEN-16-P in Ishikawa cells. ZEN, ZEN-14-P and ZEN-16-P activated the expression of estrogen-responsive genes ALPP, ALPG, PGR, ESR1, ESR2, and GPER1 at 10 μM in Ishikawa cells. Furthermore, ZEN and phosphorylated ZENs induced the proliferation of endometrial glands in prepubertal gilt uterine explants at 30 µM. Finally, LC-MS/MS analysis of the supernatant of IPEC-J2 and Ishikawa cell cultures exposed to ZEN or its phosphorylated metabolites showed that the latter were partially hydrolyzed back to ZEN, but their phase-I and phase-II metabolites still differ from the ones of ZEN. Overall, these results suggest that phosphorylation does not reduce the toxicity of ZEN for pigs and humans. Moreover, different metabolic fate is involved in the biotransformation of ZEN and phosphorylation products.