Dissertationen zum Thema „Deoxynivalenol“

Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses
The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination

Deoxynivalenol (DON), a mycotoxin produced in cereal grains infected by Fusarium Head Blight produced by Fusarium graminearium and Deoxynivalenol-3-?-D-glucopyranoside (DON-3G), were studied during processing using LC-MS-MS and GC. DON reduced significantly (P<0.05) 61.8% during milling into flour. Therefore, DON was concentrated mostly in the bran and germ. DON increased 40.8% during the fermentation stage of baking. DON increased in dough more than flour and mixed dough. Milling reduced by 23.7% but fermentation did not. But bread was significantly lower in DON-3G at 0.15 ppm than flour and dough at 0.31 ppm. The baking increased DON and decreased DON-3G showing a difference in stability of the mycotoxins during processing. Enzyme hydrolysis on DON using ?-amylase, cellulase, protease, and xylanase, showed a significant increase with cellulase (20.8%), protease (11.4%), and xylanase (35.6%) compared to wheat composite. DON may be bound to the cell wall or protein component of the kernel.

Deoxynivalenol (DON) is commonly found on small grains and causes food safety issues. Deoxynivalenol-3-Glucoside (DON-3-G) is a conjugate, formed as a defense response by the host plant. Past studies have shown both to be present in Fusarium infected small grains, and processed products like beer, but there is limited information on DON-3-G in malt. Objectives were to determine the levels of DON-3-G in barley and wheat, and to study its fate during malting of inoculated and commercial samples. Commercial barley and wheat samples were used to determine levels in naturally infected grain. During malting, barley DON declined 48% on average, but DON-3-G increased by 115%. Both compounds increased in malted wheat. The genotype x crop year interactions were significant for both toxins, indicating that the genotypes did not respond similarly in the two years. The potential for large amounts of DON-3-G to be formed during malting has not been reported.

The fungal disease Fusarium head blight affects cereal grains and can produce mycotoxins, like the water-soluble deoxynivalenol (DON). Wheat wet milling process begins with ground endosperm obtained by dry milling and ends with the separation of starch from gluten. Research was conducted on hard red spring wheat and durum wheat samples naturally contaminated with DON. The fate of DON in wheat dry milled fractionations (farina/semolina, shorts, and bran) during wet milling was investigated. Three wet milling processes were evaluated. DON levels were assessed by GC-ECD. Results showed that DON was present in all dry milled fractions. DON concentration in farina and semolina exceeded the safety threshold for human consumption. After wet milling farina and semolina, nearly all the DON was found in the water-soluble fraction, regardless the wet milling process. A negligible level of DON was found in the gluten extracted from HRSW with Martin wet milling process.

Das Fusarium Mykotoxin Deoxynivalenol (DON) löst in Tieren zahlreiche Krankheitssymptome aus und verursacht beträchtliche wirtschaftliche Schäden. Pflanzen besitzen einen wirksamen Verteidigungsmechanismus gegenüber diesem Toxin, indem sie Glukose an DON konjugieren. Das resultierende maskierte Mykotoxin Deoxynivalenol-3-β-D-Glukosid (D3G) wurde sowohl in Nahrungs- als auch in Futtermitteln nachgewiesen. Eine mögliche Hydrolyse von D3G im Verdauungstrakt von Säugetieren könnte zu einer Erhöhung der Gesamtbelastung an DON führen und somit gesundheitsschädigende Wirkung aufweisen. Aufgrund fehlender in vivo Daten wurde dieses maskierte Mykotoxin bislang nicht in die EU-Höchstmengenregelungen für DON inkludiert. Ziel der vorliegenden Arbeit war es daher abzuklären, ob oral verabreichtes D3G in Ratten hydrolysiert wird und ob es in der Folge zu einer Absorption von freigesetztem DON kommt. In einem Messwiederholungsdesign wurde sechs Sprague-Dawley Ratten Wasser, DON (2,0 mg/kg KG) und die äquimolare Menge an D3G (3,1 mg/kg KG) an den Tagen 1, 8 und 15 oral verabreicht. Nach jeder Applikation wurden die Tiere für 48 h einzeln in Stoffwechselkäfigen gehalten, um Kot und Urin zu sammeln. Die darin enthaltenen Mengen an D3G, DON, Deoxynivalenol-Glukuronid (DON-GlcA) und Deepoxy-deoxynivalonol (DOM-1) wurden anhand einer validierten LC-MS/MS Analysenmethode bestimmt. Nach Verabreichung von D3G konnten sowohl das maskierte Mykotoxin selbst, als auch DON, DON-GlcA und DOM-1 im Urin der Ratten detektiert werden. D3G repräsentierte hierbei lediglich 0,3 ± 0,1% der verabreichten Dosis, was eine äußerst geringe Bioverfügbarkeit indiziert. Insgesamt konnten im Urin nach Applikation von D3G und DON 3,7 ± 0,7% und 14,9 ± 5,0% der verabreichten Toxinmengen wiedergefunden werden. Der Hauptteil an verabreichtem D3G wurde in Form von DON und DOM-1 im Kot der Tiere wiedergefunden. Die Studie konnte belegen, dass D3G im Verdauungstrakt von Ratten hydrolysiert und DON freigesetzt wird. Dieses wird zum Teil zu DON-GlcA und DOM-1 metabolisiert, jedoch nur in geringen Mengen resorbiert. Unsere Daten weisen daher darauf hin, dass D3G in Ratten eine geringere toxikologische Relevanz als DON besitzt.
The Fusarium mycotoxin Deoxynivalenol (DON) leads to numerous adverse health effects in animals and causes serious economic losses. Plants can defend themselves against this toxin by conjugating glucose to DON. The resulting masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G) is frequently occurring in food and feed. There are major concerns that D3G is hydrolyzed in the digestive tract of mammals, thus increasing the total DON load of an individual. Due to a lack of in vivo data D3G has not been included in the regulatory limits established by the European Commission for DON. Therefore, the aim of our study was to clarify whether orally administered D3G is hydrolyzed in rats and liberated DON is subsequently absorbed. Using a repeated measures design, six Sprague-Dawley rats received water, DON (2.0 mg/kg body weight; bw) and the equimolar amount of D3G (3.1 mg/kg bw) by gavage on days 1, 8 and 15, respectively. After each application, the animals were housed individually for 48 h in metabolic cages to collect urine and feces. The concentrations of D3G, DON, deoxynivalenol-glucuronide (DON-GlcA) and de-epoxydeoxynivalenol (DOM-1) in the excreta were determined by a validated LC-MS/MS based method. After administration of D3G, the masked mycotoxin itself as well as DON, DON-GlcA and DOM-1 were detected in the urine of rats. In total, 3.7 ± 0.7% and 14.9 ± 5.0% of the administered dose were recovered in urine after application of D3G and DON, respectively. Urinary eliminated D3G represented only 0.3 ± 0.1% of the given dose, thus indicating a very low bioavailability of this masked mycotoxin in rats. The majority of administered D3G was recovered as DON and DOM-1 in feces. This study clearly demonstrated that D3G is hydrolyzed in the digestive tract of rats. The liberated DON is metabolized to DOM-1 and DON-GlcA, but only poorly absorbed. Our data indicate that D3G is of considerably lower toxicological relevance than DON in rats.

Les micotoxines es consideren un problema de salut pública molt important a causa dels seus efectes adversos en animals i humans. El deoxinivalenol (DON) és la micotoxina més freqüent en els cereals a tot el món. La contaminació per DON provoca grans pèrdues econòmiques a la indústria avícola degut a les seves dietes a base de cereals. El mètode més utilitzat per a contrarestar l'impacte negatiu de les micotoxines en els animals és l'addició d'agents detoxificants de micotoxines als pinsos. Aquests additius per a pinsos, els anomenats adsorbents o modificadors de micotoxines, adsorbeixen o biotransformen les micotoxines en el tracte gastrointestinal, respectivament. Aquests agents detoxificants han de provar-se en funció de la seva capacitat per unir-se o modificar micotoxines in vivo. De moment, no es disposa de models in vivo fiables en pollastres per a avaluar l'eficàcia dels detoxificants de micotoxines basats en biomarcadors específics. L'objectiu d'aquesta tesi va ser, en primer lloc, desenvolupar un model in vivo, i després, investigar l'eficàcia dels agents detoxificants, basats en indicadors específics i no específics, en pollastres d'engreix. La introducció general d'aquesta recerca doctoral comença amb una descripció general de la micotoxina DON. A continuació, s'han reportat biomarcadors específics i inespecífics de toxicitat per DON en pollastres. Finalment, s'ha realitzat una descripció general dels agents adsorbents i biotransformadors davant el DON. El primer capítol mostra els resultats d'un estudi toxicocinètic de DON en plasma de pollastres d'engreix, realitzat mitjançant bolus oral o injecció intravenosa amb 0.75 o 2.25 mg de DON / kg de pes viu. Aquest estudi toxicocinètic es va realitzar amb l'objectiu d'identificar en quin compartiment biològic era més probable que arribés el DON i especificar en quina forma ho fa (compost inicial o metabòlits). L'anàlisi de plasma per LC-MS / MS va revelar que el DON no es va poder quantificar després de l'aplicació de bolus oral, el que indica la molt baixa biodisponibilitat del DON en pollastres d'engreix. L'avaluació dels paràmetres toxicocinètics després de la injecció intravenosa va revelar la metabolització del DON al DON-3 sulfat, la seva ràpida eliminació i excreció en pollastres d'engreix. Els capítols 2 i 3 mostren un model in vivo per a estudiar l'efecte d'una dieta contaminada amb DON sobre paràmetres específics i no específics en pollastres d'engreix. Es van alimentar quaranta-cinc pollastres d'engreix mascles d'1 dia (Ross 308) durant 42 dies, distribuidos en 3 grupos experimentales: grupo control (T1),, pinso contaminat amb DON (5 mg/kg pinso) (T2) o pinso contaminat amb DON (15 mg/kg pinso) (T3). Les concentracions plasmàtiques, hepàtiques o en excretes de DON i DON-3 sulfat es van utilitzar com indicadors específics (capítol 3). Els paràmetres inespecífics avaluats van ser: paràmetres productius, pes relatiu d'òrgans, morfologia i histologia de l'intestí prim, perfil bioquímic sèric, comportament en front de la por i color de les potes (capítol 2), hematologia sanguínia, resposta a vacunes comuns, IL-8 plasmàtica, expressió gènica relativa d'IL-6, IL-1β, IFN-γ i IL-10 en jejú, índex d'estrès (proporció d’heteròfils a limfòcits) i corticosterona plasmàtica (capítol 3). El DON només es va quantificar en les excretes, el que suggereix una baixa biodisponibilitat, una acumulació limitada i una ràpida excreció. El DON-3S es va quantificar en totes les matrius biològiques, el que indica que el DON-3S és el metabòlit més adequat d'exposició al DON en pollastres d'engreix. Amb 5 mg de DON/kg pinso, la creatina quinasa va disminuir i la IL-1β, IL-6 i IFN-γ es van regular positivament. Amb 15 mg de DON/kg pinso, l’índex de transformació alimentària es va alterar negativament i el colesterol en sang i els glòbuls vermells van disminuir. A ambdós nivells assajats van augmentar els pesos relatius del pedrer i del timus, la longitud de l'intestí prim i la IL-8 plasmàtica. No obstant això, es va reduir el pes relatiu de l'intestí prim, el còlon i la borsa de Fabrici, la densitat (pes/longitud) de l'intestí prim, l'hemoglobina i la corticosterona plasmàtica. Es pot concloure que els paràmetres específics i no específics afectats pel pinso contaminat podrien ser adequats per avaluar l'eficàcia dels agents detoxificants de micotoxines en pollastres d'engreix. El capítol 4 mostra el model d'eficàcia in vivo utilitzat per provar 3 detoxificants de micotoxines (MFA, IMP i MDE) basats en biomarcadors seleccionats en els capítols 2 i 3, així com en altres biomarcadors (DOM-3 sulfat i DOM-1 en excretes, triglicèrids sèrics, expressió relativa d'IL-8 i TNF-α en teixits de jejú i corticosterona en plomes). Es van alimentar 384 pollastres d'engreix mascles (Ross308) d'1 dia d'edat, durant 42 dies, amb dietes formulades com a pinso no contaminat (control), pinso contaminat, control + 0.2% MFA, pinso contaminat + 0.2% MFA, control + 0,0125% IMP, pinso contaminat + 0,0125% IMP, control + 0,15% MDE, o pinso contaminat + 0,15% MDE. El DON va ser la principal micotoxina del pinso contaminat i les concentracions van variar al voltant dels 7 mg/kg pinso. Els biomarcadors estudiats es van avaluar als 10 i 42 dies. Es van detectar DON, DON-3S i DOM-3S a les excretes dels grups amb pinso contaminat. L'addició de MDE a la dieta contaminada va augmentar l'excreció de DON però va disminuir l'excreció de metabòlit DOM-3S. L'addició de MFA a la dieta contaminada va augmentar l'excreció de DON, la qual cosa suggereix que aquest producte és eficaç per a detoxificar aquesta micotoxina. Al dia 10, el DON va perjudicar l'índex de transformació alimentària, i va augmentar els nivells de colesterol i triglicèrids en sèrum. L'efecte sobre l'índex de transformació alimentària es va evitar mitjançant l'addició d'IMP a la dieta contaminada. L'efecte sobre el nivell de colesterol sèric es va revertir mitjançant la suplementació amb MFA o IMP a la dieta contaminada. A més, al dia 10, el DON va reduir els nivells d'hematòcrit, hemoglobina, glòbuls vermells i monòcits. L'addició d'IMP a la dieta contaminada va contrarestar l'efecte observat sobre els nivells d'hematòcrit i monòcits en sang. Als 42 dies, el DON va millorar l'índex de transformació alimentària, va reduir el pes relatiu del fetge i el nivell de limfòcits en sang. Als 42 dies, a més, el DON va augmentar els recomptes de glòbuls blancs, l'índex d'estrès (proporció d’heteròfils a limfòcits) i corticosterona en plomes. L'efecte sobre l'índex d'estrès va ser contrarestat per l'addició de MFA a la dieta contaminada. Es pot concloure que els paràmetres específics seleccionats són adequats per a avaluar l'eficàcia dels agents detoxificants de DON en pollastres d'engreix, i que el producte MFA va contrarestar parcialment els efectes negatius de DON.
Las micotoxinas representan un peligro para la salud animal y humana. El deoxinivalenol (DON) es la micotoxina que más frecuentemente contamina los cereales tanto en países desarrollados como en vías de desarrollo. Debido a sus dietas a base de cereales, la presencia del DON en el alimento de las aves genera serias pérdidas económicas. El método más utilizado para detoxificar las micotoxinas presentes en los piensos es el tratamiento con adsorbentes o la biotransformación de las micotoxinas en el tracto gastrointestinal. Su eficacia real, más allá de los posibles resultados prometedores observados en ensayos in vitro, debe ser siempre evaluada en ensayos in vivo, poniendo de manifiesto su efecto protector mediante la observación de la evolución de biomarcadores específicos relacionados con la toxicidad de las micotoxinas. El objetivo de esta tesis fue desarrollar un modelo in vivo, y luego evaluar in vivo la eficacia de productos detoxificantes de DON, en base a biomarcadores específicos y no específicos, en pollos de engorde. La introducción general de esta Tesis comienza con una descripción general del DON. A continuación, se han reportado los biomarcadores específicos y no específicos de toxicidad por DON en pollos. Por último, se ha realizado una descripción general de los productos detoxificantes de DON. El primer capítulo muestra los resultados de la cinética del DON en plasma de los pollos broilers, realizada mediante bolo oral o inyección intravenosa de 0.75 o 2.25 mg de DON/kg de peso vivo. Este estudio de toxicocinética se realizó con el objetivo de identificar a qué compartimento biológico era probable que llegara el DON y especificar en qué forma lo hace (compuesto inicial o metabolitos). El análisis de plasma por LC-MS/MS reveló que el DON no se pudo cuantificar después de la aplicación de bolo oral, indicando la muy baja biodisponibilidad del DON en pollos de engorde. La evaluación de los parámetros toxicocinéticos después de la inyección intravenosa reveló la metabolización del DON en DON-3-sulfato, su rápida eliminación y excreción en pollos de engorde. Los capítulos 2 y 3 muestran el desarrollo de un modelo in vivo en pollos de engorde, que permite evaluar los efectos tóxicos del DON, e identificar los biomarcadores más relevantes (específicos y no específicos). Se realizó un ensayo con 45 pollos broiler machos Ross 308 de un día de vida durante 42 días, distribuidos en 3 grupos experimentales: grupo control (T1), grupo alimentado con 5 mg/kg de DON (T2) y grupo alimentado con 15 mg mg/kg de DON (T3). El DON y el DON-3 S en plasma, hígado y excretas fueron como biomarcadores específicos (capítulo 3). Los parámetros no específicos evaluados fueron: parámetros productivos, peso relativo de órganos, morfología e histología del intestino delgado, bioquímica de sangre, reacción frente al miedo y el color de las patas (capítulo 2), hematología de sangre, respuesta a las vacunas comunes, IL-8 en plasma, expresión relativa de los genes IL-6, IL-1β, IFN-γ e IL-10 en yeyuno, índice de estrés (proporción de heterófilos a linfocitos) y corticosterona en plasma (capítulo 3). El DON solo se cuantificó en las excretas, lo que sugiere una baja biodisponibilidad, una acumulación limitada y una rápida excreción. El DON-3S fue cuantificado en todas las matrices biológicas, indicando que el DON-3S es el metabolito más adecuado como biomarcador de exposición al DON en pollos de engorde. Con 5 mg de DON/kg de alimento, la creatina quinasa disminuyó y la IL-1β, IL-6 e IFN-γ aumentaron. Con 15 mg de DON/kg de alimento, el índice de conversión se alteró negativamente, el colesterol en sangre y los glóbulos rojos disminuyeron. A ambos niveles ensayados, los pesos relativos de molleja y timo, la longitud del intestino delgado y la IL-8 plasmática aumentaron. Sin embargo, el peso relativo del intestino delgado, el colon y la bolsa de Fabricius, la densidad del intestino delgado (peso/longitud), la hemoglobina y la corticosterona plasmática disminuyeron. Así, se puede considerar que los parámetros específicos y no específicos afectados significamente por el pienso contaminado podrían ser relevantes para evaluar la eficacia de los agentes detoxificantes de micotoxinas en pollos de engorde. En el capítulo 4 se evaluó in vivo la eficacia de diferentes productos detoxificantes de DON (MFA, IMP y MDE), en base a los biomarcadores previamente seleccionados en los capítulos 2 y 3, así como otros biomarcadores añadidos (DOM-3 sulfato, DOM-1 en excretas, triglicéridos en suero, expresión relativa de los genes IL-8 y TNF-α en yeyuno y corticosterona en plumas). Se realizó un ensayo con 384 pollos de engorde machos (Ross 308) de 1 día de edad que recibieron dietas durante 42 días, formuladas como: alimento no contaminado (control), alimento contaminado, control + 0.2% MFA, alimento contaminado + 0.2% MFA, control + 0.0125% IMP, alimento contaminado + 0.0125% IMP, control + 0.15% MDE, o alimento contaminado + 0.15% MDE. El DON fue la principal micotoxina presente en el alimento contaminado y las concentraciones analizadas variaron alrededor de 7mg/kg de alimento. Los biomarcadores se evaluaron a los 10 y 42 d. Se detectaron DON, DON-3S y DOM 3S en las excretas de grupos con pienso contaminado. La adición de MDE al pienso contaminado aumentó la excreción de DON, pero disminuyó la excreción del metabolito DOM-3S. La adición de MFA a la dieta contaminada aumentó la excreción de DON, lo que sugiere la eficacia de este producto para desintoxicar esta micotoxina. A los 10 d, el DON perjudicó el índice de conversión, y aumentó los niveles de colesterol y triglicéridos en suero. La adición de IMP a la dieta contaminada contrarrestó el efecto negativo sobre el índice de conversión. El efecto sobre el nivel de colesterol en suero se revirtió mediante la suplementación con MFA o IMP a la dieta contaminada. Además, a los 10 d, el DON redujo los niveles de hematocrito, hemoglobina, glóbulos rojos y monocitos en sangre. La adición de IMP a la dieta contaminada contrarrestó el efecto observado sobre los niveles de hematocrito y monocitos en sangre. A los 42 días, el DON mejoró el índice de conversión, redujo el peso relativo del hígado y el nivel de linfocitos en sangre, mientras que aumentó el recuento de glóbulos blancos, el índice de estrés (relación heterófilos/linfocitos) y el nivel de corticosterona en plumas. La adición de MFA a la dieta contaminada contrarrestó el efecto sobre el índice de estrés. En conclusión, los parámetros específicos selecionados son adecuados para evaluar la eficacia de agentes desintoxicantes del DON en pollos de engorde, y el producto MFA contrarrestó parcialmente los efectos negativos de DON.
Mycotoxins are considered a very important public health issue because of their adverse effects on animals and humans. Deoxynivalenol (DON) is the most frequent mycotoxin in cereals worldwide. DON contamination leads to great economic losses in poultry industry due to their cereal-based diets. The most commonly used method to counteract the negative impact of mycotoxins on animals is the addition of mycotoxin detoxifying agents (mycotoxin detoxifiers) to feed. These feed additives, so-called mycotoxin binders or mycotoxin modifiers, either adsorb or biotransform mycotoxins in the gastrointestinal tract, respectively. These detoxifying agents should be tested not only in vitro, but also in vivo on their ability to bind or modify mycotoxins. At the time being, no reliable in vivo models in chicken are available to evaluate the efficacy of mycotoxin detoxifiers based on specific biomarkers. The aim of this thesis was first to develop an in vivo model then to investigate the efficacy of detoxifying agents, based on specific and nonspecific indicators, in broiler chickens. The general introduction of this doctoral research starts with an overview of DON mycotoxin. Next, specific and nonspecific biomarkers of DON toxicity in chickens have been reported. Finally, an overview of binding and biotransforming agents against DON has been carried out. The first chapter shows the results of a DON toxicokinetic study in broiler chickens’ plasma, performed via oral bolus or intravenous injection of 0.75 or 2.25 mg DON/kg of body weight (BW). This toxicokinetic study was done with the objective to identify which biological compartment the DON was likely to reach and to specify in what form (initial compound or metabolites) it does so. The analysis of plasma by LC-MS/MS revealed that DON could not be quantified after oral bolus application, indicating the very low bioavailability of DON in broiler chickens. The evaluation of toxicokinetics parameters after the intravenous injection revealed the metabolization of DON in DON-3 sulphate, its rapid clearance and excretion in broiler chickens. Chapter 2 and 3 show an in vivo model set-up to study the effect of a DON contaminated diet on specific and nonspecific relevant biomarkers on broiler chickens. Forty-five 1-day-old male broiler chickens (Ross 308) were fed diets during 42 d, distributed into 3 experimental groups:distribuidos en 3 grupos experimentales: control group (T1), DON contaminated feed (5 mg/kg feed) (T2), or DON contaminated feed (15 mg/kg feed) (T3). Plasma, liver or excreta concentrations of DON and DON-3 sulphate were used as specific indicators (chapter 3). The nonspecific parameters evaluated were performance parameters, relative organ weights, morphology and histology of small intestine, serum biochemistry profile, fear behavior and leg color (chapter 2), blood hematology, response to common vaccines, plasma IL-8, relative gene expression of IL 6, IL-1β, IFN-γ and IL-10, stress index (heterophil to lymphocyte ratio), and plasma corticosterone (chapter 3). DON was only quantified in excreta, suggesting low bioavailability, limited accumulation and rapid excretion of DON. DON-3S was quantified in all biological matrices, indicating that DON-3S is the most suitable metabolite of exposure of DON in broiler chickens. At 5 mg DON/kg feed, creatine kinase decreased and IL-1β, IL-6, and IFN-γ were upregulated. At 15 mg DON/kg feed, feed conversion ratio was impaired and blood cholesterol and red blood cells decreased. At both levels assayed relative weights of gizzard and thymus, the length of small intestine, and plasma IL-8 increased. However, the relative weight of small intestine, colon, and bursa of Fabricius, the density (weight/length) of small intestine, hemoglobin and plasma corticosterone were reduced. It can be concluded that specific and unspecific parameters affected by the contaminated feed could be suitable to evaluate the efficacy of the mycotoxin detoxifying-agents in broiler chickens. Chapter 4 shows the in vivo efficacy model used to test 3 mycotoxin detoxifiers (MFA, IMP and MDE) based on biomarkers selected on chapters 2 and 3, as well as other biomarkers (DOM-3 sulphate and DOM-1 in excreta, serum triglycerides, relative expression of IL-8 and TNF-α in jejunum tissues, and corticosterone in feathers). Three hundred eighty four 1-d-old male broiler chickens (Ross308) were fed for 42 d with diets formulated as non-contaminated feed (control), contaminated feed, control+0.2% MFA, contaminated feed+0.2% MFA, control+0.0125% IMP, contaminated feed+0.0125% IMP, control+0.15% MDE, or contaminated feed+0.15% MDE. DON was the main mycotoxin of the contaminated feed and concentrations varied around 7 mg/kg feed. Studied biomarkers were evaluated at 10 and 42 d. DON, DON-3S, and DOM-3S were detected in excreta from contaminated groups. The addition of MDE to contaminated feed increased the excretion of DON but decreased the excretion of the metabolite (DOM-3S). The addition of MFA to contaminated diet increased the excretion of DON, suggesting that this product is effective to detoxify this mycotoxin. At d 10, DON impaired feed conversion ratio, increased serum cholesterol and triglycerides levels. The effect on feed conversion ratio was prevented by IMP addition to the contaminated diet. The effect on serum cholesterol level was reversed by MFA or IMP supplementation to the contaminated feed. At d 10, moreover, DON reduced hematocrit, hemoglobin, red blood cells, and monocytes levels. The addition of IMP to the contaminated diet counteracted the effect observed on blood hematocrit and monocytes levels. At 42 d, DON improved the feed conversion ratio, reduced the relative weight of liver, and blood lymphocytes level. At 42 d, furthermore, DON increased white blood cells counts, stress index (heterophils to lymphocytes ratio) and feather corticosterone. The effect on stress index was counteracted by the addition of MFA to the contaminated diet.

This diploma thesis is focused on current problematics of monitoring of selected mycotoxins, DON and ZEA, in feeds in Czech Republic. The objective of my thesis was to elaborate a literature search from available books, electronic and periodical sources and service materials. Literature search is aimed at overall overview of mycotoxins, describes their characteristics, biological effects, methods of detection as well as summarizes recent legislation requirements concerning occurrence of these substances in feeds for animals. The aim of experimental part was a determination of selected mycotoxins in feed (DON and ZEA) by ELISA method and their evaluation according to the maximum limits. The diploma thesis was prepared in diagnostic laboratory SEVARON, s. r. o. in Brno.

Mycotoxins are toxic secondary metabolites produced by fungi that are a threat to the health of humans and domestic animals. The most important mycotoxin in the U.S. is deoxynivalenol (DON), which causes symptoms such as vomiting, feed refusal, and weight loss in farm animals. The fungus Fusarium graminearum produces DON in staple crops such as wheat, barley, and corn. It is estimated that the economic losses associated with DON contamination alone exceed $650 million per year in the U.S. New strategies are needed to mitigate DON and improve food safety in the U.S. The overall goal of my research is to discover and employ microorganisms and enzymes to detoxify DON. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) use a cell free protein synthesis (CFPS) system to study enzymes that modify DON, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food. In Objective 1, two mixed cultures were identified from environmental samples that converted DON into the less toxic 3-keto-deoxynivalenol (3-keto-DON). In Objective 2, a CFPS system was used to express three known acetyltransferase genes to convert DON to 3-acetyl-DON (3-A-DON). In Objective 3, we identified a potential DON transporter from a library of randomly amplified fragments from the genomes of mixed cultures of microbes isolated from the environment. In Objective 4, we developed and delivered a unique high school unit to educate high school students about potential mycotoxins in food and feed products. The work presented here represents new and improved methods for mitigating mycotoxin contamination in the United States.
Ph. D.

There is growing interest in malting and brewing with rye. However, previous research has shown a propensity for the development of deoxynivalenol (DON) in rye malts, even when levels on the grain is low. The main objective of this study was to assess the growth of F. graminearum and development of trichothecenes during malting of rye. Infected samples were obtained from 2016 variety trails in Minnesota. While DON levels were generally below 0.2 mg/kg, an average increase of 41 % was seen after malting. The most significant increases in DON were at three days of germination. Fusarium Tri5 DNA levels were observed to increase at two days. When single kernels were tested, most were free from DON. Levels in the bulk grain sample were due to a small number of highly contaminated kernels. In the malted samples, a greater portion of kernels contained DON, and overall levels were much higher.

Thesis (Ph.D.)--Michigan State University. Dept. of Food Science and Environmental Toxicology, 2008.
Title from PDF t.p. (viewed on July 31, 2009) Includes bibliographic references (p. 160-184). Also issued in print.

La fusariosi della spiga e il marciume al colletto in frumento sono malattie causate da Fusarium graminearum, F. culmorum e F. pseudograminearum. Essi determinano ingenti perdite di raccolto oltre a contaminare il prodotto con micotossine appartenenti alla famiglia dei tricoteceni. I batteri Gram-positivi appartenenti al genere Streptomyces sono ubiquitari nel suolo ed endofiti dei tessuti interni delle radici. Essi producono una vasta gamma di metaboliti secondari con proprietà antimicrobiche e possono essere utilizzati come agenti promotori della crescita delle piante, limitando lo sviluppo dei patogeni. L’obiettivo del presente dottorato di ricerca è stato quello di selezionare ceppi di streptomiceti attivi contro Fusarium spp. in grano. La prima fase dello studio ha permesso di conoscere lo stato dell’arte sull’utilizzo di streptomiceti contro specie micotossigene appartenenti al genere Fusarium (Chapter 1). Successivamente sono state sviluppate strategie innovative per la selezione degli stessi (Chapter 2) e l’efficacia dei ceppi più promettenti è stata poi saggiata in diverse condizioni (Chapter 3). Inoltre, i metaboliti secondari responsabili dell’attività antifungina sono stati caratterizzati (Chapter 4). L’influenza della variabilità dei ceppi di Fusarium spp. e dei substrati colturali sull’attività di biocontrollo è stata valutata tramite saggi di antibiosi. Questi fattori hanno avuto un’influenza significativa nel determinare il livello di attività in vitro. I mezzi standard utilizzati in laboratorio hanno diminuito infatti tale parametro. Inoltre è stata riscontrata un’assenza di correlazione con il livello di biocontrollo ottenuto in pianta. Unica eccezione è per i risultati ottenuti utilizzando un terreno a base di frumento, che ha permesso di osservare un valore di correlazione più elevato con il livello di biocontrollo riscontrato contro marciume radicale in frumento (r = 0.5). Successivamente, al fine di saggiare metaboliti limitanti la produzione di tricoteceni, è stato sviluppato un sistema in micropiastra che misura la fluorescenza emessa da un ceppo di F. graminearum trasformato con il costrutto TRI5::GFP. TRI5 è il primo gene essenziale coinvolto nella via metabolica di produzione di tricoteceni. Da questa prova si è potuto selezionare il ceppo di streptomicete DEF39 che riduce significativamente la produzione di DON. Le potenziali attività di promozione della crescita e di biocontrollo di una selezione dei ceppi più promettenti (N = 21) sono state saggiate in pianta. Gli streptomiceti testati non hanno esibito la capacità di aumentare i parametri di sviluppo dei germinelli di frumento, ma il ceppo DEF09 ha ridotto significativamente il marciume al colletto e la fusariosi della spiga ottenendo livelli di protezioni sopra al 40% e del 60% rispettivamente. Basandosi sui risultati ottenuti, quattro ceppi (DEF09, DEF20, DEF39, DEF48) sono stati applicati su grano sterilizzato testando due tempistiche di trattamento per osservarne la capacità di riduzione della biomassa fungina e della produzione di DON. Inoltre tramite qPCR si è osservata la fitness degli agenti di biocontrollo nelle condizioni testate. Gli streptomiceti, abili colonizzatori del substrato testato, sono stati efficaci nel ridurre la produzione di micotossine, limitando -quando co-inoculati con il patogeno- lo sviluppo dello stesso. I ceppi selezionati agiscono perciò sia contro lo sviluppo fugino e/o contro la produzione di DON. Ulteriori studi saranno necessari per confermarne l’attività in pianta, così come per permettere lo sviluppo di formulati efficaci per limitare la contaminazione da tricoteceni.
Fusarium head blight (FHB), root rot (FRR) and foot rot (FFR) cause important yield losses in wheat. The harvested product is often contaminated with mycotoxins, belonging to the group of trichothecenes. The main causal agents are Fusarium graminearum, F. culmorum and F. pseudograminearum. The biocontrol approach is a feasible option in order to reduce disease severity, as well as trichothecene contamination in grains. Streptomyces spp. are Gram-positive bacteria, ubiquitous in soil and endophytes of inner tissues of plant roots. They produce a wide range of secondary metabolites able to limit pathogen development and disease severity in planta, as well as to enhance plant growth. This PhD project aimed to select Streptomyces strains active within the wheat-Fusarium spp. pathosystem. To achieve this, a detailed literature and patents analysis focused on biocontrol of toxigenic Fusarium spp. was carried out (Chapter 1) and new methodological approaches for antagonist screening have been developed (Chapter 2). Furthermore, the biocontrol efficacy of a selected subset of strains obtained from the culture collection maintained at the Plant Pathology laboratory (DeFENS, University of Milan, Italy) was evaluated in different conditions (Chapter 3) and bioactive metabolites were isolated (Chapter 4). The influence of growth media and Fusarium strain diversity on streptomycete antifungal activity was assessed in dual culture assays. All the factors influenced the level of antifungal activity. The media commonly used for in vitro screening reduced the inhibitory activity of streptomycetes. Overall, results from dual culture assays and level of disease protection observed in planta did not correlate, except for those recorded on a medium based on wheat grains. Indeed, it was the most effective in eliciting antifungal activity and showed the highest correlation (r = 0.5) with FRR inhibition. Subsequently, being TRI5 the first and essential gene involved in trichothecene biosynthetic pathway in Fusarium spp., a microplate bioassay using a TRI5::GFP transformed F. graminearum strain was developed and validated in order to screen the effect of natural products on GFP fluorescence and consequently on trichothecene production. Surprisingly, culture filtrate from DEF39 strain completely suppressed deoxynivalenol (DON) production without affecting fungal growth. The most promising isolates (N = 21) were further characterized for their potential plant growth promotion ability, as well as for their activity against FRR and FFR in wheat seedlings. None of them was able to increase plant growth. However, DEF09 strain exhibited consistent efficacy to limit FRR-FFR symptom severity (protection level > 40%) in soil and soilless conditions. Therefore, a field trial was performed to test its ability to reduce FHB severity, obtaining up to 60% protection. Based on the activity observed from the previous screenings, four promising streptomycetes (DEF09, DEF20, DEF39, DEF48) were applied on sterilized wheat grains (microsilage) at two timepoints of application, in order to evaluate their ability to suppress fungal growth and DON production. Moreover, the fitness of streptomycetes in microsilage conditions was assessed by qPCR analysis. Streptomycetes were able to efficiently colonize the substrate, which resulted in reducing fungal biomass and DON accumulation only when co-inoculated with the pathogen. A pool of promising biocontrol agents has been selected against fungal development and/or DON production. This research highlighted the complexity of finding an efficient screening procedure due to multiple interactions occurring in wheat-Fusarium spp. pathosystem. Further studies will be needed to confirm the activity of the strains in planta. The identification of the mechanisms of action and the molecules involved in the bioactivity of the strains will possibly allow to develop effective treatments limiting trichothecene accumulation in wheat.

Le Cd est un métal lourd toxique très répandu. L'homme peut être exposé à ce contaminant environnemental par la fumée, la nourriture et l'eau. Le déoxynivalénol (DON) est l'une des mycotoxines les plus répandues dans les céréales. Si de nombreuses études ont étudié la toxicité du DON et du Cd individuellement, leur toxicité combinée est très peu connue. Cependant, les consommateurs peuvent être exposés à un mélange DON et Cd. Dans la présente étude, nous nous sommes concentrés sur les effets du DON et du Cd, seuls ou en combinaison, en utilisant une approche in vitro. Différentes lignées cellulaires humaines provenant du rein (HEK-293), de l'intestin (Caco-2), du sang (HL-60) et du foie (HepG2) ont été exposées à une gamme de doses de DON et Cd seuls et en combinaison. La toxicité induite a été évaluée avec le test CellTiter-Glo(r) Luminescent Assay, basé sur la mesure de la teneur en ATP, proportionnelle au nombre de cellules viables. Les interactions entre DON et Cd ont été analysées à l'aide de la méthode de l'indice de combinaison /isobologramme basée sur de l'équation à effet médian de la loi d'action de masse de Chou et Talalay (2006). Les cellules HEK-293 ont été exposées à des doses croissantes de DON, Cd et leur combinaison à différents ratios (DON / Cd de 2/1, 1/1, 1/2 et 1/8). Indépendamment du ratio, le type d'interaction observé dans les cellules HEK-293 allait de l'antagonisme modéré à presque additif. Dans les cellules Caco-2, les interactions variait de la légère synergie à l'antagonisme, quel que soit le ratio. Au ratio 1/1, dans les cellules HL-60 et HepG2, les interactions variaient de la synergie à l'antagonisme en fonction du niveau de cytotoxicité. Dans le milieu additionné de 1% de sérum de veau fœtal (FCS), la nature des interactions entre le DON et Cd sur les cellules HEK-293 et Caco-2 n'a pas montré de différence significative par rapport au milieu avec 10% de FCS. Les effets du DON et du Cd sur la fonction barrière intestinale et l'expression des gènes ont été évalués. Sur la perméabilité des monocouches de Caco-2, le DON et le mélange DON / Cd ont montré un effet dose-dépendant tandis qu'aucun effet n'a été observé pour le Cd. Le DON a induit une altération significative des cytokines inflammatoires alors que le Cd a montré une surexpression des gènes de la métallothionéine. Dans le milieu supplémenté avec 1% de FCS, nos résultats préliminaires ont montré des effets du Cd sur la fonction de barrière intestinale. Les effets combinés du DON et du Cd sur l'intégrité de barrière des cellules Caco-2 différentiées variait d'un antagonisme modéré à presque additif. En conclusion, notre étude indique que l'exposition combinée au DON et au Cd est spécifique à l'organe cible et au stade de développement de la cellule. De plus, les interactions entre le DON et le Cd devront être étudiées dans des expériences ex vivo et in vivo pour confirmer ces résultats
Cadmium (Cd) is a common and widespread toxic heavy metal. Human can be exposed to this environmental contaminant through smoke, food and water. Deoxynivalenol (DON) is one of the most prevalent mycotoxins in cereals. If numerous studies investigated the toxicity of DON and Cd individually, very little is known about their combined toxicity. However, consumers can be exposed to a cocktail DON and Cd. In the present study, we focused on the effects of DON and Cd, alone or in the mixture using in vitro approach. Different human cell lines from kidney (HEK-293), intestine (Caco-2), blood (HL-60) and liver (HepG2) were exposed to a range of doses of DON and Cd alone and in combination. The induced toxicity was evaluated with CellTiter-Glo(r) Luminescent Assay, based on the measure of ATP content, proportional to the number of viable cells. Interactions between DON and Cd were analyzed with isobologram-combination index method derived from the Median-Effect Equation of the Mass Action Law of Chou and Talalay (1984). HEK-293 cells were exposed to increasing doses of DON, Cd and their combinations at different ratios (DON/Cd of 2/1; 1/1; 1/2 and 1/8). Regardless of the ratio, the type of interaction observed in HEK-293 cells ranged from moderate antagonism to nearly additive. In Caco-2 cells, the interactions ranged from slight synergy to antagonism whatever the ratio. At ratio 1/1, in HL-60 and HepG2 cells, interactions ranged from synergy to antagonism depending on the cytotoxicity level. In the medium supplemented with 1% Fetal Calf Serum (FCS), the interaction of DON and Cd on HEK-293 and Caco-2 cells did not show a significant difference compared to medium with 10% FCS. Then, the effects of DON and Cd on the barrier function and gene expression were evaluated. On Caco-2 monolayers permeability, DON and DON/Cd mixture showed a dose- dependent effect while no effect was observed with Cd. DON-induced a significant alteration of inflammatory cytokines whereas Cd showed overexpression of metallothionein genes. In medium supplemented with 1% FCS, our preliminary results showed effects of Cd on intestinal barrier function. The combined effects of DON and Cd on Caco-2 cells barrier function ranged from moderate antagonism to nearly additive. In conclusion, our study indicates that the combined exposure to DON and Cd is specific to the target organ and development stage of the cells. Moreover, the interactions between DON and Cd will have to be investigated in ex vivo and in vivo experiments to confirm these results

Chapter 1 describes deoxynivalenol and zearalenone mycotoxins as secondary metabolites of Fusarium fungi. Metabolism of Fusarium fungi is discussed in the light of deoxynivalenol and zearalenone biosynthesis. Consequences of the presence of toxic fungal metabolites in cereal products are highlighted. Chapter 2 focuses on production of material for degradation and binding studies i.e. gram quantities of both mycotoxins. Procedures of deoxynivalenol and zearalenone production by fermentation were successfully designed and executed. The identities of the compounds were confirmed by NMR. Additionally, radio labelled deoxynivalenol was produced using developed fermentation methods. Chapter 3 investigates degradation of deoxynivalenol and zearalenone under typical cereal processing conditions. Acidic and basic degradation models were developed and executed. Degradation products of deoxynivalenol and zearalenone were isolated and characterised by LC-MS and NMR. NMR time course experiments were successfully performed resulting in mechanistic and kinetic information of deoxynivalenol and zearalenone degradation. Chapter 4 presents studies on binding of deoxynivalenol to food components such starch and flour. Previously produced 14C-labelled deoxynivalenol served as a tracer during binding studies. Influence of extraction procedures on deoxynivalenol bound to starch was investigated. The existence of binding of deoxynivalenol to flour was observed. Speculations on the nature of binding were made.

The fungal plant pathogen Fusarium graminearum Schwabe (teleomorph Gibberella zeae¬) produces a dangerous trichothecene mycotoxin called deoxynivalenol (DON) and causes a devastating disease of barley (Hordeum vulgare L.) called Fusarium head blight (FHB). Food and feed products derived from barley, such as dried distillers grains with solubles (DDGS), may be contaminated with DON and pose a threat to the health of humans and domestic animals. New methods to mitigate the threat of DON in barley need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that modify DON and reduce its toxicity. The first objective of this research was to isolate unique TRI101 and TRI201 enzymes that modify DON efficiently. We hypothesized that TRI101/TRI201 enzymes from different species of Fusarium would have varying rates and abilities to modify DON. Using degenerate primers, an internal portion of TRI101 or TRI201 was identified in 54 strains of Fusarium. Full-length sequences of seven TRI101 or TRI201 genes were cloned and expressed in yeast. All seven genes acetylated DON, but at different rates. The second objective of this research was to utilize transformed yeast expressing TRI101 or TRI201 to reduce DON levels in barley mashes and ultimately in DDGS. We hypothesized that DON levels would be reduced in DDGS derived from mashes prepared with transformed yeast. Five different barley genotypes were used to prepare the fermentation mashes and DON levels were reduced in all DDGS samples derived from mashes prepared with transformed yeast. The third objective of this study was to characterize barley genotypes developed at Virginia Tech for resistance to FHB and DON. We hypothesized that significant differences in resistance would be observed among barley genotypes and FHB resistance would be associated with reduced DON accumulation. From 2006 to 2010, FHB resistance was assessed in hulled (22 to 37) and hulless (13 to 32) barley genotypes by measuring incidence and index, and DON resistance was determined by quantifying DON levels in ground grain using gas chromatography-mass spectrometry. Our study showed that FHB and DON resistance is significantly determined by genotype. The final objective of this study was to develop a robust tissue culture system necessary for future development of transformed barley plants with FHB resistance gene(s). We hypothesized that callus production would vary among barley genotypes. In our analysis of 47 Virginia barley genotypes, 76% (36/47) of the genotypes produced callus tissue and there were significant differences in callus size. Our work sets the stage for identifying and characterizing DON detoxification genes in the future. The development of commercial barley lines that do not accumulate DON and that are resistant to FHB will directly impact growers and producers of small grains in the eastern U.S.
Ph. D.

Food safety related problems are one of the biggest challenges worldwide. DON is produced by Fusarium species which causes the well-known Fusarium Head Blight (FHB) of wheat and barley. FHB outbreaks have led to variability in yield and revenue losses over the years. The main objective of my thesis was to quantify risk premiums at the farm level and with industry impact, to determine the effectiveness of FHB/DON mitigation strategies over time from 1997 to 2014. Data on revenue losses ($million) were obtained from USDA-ERS and was simulated using a risk analysis software called @RISK 7.5. The sample data was simulated 10,000 times to obtain a population. Risk premiums were calculated for each year and for each crop over time and graphs were plotted. Trends in risk premiums showed an overall decrease from 1997 to 2014, indicating that variability of losses have reduced and that the management practices have been effective.

Doctor of Philosophy
Department of Animal Sciences and Industry
Duane L. Davis
Jim L. Nelssen
A total of 188 sows and their litters were used in 2 experiments to evaluate methods to induce estrus and ovulation in lactating sows and effects on pig growth. In Exp. 1, an altered suckling method (ALT) was designed to combine split-weaning and intermittent suckling as a means to reduce the suckling stimulus in primi- and multiparous sows during the last week of lactation (d 18 to 25). The ALT sows were also removed for daily boar exposure. The ALT treatment produced lactational estrus in 75% and 95% of primiparous and multiparous sows, respectively. The ALT sows were in estrus earlier (P < 0.01) than controls post-farrowing, with no effect on subsequent reproductive performance. From d 18 to 32, the ALT treatment benefited (P < 0.01) growth of lightweight pigs but decreased (P < 0.01) BW gain of heavyweight pigs, resulting in overall similar growth. However, variation in BW was reduced (P < 0.01) by 50% for ALT litters. In Exp. 2, varying suckling reduction strategies were applied to boar-exposed lactating sows. Overall, 76% of sows in suckling reduction treatments expressed estrus in lactation. Split-weaned and ALT sows performed reproductively similar to controls, whereas sows with daily litter separation or a single 24 h litter removal tended (P < 0.10) to have reduced conception rates versus controls or split-weaned sows. Reduced suckling treatments differed in their ability to induce lactational estrus and impact on pig BW gain immediately post-weaning. However, no evidence was found of benefit for pig growth to market weight or litter BW variation. Four additional experiments using 902 nursery pigs were conducted to test the efficacy of potential detoxifying agents against deoxynivalenol (DON) in swine diets. The effects of DON were not offset by adding an algae-modified montmorillonite clay nor by a proprietary blend of preservatives and clays. However, hydrothermally treating DON-contaminated diets with sodium metabisulfite modified the structure of DON to a non-toxic DON-sulfonate adduct and restored nursery pig growth via improved (P < 0.05) ADG, ADFI and G:F.

Deoxynivalenol (DON) contamination of grain destined for animal feeds is a major toxicological risk to monogastrics and is suspected of restricting productivity in ruminants. Whereas bacterial additives have been developed that can detoxify DON in the rumen and lower intestine, there are currently no commercial inoculants able to perform this task in crimped grain (CG) silage, a regionally important method of moist grain preservation based on homo- and heterofermentative lactic acid bacteria or chemical additives. Determining whether this ensiling process alongside the action of detoxifying bacteria has the potential to remove DON in CG prior to ingestion, was explored in mini-silo ensiling experiments. CG was heat treated (100 °C, 60 min) or ensiled fresh in triplicate 50 g silos, spiked with 5 mg/kg DON and inoculated with lactic acid bacteria derived from wild birds, natural epiphytic inoculants and commercially sourced silage additives (21 d). DON recovery was only significantly reduced (31.2 ± 14.4% recovery, p < 0.001, n= 30) by heat treatment, as determined by IAC-RP-HPLC-UV. Bacterial assemblage analysis by 16S rRNA PCR-DGGE-SEQ identified Weissella cibaria, Pantoea agglomerans, Bacillus subtilis, B. licheniformis and Hafnia alvei as candidate detoxification agents, of which W. cibaria and H. alvei decreased DON recovery in vitro (11.3 and 6.2% recovery respectively, p < 0.05, n = 18), which translated to inoculated W. cibaria yielding a decrease in DON recovery (67.2± 14.4%, 28 d) in naturally contaminated crimped wheat (13.5 ± 1.0 mg/kg, 35-40% moisture, p < 0.05, n =15). As W. cibaria is a lactic acid bacteria already associated with fermented CG by default it has promise as a novel DON detoxification agent in CG silage. DON is however just one of many hepatotoxic co-contaminants. Retrorsine, a DNA-crosslinking pyrrolizidine alkaloid derived from Ragwort (Senecio sp.) was investigated for interactive toxicity with DON in an in vitro co-exposure experiment. HepG2 cells were exposed to Log10 multifactorial binary exposures for 48 h followed by a suite of assays to elucidate mechanisms of interactive cytotoxicity, genotoxicity and modulation of the proteome. Retrorsine was tentatively confirmed to form DNA/protein crosslinks in the comet, micronucleus and crosslinking assays, whilst DON was found to potently induce cytotoxicity and apoptosis. Co-exposure yielded a complex toxicity response, with low doses yielding antagonistic effects and high doses trending towards additive effects, although DON dose was generally the principle component. The difficulties associated with undertaking an interactive toxicity study where both toxins have multiple metabolic and cellular targets are highlighted.

Einleitung: Ziel der vorliegenden Arbeit war es, mögliche Zusammenhänge zwischen einer Exposition von Milchkühen mit Mykotoxinen (DON, ZON und deren Metaboliten) und dem Entwickeln und Bestehen einer LMV sowie weiteren Primär- und Sekundärerkrankungen zu prüfen. Es bestand die Frage nach einer möglichen Beziehung zu klinischen Störungen, nach einer möglichen Korrelation von Veränderungen der Blutparameter und nach der Eignung von Gallensaft zum Mykotoxinnachweis. Material und Methoden: Für die Untersuchungen wurden 61 Kühe der Rasse Schwarzbunte aus dem Patientengut der Medizinischen Tierklinik mit der Diagnose LMV genutzt. Die gesunden Kontrollkühe (n = 13) stammten aus zwei Betrieben im Leipziger Umland. Bei der Aufnahme in die Klinik bzw. der Probenentnahme wurden alle Kühe klinisch untersucht. Für die Mykotoxinbestimmungen wurden von 20 Patienten- und 13 Kontrollkühen Futter, Serum, Milch und Galle sowie von 41 Patientenkühen Serum und Galle entnommen. Es wurden klinisch-chemische Parameter im Serum untersucht. Die Mykotoxine wurden mittels High-Performance-Liquid-Chromatography (HPLC) analysiert. Ergebnisse: Bei 36 von 61 Patienten konnten keine Mykotoxine in der Galle nachgewiesen werden. Bei den anderen 25 Tieren war ein positiver Mykotoxinnachweis erfolgt. Von den 36 Kühen mit negativem Mykotoxinnachweis konnten 20 Kühe (56 %) mit linksseitiger LMV und 8 Kühe (22 %) mit rechtsseitiger LMV als geheilt entlassen werden. Sechs Kühe (17 %) mit LMV nach links und negativem Mykotoxinnachweis und zwei Kühe (6 %) mit LMV nach rechts und negativem Mykotoxinnachweis hatten einen Exitus letalis. Von 25 Kühen mit positivem Mykotoxinnachweis wurden zwölf Kühe (48 %) mit der Diagnose LMV nach links und sechs Kühe (24 %) mit LMV nach rechts geheilt. Fünf Kühe (20 %) mit LMV nach links und zwei Kühe (8 %) mit LMV nach rechts hatten einen Exitus letalis. Die makroskopische Untersuchung der Futterproben ergab keine grobsinnlichen Veränderungen. Mykotoxine konnten hingegen regelmäßig in den Futterproben aus den Herkunftsbetrieben der Patienten und der gesunden Kühe nachgewiesen werden. Der Median betrug für DON 0,161 mg/kg (0,086-0,191) und für ZON 6,35 µg/kg (4,88-7,85). Im Serum enthielten eine von 61 Proben DON (0,002 µg/mL) und weitere vier Proben DOM-1 (0,002-0,003-0,004-0,009 µg/mL). Im Gallensaft konnte bei 61 Proben nur einmal DOM-1 (37,6 µg/mL) und kein DON nachgewiesen werden. Die untersuchten Galleproben waren zu 39 % mit ZON bzw. dessen Metabolite belastet. Dabei lag ZON (ng/g) im Mittel bei 9,85 (8,10-16,33), α-ZOL bei 59,9 (5-78) ng/g und ß-ZOL bei 37,6 ng/g. Bei Patienten mit positivem Nachweis von ZON und seinen Metaboliten α- und ß-ZOL konnten keine Veränderungen am Genitaltrakt festgestellt werden. Bei den gesunden Kühen waren keine Mykotoxine in Serum, Milch oder Gallensaft nachweisbar. In den eigenen Untersuchungen ließen sich klinisch sowie biochemisch weder DON- noch ZON-spezifische Effekte nachweisen. Entweder waren die geprüften Parameter im physiologischen Bereich, wie z.B. Protein, Kreatinin, TEAC und im Blutbild, oder die Veränderungen waren auch bei den Kühen ohne nachweisbare Mykotoxine vorhanden, wie z.B. bei Bilirubin, Glucose, FFS, Cholesterol, BHB, CK, GGT, Mg, Ca, Na, K, Cl, FFS und im Säure-Basen-Status. Die mittleren GLDH-Aktivitäten aller Patienten mit DON-Nachweis waren geringgradig höher als bei Patienten ohne Mykotoxinnachweis. Schlussfolgerung: Mykotoxine konnten in dieser Untersuchung weder für spezifi-sche Symptome oder hämatologische sowie klinisch-chemische Abweichungen im Blut noch für den Krankheitsausgang, d.h. Heilung oder Exitus letalis, verantwortlich gemacht werden. Eine Ausnahme stellt die Frequenz und Intensität der Pansenkontraktionen dar, die bei Patienten mit positivem Mykotoxinnachweis im Futter reduziert waren. ZON- bzw. ZOL-assoziierte Veränderungen an den Ovarien und dem Uterus wurden makroskopisch in keinem Fall festgestellt. Als zusätzliche Erkenntnis stellte sich im Verlaufe dieser Arbeit unabhängig von der eigentlichen Fragestellung heraus, dass die Gallensaftentnahme in der Praxis eine leicht durchführbar, diagnostisch wertvolle Maßnahme darstellt. Abschließend kann festgestellt werden, dass die DON- und ZON-Kontaminationen in geringen Konzentrationen beim Milchrind keine besondere Gefahrenquelle darstellen, wenn die Haltungs- und Fütterungsbedingungen optimal gestaltet werden.

Le déoxynivalénol (DON) est une mycotoxine de type trichothécène de type B, principalement produite par le genre Fusarium. C'est l'une des mycotoxines les plus répandues, elle est largement trouvée dans les céréales et les produits dérivés des céréales. Le cadmium est un composant de la croûte terrestre et un polluant environnemental courant. C'est un métal trace non essentiel et toxique pour la santé des humains et des animaux. Bien que la toxicité individuelle du DON et du Cd ait été bien étudiée, leur effet combiné est peu étudié. L'intestin étant le premier organe ciblé par les contaminants alimentaires, le but de cette étude est d'explorer l'effet combiné du DON et du Cd sur la fonction de barrière intestinale à l'aide de modèles in vitro, in vivo et ex vivo. In vitro, des cellules épithéliales intestinales humaines Caco-2 ont été traitées avec une série de concentrations de DON et de Cd (0-30 μM) seules ou en combinaison. La fonction de barrière des cellules Caco-2 a été évaluée par la mesure de la résistance électrique transépithéliale (TEER), de la perméabilité paracellulaire et des protéines jonctionnelles. Le mélange DON, Cd et DON+Cd a diminué le TEER et augmenté la perméabilité paracellulaire de manière dépendante de la concentration. L'abondance des protéines jonctionnelles E-cadhérine et occludine a été considérablement réduite dans les cellules exposées au DON, au Cd et au DON+Cd, tandis que l'expression de ZO-1 et de claudine-3 et -4 est restée inchangée. Le mélange DON Cd a eu des effets légèrement supérieurs ou similaires à ceux du contaminant le plus toxique. In vivo, les rats ont été exposés à des aliments contaminés par du DON (8,2 mg / kg), et à de l'eau de boisson contaminée par du Cd (5 mg / L) ou au mélange DON+Cd pendant 4 semaines. Les résultats n'ont montré aucun effet sur la prise de poids corporel au cours de l'expérience. Des dommages morphologiques légers caractérisés par un oedème au niveau de la lamina propria et un aplatissement et une fusion des villosités ont été découverts chez le rat exposé à chaque contaminant. Le score lésionnel du jéjunum était plus élevé chez tous les animaux traités que chez les animaux témoins. Une diminution significative de la profondeur de la crypte jéjunale a été observée chez les rats exposés au DON, au Cd et au DON+Cd, alors que la hauteur des villosités n'était pas affectée. Une immunomarquage plus faible de l'E-cadhérine dans le jéjunum de rats exposés à des contaminants seuls ou en association a également été observée, alors que l'occludine n'a diminué que chez les rats exposés au DON et au DON+Cd. Comme indiqué in vitro, l'exposition in vivo au DON et au Cd a induit des effets similaires à ceux du contaminant le plus toxique. Des explants jéjunaux de porcs ex vivo ont été exposés au DON (0-24 μM), au Cd (0-96 μM) et à la combinaison de DON+Cd. Le DON seul et le mélange DON Cd ont stimulé la réponse immunitaire chez le jéjunum en régulant positivement l'expression d'ARNm de IL-1, IL-1, IL-8 et TNF- de manière dose-dépendante, tandis que le Cd seul n'a pas affecté ces gènes. L'expression génique des métallothionéines (MT), y compris MT1A et MT2A, était régulée positivement de manière dose-dépendante par le Cd seul et le mélange, mais n'était pas affectée par le DON seul. La régulation à la hausse des gènes de cytokines et de MT induite par le DON+Cd était similaire à celle obtenue par le DON ou le Cd seul. En conclusion, le DON et le Cd modifient tous deux la fonction de barrière intestinale et l'effet combiné est similaire avec leur effet individuel. L'effet du mélange n'a démontré aucune synergie, ce qui suggère que la réglementation sur chaque contaminant protège suffisamment les consommateurs exposés aux mélanges de DON et de Cd
Deoxynivalenol (DON) is a type B trichothecene mycotoxin mainly produced by Fusarium genus. It is one of the most prevalent mycotoxins widely found in cereals and cereal-derived products. Cadmium is a component of earth’s crust and also a common environmental pollutant. It is a nonessential trace metal and toxic for humans and animals health. Although the individual toxicity of DON and Cd has been well investigated, their combined effect is poorly studied. As intestine is the first organ targeted by food contaminants, the aim of this study is to explore the combined effect of DON and Cd on the intestinal barrier function using in vitro, in vivo and ex vivo models. In vitro, the human intestinal epithelail cells Caco-2 were treated with a series of concentrations of DON and Cd (0-30 μM) alone or in combination. The barrier function of Caco-2 cells was assessed through the measurement of transepithelial electrical resistance (TEER), paracellular permeability and junctional proteins. DON, Cd and DON+Cd mixture decreased the TEER and increased the paracellular permeability in a concentration-dependent manner. The abundance of junctional proteins E-cadherin and occludin was considerably reduced in cells exposed to DON, Cd and DON+Cd, while the expression of ZO-1, and claudin-3 and -4 remained unchanged. The mixture DON+Cd induced slightly higher or similar effects than the most toxic contaminant. In vivo, rats were exposed to DON-contaminated feed (8.2 mg/kg feed), and Cd-contaminated drinking water (5 mg/L) or to the mixture DON+Cd for 4 weeks. The results showed no effect on body weight gain during the experiment. Mild morphological damage characterized by edema in lamina propria and villi flattening and fusion was found in rat exposed to each contaminant. The lesional score of jejunum was higher in all the treated animals than that in control animals. A significant decrease of jejunal crypt depth was observed in rats exposed to DON, Cd and DON+Cd, whereas villi height remained unaffected. A lower immunostaining of E-cadherin in the jejunum of rats exposed to contaminants alone or in combination was also observed, whereas occludin was only decreased in rats exposed to DON and DON+Cd. As shown in vitro, in vivo exposure to both DON and Cd induced similar effects than the most toxic contaminant. Ex vivo, jejunal explants of pigs were exposed to DON (0-24 μM), Cd (0-96 μM) and in combination DON+Cd. DON alone and mixture DON+Cd stimulated immune response in jejunum by upregulating mRNA expression of IL-1, IL-1, IL- 8 and TNF- in a dose-dependent manner, while Cd alone did not affect these genes. Gene expression of metallothioneins (MTs) including MT1A and MT2A was dose-dependently upregulated by Cd alone and mixture, but not affected by DON alone. The upregulation of cytokine and MTs genes induced by DON+Cd was similar than by DON or Cd alone. In conclusion, both DON and Cd alter intestinal barrier function and the combined effect is similar with their individual effect. The effect of the mixture did not demonstrate any synergy, suggesting that regulation on individual contaminant is protective enough for consumers exposed to DON and Cd mixtures

Déoxynivalenol (DON) et nivalénol (NIV), fusariotoxines majeures des céréréales peuvent endommager l'intestin. Les effets de DON et NIV sur la muqueuse intestinale ont été étudiés chez le porc, après exposition aigüe d'explants jéjunaux après 4 heures (0 à 10 µM), d'anses jéjunales après 4 et 24 heures (0 à 10 µM), et après exposition d'animaux pendant 28 jours, à des aliments naturellement contaminés. Ex vivo sur les explants, des modifications histologiques dose-dépendantes ont été induites. In vivo, la diminution du nombre de cellules villositaires en prolifération a été concordante pour les anses jejunales et chez les animaux, atteignant, pour DON et NIV respectivement, 13 et 30% après 4h, et après 24 heures 35% et 40%, à 10 µM. Dans les anses, l'apoptose a été induite au niveau des villosités à 10µM de DON et de NIV. Après 24 heures, le nombre d'entérocytes apoptotiques a augmenté de façon dose dépendante avec DON, NIV, et DON+NIV (1:1), et l'analyse d'interaction a montré une synergie pour ce paramètre
Deoxynivalenol (DON) and nivalenol (NIV), major fusariotoxins and worldwide cereal contaminants, raise concerns for intestinal health. The impact of DON and NIV on pig intestinal mucosa was investigated after acute exposure on jejunal explants after 4 hours (0 to 10 µM), on jejunal loops after 4 hours and 24 hours (0 to 10 µM), and after 28-day natural contamination feeding of animals. On explants, dose-dependent increases in the histological changes were induced. The decrease in the overall proliferative villus cells was concordant between animal experiment and loops, reaching after 4 hours in loops 13% and 30%, and after 24 hours 35 and 40 % for DON and NIV respectively, at 10 µM. In loops, villus apoptosis increased after DON and NIV at 10 µM. After 24 hours, apoptotic enterocytes increased dose-dependently by DON, NIV, or the combination DON+NIV (1:1). The interaction analysis showed synergism between DON and NIV for villus enterocyte apoptosis

Increased interest in value-added traits of soft winter wheat (SWW; Triticum aestivum L.), such as white-seed coat and gluten strength, has resulted from economic incentives for these traits. The first objective of this study was to determine whether differences existed between red- and white- seeded progeny of 17 populations. When abiotic and biotic stresses were negligible, significant differences were not detected between red- and white-seeded progeny, except for yield: red-seeded progeny had a significantly higher yield than the white-seeded progeny. However, when abiotic and biotic stresses were larger, the yield of white-seeded progeny was not significantly different from red-seeded progeny and the white-seeded progeny accumulated a significantly greater amount of deoxynivalenol (DON) than red-seeded progeny. Therefore, Kentucky producers should be cautious when considering production of white-seeded cultivars. The second objective of this study was to determine whether early- or late- generation selection for white-seeded progeny produced a higher frequency of superior white-seeded lines. Three selection methods were studied. Late-generation bulk selection produced a significantly lower frequency of superior white-seeded lines (1.7%) than single seed descent (SSD; 13.9%); the early-generation bulk (9.6%) did not differ statistically from either method. Although SSD produced the most superior lines, the utility of SSD breeding will have to be assessed by SWW breeders to justify additional labor and space requirements. The final objective was to determine whether early-generation selection of wheat quality, as determined by wheat meal-based assays, was effective. A cross between a strong gluten soft red winter and a weak gluten soft white winter wheat was examined. Significant correlations and regressions between wheat meal assays and flour-based assays were found. High heritability and realized genetic gains were also observed. Therefore, early-generation selection for quality characteristics appears to be effective.

Fumonisins, zearalenone and trichothecenes, such as deoxynivalenol (DON) and T-2 toxin, are the major Fusarium mycotoxins occurring throughout the world in cereal grains and animal feeds. Direct ovarian effects of fumonisin B1 (FB1) and its interaction with DON or α-zearalenol (α-ZOL), zearalenone major active metabolite, have so far not been investigated. Thus, the goal of this thesis was to determine if FB1, alone or combined with DON or α-ZOL, can impair swine reproductive activity via affecting granulosa cell function. To this aim, two different studies were designed. In the first study the effects of FB1 alone and combined with DON or α-ZOL on granulosa cell proliferation were evaluated. Porcine granulosa cells from small ovarian follicles (1-5 mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing FB1 at various doses (0, 0.01, 0.4, 10 and 14 μM) and combinations. At the end of the experiments, numbers of granulosa cells were determined using a Coulter counter. The results revealed that FB1 had inhibitory effects on granulosa cell proliferation at doses ≥ 10 μM. DON (3.4 μM) strongly inhibited (by 80%; P < 0.0001) granulosa cell proliferation and no significant difference was detected in combination with FB1 (10 μM). α-ZOL (9.4 μM) showed a stimulatory effect (P < 0.01) on granulosa cell numbers, even when treated in combination with FB1 (10 μM). In the second study the effects of FB1 alone and combined with DON or α-ZOL on granulosa cell steroid production and gene expression were investigated. Porcine granulosa cells from small follicles (1-5 mm) were cultured as described above. At the end of the experiments, concentrations of progesterone and estradiol in culture medium were determined by radioimmunoassays. Real-time RT-PCR was used to elucidate the effects of FB1 on gene expression of P450scc (CYP11A1) and aromatase (CYP19A1). All doses of FB1 (i.e., 0.01, 0.4, 10 and 14 µM) had no significant effect on estradiol production, whereas FB1 showed a stimulatory effect on progesterone production induced by FSH plus insulin-like growth factor-I (IGF-I) at 10 and 14 µM. α-ZOL (9.4 µM) increased (P < 0.0001) FSH plus IGF-I-induced progesterone production by 51%. Combination of FB1 with α-ZOL resulted in an increase of progesterone production (91%; P < 0.0001) that was significantly higher than that induced by either Fusarium mycotoxin alone. DON drastically inhibited (by 74%; P < 0.0001) progesterone production and FB1 had little effect on this response. α-ZOL had no effect on FSH plus IGF-I-induced estradiol production, whereas decreased (P < 0.05) estradiol production when co-treated with FB1. DON (3.4 µM) strongly inhibited (by 67%; P < 0.0001) estradiol production and no difference was detected in combination with FB1 (10 µM). FB1 (10 µM) had no effect on granulosa cell CYP19A1 mRNA abundance, whereas decreased (by 23%; P < 0.0001) granulosa cell CYP11A1 mRNA abundance induced by FSH plus IGF-I. In conclusion, the present thesis indicates that FB1 has direct effects on porcine granulosa cell proliferation, steroid production and gene expression and provides information on the interactions between FB1 and DON or α-ZOL in granulosa cells. These interactions and direct ovarian effects should be considered in swine reproductive failures.

Fusarium graminearum, the causative agent of Fusarium head blight, is an economically important pathogen of wheat (Triticum aestivum). Breeding Fusarium head blight (FHB) resistant wheat requires knowledge of the underlying genetic control of FHB resistance. Genetic parameters for FHB resistance and five related traits were estimated in three populations at two locations and in two years. Moderate broad sense heritabilities for FHB severity and Fusarium damaged kernels (FDK) were observed. Incidence of FHB and the toxin deoxynivalenol (DON) accumulation had low to moderate broad sense heritabilities. Correlations between FDK and severity and FDK and DON were moderate to high in the three populations and do support indirect selection for FHB severity or DON based on FDK data alone, but it is important to be cautious in years with a high disease pressure when FHB resistance could be masked. A cycle of among-family and within-family selection cycle was conducted in 2003. Actual selection gain was higher than predicted gain based on variance components in 2003 in the within-family selection study. One population had also a strong response for low DON in the among-family selection study. The observed results suggest that selection for FHB resistant genotypes could be achieved with a recurrent selection scheme. Along with conventional breeding, molecular techniques are being used in breeding for FHB resistance. A first genotypic screening of the three populations showed Population 2 had the presence of a resistance allele form the resistant Chinese cultivar Sumai 3. Although Populations 1 and 3 did not have the resistance allele, the results suggest other sources of resistance might be present in these two populations.

Fusarium graminearum, the causative agent of Fusarium head blight, is an economically important pathogen of wheat (Triticum aestivum). Breeding Fusarium head blight (FHB) resistant wheat requires knowledge of the underlying genetic control of FHB resistance. Two nine-parent diallel analyses were completed in greenhouse and field environments. Combining abilities, variance component ratios, and narrow sense heritabilities for FHB resistance and deoxynivalenol levels were calculated. Significant general and specific combining ability effects were observed. Resistance to FHB seems to be mostly controlled by additive genetic effects with some dominance noted in the field. Resistance noted in the greenhouse environment may not hold up in the field. Genetic parameters for FHB resistance and four related traits were estimated in three populations. Moderate to high broad sense heritabilities for FHB severity and Fusarium damaged kernels (FDK) were observed. Incidence of FHB had low to moderate broad sense heritabilities. Correlations between FDK and severity and FDK and incidence were moderate and low, respectively, and do not support indirect selection for FHB severity or incidence based on FDK data alone. Substantial predicted gains from family selection were observed and therefore selection of FHB resistant wheat lines should be based on family means and not individual selection.

This master thesis deals with monitoring of a content of deoxynivalenol, its metabolite deoxynivalenol-3-b-D-glucopyranoside and ochratoxin A in beer samples collected from retail market in the Czech Republic, Poland and Slovakia. The theoretical part describes general characteristics of mycotoxins, its transfer from field barely through malt to beer and its occurrence in beers. Malting process and brewing technology were also mentioned. Subsequently possibilities for a determination of the mycotoxins by the chromatografic and immunochemical method were presented. The experimental section describes analysis of 30 samples of beer. The analyses were conducted using ultra high-performance liquid chromatography with fluorimetric detection (UPLC/FLR) for ochratoxin A and high-performance liquid chromatography coupled with mass spectrometer (HPLC/MS) for deoxynivalenol and its metabolite. Ochratoxin A was detected in 25 of the 30 samples in concentration range of 0,6 - 82,5 ng·l-1. Deoxynivalenol was found in 24 of the 30 samples with concentration range of 2,29 - 12,57 ug·l-1 and deoxynivalenol-3-b-D-glucopyranoside was occure in 19 of the 30 samples in concentration range of 2,45 - 12,47 ug·l-1. It was also assessed the relationship between beer gushing and presence of mycotoxins in beer. No connection between the parameters has been found. Consequently it is not possible to predict beer gushing from the presence of mycotoxins.