Muitos fungos filamentosos podem produzir metabólitos secundários, denominados micotoxinas, podendo ser encontradas em produtos alimentícios e produtos agrícolas. Um dos principais gêneros de fungos micotoxigênicos relacionados à cadeia alimentar é o Aspergillus spp. Existem mais de 400 micotoxinas descritas na literatura, sendo as mais comuns as aflatoxinas B1, B2, G1 e G2. As micotoxinas são frequentemente encontradas em alimentos e são consideradas como um dos mais perigosos contaminantes, sendo a aflatoxina B1 classificada no Grupo 1 pela International Agency of Research on Cancer. As aflatoxinas são termorresistentes, resistindo por mais de uma hora em autoclave, fazendo-se necessário outro meio de degradação dessas toxinas. Este trabalho teve como objetivo observar os efeitos da radiação gama de 60Co e de feixes de elétrons na degradação das aflatoxinas e comparar os danos causados na morfologia do fungo Aspergillus flavus. O fungo foi cultivado em agar batata dextrose (PDA) por 10 dias e posteriormente foi transferido para o meio agar coco, sendo mantido por 14 dias à 25 °C. Após esta etapa o agar coco foi triturado até se tornar um meio pastoso homogêneo e foi irradiado com doses de 2,5, 5,0, 10 e 20 kGy. As amostras utilizadas na microscopia eletrônica de varredura foram irradiadas com as doses de 0, 2,5, 5,0, 10 e 20 kGy com fontes de 60Co e de feixes de elétrons. A irradiação com acelerador de elétrons apresentou uma degradação ligeiramente superior à radiação gama, reduzindo 29,93 %, 34,50 %, 52,63 % e 72,30 % para as doses de 2,5, 5,0, 10 e 20 kGy, respectivamente. A microscopia eletrônica de varredura demonstrou que as doses de 2,5 até 10 kGy não causaram danos no fungo, porém com a dose de 20 kGy pode-se observar danos nas estruturas fúngicas. / Many filamentous fungi can produce secondary metabolites, called mycotoxins, which can be found in food and agricultural products. One of the main genera of mycotoxigenic fungi related to the food chain is the Aspergillus spp. There are over 400 mycotoxins described in the literature, the most common the aflatoxins B1, B2, G1 and G2. The mycotoxins are commonly found in foods and are considered one of the most dangerous contaminants. The aflatoxin B1 is classified in group one by the International Agency of Research on Cancer. Aflatoxins resisting for more than one hour in autoclave making it necessary to other means of degradation of these toxins. This work aimed to observe the effects of gamma radiation of 60Co and electron beams in the degradation of aflatoxins and compare the damage caused on the morphology of the Aspergillus flavus. The fungus was grown on potato dextrose agar (PDA) for 10 days and was subsequently transferred to coconut agar medium, and maintained for 14 days at 25 °C. After this step the coconut agar was ground to become a homogeneous pasty and was irradiated with doses of 2.5, 5.0, 10 and 20 kGy. The samples used in scanning electron microscopy were irradiated with doses of 0, 2.5, 5.0, 10 and 20 kGy with sources of 60Co and electron beams. Irradiation with electron accelerator showed a slightly higher degradation to gamma radiation, reducing 29.93 %, 34.50 %, 52.63 % and 72.30 % for doses of 2.5, 5.0, 10 and 20 kGy, respectively. The Scanning Electron Microscopy showed that doses of 2.5 to 10 kGy did not cause damage to the fungus, but with a dose of 20 kGy it can be observed fungal damage to structures. SUMÁRIO

Aspergillus fumigatus est un agent pathogène respiratoire majeur chez les oiseaux d’ornement comme de production. L’aspergillose qui peut être responsable de mortalités importantes et de chutes de performances est difficile à traiter. Nous avons développé un modèle d’aspergillose aiguë chez le dindonneau en inoculant différents lots d’oiseaux âgés de moins d’une semaine via une aérosolisation intratrachéale de doses croissantes de conidies (105 à 108/animal) en utilisant un MicroSprayer®. Le développement de la masse fongique a été évalué par qPCR, dosage du galactomannane (GM), culture fongique et évaluation histopathologique dans le but de comparer les résultats obtenus en fonction du nombre de conidies inoculées. Une mortalité significative a été observée dans les 4 jours suivant l’inoculation uniquement pour l’inoculum le plus concentré. Les résultats des différents marqueurs du développement du champignon (culture, qPCR et GM), sont très bien corrélés avec la dose de l’inoculum administrée. Les moyennes d’équivalents conidies/g de poumon obtenues par qPCR étaient 1,3 log10 plus importantes que les numérations obtenues par culture sur gélose. Ce nouveau modèle incluant une combinaison inédite de biomarqueurs chez la dinde a été utilisé pour évaluer l’efficacité de l’énilconazole, seule molécule utilisée en élevage avicole pour lutter contre l’aspergillose / Aspergillus fumigatus remains a major respiratory pathogen in both ornamental and poultry. Aspergillosis can be responsible for high mortality rates and induces significant economic losses, particularly in turkey production, and it is still difficult to treat. We developed a new model of acute aspergillosis in young turkeys by inoculating few-days-old turkeys via intratracheal aerosolization with increasing concentrations (105 up to 108) of conidia using a MicroSprayer® device. The fungal burden was assessed and compared by real-time PCR, galactomannan (GM) dosage, fungal colony (CFU) counting and by histopathology. Early death occurred in the first 96 h post-inoculation only at the highest inoculum dose. We observed a correlation between inoculum size and results obtained by real-time PCR, GM dosage and CFU counting. The mean fungal burden detected by qPCR was 1.3 log10 units higher than the mean values obtained by CFU measurement. Furthermore, this new model, with its unique combination of markers, has been used to evaluate the efficacy of enilconazole

O projeto teve por finalidade isolar e identificar fungos, avaliar o potencial toxigênico dos isolados de Aspergillus spp. e Fusarium spp., detectar e quantificar aflatoxinas B1, B2, G1, G2 e fumonisinas B1 e B2 em amostras de misturas de cereais. Foram analisadas 15 marcas de misturas de cereais, prontas para o consumo, adquiridas de supermercados e de empresas que comercializam o produto nacionalmente via internet. Foram adquiridas amostras por sete meses, totalizando 105 amostras ao final do experimento. A contagem de bolores nas amostras variou de 1,0 x 101 a 2 x 105 unidades formadoras de colônias (UFC)/g, com isolamento de sete cepas de Aspergillus flavus. As aflatoxinas B1 e G1 foram detectadas em poucas amostras e em baixos níveis, sendo que estes resultados podem ser devidos à baixa atividade de água nos produtos avaliados, a qual foi inferior a 0,63. A fumonisina B1 foi detectada em 84,8% das amostras, no entanto, a ingestão diária provável calculada para as fumonisinas esteve abaixo da recomendação do JECFA. Apenas uma amostra apresentou níveis de fumonisinas acima do limite esperado para 2016. Adicionalmente, foi observado que 21% das amostras apresentaram mais de um tipo de micotoxina, o que poderia conduzir à potencialização de efeitos tóxicos. / The project aimed to isolate and identify fungi, evaluate the toxigenic potential of isolates of Aspergillus spp. and Fusarium spp. detect and quantify aflatoxins B1, B2, G1, G2 and fumonisins B1 and B2 in samples of cereal mixtures. We analyzed 15 brands of cereal mixtures ready to eat adding up to 105 samples at the end of the experiment. Samples were acquired in supermarkets and from companies that market the product nationally by internet. The mold count in the samples ranged from 1.0 x 101 to 2 × 105 colonies forming units (CFU)/ g, with isolation of seven strains of Aspergillus flavus. Aflatoxin B1 and G1 were detected in a few samples and at low levels, what might be due to the low water activity in the product reviews, which was less than 0.63. Fumonisin B1 was detected in 84.8% of the samples, however, the probable daily intake calculated for fumonisin was bellow the JECFA recommendation. Only one sample showed fumonisin levels above the expected limit for 2016. Additionally, it was observed that 21% of the samples presented more than one type of mycotoxin, which could lead to enhancement of toxic effects.

Le champignon microscopique Aspergillus fumigatus provoque un nombre important de maladies graves. Parmi elles, l’aspergillose pulmonaire chronique (APC) et l’aspergillose broncho-pulmonaire allergique (ABPA) affectent 3 et 4,8 millions de personnes dans le monde, respectivement.L’APC est très souvent mortelle si elle n’est pas soignée. Elle se développe très souvent après une tuberculose. C’est donc une maladie des pays émergents, où il n’est souvent pas possible de la diagnostiquer à cause du coût trop important des techniques existantes.L’ABPA est une complication très grave de l’asthme et de la mucoviscidose, qui complique fortement ces maladies. Elle est très difficile à diagnostiquer.Notre travail a donc consisté à développer et évaluer deux tests, un test rapide permettant de poser le diagnostic d’APC sans avoir à utiliser de matériel de laboratoire à destination des pays émergents et un western blot qui permet la confirmation du diagnostic d’ABPA. / Aspergillus fumigatus is a microscopic fungus that can cause numerous diseases. Among them, chronic pulmonary aspergillosis (CPA) and allergic broncho-pulmonary aspergilloses (ABPA) affect 3 and 4.8 million people, respectively.CPA is often fatal if left untreated. It is often a complication of tuberculosis and therefore affect low and middle income countries. However, it is difficult to diagnose it in those countries, as the tests are too expansive.ABPA is a severe complication of asthma and cystic fibrosis, worsening those diseases. It’s very hard to diagnose it.Our work was to develop and evaluate two tests, a rapid test for the diagnosis of CPA that does not require laboratory equipment designed for low and middle income countries and a western blot for confirmation of ABPA diagnosis.

No description available.

La presente Memoria de Tesis Doctoral, presentada en forma de compendio de publicaciones, recoge el estudio fisiológico y molecular realizado con especies del género Aspergillus sección Nigri productoras de ocratoxina A (OTA). Esta micotoxina está recibiendo una especial atención en todo el mundo debido a su marcado carácter nefrotóxico. Además, es una sustancia carcinógena, teratógena e inmunotóxica y desde el año 2002, la UE ha comenzado a establecer niveles máximos de esta micotoxina en algunos alimentos. Estudios muy recientes han puesto claramente de manifiesto la implicación de algunas especies del género Aspergillus de la sección Nigri (A. carbonarius y las incluidas en el "agregado A. niger") en la presencia de OTA en alimentos como la uva, las pasas y el vino, entre otros. No obstante, no se conocen hasta el momento las condiciones ambientales que favorecen la producción de la micotoxina por estas especies.En los modelos fisiológicos utilizados, se han incluido cepas de A. carbonarius y del agregado A. niger, seleccionadas en base a su diferente origen y capacidad ocratoxígena. Se ha estudiado el efecto del sustrato (medios CYA y YES), la temperatura (5-45ºC), la actividad de agua (aw) (0,78-0,99) y el pH (2-10) en el crecimiento y la producción de OTA. Asimismo, se ha analizado la relación filogenética de las cepas estudiadas mediante diferentes técnicas de biologíamolecular (RFLP, RAPD, secuenciación, ERIC-PCR, AFLP y microsatélites). Los resultados obtenidos han permitido determinar las condiciones óptimas para la producción de OTA en las cepas del agregado A. niger (medio YES a 20-25ºC en el intervalo 0,96-0,99 aw y pH 5-10) y en las de A. carbonarius (medio CYA a 15-20ºC en el intervalo 0,98-0,99 aw y pH 5-7). En relación a la caracterización molecular de las cepas estudiadas, las técnicas de RAPD y secuenciación confirman la separación del agregado A. niger en dos grupos que se corresponden con los patrones de RFLP (N y T) establecidos previamente. En el caso de A. carbonarius estas técnicas han permitido la diferenciación molecular de una de las cepas estudiadas. Esta cepa pertenecería a la nueva especie propuesta dentro de la sección Nigri, denominada "A. ibericus".Las técnicas ERIC-PCR, AFLP y microsatélites han resultado útiles para la caracterización molecular de las especies estudiadas. Los resultados más significativos se han obtenido mediante AFLP y el análisis de microsatélites, permitiendo ambos la diferenciación de las cepas del agregado A. niger en dos grupos que se corresponden con los patrones de RFLP N y T.Los resultados obtenidos ponen de manifiesto la capacidad de las cepas del agregado A. niger y A. carbonarius para crecer y elaborar OTA en amplios márgenes de temperatura, pH y actividad de agua. Esta capacidad permite explicar el papel que juegan estas especies ocratoxígenas en la presencia de esta micotoxina en los alimentos y especialmente en aquellos donde constituyen la micobiota predominante. / The present work, presented as compendium of publications, is focused on the physiology and molecular study of species within Aspergillus section Nigri (black aspergilli) able to produce ochratoxin A (OTA). This mycotoxin is achieving an increasing attention worldwide due to its acute nephrotoxicity. It is also carcinogenic, teratogenic and is clearly an immunosuppressive agent. Since 2002 the European Commission has established limits for OTA in some food commodities. Recently, several studies have highlighted the involvement of several species of black aspergilli (A. carbonarius and A. niger aggregate) in the presence of OTA in food commodities such as grapes, raisins and wine. However, the environmental conditions which support the production of this mycotoxin by these species are still unknown.Strains of A. carbonarius and A. niger aggregate, selected to include different sources and ochratoxigenic abilities, have been included in the physiology study. The effect of substrate (CYA and YES media), temperature (5-45ºC), water activity (aw) (0.78-0.99) and pH (2-10) has been studied. Analysis of genetic relationships within the studied strains has been also performed through different molecular techniques (RFLP, RAPD, sequencing, ERIC-PCR, AFLP and microsatellites).The results obtained show the optimal conditions for the production of OTA in A. niger aggregate strains (YES medium, 20-25ºC, 0.96-0.99 aw and pH 5-10) and in A. carbonarius strains (CYA medium 0.98-0.99 aw and pH 5-7). With regard to the molecular characterization of the studied strains, RAPD and sequencing techniques confirm the division of the A. niger aggregate into two groups which correspond with the previously described RFLP patterns (N and T). In the case of A. carbonarius one strain differs from the rest and it may belong to the new species proposed in section Nigri, "A. ibericus". The molecular markers ERIC-PCR, AFLP and microsatellites have been useful to characterize the studied species. The most significant results have been obtained through AFLP and microsatellites. Both markers make possible the differentiation of the A. niger aggregate strains in two groups which correspond with the two RFLP patters N and T.The results obtained state the ability of A. niger aggregate and A. carbonarius strains to grow and produce OTA in wide ranges of temperature, pH, and water activity. This ability let understand the role of these ochratoxigenic species in the presence of this mycotoxin in food commodities, especially in those where they are the predominant isolated fungi.

The combined effect of Modified Atmosphere Packaging (MAP) involving gas packaging, oxygen absorbent and other environmental factors to control aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in both synthetic media and peanuts were studied using a process optimization technique termed Response Surface Methodology (RSM). Regression analysis of the data indicated that water activity (a$ sb{ rm w}$), pH, storage temperature, initial concentration of headspace oxygen and inoculum level were all highly significant factors (p 0%). These changes in the barrier characteristics influenced the headspace gas composition within the product and under modified atmospheres hence the level of aflatoxin detected in these stored products. / In conclusion, this study has shown that the combined effect of several "barriers" can be used in conjunction with low oxygen modified atmosphere and high barrier packaging films to inhibit or reduce aflatoxin to safe and acceptable levels, particularly at abusive temperatures encountered during storage.

A.fumigatus ist der häufigste Erreger invasiver, meist tödlich verlaufender Aspergillosen bei stark immunsupprimierten Patienten. Eine natürliche Resistenz gegen den Pilz wird durch das angeborene Immunsystem vermittelt, jedoch wurde im Tierversuch auch eine erworbene Immunität nach einer überlebten systemischen Infektion festgestellt. Deshalb sollten Antigene, die im Verlauf der Erkrankung vom Pilz exprimiert werden, eine spezifische und protektive Immunantwort hervorrufen können. Für die Entwicklung eines Impfstoffes ist die Identifikation solcher Antigene Voraussetzung. Im Mausmodell wurden insgesamt 31 rekombinante Aspergillus-Antigene in Kombinationen getestet. Als besonders erfolgversprechend stellte sich ein trivalenter Impfstoff, bestehend aus Enolase, HSP90 und TBP (Thiamin-Biosyntheseprotein), dar. Im Vergleich zu einer ungeimpften Kontrollgruppe fand sich bei der Impfgruppe eine signifikante Protektion gegenüber einer an sich letalen Aspergillus-Infektion. Bei einer Wiederholung des Versuchs ließ sich der Schutz bestätigen, war jedoch nicht mehr im Signifikanzbereich. Mögliche Gründe hierfür sind die unsichere Aufnahme der Infektionsdosis durch nasale Verabreichung oder auch eine „Stille Feiung“ der Mäuse durch Einatmung von Konidien aus der Umgebung vor der eigentlichen Infektion. Eine Verbesserung der Versuchsbedingungen könnte durch die Verwendung individuell ventilierter Käfige (ohne Sporenbelastung aus der Raumluft) und/oder eine Vergrößerung der Versuchsgruppen erreicht werden. Da die Konidienoberfläche den ersten Kontakt mit dem Wirt vermittelt, könnten Konidienoberflächenproteine sowohl vielversprechende Impfstoffkandidaten als auch Virulenzfaktoren sein. Im Tierversuch wurde hier nachgewiesen, dass das stark exprimierte Konidienoberflächenprotein HP16 entscheidend an der Virulenz von A.fumigatus beteiligt ist. Dabei stellte sich der Virulenzunterschied zwischen der Deletionsmutante ΔHP16 und dem Wildtyp D141 als hoch signifikant dar. Der Vergleich zwischen der Deletionsmutante ΔHP16 und der Komplementmutante ΔHP16K erbrachte eine komplett restaurierte Virulenz. Da HP16 zumindest in vitro vorwiegend auf Konidien lokalisiert ist, könnte sich hier ein Angriffspunkt für eine präemptive Therapie bzw. für eine Prophylaxe der invasiven Aspergillose ergeben. HP16 wurde auch als rekombinantes Protein hergestellt. Das rekombinante Antigen soll als Impfstoffkandidat in zukünftigen Projekten eingesetzt werden. Letztlich wurde noch Aspf3, eine Peroxireduktase von A.fumigatus, als ein weiterer Target- und Impfstoffkandidat untersucht. Die stark verminderte Virulenz der Deletionsmutante ΔAspf3 und eine zumindest teilrestaurierte Virulenz durch die hergestellte Komplementmutante ΔAspf3K lassen vermuten, dass Aspf3 während der Pathogenese ebenfalls eine entscheidende Rolle spielt.

Univ., Diss.--Regensburg, 1997.

Muitos fungos filamentosos podem produzir metabólitos secundários, denominados micotoxinas, podendo ser encontradas em produtos alimentícios e produtos agrícolas. Um dos principais gêneros de fungos micotoxigênicos relacionados à cadeia alimentar é o Aspergillus spp. Existem mais de 400 micotoxinas descritas na literatura, sendo as mais comuns as aflatoxinas B1, B2, G1 e G2. As micotoxinas são frequentemente encontradas em alimentos e são consideradas como um dos mais perigosos contaminantes, sendo a aflatoxina B1 classificada no Grupo 1 pela International Agency of Research on Cancer. As aflatoxinas são termorresistentes, resistindo por mais de uma hora em autoclave, fazendo-se necessário outro meio de degradação dessas toxinas. Este trabalho teve como objetivo observar os efeitos da radiação gama de 60Co e de feixes de elétrons na degradação das aflatoxinas e comparar os danos causados na morfologia do fungo Aspergillus flavus. O fungo foi cultivado em agar batata dextrose (PDA) por 10 dias e posteriormente foi transferido para o meio agar coco, sendo mantido por 14 dias à 25 °C. Após esta etapa o agar coco foi triturado até se tornar um meio pastoso homogêneo e foi irradiado com doses de 2,5, 5,0, 10 e 20 kGy. As amostras utilizadas na microscopia eletrônica de varredura foram irradiadas com as doses de 0, 2,5, 5,0, 10 e 20 kGy com fontes de 60Co e de feixes de elétrons. A irradiação com acelerador de elétrons apresentou uma degradação ligeiramente superior à radiação gama, reduzindo 29,93 %, 34,50 %, 52,63 % e 72,30 % para as doses de 2,5, 5,0, 10 e 20 kGy, respectivamente. A microscopia eletrônica de varredura demonstrou que as doses de 2,5 até 10 kGy não causaram danos no fungo, porém com a dose de 20 kGy pode-se observar danos nas estruturas fúngicas. / Many filamentous fungi can produce secondary metabolites, called mycotoxins, which can be found in food and agricultural products. One of the main genera of mycotoxigenic fungi related to the food chain is the Aspergillus spp. There are over 400 mycotoxins described in the literature, the most common the aflatoxins B1, B2, G1 and G2. The mycotoxins are commonly found in foods and are considered one of the most dangerous contaminants. The aflatoxin B1 is classified in group one by the International Agency of Research on Cancer. Aflatoxins resisting for more than one hour in autoclave making it necessary to other means of degradation of these toxins. This work aimed to observe the effects of gamma radiation of 60Co and electron beams in the degradation of aflatoxins and compare the damage caused on the morphology of the Aspergillus flavus. The fungus was grown on potato dextrose agar (PDA) for 10 days and was subsequently transferred to coconut agar medium, and maintained for 14 days at 25 °C. After this step the coconut agar was ground to become a homogeneous pasty and was irradiated with doses of 2.5, 5.0, 10 and 20 kGy. The samples used in scanning electron microscopy were irradiated with doses of 0, 2.5, 5.0, 10 and 20 kGy with sources of 60Co and electron beams. Irradiation with electron accelerator showed a slightly higher degradation to gamma radiation, reducing 29.93 %, 34.50 %, 52.63 % and 72.30 % for doses of 2.5, 5.0, 10 and 20 kGy, respectively. The Scanning Electron Microscopy showed that doses of 2.5 to 10 kGy did not cause damage to the fungus, but with a dose of 20 kGy it can be observed fungal damage to structures. SUMÁRIO