As aflatoxinas são substâncias naturais que apresentam efeitos tóxicos aos humanos e são reconhecidamente carcinogênicas. Estas substâncias podem estar presentes na dieta humana ou, em casos específicos, no ar respirado. Desta maneira, a exposição humana às aflatoxinas é objeto de muita preocupação. Uma das maneiras mais eficazes de avaliar a exposição humana as aflatoxinas é através da mensuração da presença de biomarcadores da exposição a estas substâncias em fluidos biológicos. Dentre as possibilidades de biomarcadores de exposição às aflatoxinas tem-se que aflatoxina M1 (AFM1), presente na urina e leite humano, é considerada um biomarcador válido. Assim sendo, o objetivo deste trabalho de pesquisa foi avaliar a presença de AFM1 em amostras de urina provenientes de indivíduos residentes na região urbana e rural da cidade de Piracicaba-SP, assim como, de leite de gestantes de Piracicaba e cidades da região. Nos indivíduos doadores de amostras de urina foi levantado também o padrão de ingestão de alimentos com alto risco de conter aflatoxinas, através da aplicação de inquéritos de freqüência alimentar e recordatórios 24 horas. A análise de AFM1 em urina e leite foi realizada por cromatografia liquida de alta eficiência (CLAE) com detecção por fluorescência. A extração e purificação do extrato foram realizadas com auxílio de colunas de imunoafinidade. No total 69 amostras de urina e 18 de leite foram analisadas. Entre as amostras de urina detectou-se a presença de AFM1 em 54 (78%) das amostras, com concentrações variando de 1,8 até 39,9 pg/mL. Não foi observada diferença estatística entre as concentrações médias detectadas entre urinas de indivíduos da zona urbana e rural, bem como no nível de consumo de produtos de risco. Apesar das concentrações de AFM1 detectadas serem inferiores as concentrações médias reportadas em outros países a freqüência de amostras positivas foi bastante elevada mostrando que as populações estudadas estão sendo expostas às aflatoxinas. Assim, melhores avaliações dos níveis de exposição necessitam ser realizados considerando que a amostragem utilizada foi pontual, pode existir variação de contaminação sazonal com aflatoxinas na dieta e a contaminação é heterogênea dentro no alimento. Não foi observada uma correlação entre o nível do consumo de produtos de risco e as concentrações detectadas em amostras de urina. Apenas uma amostra de leite apresentou contaminação detectada; entretanto, o nível de contaminação estava entre o limite de detecção (LD) e o limite de quantificação (LQ). / Aflatoxins are natural substances that present toxic and carcinogenic effects to humans. These substances may be present in human diet or, in specific cases, in the breathing air. Thus, the human exposition to aflatoxins is object of concern. One of the most effective ways to evaluate human exposition to aflatoxins is to measure the presence of biomarkers in biological fluids. Among the possibilities of aflatoxin presence biomarkers, the aflatoxin M1 (AFM1), present in human urine and milk, is considered a valid biomarker. The objective of this work was to evaluate the presence of AFM1 in urine samples from individuals who live in urban and rural areas in the county of Piracicaba, state of São Paulo, Brazil, and in milk of pregnant women from Piracicaba and neighbor cities. Urine-donor individuals were researched in relation to the ingestion of food with high risk of containing aflatoxins through the application of a food frequency questionnaire and 24-hour recall. The analysis of AFM1 in urine and milk was performed through high-performance liquid chromatography (HPLC) with fluorescence detection. The extract purification and extraction were performed with the aid of immunoaffinity columns. Overall, 69 urine and 18 human breast milk samples were analyzed. Among urine samples, the presence of AFM1 was detected in 54 (78%), with concentrations ranging from 1.8 to 39.9 pg/mL. No statistical difference was observed between average concentrations detected in the urine of individuals from urban and rural areas, as well as the consumption of aflatoxin risky food. Although the AFM1 concentrations detected are lower than those reported for other countries, the frequency of positive samples was quite high, showing that the populations studied are exposed to aflatoxins. Thus, further evaluations on the exposition levels should be performed, and considering that the sampling used in this work was punctual, there may be seasonal contamination variations in diet and the contamination level is heterogeneous within a food. No correlation between the consumption of risky food and concentrations detected in urine samples was observed. Only one milk sample presented detected contamination; however, the contamination level was between the limit of detection (LOD) and the limit of quantification (LOQ).

O presente trabalho teve como objetivos verificar os efeitos da radiação gama em grãos de milho contaminados artificialmente com Aspergillus flavus Link produtor de aflatoxinas; demonstrar a aplicação da técnica da Reação em Cadeia da Polimerase (PCR) no diagnóstico de A. flavus, bem como verificar o efeito da radiação no perfil das bandas de DNA. Vinte amostras de grãos de milho com 200 g cada foram irradiadas individualmente com 20 kGy, para eliminar a contaminação microbiana. Em seguida, as amostras foram inoculadas com A. flavus toxigênico (1 x 106 esporos / ml), incubadas por 15 dias a 25 °C em ambiente com umidade relativa ao redor de 97,5% e irradiadas com 0; 2; 5 e 10 kGy. As amostras, 5 para cada dose de irradiação, foram analisadas individualmente quanto ao número de células fúngicas, atividade de água, teste de viabilidade (diacetato de fluoresceína e brometo de etídio), PCR e detecção de aflatoxinas (AFB). Os resultados demonstraram que as doses utilizadas foram efetivas na redução do número de Unidades Formadoras de Colônias (UFC/g), principalmente as doses de 5 e 10 kGy. Em adição, o teste de viabilidade mostrou uma diminuição de células viáveis com o aumento das doses de irradiação. A redução de AFB1 e AFB2 foi mais eficiente com o emprego de 2 kGy, comparativamente à dose de 5 kGy, enquanto a dose de 10 kGy degradou totalmente as aflatoxinas. Além disso, observou-se que AFB2 apresentou-se mais radiosensível. O emprego da técnica de PCR revelou a presença de bandas de DNA em todas as amostras. / The aim of this present study was to verify the effects of gamma radiation on the growth of Aspergillus flavus Link aflatoxins producer; to demonstrate the application of Polymerase Chain Reaction (PCR) technique in the diagnostic of A. Flavus, as well to verify the effect of radiation in the profile of DNA bands. Twenty samples of grains maize with 200 g each were individually irradiated with 20 kGy, to eliminate the microbial contamination. In following, the samples were inoculated with an toxigenic A. flavus (1x106 spores/ml), incubated for 15 days at 25 °C with a relative humidity of around 97,5% and irradiated with 0; 2; 5 and 10 kGy. The samples, 5 to each dose of irradiation, were individually analyzed for the number of fungal cells, water activity, viability test (fluorescein diacetate and ethidium bromide), PCR and aflatoxins (AFB) detection. The results showed that the doses used were effectives in reducing the number of Colony Forming Units (CFU/g) mainly the doses of 5 and 10 kGy. In addition, the viability test showed a decrease of viable cells with increase of irradiation doses. The reduction of AFB1 and AFB2, was more efficient with the use of 2 kGy in comparison with the dose of 5 kGy, while the dose of 10 kGy, degraded the aflatoxins. Thereby, it was observed that AFB2 showed to be more radiosensitive. The use of PCR technique showed the presence of DNA bands, in all samples

Há relatos na literatura de alguns compostos naturais de plantas que são usados para preservação de alimentos e no controle do desenvolvimento de fungos e bactérias que ocorrem em plantas, grãos, cereais e derivados. O presente trabalho teve como objetivo avaliar "in vitro" o efeito do óleo essencial de Eucalyptus globulus e seu composto majoritário sobre o crescimento micelial dos fungos Aspergillus flavus e Aspergillus parasiticus e a produção de aflatoxinas. A composição química do óleo analisado por cromatografia gasosa acoplada ao espectrofotômetro de massa mostrou-nos que o composto majoritário com 89,95% é o 1,8-cineol. Assim foi avaliada a ação por contato e por compostos voláteis do óleo essencial e do 1,8-cineol. O modo de ação por compostos voláteis, do óleo e do composto, foi estatisticamente mais eficiente do à ação por contato, para ambos os fungos. Independente do modo da ação a partir da dose de 500µL do óleo os fungos tiveram comportamentos semelhantes, com mais de 90% de inibição micelial. O composto 1,8-cineol não demonstrou a mesma eficiência que o óleo, produzindo algum efeito inibitório apenas na dose de 1.500µL, com apenas 5,5% de inibição de crescimento micelial. Foi verificado que o óleo essencial e o composto 1,8-cineol não cessaram a produção de aflatoxinas de ambos os fungos mesmo inibindo o crescimento micelial. / Literature describes some natural plants composites that are used to preserve food and to control fungi and bacteria development in plants, grains, cereals and derivatives. The objective of this research was evaluate the effect in vitro of the Eucalyptus globulus essential oil and its major component over mycelial growth and aflatoxin production by Aspergillus flavus and Aspergillus parasiticus. The chemical oil composition, analyzed by gas chromatography connected to mass spectra, showed that 1,8-cineole is the major compound with 89.95%. Thus, the essential oil and the 1,8-cineole were evaluated by the contact and volatiles action. For both analyzed fungi, the oil and the compound action promoted throughout volatile compound were statically more efficient than the action promoted throughout contact. Considering the oil dose of 500µL and so forth, the fungi behaviors were similar independently of the action modes, with more than 90% of mycelial inhibition. The 1,8-cineole compound did not demonstrate the same efficiency that the essential oil did, producing some inhibitory effect only in the dose of 1.500µL, with only 5.5% of inhibition of mycelial growth. It was verified that the essential oil and 1,8-cineole compound did not cease the aflatoxins production in both fungi, even with the inhibition of mycelial growth.

O trabalho teve como objetivo avaliar a presença de fungos e aflatoxinas e ácido ciclopiazônico (ACP) em castanhas-do-brasil a campo e armazenadas, no solo e ar a fim de estabelecer vias de contaminação fúngica. A pesquisa de fungos foi feita pela técnica de semeadura em superfície em meio Ágar batata dextrose e Ágar Aspergillus flavus parasiticus e a determinação de micotoxinas, por cromatografia líquida de alta eficiência. As amostras a campo foram analisadas em: Dia 0; amostras na árvore, Dias 5, 10 e 15; em contato com solo 5, 10 e 15 dias, respectivamente. As armazenadas foram analisadas mensalmente por 11 meses. A campo, os fungos mais prevalentes foram: A. flavus em ouriços e amêndoas; Fusarium spp. em cascas. No solo foram isolados Penicillium spp. e Aspergillus flavus e no ar, Fusarium spp. e Penicillium spp. No armazenamento, em amêndoas, foi constatado A. flavus, Fusarium spp. e A. nomius; e em cascas, Fusarium spp. e A. flavus. Aflatoxinas e ACP não foram detectados. O aumento do tempo de contato das castanhas-do-brasil com o solo foi acompanhado de maior isolamento de A. flavus; sugerindo contaminação da castanha durante etapa a campo. A. nomius e A. parasiticus foram isolados apenas no armazenamento, indicando contaminação no estoque das amêndoas. / The objective of this present study was analyse, in the field and in the storage, mycobiota and the contamination by (aflatoxins and cyclopiazonic acid from Brazil nuts, soil and air samples to determine route of fungi contamination. The Brazil nuts mycobiota was determine by diluition plating method in Potate Dextrose Agar and Aspergillus flavus parasiticus agar and, micotoxins was done by high performance liquid chromatography. Field samples were collected in: day 0, samples still on the tree; days 5, 10 and 15, samples in contact with soil for 5, 10 and 15 days, respectively. The most prevalent fungi were Aspergillus flavus in fruit pods and nuts and Fusarium spp. in shells. Penicillium spp. and A. flavus were isolated from soil, and Fusarium spp. and Penicillium spp. from air. The storage samples were analysed montly during 11 months; and showed predominance A. flavus, Fusarium spp. e A. nomius in nuts, and Fusarium spp. and A. flavus in shells. Aflatoxins and cyclopiazonic acid were not detected. These findings indicate soil as the main source of fungal contamination of Brazil nuts. A. nomius e A. parasiticus were isolated only in storage, suggesting Brazil nuts contamination occurs in this period.

Certains champignons, notamment des Ascomycètes, peuvent synthétiser des métabolites secondaires toxiques pour les hommes et les vertébrés, appelés mycotoxines. Étant donné que la présence de ces champignons dans les aliments de base constitue un risque potentiel pour la santé humaine et animale, les aliments de base sont éliminés lorsqu'ils sont contaminés. La section Flavi est un des groupes de champignons les plus importants du point de vue économique et sanitaire car il comprend des espèces productrices de mycotoxines. Parmi les mycotoxines produites par ce groupe se trouvent les aflatoxines (AF), considérées comme une préoccupation majeure en raison de leurs effets délétères chez les vertébrés. Les espèces de la section Flavi se développent principalement dans les régions tropicales et subtropicales car elles bénéficient de conditions environnementales optimales. De plus, les conditions de récolte et de stockage sont souvent inappropriées, favorisant ainsi leur développement. Dans les régions tempérées, ces espèces se rencontrent moins fréquemment. Cependant, le réchauffement climatique pourrait favoriser leur colonisation. L'identification des espèces d'Aspergillus de la section Flavi est un défi, en raison de l'inter- et intra-variabilité des caractères. Par conséquent, l'utilisation d'une seule méthode d'identification (caractérisation morphologique, moléculaire ou du profil des métabolites secondaires) est insuffisante. Inversement, le développement d'outils moléculaires a facilité la tâche. Le but de notre étude était de déterminer les relations entre les espèces d'Aspergillus de la section Flavi à partir de différents marqueurs moléculaires (ITS, benA, cmdA, amdS, préA, perB, ppgA, aflP, gènes Mat1), puis d'identifier ceux qui permettent une classification des espèces par inférence phylogénétique. L'utilisation de l'inférence phylogénétique dans cette étude a montré qu'il s'agit d'une approche robuste pour identifier les espèces d'Aspergillus de la section Flavi, notamment en confirmant certaines hypothèses déjà proposées pour les espèces de la section Flavi. En effet, l'ajout de marqueurs moléculaires a permis de confirmer le placement phylogénétique des espèces dans la section Flavi. De plus, une nouvelle espèce cryptique a pu être décrite : Aspergillus korhogoensis (appartenant au clade A. flavus). Notre étude a également pu mettre en évidence que les marqueurs moléculaires sélectionnés (benA, cmdA, mcm7, rpb1, preB, preA et ppgA) sont de bons candidats pour l'étude d'autres sections d'Aspergillus. L'utilisation de l'inférence phylogénétique est une méthode élégante permettant d'identifier de façon précise les espèces. Sur la base de nos résultats, il est recommandé d'utiliser des matrices concaténées pour effectuer une inférence phylogénétique dans cette section, et la meilleure combinaison inclut les gènes benA, cmdA, et l'inclusion d'un autre gène : mcm7, rpb1, preB, preA ou ppgA. A l'inverse, l'utilisation du gène ITS chez Aspergillus peut conduire à une sous-estimation de la diversité car le gène est très fortement conservé. L'étude des gènes du loci Mat1 dans la section est utile pour accroître les connaissances sur la reproduction sexuée chez les ascomycètes. De plus, plusieurs fonctions de la machinerie biologique fongique sont liées aux gènes du loci Mat1. La caractérisation du profil métabolique secondaire chez les souches d'Aspergillus de la section Flavi doit être utilisée, non seulement comme outil d'identification, mais également pour discriminer les souches toxinogènes et atoxinogènes. La section Flavi renferme des espèces capables de produire à la fois de mycotoxines et de composés bénéfiques. Parmi les mycotoxines qui devraient faire l'objet d'une attention particulière figurent les AF, l'acide cyclopiazonique, les versicolorines a et b, la stérigmatocystine. Une étude plus approfondie du métabolisme secondaire sera également utile pour la recherche de nouveaux composés bénéfiques. / Some fungi, mostly Ascomycota, are able to synthesize secondary metabolites that are toxic to humans and vertebrates, called mycotoxins. Since the presence of these fungi in staples represents a potential risk to human and livestock health, staples are eliminated when they are contaminated. The section Flavi is one most important group of fungi from an economic and public health point of view because it comprises several mycotoxin producer species. Amongst the mycotoxins produced by this group are aflatoxins (AFs), considered a main concern because of their deleterious effects on humans and vertebrates. Species from section Flavi grow mainly in tropical and subtropical regions where environmental conditions are optimal, and harvest and storage conditions are not always appropriate to avoid production of mycotoxins, which enhance their growth. In temperate regions, these species are less frequent; however, climate changes can favor their colonization. Species identification in Aspergillus section Flavi is challenging because of inter- and intra- variability of traits. Therefore, the use of one identification method (morphological, molecular or secondary metabolite profile characterization) is futile. Conversely, the development of molecular tools has facilitated the task. The aim of this study was to screen the species relationships in Aspergillus section Flavi based on different molecular markers (ITS, benA, cmdA, amdS, preA, preB, ppgA, aflP, Mat1 genes), and subsequently identify which ones allow a fine species classification in the section Flavi by phylogenetic inference. The use of phylogenetic inference in the present study showed that it is a robust approach to identify Aspergillus section Flavi species. The use of this technique confirmed some of the hypotheses proposed in the Flavi section, since more genetic information was added, thus strengthening the placement of the species in the Flavi section. In addition, we described a new cryptic species in this section Aspergillus korhogoensis that is nested in A. flavus clade as the sister taxon of A. parvisclerotigenus. Likewise, the molecular markers (benA, cmdA, mcm7, rpb1, preB, preA or ppgA) were good candidates for studying other sections in Aspergillus. The use of phylogenetic inference is a good method for fine-scale species identification; however, it should be used carefully, and the morphological approach and characterization of secondary metabolites should also be carried out. Based on our results, concatenated matrices are recommended to perform phylogenetic inference in this section, and the best combination includes benA, cmdA, and the inclusion of at least one another gene (preB, mcm7, rpb1, preA or ppgA). Conversely, the use of ITS in Aspergillus may lead to an underestimation of the diversity because the gene is highly conserved. Studying mating type MAT1 loci in the section is helpful to increase the knowledge of sexual reproduction in ascomycetes. In addition, several functions of fungal biological machinery are linked to Mat1 loci genes. Secondary metabolic profile characterization of Aspergillus section Flavi strains should be performed, not only as an identification tool, but also to discriminate toxinogenic and atoxinogenic strains. Section Flavi encloses species able to produce a mixture of mycotoxins and beneficial compounds. Amongst mycotoxins that should be screened are AFs, cyclopiazonic acid, A and B versicolorin, sterigmatocystin, tenuazonic acid. An exhaustive study of the secondary metabolism can also be useful to investigate novel beneficial products.

A esterigmatocistina (ST) é uma micotoxina policetônica produzida por diferentes espécies de Aspergillus e outros gêneros fúngicos como Bipolaris e Chaetomium. Esta toxina é caracterizada como uma bifuranoxantona com fórmula molecular C18H12O6, freqüentemente encontrada como contaminante de diversos produtos alimentícios como sementes oleaginosas e cereais. Possui propriedades carcinogênicas, toxigênicas, mutagênicas e teratogênicas, entretanto, ela é menos tóxica do que a aflatoxina. O fungo Emericella nidulans (Eidam) Vuillemin, cujo anamorfo é o Aspergillus nidulans (Eidam) Winter, foi usado como modelo genético para a investigação de genes envolvidos na via biossintética da esterigmatocistina. Linhagens estoque de Utrecht (originárias de Glasgow) foram analisadas quanto à produção de ST e, entre elas, somente a linhagem UT196 [yA2 (I); metA17 (II); piroA4 (IV)] apresentou produção de 4 ppm de ST (stc+), enquanto que as linhagens UT448 e UT184 mostraram-se não produtoras dessa micotoxina (stc). Embora a linhagem UT196 seja muito bem caracterizada geneticamente, este foi o primeiro relato da produção de ST nessa linhagem. Os índices de segregação alélica e todas as freqüências de recombinação entre os marcadores genéticos ligados e não ligados foram determinados tanto pelo cruzamento meiótico UT448 (stc) x UT196 (stc+) como pelo cruzamento mitótico UT448//UT184. 175 segregantes meióticos e 140 segregantes mitóticos foram analisados quanto à produção de ST, aos marcadores de auxotrofia e de resistência a acriflavina. Essas linhagens cruzadas apresentavam vários marcadores em heterozigose e isso permitiu o mapeamento de um gene estrutural da ST (stcZ+), localizado no braço esquerdo do cromossomo I, 4% distante do gene da riboflavina (riboA1). Como um subproduto deste trabalho, foi detectado um pigmento vermelho, de Rf = 0,90, em todos os segregantes meióticos e mitóticos, produtores ou não de ST, indicando tratar-se, provavelmente, de um pigmento policetônico produzido pelos ascosporos do fungo. A baixa expressão da produção de ST em 13 segregantes meióticos e a alta expressão dessa toxina nos diplóides UT448//UT196 e UT448//UT184 (40 ppm) permitiram concluir a existência de um fator regulador, compreendido na região w-met do cromossomo II, responsável pela expressão do gene estrutural stcZ+. A análise desses genes através do ciclo parassexual sugeriu um comportamento epigenético típico envolvendo esses genes. Com base nos dados obtidos, hipóteses para explicar o controle da expressão desses genes e suas inter-relações foram aqui apresentadas. / Sterigmatocystin (ST) is a polyketide produced by different species of Aspergillus as well as by other fungus genera such as Bipolaris and Chaetomium. This toxin is characterized as a bifuranoxanthone, whose molecular formula is C18H12O6 and which is frequently found as a contaminant in several food products such as oil-seed grains and cereals. It has carcinogenic, toxigenic, mutagenic and teratogenic properties; however, it is less toxic than aflatoxin. The fungus Emericella nidulans (Eidam) Vuillemin, whose anamorph is Aspergillus nidulans (Eidam) Winter, was used as a genetic model to investigate the genes involved in the sterigmatocystin biosynthetic pathway. Strains from Utrecht stocks (originally from Glasgow) were analyzed in order to detect ST production and, among them, only the UT196 strain [yA2 (I); metA17 (II); piroA4 (IV)] showed the production of 4 ppm of ST (stc+), whereas the UT448 and UT184 strains showed to be nonproducers of such toxin (stc). Although the UT196 strain is very well characterized genetically, this has been the first report on its production of ST. The allelic segregation rates and all the recombination frequencies between linked and non-linked genetic markers were determined by both the meiotic crossing UT448 (stc) x UT196 (stc+) and mitotic crossing UT448//UT184. 175 meiotic segregants and 140 mitotic segregants were analyzed as to ST production, auxotrophy markers and resistance to acriflavine. These crossed strains presented various markers in heterozygous configuration, which allowed to map a structural gene of ST (stcZ+) located on the left arm of chromosome I, 4% distant from the riboflavin gene (riboA1). As a byproduct of this work, a red pigment of 0.90 Rf was detected in all meiotic and mitotic segregants, whether they were ST producers or not, which indicated that was probably a polyketide produced by the fungus ascopores. The low expression of ST production in 13 meiotic segregants and the high expression of such toxin in the UT448//UT196 and UT448//UT184 diploids (40 ppm) allowed to conclude that a regulating factor existed in the w-met region of chromosome II, which is responsible for the expression of the structural gene stcZ+. The analyses of those genes through the parasexual cycle suggested a typical epigenetic behavior which involved them. Based on the data obtained, hypotheses to explain the expression control of these genes as well as their inter-relationships were here presented.

In this thesis, two different approaches are used to extract the relevant features for classifying the aflatoxin contaminated and uncontaminated scaled chili pepper samples: Statistical approach and Local Discriminant Bases (LDB) approach. In the statistical approach, First Order Statistical (FOS) features and Gray Level Cooccurrence Matrix (GLCM) features are extracted. In the LDB approach, the original LDB algorithm is modified to perform 2D searches to extract the most discriminative features from the hyperspectral images by removing irrelevant features and/or combining the features that do not provide sufficient discriminative information on their own. The classification is performed by using Linear Discriminant Analysis (LDA) classifier. Hyperspectral images of scaled chili peppers purchased from various locations in Turkey are used in this study. Correct classification accuracy about 80% is obtained by using the extracted features.

Thesis (M.Tech.: Biotechnology)- Dept. of Biotechnology & Food Technology, Durban University of Technology, 2007 xv, 102 leaves / For decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.

In this thesis, it is shown how aflatoxins can be determined in biological material. The thesis is a summary of five papers. Aflatoxins are carcinogenic mycotoxins produced by Aspergillus moulds. Methods were developed for the determination of aflatoxins in samples of airborne dust and human urine collected at feed factories. For the dust samples from such agricultural products as copra, cotton seed and maize, methods were developed for the determination of aflatoxins B1, B2, G1 and G2. For urine samples, methods were developed for analysing the four aflatoxins above that naturally occur in dust, and the metabolites aflatoxins M1 and Q1. Sample preparation of dust samples included solvent extraction, filtration and immunoaffinity column extraction. Urine samples were cleaned up using immunoaffinity column extraction or solid-phase extraction using ethyl bonded-phase columns. All extractions with these columns were automated by means of a laboratory robot. Reversed-phase liquid chromatography was used to separate the aflatoxins in the cleaned-up extracts. Detection was performed by fluorescence after post-column derivatization by addition of bromine. Parameters for the derivatization were studied using factorial designs. To confirm the identity of aflatoxins in naturally contaminated airborne dust samples and spiked urine, liquid chromatography was combined with electrospray mass spectrometry. The detection limits of the aflatoxins in dust samples were in the range 1.8-3.1 ng/g in 10-mg dust samples using fluorescence detection. Aflatoxins were determined in spiked urine down to the 6.8-18 pg/ml level. In naturally contaminated dust of copra and cotton seed, aflatoxins were detected with a content of 9-50 pg/mg of aflatoxin Bi. No aflatoxins could be detected in any urine sample obtained from feed factory workers that were less than 6.8 pg/ml of aflatoxins B1, B2, G1 and G2 and less than 18 pg/ml of aflatoxins M1 and Q1. / <p>Diss. (sammanfattning) Umeå : Univ., härtill 5 uppsatser</p> / digitalisering@umu

The aim of this research was to evaluate the performance of turkey poults fed crescent doses of aflatoxins in the diet during the first 42 days of life. Three hundred and thirty six one-day-old, male turkey poults were randomly divided into seven treatments, as follows: T1= control; T2= 20 ppb of total aflatoxins; T3= 50 ppb; T4= 100 ppb; T5= 200 ppb; T6= 500 ppb; T7= 1,000 ppb. Turkeys were kept in cages and euthanized in two periods: half the birds at 21 days of experiment and the remaining birds at 42 days of experiment. Parameters evaluated were feed consumption, body weight, organs relative weight (liver, heart, gizzard and bursa of Fabricius), relative weight of meat (thighs and breast), and clinical biochemical parameters (total plasmatic proteins, albumin, uric acid, cholesterol, alanine aminotransferase, aspartate aminotransferase, and alkaline Phosfatase). At 21 days of age, both feed consumption (R= -0.98) and body weight (R= -0.85) were significantly affected (P=0.00) by the presence of aflatoxins in the diet. Relative weight of gizzard (R=0.50), total proteins (R= -0.87), and cholesterol (R= -0.73) levels were also significantly affected by the aflatoxins present in the diet. At 42 days of age feed consumption (R= -0.96), body weight (R= -0.84), relative weight of gizzard (R=0.64), relative weight of liver (R=0.63), and cholesterol levels (R=0.71) were all significantly affected by the presence of aflatoxins in the diet. These results show that aflatoxins interfere significantly in several productive and biological parameters in turkey poults. / O objetivo deste trabalho foi avaliar o desempenho de perus de corte alimentados com dietas contendo sete diferentes doses de aflatoxinas, durante os primeiros 42 dias de vida. Foram utilizados 336 perus de corte, machos, com 1 dia de vida, divididos aleatoriamente em 7 tratamentos, conforme a dose de aflatoxinas utilizada (T1= controle; T2= 20 ppb de aflatoxinas totais; T3= 50 ppb; T4= 100 ppb; T5= 200 ppb; T6= 500 ppb; e T7= 1.000 ppb). As aves foram alojadas em gaiolas e eutanasiadas em dois períodos: metade das aves aos 21 dias de experimento e o restante aos 42 dias. Os parâmetros avaliados em cada período foram consumo de ração, peso corporal, peso relativo de órgãos (fígado, coração, moela e bursa de Fabricius), peso relativo de cortes da carcaça (coxa+sobrecoxa e peito) e parâmetros de bioquímica clínica (proteínas plasmáticas totais, albumina, ácido úrico, colesterol, alanina aminotransferase, aspartato aminotransferase e fosfatase alcalina). Aos 21 dias de experimento, tanto o consumo de ração (R= -0,98), quanto o peso corporal (R= -0,85) foram significativamente afetados (P=0,00) pela presença das aflatoxinas na dieta. Além disto, o peso relativo de moela (R=0,50) e os níveis de proteínas plasmáticas totais (R= -0,87) e de colesterol (R= -0,73) também foram significativamente afetados pelas aflatoxinas presentes na dieta. Aos 42 dias, além do consumo de ração (R= -0,96) e do peso corporal (R= -0,84), foram afetados significativamente os pesos relativos de moela (R=0,64) e de fígado (R=0,63) e os níveis séricos de colesterol (R=0,71) pela presença de aflatoxinas na ração. Diante destes resultados, conclui-se que as aflatoxinas interferem significativamente em diversos parâmetros produtivos e biológicos em perus de corte.