Dissertations / Theses on the topic 'Mycotoxicosis'

Poisonous plants are one of the most important causes of economic losses in the livestock industry throughout the world, especially in those areas where rangeland and pasture grazing are practiced. In the livestock industry in South Africa, total annual costs of plant and fungal poisoning (mycotoxicosis) are conservatively estimated to be R104 506 077, 8% of which is due to D. cymosum poisoning. There is no antidote for D. cymosum poisoning of livestock and wide scale eradication by conventional control methods are labour intensive, expensive and often impractical. In South Africa, the communal or emerging farming sectors are the most affected. This is mainly due to the high costs associated with control measures such as fencing, supplementary feeding and veterinary expenses, and / or lack of grazing management with livestock numbers exceeding the biological carrying capacity of the rangeland due to lack of grazing lands. Proper rangeland management is the surest and most economical means of reducing plant poisoning of livestock. Focused research on the ecology of these poisonous plants in rangeland communities to improve rangeland management would assist in the development of these strategies. This study investigated D. cymosum infested savanna communities, focusing on understanding how negative (inter- and intraspecific competition) interactions influence community structure, dynamics and productivity and how plants in D. cymosum communities avoid these negative interactions by investigating their seasonal phenological patterns. Dichapetalum cymosum coexists with trees, such as Burkea africana, Ochna pulchra and Terminalia serecia, as well as shrubs species, such as Pygmaeothamnus zeyheri and Perinari capensis, in well drained, nutrient poor soils. Some plant ecologists contend that in an environment where water is limited, competition is inevitable amongst plants occupying the same above-ground stratum and the same soil horizon. Others contend that plants avoid competition with each other by sharing resources spatially and temporarily. Field experiments were conducted to investigate how the above species interact and coexist with each other in two South African savanna communities. Fourteen (100 m x 100 m) D. cymosum infested sites were identified in each community. Disperal analysis using nearest neighbour distance was used to investigate competition among species, and above ground flowering phenology along niche axes to determine temporal and spatial sharing of resources. The dispersal analysis revealed aggregated populations among species when intraspecific and combined (all individuals independent of species) analyses were conducted. However, in all instances, aggregation among species was not significant. No interspecific competition was observed among species when correlation analysis was performed between nearest neighbour distance and combined canopy cover of the nearest neighbour pair. Intraspecific competition was, however, observed for tree species T. sericea (n = 128; r = 0.3952; P < 0.0001) and B. africana (n = 166; r = 0.49926; P <0.0001) and a shrub species, D. cymosum (n = 391; r = 0.39788; P <0.0001). Segregation was found between O. pulchra and both B. africana (S = 0.999, χ² = 102.7588, P <0.0001) and T. sericea (S = 0.999, χ² = 57.8571, P <0.0001). Shrub species were also segregated, all with interspecific nearest neighbour pairs occurring less often than expected. The vegetative phenology of all experimental plant species followed the rainfall gradient. Differences in reproductive phenologies were observed between O. pulchra and both B. africana and T. sericea. Dichapetalum cymosum also differed from P. capensis and P. zeyheri in their reproductive phenologies. The differences in the reproductive strategies of at least one of the species in each growth form account for the observed spatial distribution amongst species in these communities. The observed growth patterns shown by the vegetative phenologies, however, suggest that lengthy retention of nutrients is a strategy to avoid competition for nutrient uptake with other species in these communities. Segregation between species and positive correlation iii between nearest neighbour distance and combined canopy cover of the nearest neighbour pairs suggest that intraspecific competition and interspecific facilitation determine D. cymosum woody plant community structure. This study had limited application to rangeland management. However, it can be concluded that grazing of D. cymosum communities should take place during mid-summer, when enough grazing material is available to allow animals to vary their diet. The introduction of animals in poor condition or naïve animals into these lands should be avoided in winter and spring as they will graze non-selectively resulting in D. cymosum poisoning. To utilize these areas as grazing lands, supplements need to be provided to assist in the detoxifications of toxins once ingested.<br>Dissertation (MSc Agric)--University of Pretoria, 2013.<br>gm2014<br>Plant Production and Soil Science<br>unrestricted

Conselho Nacional de Desenvolvimento Científico e Tecnológico<br>In the first part of the thesis, the spontaneous occurrence of an outbreak of chronic aflatoxicosis is reported in dairy calves. Forty 4-month-old male Holstein calves of approximately 100kg were fed a ration constituted by alfalfa hay, broken corn and milk substitute. Six calves (15%) died after presenting a disease characterized by general unthriftiness, diarrhea, rough hair coats, abdominal pain, prolapsed rectum and grinding of teeth. The clinical course, was 2-3 days; however many calves in this lot that did not die, remained underdeveloped. Three calves were necropsied. Necropsy findings included firm, light tan livers and marked hydrothorax, ascites and edema of the mesentery, mesocolon and of the mucosal folds of the abomasum. Main histopathological changes were restricted to the liver and consisted of fibrosis, moderate megalocytosis, biliary duct hyperplasia and venoocclusive disease. The analysis by thin layer chromatography of the corn fed to calves revealed 5,136 ppb of aflatoxin B1. A diagnosis of aflatoxicosis was made based on the characteristic clinical signs and pathology, on the absence of Senecio spp. in the food and on the presence of high levels of aflatoxin in the corn fed to the calves. In the second part of the thesis, two experiments were performed in order to determine the toxic effects of varying doses of aflatoxins in calves. Clinical, productive and pathologic aspects of affected calves were considered. In the first experiment, nine 2-4- month old calves Holstein Friesian calves were fed, for two months, daily amounts corresponding to 1.5% of their body weight of a ration containing 500±100 ppb of aflatoxins. Three calves were used as controls. In the second experiment, three 4-5-month old Holstein Friesian calves, were orally fed daily small parcels of a concentrate of aflatoxins diluted in 500 ml of water corresponding to 1,250, 2,500 e 5,000 ppb of B1 aflatoxin (AFB1). A male calf was used as control. During all the experimental period of the first experiment, the weight gain of the calves receiving AFB1 was equivalent to that of the control group and no differences were observed between treated and control calves when the values of serum activity of aspartate transaminase (AST), serum albumin (SA), total serum protein (TP), and PVC, determined weekly, were compared. A significant difference in the serum activities of alkaline phosphatase (AP) and gamma glutamyl transferase when the serum sampled on the 63th day of the experiment was considered. During the whole experimental period and up to three weeks after the final of the experiment, no clinical signs or histopathological changes associated with the consumption of aflatoxins were observed in any of the calves of the first experiment. In the second experiment, clinical signs observed in three treated calves included loss of appetite, decrease in weight gain, and loss of weight Jaundice, intermittent diarrhea, tenesmus and apathy were only observed in the calf receiving 5,000 ppb of AFB1. Increased activity of AF and GGT were observed in all the calves of the treated group. No changes were observed regarding PCV, TP, total bilirubin, direct bilirubin and in the serum activity of AST in any of the calves of the second experiment. Histopathological changes in intoxicated calves included bile duct proliferation, cytoplasmic vacuolar hepatocelular degeneration consistent with hepatocelular deposit of lipids, periportal to bridging fibrosis, megalocytosis, subendothelial edema and fibrosis in terminal hepatic veins. Necropsy findings in the euthanatized calf which receive de largest doses of AFB1 included slight enlargement of the liver which was firm and diffusely light-yellow, mild ascites, and edema of the mesentery and of abomasal folds.<br>Na primeira parte dessa tese, relatamos a ocorrência de um surto de aflatoxicose crônica bezerros de raça leiteira. Quarenta bezerros holandeses, machos, de quatro meses de idade e aproximadamente 100 kg eram alimentados com feno de alfafa, milho quebrado e substituto de leite. Seis bezerros (15%) morreram após apresentar uma doença caracterizada por mau desenvolvimento geral, diarreia, pelagem áspera, dor abdominal, tenesmo, prolapso de reto e bruxismo. A duração do curso clínico foi de 2-3 dias; muitos terneiros desse lote que não morreram permaneceram pouco desenvolvidos. Os achados de necropsia de três bezerros incluíam fígado firme e castanho-claro, marcados hidrotórax e ascite, e edema do mesentério, mesocólon e das dobras da mucosa do abomaso. Os principais achados histopatológicos estavam restritos ao fígado e consistiam de fibrose, moderada megalocitose, hiperplasia de ductos biliares e lesão veno-oclusiva. A análise do milho do alimento dos bezerros por cromatografia de camada delgada revelou 5.136 ppb de aflatoxina B1 (AFB1). O diagnóstico de aflatoxicose foi feito baseado nos sinais clínicos e patologia característicos, na ausência de Senecio spp. na alimentação dos terneiros e na presença de altos níveis de aflatoxina no milho da alimentação dos bezerros. Na segunda parte da tese, dois experimentos foram realizados para determinar os efeitos tóxicos de diferentes doses de aflatoxinas em bezerros, considerando-se aspectos clínicos, produtivos e patológicos. No primeiro, nove bezerros, Holandês, com 2-4 meses de idade, receberam ração contendo 500±100 ppb de aflatoxina, na quantidade equivalente a 1,5% do peso vivo/dia, durante dois meses. Três bezerros foram usados como controle. No segundo experimento, três bezerros, Holandês, com 4-5 meses de idade, receberam, por via oral, pequenas porções diárias de um concentrado de aflatoxinas, diluídas em 500 ml de água, correspondendo a doses de 1.250, 2.500 e 5.000 ppb de AFB1. Um bezerro foi usado como controle. No primeiro experimento, o ganho de peso dos bezerros recebendo AFB1 foi equivalente ao do grupo controle durante todo período experimental e não foram observadas alterações na atividade sérica da enzima aspartato transaminase (AST), da albumina sérica (AS), da proteína total (PT) e no hematócrito, quando comparados os resultados semanais do grupo tratamento e controle. Diferenças significativas nas atividades séricas das enzimas fosfatase alcalina (FA) e gama glutamil transferase (GGT) ocorreram na coleta do 63º dia do experimento. Não foram observados sinais clínicos e alterações histopatológicas de aflatoxicose em qualquer dos bezerros do grupo tratamento desse experimento. No segundo experimento, sinais clínicos observados nos bezerros intoxicados incluíram perda de apetite, diminuição do ganho de peso e emagrecimento. Icterícia, diarreia intermitente, tenesmo e apatia severa, foram observadas apenas em um bezerro (5.000 ppb de AFB1). Níveis alterados da atividade sérica de FA e GGT foram observados em todos os bezerros do grupo tratamento. Não foram observadas variações no hematócrito e na atividade sérica de AST, nem nos níveis séricos de PT, bilirrubina total e bilirrubina direta em qualquer dos bezerros desse experimento. Alterações histopatológicas nos bezerros intoxicados incluíram proliferação de ductos biliares, degeneração citoplasmática vacuolar consistente com acumulação hepatocelular de lipídios, fibrose periportal, ou em ponte, megalocitose, fibrose subendotelial das veias hepáticas terminais e edema. Achados de necropsia do bezerro recebendo a maior dose de AFB1 incluíram fígado levemente aumentado de tamanho, difusamente amarelo-claro e firme, discreta ascite, edema de mesentério e submucosa do abomaso.

Fumonisins B1 6 and B3 8 are toxic secondary metabolites of the fungus Fusarium moniliforme that inhibit enzymes of sphingolipid biosynthesis. This dissertation describes work towards the stereocontrolled total synthesis of the fumonisin natural products. The proposed highly convergent strategy allows for the late stage stereocontrolled coupling of the two fragments C1-C10 58 and C11-C20 57 with concomitant installation of the C10 hydroxyl using key boron aldol methodology. A directed hydrogenation installs the final methyl-bearing stereocentre at C12. Syntheses of the left- and right-hand fragments 57 and 58 by means of substrate-based stereocontrol and asymmetric catalytic methods is reported. A completed synthesis of the protected FB3 carbon backbone 59 is achieved in a linear reaction sequence of 14 steps. Tentative assignment of stereogenic centres within 59 was made by analogy to the C4-C20 fragment 190 of fumonisin B3. Synthesis of C4-C20 190 by the coupling of C11-C20 57 with heptaldehyde is also described.

Doctor of Philosophy<br>Fumonisins B1 6 and B3 8 are toxic secondary metabolites of the fungus Fusarium moniliforme that inhibit enzymes of sphingolipid biosynthesis. This dissertation describes work towards the stereocontrolled total synthesis of the fumonisin natural products. The proposed highly convergent strategy allows for the late stage stereocontrolled coupling of the two fragments C1-C10 58 and C11-C20 57 with concomitant installation of the C10 hydroxyl using key boron aldol methodology. A directed hydrogenation installs the final methyl-bearing stereocentre at C12. Syntheses of the left- and right-hand fragments 57 and 58 by means of substrate-based stereocontrol and asymmetric catalytic methods is reported. A completed synthesis of the protected FB3 carbon backbone 59 is achieved in a linear reaction sequence of 14 steps. Tentative assignment of stereogenic centres within 59 was made by analogy to the C4-C20 fragment 190 of fumonisin B3. Synthesis of C4-C20 190 by the coupling of C11-C20 57 with heptaldehyde is also described.

Thesis (Ph. D.)--University of Sydney, 2004.<br>Title from title screen (viewed April 6, 2009). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Chemistry, Faculty of Science. Degree awarded 2004; thesis submitted 2003. Also available in print form.

The mycotoxin zearalenone is known for its harmful effects on livestock reproduction. Animal exposure occurs through feed sources colonized by Fusarium species which produce the mycotoxin. Since regular screening procedures for zearalenone are not conducted on Western Canadian barley, a survey was carried out to test for possible significant levels of contamination. All samples were found to be negative at a detection level of 500 ppb; therefore, feeds formulated from the barley samples sources would not likely cause zearalenone toxicosis problems in livestock. Also, an ELISA method, Agri-Screen™, developed by Neogen Corporation (Lansing, Michigan) was tested and found to be a simple and economical method for pre-screening of feed samples in the field. To study direct toxicological effects of zearalenone on in vitro murine blastocyst development, murine embryos were cultured in medium (Ham's F-10 + estrous cow serum) containing various levels of the mycotoxin. The critical concentration range for zearalenone to cause detrimental effects on blastocyst development was determined to be between 70-160 μg/ml medium. Additionally, a concentration effect on the length of time required to exert deleterious actions was demonstrated. At mycotoxin concentrations of 500 μg/ml medium and above, blastocysts degenerated after 6 h of culture. At a lower concentration level of 160 μg/ml, blastocysts were not affected until 28 h of culture. In order to investigate the direct toxicological effects of zearalenone on in vitro porcine pre-implantation embryo development, attempts were made to develop a successful culture system. Since a suitable system was not developed, toxicological studies were not possible. Possibly, steps in the recovery process could have resulted in detrimental effects before the embryos were placed in culture. Alternatively, the media chosen (Ham's F-10 + estrous cow serum; Minimum essential medium + fetal calf serum) may not be suitable for in vitro culture of porcine pre-implantation embryos. Finally, at a zearalenone concentration level (250 μg/ml medium) found to cause degeneration of murine blastocysts, the in vitro maturation of bovine oocytes in Tissue Culture Medium 199 was not affected. It was suggested that the surrounding cumulus layer acts as a barrier to prevent the mycotoxin from directly acting on the oocyte.<br>Land and Food Systems, Faculty of<br>Graduate

Thesis (MSc)--Stellenbosch University, 2002.<br>ENGLISH ABSTRACT: Mycotoxins have assumed worldwide importance due to the ubiquitous occurrence of toxigenic fungi, their infestation of plant-based foods and feeds and the subsequent economical and health impact it because of their contamination of commercial products. Ochratoxin A (OA) is a nephrotoxic mycotoxin produced by isolates of Aspergillus ochraceus and Penicillium verrucosum and occurs frequently in nature. The major target for toxicity of OA in mammalian species is the kidneys and it has been the major cause of Danish Porcine Nephropathy. OA has also been extensively implicated in the aetiology of Balkan Endemic Nephropathy and Chronic Interstitial Nephropathy in Northern-Africa. Furthermore, OA has been identified as a carcinogen, an immunosuppressant and a teratogen with respect to the foetal central nervous system. Although a large amount of research has been conducted into the chemical nature of the toxicity of OA, the exact molecular mechanism of action of OA is not yet conclusive. Numerous structure activity relationship studies have suggested that the toxicity of OA may be assigned to three major processes: (i) inhibition of ATP production; (ii) inhibition of protein synthesis; and (iii) the disruption of hepatic microsomal calcium homeostasis through the promotion of membrane lipid peroxidation. It is the aim of this thesis to gain a better understanding, through the synthesis ofOA analogues, of the chemical structure responsible for the toxic function of the ochratoxins. The halogen-group has extensively been implicated in the toxicity of the ochratoxins. This is evident in ochratoxin B (OB), the dechloro analogue of OA, which is approximately ten times less toxic than OA. Preliminary tests have indicated that bromo-ochratoxin B(BrOB), the bromo analogue of OA, is more toxic than ochratoxin A to renal cells. Fluoro-ochratoxin B and other analogues of OA, where other amino acids are incorporated, should provide invaluable information on the structure-activity relationships and the mode of action of the ochratoxins. Our research effort addresses both these aspects (i) fluorination of the dihydroisocoumarin moiety and (ii) the coupling of different amino acids and dipeptides to the non-toxic hydrolysed product of OA, ochratoxin a. Chapter one includes a review of the important biological aspects of OA that has served as a guideline to the synthesis of effective OA analogues. An overview of the relevant chemistry involved in the modification of OA will conclude the chapter. Chapter two entails a discussion of fluorine in bio-organic chemistry. This includes an overview of the impact that fluorine substitution has on the biological reactivity of molecules. A review on the synthesis of organofluorine compounds, which forms the emphasis of this study, concludes the chapter. Chapter three elaborates on the different methodologies used in our attempts to synthesise fluoro-ochratoxin B and other analogues. These included the direct electrophilic fluorination of OB and different analogous aromatic model compounds by xenon difluoride, N-fluorobenzenesulfonimides and Selectfluor™ as fluorinating agents. Also involved is an investigation into an alternative route for the synthesis of fluoro aromatic compounds from bromo and chloro analogues by means of palladium catalysed trimethyl- and tributylstannyl and trimethylsilylation which in tum may be substituted with fluorine by means of xenon difluoride. Efforts towards the direct catalytic fluorosubstitution of aryl halides are also investigated. The synthesis of a key intermediate, fluoroacetoacetaldehyde, in a de nova synthetic route to fluoroochratoxin B is also discussed. Furthermore, the synthesis of novel OA analogues with respect to the replacement of the L-phenylalanine moiety is addressed. This includes the conversion of OA to Oa, by acid hydrolysis, followed by the coupling of ortho-, meta- and para- substituted DL-fluorophenylalanine to the lactone acid. This is followed by the synthesis of histidylhistidine methyl ester and attempted coupling to Oa. The coupling of halosalicylic acids and salicylic acid to L-phenylalanine, for use as model aromatic substrates for fluorination, IS discussed. Peptide coupling by dicyclohexylcarbodiimide carboxyl activation, with reference to the protection of the phenolic hydroxyl group in 5-chlorosalicylic acid for application to Oa, concludes this work.<br>AFRIKAANSE OPSOMMING: Mikotoksiene is van wêreld-wye belang as gevolg van die alomteenwoordige voorkoms van toksigeniese fungi, hul besmetting van plantaardige kossoorte en voerstowwe en die gevolglike ekonomiese en gesondheidsimpak deur die besoedeling van kommersiële produkte. Ochratoksien A (OA) is 'n nefrotoksiese mikotoksien wat geproduseer word deur isolate van Aspergillus ochraceus en Penicillium verrucosum en kom algemeen in die natuur voor. Die niere is die hoof teiken vir vergifiting deur OA in soogdierspesies en is as die vername oorsaak van "Danish Porcine Nephropathy" aangewys. OA word verder aangedui as die oorsaak vir "Balkan Endemic Nephropathy" en "Chronic Interstitial Nephropathy" in Noord- Afrika. OA is verder geïdentifiseer as 'n karsinogeen, immuno-onderdrukker en is teratogenies ten opsigte van die sentrale senuweestelsel van fetusse. Alhoewel aansienlike navorsing alreeds gewei is aan die chemiese natuur van die toksisiteit van OA, is die presiese molekulêre meganisme van OA reaktiwiteit onbeslis. Verskeie struktuur-aktiwitweit verwantskaps studies dui daarop dat die toksisiteit van OA hoofsaaklik toegeskryf kan word aan drie hoof prosesse: (i) inhibisie van ATP produksie; (ii) inhibisie van proteïen sintese; en (iii) die ontwrigting van hepatiese mikrosomale kalsiumhomeostase deur die bevordering van membraanlipiedperoksidasie. Hierdie tesis het ten doel, deur die sintese van OA analoë, om 'n beter insig oor die chemiese struktuur wat verantwoordelik is vir die toksiese funksionaliteit van ochratoksiene te verkry. Die halogeen substituent is grootliks geïmpliseer in die toksisiteit van OA. 'n Bewys hiervan is ochratoksien B (OB), die dechlooranaloog van OA, wat ongeveer tien maal minder toksies is as OA. Voorlopige ondersoeke het aangetoon dat bromoochratoksien B (BrOB), die broomanaloog van OA, meer toksies is vir nierselle as OA. Fluoorochratoksien B en ander analoë van OA, waar ander aminosure geïnkorporeer word, behoort waardevolle inligting te voorsien met betrekking tot die struktuur-aktiwiteitsverwantskappe en die wyse waarop ochratoksiene funksioneer. Hierdie navorsingspoging spreek beide aspekte aan; (i) die fluorering van die dihidroïsokumarien gedeelte en, (ii) die koppeling van verskillende armnosure en dipeptiede aan die nie-toksiese hidrolieseproduk van OA, nl. ochratoksien a. Hoofstuk een vervat 'n oorsig van die belangrike biologiese aspekte van OA wat dien as riglyn vir die sintese van doeltreffende OA analoë. Die hoofstuk word afgesluit met 'n oorsig van die relevante chemie betrokke by die modifisering van die struktuur van OA. Hoofstuk twee bevat 'n bespreking van die aanwending van fluoor in bio-organiese chemie. Dit bevat 'n oorsig van die impak wat fluoorsubstitusie het op die biologiese reaktiwiteit van molekules. 'n Opsomming oor die sintese van organofluoorverbindings, wat die essensie van hierdie studie is, beëindig die hoofstuk. Hoofstuk drie handeloor die veskillende metodes wat toegepas is in pogings om fluoorochratoksien B en ander analoë te sintetiseer. Dit sluit in die direkte elektrofiliese fluorering van OB en ander verwante aromatiese modelverbindings deur gebruik te maak van xenondifluoried, N-fluoorbenseensulfonimied en Selectfluor™ as fluoreringsreagense. Dit behels verder ook 'n ondersoek na 'n alternatiewe roete tot die sintese van fluooraromatiese verbindings vanaf broom- en chlooranaloë. Vir die doel word palladiumgekataliseerde trimetiel- en tributielstannilering, en trimetielsililering wat vervolgens deur middel van xenondifluoried met fluoor gesubstitueer kan word, aangewend. Pogings tot die direkte katalitiese fluoorsubstitusie van arielhaliede word ook bespreek. Die sintese van 'n sleutelintermediêr, fluoroasetoasetaldehied, in 'n de nova sintese roete tot fluoorochratoksien B word bespreek. Die sintese van nuwe OA analoë, met betrekking to die vervangmg van die Lfenielalanien (L-Phe) groep word ondersoek. Dit bevat die omsetting van OA na Oa, deur suurhidrolise, gevolg deur die koppeling van orto-, meta- en paragesubstitueerde DL-fluoorfenielalanien aan die laktoonsuur, Oa. Daarna word die sintese van histidielhistidienmetielester en die verdere pogings aangaande koppeling met Oa bespreek. Die koppeling van halosalisielsure en salisielsuur aan L-Phe wat dien as model aromatiese verbindings vir fluorering, word behandel. Peptiedkoppeling met behulp van disikloheksielkarbodiimied-karboksielaktivering, met inbegrip van die beskerming van die fenoliese hidroksiel groep m 5-chloorsalisielsuur Vir die toepassing op Oa, beëindig hierdie werk.

Les trichothécènes, mycotoxines produites par différentes espèces de Fusarium, ont une incidence rapportée dans le monde entier. Ces composés contaminent les produits agro-alimentaires, et sont à l'origine de mycotoxicoses humaines et animales, dont les symptômes se caractérisent entre autres par des troubles hématologiques. L’origine de ces troubles a été recherchée à l'aide d'un modèle de culture de progéniteurs hématopoïétiques granulo-monocytaires (CFU-GM) humains et de rat. Les trichothecenes les plus fréquemment détectes, les toxines T-2, HT-2, le diacétoxyscirpénol (DAS) et le déoxynivalénol (DON) ont été choisis pour mener cette étude. Leurs effets sur la prolifération des CFU-GM ont permis de mettre en évidence leur forte myélotoxicité. Les effets de T-2, HT-2, DAS et DON ont ensuite été recherchés sur la lignée érythroide humaine (BFU-E/CFU-E). Chez l'homme, les BFU-E sont beaucoup moins sensibles aux trichothécènes que les CFU-GM. Néanmoins, la différenciation des BFU-E est affectée par les trichothécènes. Afin de rechercher les cibles cellulaires des trichothécènes, de nouveaux outils d'étude de l'hématotoxicite ont été développés : une technique de culture de CFU-GM permettant la récupération des cellules après 14 jours ; le test MTT, jusqu'a présent non utilisable pour les CFU-GM ; le dosage des protéines totales et de l'activité LDH ; deux techniques d'évaluation des péroxydations lipidiques ; l'évaluation du pouvoir antioxydant de xénobiotiques, appliquée aux CFU-GM. Si la cible cellulaire majeure de T-2 sur les CFU-GM semble être la membrane plasmique, l'activité mitochondriale et les contenus protéiques de ces progéniteurs sont également affectés. Les atteintes membranaires de T-2 ne s'exercent pas par l'intermédiaire de péroxydations lipidiques. D’autre part, les différences de sensibilité entre les CFU-GM humains et de rat semblent s'expliquer par des mécanismes cellulaires différents.

L'étude présentée comporte deux parties distinctes ayant la même objectif : préserver le bois face aux organismes lignivores par l'utilisation de biocides naturels. La première formulation est basée sur l'association du cuivre, fongicide puissant mais facilement lessivable, avec les fonctions acides carboxyliques des acides résiniques de la rosine de Tall-Oil, sous-produit industriel de la fabrication des pâtes à papier. L'imprégnation du bois s'effectue successivement en présence de rosine en milieu alcalin puis en présence d'une solution de sulfate de cuivre. Le complexe cuivre-acides résiniques est alors directement formé à l'intérieur du bois. Cette formulation est faiblement lessivée et permet une bonne rétention du cuivre à l'intérieur du bois. Le bois traité montre aussi une excellente résistance face aux basidiomycètes et face à réticuliterme santonensis. La deuxième formulation étudiée est liée à la durabilité naturelle du Thuja Plicata qui est due à la présence dans ses extraits d'une certaine catégorie de molécules : les tropolones et plus particulièrem̀ent les Thujaplicines. Dans un premier temps, nous avons vérifié l'activité antifongique de la tropolone de synthèse et de la -Thulaplicine commerciale. Cependant, afin d'éviter une utilisation importante de copeaux de bois pour l'extraction des tropolones, nous avons développé des cultures cellulaires de Thuja Plicata à partir de cals provenant de méristèmes foliaires de plantules cultivés sur un milieu Gamborg. L'extraction des tropolones intracellulaires (avec une majorité de -Thujaplicine) est effectuée en présence d'acétate d'éthyle. La culture de cals en présence d'éliciteurs (activateurs de biosynthèse) et de glucose conduit à des rendements de biosynthèse en Thujaplicines très intéressants. Par ailleurs, la culture de suspension cellualire de Thuja Plicate en milieu liquide est prometteuse. Les extraits bruts de Thujaplicines obtenus à partir de l'extraction des cals ont une forte activité antifongique vis-à-vis de plusieurs basidiomycètes. Cette activité est comparable à celle d'extraits éthanoliques de sciure de Thuja Plicata. Ces formulations basées sur l'utilisation de biocides naturels, faiblement toxiques pour l'environnement, peuvent être considérées à long terme comme des agents de substitution des produits chimiques de préservations du bois qui posent des problèmes sur le plan toxicologique.

Thesis (D. Tech.) -- Central University of Technology, Free State, 2008<br>The purpose of this study was to evaluate brewing and consumption practices and to screen for micro-organisms and mycotoxins associated with traditional beer produced and consumed in the marginal urban settlements of the city of Kimberley in the Northern Cape Province of South Africa. The survey study revealed that traditional beer is no longer being brewed for traditional purposes only, as was the case in the past, but rather for commercial gain. Both brewers and consumers, however, appeared to be largely unaware of disease-causing micro-organisms present on the hands or bodies of handlers that can be transferred to the beverage during the handling process, and were seemingly not conversant with regard to the effects of hazardous ingredients sometimes incorporated during the brewing process. Unemployment and a lack of education emerged as pivotal factors related to the production of traditional beer and the ignorance of the associated safety thereof. The survey further indicated that although facilities such as the availability of potable water (taps in yards) and flushing toilets were sometimes in place, other facilities such as basins with hot running water were often not available. In commercially produced and homebrewed traditional beer the mean counts for total coliforms and Staphylococcus spp. were circa 105 cfu.ml-1 whereas the TVC (Total Viable Counts) and total fungi counts were 106 and 107 cfu.ml-1 respectively. The total coliforms and Staphylococcus spp. counts for homebrewed traditional beer were approximately one log-phase higher than the commercial version. The counts in the homebrewed beer probably originated from contamination during handling, while in the commercial product contamination originated either in the raw ingredients or during postprocessing and consumption. Apart from staphylococci, considerable numbers of total coliforms indicating faecal contamination were noted. A rapid, easy, reliable and accurate technique that could be used to quantify the level of mycotoxins (deoxynivalenol and citrinin) in the beer was developed through validation of the ELISA Ridascreen methodology. Using this method, the deoxynivalenol (DON) level in the beer samples was found to exceed the recommended levels suggested by the European Union, while citrinin levels in the samples varied between 35.6 ppb and 942.2 ppb. In the case of citrinin there were statistically significant differences between spring, summer and winter samples, confirming the seasonal impact on fungal growth and consequent mycotoxin production. An R2-value of 0.409 was noted between DON and citrinin, indicating a weak positive association. Finally, an awareness programme in the format of a poster with accompanying subscripts was developed to address issues of safety and hygiene of traditional beer in the study area. The poster utilises animatedstyle colour images of selected practices that need to be addressed, accompanied by slogans summarising the particular image in English, Afrikaans and Setswana. It is envisaged that, as part of a comprehensive awareness programme, the poster will contribute greatly to the quality, safety and promotion of traditional beer in the area.

L'aflatoxine B1 (AFB1) et l'ochratoxine A (OTA) sont des mycotoxines pouvant contaminer une large variété de denrées alimentaires. L'ingestion d'aliments contaminés par des animaux d'élevage peut entraîner l'altération de leur santé et de leurs performances zootechniques ainsi qu'un problème de sécurité alimentaire lié à la présence de résidus de mycotoxines dans les produits animaux, notamment le lait. Des traitements de détoxication basés sur l'addition d'adsorbants organiques ont été développés pour fixer les mycotoxines dans le tube digestif et ainsi réduire l'exposition des animaux. L'objectif de ce travail de thèse était d'évaluer l'efficacité d'un adsorbant à base d'extraits de parois modifiées de levures (Mycosorb®) sur deux modèles animaux, le rat et la brebis. L'efficacité a été déterminée en réalisant un suivi des mycotoxines et/ou de leurs métabolites dans 3 matrices : l'excrétion urinaire et fécale et la cinétique sanguine. Sur les 2 modèles animaux, nous avons ainsi étudié les effets de l'apport en Mycosorb sur l'excrétion urinaire et fécale et la cinétique sanguine des 2 mycotoxines (AFB1 et OTA). Chez le rat, le suivi de la radioactivité a montré que les fèces d'animaux supplémentés en parois de levures contiennent significativement plus de mycotoxines. Cette augmentation de la radioactivité dans les fèces s'est accompagnée d'une diminution marquée de la radioactivité dans le sang et dans les urines. Chez la brebis laitière, en plus de ces paramètres, nous avons évalué l'effet de l'adsorbant sur les paramètres de production de l'animal et l'excrétion des mycotoxines dans le lait. L'addition de la paroi de levure a entraîné une augmentation significative de l'excrétion de l'AFB1 et de son métabolite, l'aflatoxine M1(AFM1) dans les fèces du ruminant. Cette augmentation de l'excrétion fécale s'accompagne de la réduction du taux d'AFM1 excrété dans l'urine mais pas dans le lait. Les effets observés chez les deux modèles expérimentaux semblent être liés à la séquestration des mycotoxines dans le tractus digestif des animaux et permettent de conclure à la capacité de l'adsorbant organique à réduire la biodisponibilité des mycotoxines testées. L'ajout de la paroi de levure pourrait, par conséquent, réduire les risques sanitaires chez les animaux d'élevage exposés à une alimentation contaminée par les mycotoxines. Cependant, nous n'avons pas observé d'effet sur la santé et les paramètres zootechniques des animaux dans les conditions expérimentales utilisées.

The work presented here represents research done on mycotoxins and plant toxins by the author and his postgraduate students over a period from 1964 to date. The first phase, which ends at 1980, mainly addresses the biosynthesis of the aflatoxins. The involvement of anthraquinone derivatives in this process was investigated and the role of versicolorin A and its derivatives was partially elucidated. Novel active enzymes systems were derived from protoplasts and used in these studies. The period lasting from 1980 to 1992 concentrates on the occurrence of mycotoxins in agricultural commodities and effects on animals and their systems. Over 7000 samples were analysed using a multimycotoxin analytical method and a fungal screen. The most common mycotoxin found was aflatoxin B₁ and prevalent fungus was Fusarium moniliforme. Later work is indicating that fumonisin B₁ is the most commonly occurring mycotoxin. As this was only discovered in 1988, its presence was only looked from 1995 onwards. It was also found that rumen fluid could metabolise trichothecenes. During this period (1980-1992) further work on aflatoxin metabolism was done and a novel dehydrogenase involved in aflatoxin B₁ was isolated and characterised. An Elisa assay was developed for atractyloside, a toxin found in a plant (Callilepis laureola) used in tradition medicine. The site of atractyloside storage was found to be in the plant vacuole. The final period covers 1992 to the present, where the occurrence and effects of mycotoxins in human disease were studied. The major and most important finding is that fumonisin B₁ is present in the blood and tissues of many of the Black population examined in Kwazulu Natal. This includes, oesophageal cancer patients, eclamptic patients, school children and members of the rural population. A similar circumstance also appertains for the presence of aflatoxin B₁. It seems likely from these results that chronic mycotoxicoses are a common occurrence, particularly in the Black rural population and are not the sporadic rare event that is found in the first world countries.<br>Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

M.Tech.<br>A study to determine the occurrence of fungi and their mycotoxins in rural food and their effect on human health was carried out at N’wamitwa (Tzaneen), a rural area of Limpopo province (South Africa). Fifty-eight maize and twenty-nine bambara nuts samples were collected from selected house holds and taken to the storage facilities of the Food, Environment and Health research group (FEHRG) laboratory at the University of Johannesburg for analysis. The samples were analysed for moisture content, fungal infestation, mycotoxin contamination and their toxicity. The moisture content of the samples were at a range of 3-20% moisture. Fungi which included species of the genus Aspergillus, Fusarium and Penicillium were detected at all moisture ranges but more dominant in samples with higher moisture levels. Fungi in this study were able to produce mycotoxins which included deoxynivalenol (DON), zearelenone (ZEA), aflatoxins (AFs), T2- toxin, fumonisins (FBs), ochratoxin A (OTA) and citrinin. The most dominant toxins in maize samples quantified by VICAM were AFs followed by DON, FBs and lastly ZEA and in bambara nuts were FBs followed by DON, AFs and ZEA. HPLC was able to detect higher concentrations of FBs than VICAM. The toxins were then tested for their toxicity using human lymphocytes and the most toxic was DON followed by AFs, FBs and lastly ZEA. Three vials of the same toxin with different concentrations, one with the highest and others with the middle and the lowest concentrations were used to treat the human lymphocytes.

Mycotoxins are secondary metabolites of fungi, which may contaminate animal feed and human food at all stages of the food chain. This has become a global concern and considered an important risk factor mostly for human and animal health. The aim of this project was to elucidate the general health and productivity of domesticated animals in selected rural areas of the Limpopo Province in relation to fungi and mycotoxin and find out possible solutions to avoid in the future further exposure and to improve animal production in rural areas. A total of 95 animal fresh faeces (50 from Mapate and 45 from Nwanedi districts), 50 feed samples (24 from Mapate and 26 from Nwanedi) and 50 fresh milk samples from cattle and goats were screened for fungi and mycotoxin contamination. The multi mycotoxin extraction method was used, followed by thin layer chromatography, also the VICAM immunoaffinity clean up, high performance liquid chromatography, gas chromatography mass spectrophotometry and the ELIZA enzyme linked kit method were used for further mycotoxin determination and quantification. The results obtained from this study revealed that species of Aspergillus, Fusarium and Penicillium fungi contaminated both feed and animal faeces samples. The species Aspergillus niger, A. clavatus, A. flavus, A. fumigatus, Fusarium verticillioides, F. graminerium and F. proliferatum were the most prevalent fungi. Fumonisin B1 and B2, aflatoxins B1, zearalenone and deoxynivalenol (DON) were found in animal feed. Fumonisins B1 and B2 were also found in faecal samples which indicated animal exposure to these mycotoxins. Cattle were the most exposed as compared to goats and pigs. In addition, aflatoxin M1 and traces of fumonisin B1 was detected in cattle and goats milk samples collected from both Mapate and Nwanedi districts. Late harvesting and poor handling of crops during storage seemed to be the reason for the results indicating feed contamination with high levels of fungi and mycotoxins. Daily exposure to this contaminants may influence or/and induce several symptoms such as dermatosis, immunosupression, liver and oesophageal cancer in both animal and human being. There is an urgent necessity to teach rural populations simple and cheap methods of crops storage and techniques to prevent feed and food contamination.<br>Prof. Mike F. Dutton Mr. F. Eric Van-Zyl

Maize (Zea mays L.) is an important cereal world-wide, serving as seed for growers, food for man and livestock as well as an industrial raw material. Unfortunately, it is also a suitable substrate for growth, development and activity of spoilage fungi. Fungal growth is a major problem in cereal grains throughout the world and may lead to poor quality of the products, as well as adverse effects to human and animal health due to mycotoxin production. Maize is usually harvested at high moisture content and then dried to bring down the moisture content to a safe level before storage. Delay in drying to safe moisture levels increases risks of mould growth and mycotoxin production. In rural villages maize is dried using only sun drying and they rely on sacks, thatched silo and drums as their storage facilities. This is insufficient to prevent damage by insects, rain, and rodents, which in turn allows fungi to invade these storage facilities. Maize was sampled in two rural areas of Venda (Limpopo Province) and the percentage moisture content was determined and then screened for total fungal contamination. The samples were also analysed for mycotoxins that have been reported to commonly occur in maize. There was no significant difference in the extent of fungal contamination in Mapate and Folovhodwe villages. Of the fungal species detected, Aspergillus species were the most common with Aspergillus flavus being the most predominant. On analysis by the multi-mycotoxin screen, aflatoxin had the highest incidence amongst mycotoxin, followed by T-2 toxin. However on using the VICAM method of analysis aflatoxins, deoxynivalenol and fumonisin were the most predominant mycotoxins in the samples, while zearalenone toxin was also amongst predominant mycotoxins but with the highest level of 0.1 ppm. Most of the mycotoxin-containing extracts were found to reduce the % cell viability of human lymphocytes, after 24 hours of incubation as determined by the methyl thiazole tetrazolium salt assay. vii In conclusion the co-occurrence of these toxins in maize and maize meal may highlight the problems associated with the intake of numerous toxins that could in turn lead to more adverse health effects such as liver, oesophageal, breast and cervical cancer, male reproductive tract damage and gynacomastasia. There is, therefore, need to disseminate information to these people, using simplified methods such as programs on radio and televisions on mycotoxin hazards and discussion on the issue should also feature regularly on daily newspapers and magazines, about the dangers and management aspects of mycotoxins, and the susceptible produce.<br>Prof. M. F. Dutton Mr. F. E. van Zyl

Several countries have enacted regulations on tolerance limits for common mycotoxins because of the hazardous nature and widespread occurrence of these fungal secondary metabolites in agricultural commodities. Screening of agricultural commodities destined for animal consumption for the presence of mycotoxins is now becoming a prerequisite in several countries as a means of minimizing ingestion of these toxins. Silage samples were analyzed for pH, % dry matter (DM) content, and the presence of total fungi, yeasts and the types of lactic acid bacteria present. The samples were also analyzed for mycotoxins that have been reported to commonly occur in silage. The pH of the samples was found to be acidic ranging from pH 3.4 to 4.7, with few samples having pH values above 6. There was a significant difference in the % DM content amongst the sampling regions. There was no significant difference in the extent of fungal contamination amongst the different regions. Aspergillus fumigatus was the predominant species from all the samples. Most of the yeast species were isolated from the Bergville region. The yeast species isolated from all samples were Trichosporon, Cryptococcus and Candida species, which are all regarded as nonlactate fermenters. Lactobacillus plantarum and Lactobacillus buchneri were the only two lactic acid producers isolated from the samples. Aflatoxins, citrinin and patulin were the most predominant toxins in the samples. Ochratoxin A and deoxynivalenol was not detected in all samples using thin layer chromatography, while the latter two toxins were only detected in two samples using VICAM fluorometry. The level of fumonisins that was found in the forage crops used for silage production was fairly low with the highest level being 9.36 ppb. Most of the mycotoxin extracts were found to reduce the % cell viability of human lymphocytes after 18 hours of incubation as determined by the MTT assay.<br>Professor Mike Dutton Mr. F. E. Van Zyl

M. Tech.<br>Fusarium species are common contaminants of maize and are also capable of producing mycotoxins, in particular the fumonisin. These are implicated in animal and human mycotoxins fumonisin B1 (FB1) for example, has been associated in the aetiology of oesophageal cancer in South Africa and other parts of the world, i.e., China and Iran. Because maize is the staple diet of the South African rural population, this study was designed with the aim of monitoring Fusarium spp. and FB1 in the food of rural people of Venda, Limpopo province of South Africa, during the course of processing maize into porridge which gave a means of estimating dietary exposure to this mycotoxin. Measurement of fumonisin in the excreta of these people allowed a determine of the extent to which FB1 the body is actually exposed to the mycotoxin.Fumonisin B1 has been identified as a major fungal contaminant on maize, especially in the home grown crops intended for human consumption. Thus the rural population of Limpopo Province is at high risk from FB1 exposure and it is therefore of importance to assess this exposure by the analysis of suitable samples.It can be seen that levels of FB1 in maize from Venda are quite high, as several of these samples had exceeded levels above 1750 μg/kg as recommended as maximum tolerance levels by theEuropean Commission. It is equally seen that a much higher proportion of this mycotoxin was destroyed by processing maize to porridge. And because porridge and other maize-based products are usually consumed on a daily basis, the low levels found in the present study must not be under-estimated, as such levels may accumulate over time and cause more severe chronic effects in humans. When setting daily tolerable levels of FB1 in foods in South Africa, it is imperative to take into account the food habits, especially those within the rural communities

Tan spot of wheat is an economically significant disease caused by the fungal pathogen, Pyrenophora tritici-repentis. Certain races of the fungus secrete Ptr ToxA (ToxA), a 13.2 kDa proteinaceous host-selective toxin that is responsible and sufficient to cause disease in susceptible wheat varieties. Disease symptoms develop only when the ToxA gene in the fungus and a single gene in the wheat host are expressed. The understanding of this gene-for-gene interaction could be instrumental towards control of the disease and is also being developed as a model system for understanding host-pathogen interactions. Here, this effort is given a solid structural foundation through crystallographic analysis of the ToxA structure. The ToxA structure was solved at 1.65 Å resolution using the anomalous signal from inherently present sulfur atoms. The monomeric toxin adopts a β-sandwich fold of two anti-parallel β-sheets composed of four strands each. The mapping of existing mutation data onto the structure reveals that a sequence of Arg- Gly-Asp(RGD) and surrounding residues required for activity are present on a solvent-exposed loop thereby making them potential candidates for recognition events that are required for ToxA activity. Unexpectedly, after a simple circular permutation, the ToxA structure is topologically identical to the classic mammalian RGD containing fibronectin type III (FnIII) domain, and furthermore the RGD residues are topologically equivalent. These results support the hypothesis that ToxA, like FnIII, interacts with an integrin-like receptor on the host plant cell surface. There has been a renewed interest in the method of using the anomalous signal from sulfur atoms to solve protein structures. As a spin-off of the structure solution work, the data were systematically analyzed to study the effects of crystal decay, resolution and data redundancy on the ability to locate the sulfur positions and subsequent phasing of the protein. The analyses show that the choices made about data redundancy and resolution limits may be crucial for the structure determination and that anomalous correlation coefficients are helpful indicators in making these choices.<br>Graduation date: 2006