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The trichothecene mycotoxins are secondary metabolites produced by a variety of fungal genera including Fusarium, Myrothecium, Trichothecium, and Stachybotyris, that are toxic to humans and animals that ingest them by consumption of contaminated grain. This work details the characterization of a novel regulatory gene from Fusarium sporotrichioides, Tri10, which is located in the trichothecene gene cluster. Northern analysis of Tri10 deletion strains, Tri10 overexpressing strains, and a Tri6 deletion strain indicated that Tri10 is required for wild-type trichothecene gene expression and for wild-type expression of a primary metabolic gene, Fpps. Analysis of these mutants also provided evidence for a regulatory feedback loop where Tri10 is required for the expression of Tri6 and Tri6 negatively regulates Tri10. Furthermore, under certain growth conditions the sensitivity of ΔTri10 and ΔTri6 strains to T-2 toxin was increased. Analysis of mutants altered in the expression and genomic position of Tri10 revealed that placing Tri10 under the control of an exogenous promoter resulted in the overexpression of Tri10 and the other Tri genes whether this construct was located inside or outside of the Tri gene cluster. Work outside of this study has shown that in addition to Fpps, three other primary metabolic genes from the isoprenoid pathway feeding into trichothecene biosynthesis (Acat, Mk, Hmgs) are also influenced by the expression of Tri10 and Tri6. In the present study, targeted cDNA microarrays were used in conjunction with multiple mutants to reveal a large group of genes, containing both trichothecene and primary metabolic genes, which were positively influenced by Tri10 expression. At the same time, a small group of genes negatively influenced by Tri10 expression was observed. These results were in agreement with observations made outside of this study and validated the use of targeted cDNA microarrays for further studies. Additional analysis of the regulatory network linking trichothecene secondary metabolism and isoprenoid primary metabolism revealed that in a mutant blocked in the first step of the pathway, and therefore in the absence of trichothecene production, this regulatory link is mediated by Tri10 and Tri6.

A method was developed for monitoring the growth of a range of bacteria and fungi in the Bactometer 32 impedimeter. 195 yeast strains, 74 strains of mould and 20 strains of bacteria were screened for sensitivity to 1 mug ml[-1] of T-2 toxin. Growth inhibition was assessed impedimetrically. Twelve bacteria, 20 moulds and 38 yeast strains were tested against roridin A, verrucarin A, deoxynivalenol, and diacetoxyscirpenol each at 1 mum ml[-1] . Deoxynivalenol was found to be only slightly toxic. Roridin A and verrucarin A were markedly more toxic than the others to the fungi, but not to the bacteria. Four microorganisms (Bacillus subtilis, Candida albicans. Hansenula fabianii and Pichia burtonii)were selected for further study. No obvious pattern of response could be discerned for the effects of different carbohydrates on microbial sensitivity to T-2 toxin. The effects of T-2 toxin on B. subtilis and C. albicans were greater if chloroform rather than dichloromethane and methanol (95:5, v/v) was used as the toxin carrying solvent, the reverse was true for H. fabianii and P. burtonii. Dose-response curves were constructed, based on impedimetric responses, and no-effect levels were determined for each species. For B. subtilis the no-effect level was 0.35 mum ml[-1], for C. albicans it was 0.40 mum ml[-1], for H. fabianii it was 0.012 mum ml[-1] and for P. burtonii it was 0.018 mum ml[-1]. H. fabianii was selected for further studies. This strain was found to be auxotrophic for proline. The effect of T-2 toxin and verrucarin A on the uptake of radiolabelled proline in H. fabianii cultures was studied. Dose-response curves were constructed for each toxin. It was found that both toxins reduced proline uptake in a dose dependent manner, the no-effect levels were 0.025 mum ml[-1] for T-2 toxin and 0.0125 mum ml[-1] for verrucarin A.

<div>Ochratoxin A (OTA) is a ubiquitous mycotoxin produced by some species of <i>Aspergillus</i> and <i>Penicillium</i>. It has been detected in a variety of foods such as cereals, coffee, grapes, cocoa, wine, and spices. Consumption of OTA has been linked to kidney and liver diseases. The aims of this study were to determine the effects of (1) water activity and temperature (2) oil content and grinding and (3) probiotic bacteria on fungal growth and OTA production by <i>Aspergillus</i> <i>fresenii</i> and <i>A. sulphureus</i>. In the first study, the two fungi were grown on ground Niger seeds with 0.82, 0.86, 0.90, 0.94 or 0.98 a_w and incubated at 20, 30 or 37°C individually. The two species showed similar growth patterns on Niger seeds under all of the testing conditions. There was no fungal growth on ground Niger seeds with 0.82 a_w and the optimal growth condition for the two species on ground Niger seeds was 0.94 a_w at 30°C. However, the optimal conditions for OTA production by <i>A. fresenii</i> and <i>A. sulphureus</i> were different. The optimal conditions for <i>A. fresenii</i> to produce OTA on ground Niger seeds was 0.90-0.94 a_wat 37°C; whereas, <i>A. sulphureus</i> produced OTA optimally with 0.90-0.94 a_w at 30°C as well as 0.94-0.98 a_w at 20°C. Overall, <i>A. sulphureus</i> produced higher levels of OTA than did <i>A. fresenii</i>. The highest concentration of OTA (643 μg/kg) produced by <i>A. fresenii</i> was detected on seed samples with 0.90 a_w incubated at 37°C for 15 days, while the highest concentration of OTA (724 μg/kg) produced by <i>A. sulphureus</i> was detected on samples with 0.98 a_w incubated at 20°C for 10 days.</div><div>In the second study, growth and OTA production by the two fungi on ground Niger seeds with different oil content (10, 25 and 35%) and on whole Niger seeds at 30°C were compared. All seed samples were adjusted to 0.94 a_w in this study. The two fungi grew most rapidly on ground seeds with 35% oil content, producing high concentrations of OTA (229-453 μg/kg). On whole seeds, <i>A. sulphureus</i> and <i>A. fresenii </i>displayed slow growth until day 5 or 10, respectively, growing rapidly after that. The two species produced either non-detectable or below the limit of quantitation (<4 μg/kg) of OTA in ground seeds with 10 or 25% oil or in whole seeds during the 30-day incubation at 30°C.</div><div>In the third study, growth inhibition of <i>A. fresenii</i> and <i>A. sulphureus</i> by probiotic bacteria <i>Bacillus coagulans</i>, <i>B. coagulans</i> (strains unique IS2TM and GBI-306086), <i>Lactobacillus acidophilus</i> (strains LA-5 and LA-14), <i>L. plantarum</i> (strains 299V and LP115), and <i>L. rhamnosus </i></div><div>was evaluated. Results of co-cultured method revealed that <i>L. plantarum</i> 299V had the highest levels of inhibition against the two fungal species; whereas, <i>L. plantarum</i> LP115, and <i>L. rhamnosus</i> showed only some inhibition effect against <i>A. sulphureus</i> and very little inhibition against <i>A. fresenii</i>. The two fungal species were not inhibited by <i>L. acidophilus</i> or <i>B. coagulans</i>.</div><div>Results from double-layer testing showed that the two <i>L. plantarum</i> strains and<i> L. rhamnosus</i> inhibited fungal growth completely when there were as few as 40-70 CFU probiotic bacterial colonies in the bottom layer of MRS agar; whereas, <i>L. acidophilus</i> inhibited fungal growth completely when the probiotic colonies were >125 CFU/plate. The three <i>B. coagulans</i> strains showed only partial growth inhibition against<i> A. fresenii</i> with 103 CFU/plate. <i>Bacillus coagulans</i> (unique IS2TM and GBI-306086) completely inhibited growth of <i>A. sulphureus</i> when there were as few as 40-70 CFU/plate; while <i>B. coagulans</i> completely inhibited the growth of <i>A. sulphureus</i> but only when there were >103 CFU plate. Even though the two fungal species were inhibited by some probiotic bacteria on MRS plates, the OTA production was not influenced.</div>

The occurrence of mould in food and animal feed is a severe problem due to the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored animal feed and the abundance of spores of the fungus is frequently spread into the air. Gliotoxin has been identified as one of the most toxic second metabolites produced by A. fumigatus. Although A. fumigatus is known to produce mycotoxins that induce neurological syndromes, the neurotoxic properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The major findings were as follows: i. Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status. ii. Gliotoxin reduces the number of neurites, but does not affect the cell bodies morphologically, at non-cytotoxic concentrations. This indicates that the toxin may induce peripheral axonopathy in vivo. iii. The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated. iv. The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity. v. Gliotoxin induces a decreased rate of protein synthesis at non-cytotoxic concentration, which may contribute to the degeneration of neurites. vi. We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.

Mould is a common contamination in cereals. The growth of mould can stimulate mycotoxins production andsome of which at critical concentrations cause health problems in humans and animals. Fusarium is one of thefungus species that has been found in crops and can cause major problems for farmers such as reduced harvestand economic losses. A group of Fusarium species, Fusarium avenaceum, Fusarium poae and Fusariumtricinctum express a mycotoxin, enniatin. The limited information available today about enniatin-forming fungiis that they grow out on fields of wheat in colder climates. This project aims at developing methods for detection,quantification and identification of known and unknown fungi present in Swedish cereals during 2009-2011. Theproject was carried out using two previously published methods, TMAV and MGB, which both use TaqManprobes with realtime-PCR detection. The methods were evaluated for robustness, efficiency, accuracy, inclusionand exclusion. The results showed that both methods, TMAV and MGB, could be used to detect Fusariumavenaceum. The results for the TMAV method were that it could be used with custom annealing temperatureand chemical concentrations for the best detection. The MGB method can be used to detect Fusarium avenaceumwith Fusarium tricinctum in the same analysis. Both methods can be included in future mapping projects when itis of interest to quantify enniatin producing moulds.

Fumonisin B1 is the most prevalent congener of the fumonisin mycotoxins produced by Fusarium verticilliodies and is considered by many to be the most toxic. Fumonisin B1 has been classified by IARC as a class 2B carcinogen. This is primarily due to evidence suggesting increased exposure to FB1 through contaminated foodstuffs is responsible for increased incidence of esophageal cancer in regions of China and South Africa. Fumonisin B1 exposure has also been implicated in the increased incidence of neural tube defects along the Texas/Mexico border. Therefore the principal goals of this research were to 1) Identify sorbent materials that would be compatible with the chemical characteristics of fumonisin B1 and evaluate their ability to sequester the toxin using established in vitro techniques; 2) evaluate urinary FB1 as a biomarker of exposure from a fumonisin contaminated diet; 3) utilize urinary FB1 as a diagnostic tool to evaluate the efficacy of NS in reducing biomarkers of FB1 bioavailability in a Ghanaian population suspected to be co-exposed to aflatoxins and fumonisins. Isothermal analysis and an alternative animal model were examined and compared to previously published results to determine the sorbent toxin interaction activity in vitro as a predictor of in vivo efficacy. An HPLC method for detection and quantitation of urinary FB1 was developed based on methods previously adapted for primary amine and biomarker analysis. Urinary FB1 was evaluated as an HPLC detectable biomarker using a rodent model. Calcium and sodium montmorillonite clays were selected to interact with the positive charge on FB1 at low pH and sorb the molecule. Ferrihydrite was selected to interact with the negative charge on the FB1 molecule at neutral to high pH. While both polarities of sorbent were effective, montmorillonite clays demonstrated a higher capacity for sorption of FB1 than ferrihydrite. These in vitro results were confirmed in a rodent model where urinary FB1 was reduced 27% in NovaSil treated rats vs. controls. Finally, in a Ghanaian population co-exposed to aflatoxins and fumonisins, urinary FB1 was significantly reduced at 2 time points when the NovaSil treatment was compared to placebo.

Mikromicetai yra labai įvairi, gyvybinga, aktyvi, įvairiomis veiklos galiomis pasižyminti mikroorganizmų grupė, gebanti aktyviai sintetinti ir išskirti įvairios cheminės prigimties metabolitus. Toksikologiniu ir cheminiu atžvilgiu tai yra heterogeniška medžiagų grupė, kurią sunku klasifikuoti ir apibūdinti. Mikotoksinai aptinkami tiek gyvulių pašare, tiek ir žmonių maisto produktuose. Jie sukelia įvairius žmogaus bei gyvūnų sveikatos sutrikimus. Toksinis šių junginių aktyvumas siejamas su jų chemine struktūra. Darbas skirtas mikotoksino deoksinivalenolio toksinio poveikio tyrimui, panaudojant dvi modelines tyrimo sistemas - laboratorinius gyvūnus (in vivo) ir ląstelių kultūras (in vitro). Deoksinivalenolis - dažniausiai Fusarium genties mikromicetų produkuojamas metabolitas, savo struktūroje turintis epoksido grupę, kuri, manoma, ir lemia jo toksiškumą. Panaudojus laboratorinius gyvūnus (peles), kaip tradicinę toksikologinių tyrimų sistemą in vivo, nustatėme, kad deoksinivalenolis po 14-kos parų kasdienio 15 µg/kg koncentracijos poveikio nesukėlė pastebimų organizmo pakitimų, gyvūnai buvo žvalūs, smalsūs, jų svoris augo. Tačiau po išsamesnių tyrimų buvo registruoti kraujo pakitimai, IgA koncentracijos padidėjimas. Panaudojus kitą modelinę toksinio poveikio tyrimo sitemą - ląstelių analizę kultūroje in vitro - nustatėme, kad labai mažos (5 ir 10 µg/ml) tiriamo mikotoksino koncentracijos sukėlė ląstelių pažeidimus. Šiame darbe deoksinivalenolio toksiškumo tyrimui mes... [toliau žr. visą tekstą] / Our study is devoted to the analysis of mycotoxin deoxinyvalenol toxicity and comparison of toxicity tests in the model systems in vivo and in vitro. It is known that mycotoxin-producing mold species grow on a wide range of substrates under a wide range of environmental conditions, they are pharmacologically active mold metabolites characterized by vertebrate toxicity. They fall into several chemically unrelated classes, are produced in a strain-specific way and vary in specificity and potency for their target organisms, organs or cells. We have used two model systems for the analysis of deoxinyvalenol effect. One of them is laboratory animal (mice) study or test system in vivo. It is as a traditional method for the evaluation of the toxic substances. We have found that animal diet containing 15 µg/kg deoxynivalenol during short-term (14 days) repeated toxicity test didn‘t influence noticeably animal health. Such small concentration of tested mycotoxin did not appear to depress animals, they were alive and curious, animal weight didn‘t fall behind control. Howewer, at the end of experiment we have found the decrease of mice blood parameters and dramatic increase of Ig A concentration in blood serum. Another test system used in our study was cell culture or test system in vitro. We have used permanent cell lines and newly established primary tissue culture cell lines, derived from potential mycotoxin-target organs: renal and lung. The results presented in this study describe... [to full text]

Master of Science / Department of Animal Sciences and Industry / Cassandra Jones / Three experiments were conducted to analyze the average mycotoxin concentration that may be reduced by cleaning corn, and to determine how removing broken kernels may affect nursery pig growth performance. A fourth and fifth experiment evaluated pellet processing parameters and their effects on gelatinized starch, phytase stability, pellet quality, and nursery pig growth performance. In Exp. 1 and 2, corn was divided into twenty 150 kg runs then cleaned by mechanical sieving. Run were randomly assigned to 1 of 4 experimental treatments: 1) no screen 2) 12.7 mm screen, 3) 4.8 mm screen, and 4) 12.7 + 4.8-mm screen. Across both experiments, cleaning reduced (P < 0.05) aflatoxin and fumonisin concentration by an average of 26% and 42.5%, respectively, compared to the original uncleaned corn level. In Exp. 3, 360 nursery pigs were evaluated to determine the impact of cleaning or pelleting on growth performance. Treatments were arranged in a 2 × 3 factorial with corn type (uncleaned vs. cleaned) and feed form (mash vs. pelleted from either mill A or B). Neither cleaning corn nor pellet mill type affected (P > 0.19) nursery pig growth performance. Pelleting improved (P < 0.0001) G:F by 7.6% compared to mash diets. This improvement in G:F is consistent when pelleting diets, however pellet processing parameters can influence this improvement percentage. For these reasons, Exp. 4 was a 3 × 4 factorial design with 3 pellet mills (model 3016-4, 1000 HD, or CL-5, California Pellet Mill Co., Crawfordsville, IN), that produced samples collected at 4 locations (initial, post-conditioner, post-die, or post-cooler). Across each pellet mill, the greatest gelatinized starch increase (P < 0.05) was found post-pellet die, while phytase stability decreased (P < 0.05) by 70% after conditioning feed to 85˚C. This decrease led to substituting phytase in the diet for other sources of phosphorus for Exp. 5, which was a 2 × 3 factorial design plus a control, with pellet diameter (4.0 or 5.2 mm), conditioning temperature (low, medium, or high), and mash, created seven experimental treatments. Overall, neither the pellet diameter × conditioning temperature interaction, nor the main effects, affected (P > 0.06) nursery pig growth performance, even though pellet quality improved (P < 0.0001) when increasing conditioning temperature. These data suggest that cleaning is an effective method to legally reduce aflatoxin and fumonisin concentration, and that increasing conditioning temperature improves pellet quality, but neither impacts nursery pig growth performance.