The mycotoxin, fumonisin B1 is produced by the mould Fusarium moniliforme, a common contaminant of maize and maize products. Small doses (mg/kg) of ingested fumonisin B1 have been shown to cause diseases and even death in animals, including non-human primates. Thus highly sensitive methods have been employed to detect fumonisin B1 presence in foods, feeds and in animals. This study comprised two parts.The initial part focused on establishing reliable extraction, purification and quantitation of fumonisin B1 using high-performance liquid chromatography (HPLC) on culture extracts. The second part was to analyse sera of Black African women with pre-eclampsia for the presence of fumonisin B1 using HPLC. Maize patty cultures and broth cultures were inoculated with Fusarium moniliforme PPRI 1059 and incubated. Fumonisin B1 was extracted and purified by centrifugation strong anion exchange chromatography (SAX). Eluents from SAX cartridges were analysed using Thin-layer chromatography (TLC) and fluorescence HPLC after o-phythadialdehyde (OPA) derivatisation. Fumonisin B1 standards on HPLC gave a retention time of 7.5 minutes using methanol/0.1 M sodium dihydrogen phosphate (68 + 32, pH 3.3) as mobile phase and a 25 cm C8 column. Patty cultures produced the highest yields of fumonisin B1. In the case of serum samples, a double-blind study was carried out using women attending the obstetric clinic at a large city teaching hospital. The population comprised normal, pre-eclamptic and eclamptic women. On HPLC analysis a significantly higher mean concentration of fumonisin B1 concentration was found in the eclamptic group (P<0,005) as compared to the other two groups.Thus fumonisin B1 may have a role to play in eclampsia for which the aetiology is still unknown. / Thesis (M.Med.Sc.)-University of Natal, 1999.

Chemoprevention may be defined as the inhibition, delay or reversal of carcinogenesis by dietary compounds or their derivatives. "Imifino" is a collective name for many wild plants used predominantly by rural people as herbs in cooking. Many of these herbs possess medicinal properties. As the rural population is at higher risk of exposure to dietary carcinogens, such as mycotoxins, this pilot study was undertaken to determine whether the Amaranthus lividus plant held potential for use in chemopreventive strategies. The plant leaves were extracted to obtain individual solvent fractions. Cytotoxic profiling of the fractions using the SNO oesophageal adenocarcinoma cell line and normal human lymphocytes was achieved using the methylthiazol tetrazolium salt bioreduction assay. The SNO cell line, the A549 lung adenocarcinoma cell line and normal human lymphocytes were utilised for the evaluation of the anti-mycotoxigenic potential of the plant fractions in combination with two important dietary carcinogens, aflatoxin B1 and fumonisin B1. A specific biomarker assay (the induction of reduced glutathione) was employed using the SNO cell line. Flow cytometry was also conducted to determine the apoptotic properties of the acetone fraction on normal human lymphocytes. The results of the anti-mycotoxigenic study showed that certain fractions did have protective effects against both of the carcinogens tested. In addition, these effects were noted in the two cancerous cell lines, which were of different tissue origin. None of the fractions tested were toxic towards the normal human lymphocytes. The glutathione assay indicated that certain acetone fraction dilutions were inducive to reduced glutathione production. This plant is a promising candidate for further investigation concerning chemoprevention and the rural community could be educated on the possible benefits of this herb. / Thesis (M.Med.)-University of KwaZulu-Natal, 2005.

Fumonisins, mycotoxins produced by the fungus, Fusarium moniliforme, which grows on maize, are a major worldwide agricultural problem. Consumption of contaminated maize feeds causes a wide variety of toxic effects in animals depending on the species of animal. In humans, high concentrations of fumonisins have been shown to correlate with increased incidence of oesophageal cancer (OC). Most analyses for fumonisins are done using high performance liquid chromatography (HPLC) which requires time-consuming extraction and clean-up prior to preparation of a fluorescent derivative. Enzyme-linked immunosorbent assays (ELISA), which are sensitive and specific, are a viable alternative but commercially available antibodies and kits are extremely expensive. Polyclonal antibodies against fumonisin B, (FB,) were raised in chickens and rabbits; all animals produced antibodies from week 2 onwards, the highest titre was at week 8 from one of the chickens. Cross-reactivities with FB, analogues were checked. A sensitive, quantitative competitive indirect ELISA (CI-ELISA) was developed and optimised; range 0.2 to 20 ng/ml (in buffer), detection limit 0.2 nglml (in buffer), intra-assay coefficient of variation (CV) was 5.33 % and inter-assay 7.04%. This method was adapted to analyse human plasma and urine samples. After removal of proteins by boiling, the range of recoveries of FBI were 94.7% toI12.4% at 4 ng/ml; and 94.6% to 108.7% at 8 ng/ml. Blood and urine samples from patients with OC (40 plasma, 17 urine), controls (21 plasma, 12 urine) and patients with other forms of cancer (20 plasma, 10 urine) were collected from hospitals in the Durban Metropolitan area and analysed for fumonisins. Detectable levels (>0.4 nglml) were found in 86.9% of plasma samples and 94.9% of urine samples. Statistical evaluation showed a highly significant difference between plasma results for OC and controls (p<0.000 1) but no significant difference between the urine results. Comparison of other forms of cancer and controls showed no significant differences for either the plasma or the urine samples. However, there was a highly significant difference between the OC and other forms of cancer results for both plasma (p<0.005) and urine (p<0.05) samples. Some samples (9 plasma, 8 urine) were checked by HPLC. For plasma samples there was correlation between the ELISA and HPLC methods (r = 0.656, p<0.005) but not for urine samples.

Consumption of feedstuffs contaminated by Fusarium mycotoxins and the development of equine gastric ulcer syndrome (EGUS) affect the overall health of horses. A study was conducted to determine if feed-borne mycotoxins have an effect on EGUS, and the efficacy of a glucomannan mycotoxin adsorbent (GMA) was determined. Feed intake was decreased with the GMA diet compared to control horses. Body weight, ulcer score and gastrin concentration were unaffected by diet. Some significant changes in blood parameters potentially indicate liver damage and inhibition of protein synthesis. Histological evaluation showed an increase in mononuclear cells in the glandular region of the contaminated group likely indicating signs of gastritis. The incorporation of the GMA may reduce these negative effects on the horse based on our findings. It can be concluded that horses are susceptible to Fusarium mycotoxins with a possible effect on EGUS, as seen in the cells of the gastric glandular mucosa. / Alltech Inc., Nicholasville, Kentucky and OMAFRA

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. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

The central nervous system is a potential site of action for the Fusarium mycotoxin Fumonisin B1 (FB1), and is exemplified in horses by the disease equine leukoencephalomalacia. Structurally resembling sphingoid bases, FB1 inhibits ceramide synthase, an enzyme involved in sphingolipid metabolism, leading to accumulation of free sphinganine (Sa) and sphingosine (So). This investigation focused on FB1, Sa, So and the Fusarium mycotoxins fusaric acid (FA), moniliformin (MaN), zearalenone (ZEA), deoxynivalenol (DON), and T-2 toxin (T2). Effects of the Fusarium mycotoxins and sphingoid bases on the N2a neuroblastoma cell line were assessed using the methylthiazol tetrazolium (MIT) and ApoGlow™ assays. The MIT assay revealed significant differences between the viability of N28 control cells and the cytotoxic effects of FB1 (p=0.001), So (p=1.1 x10-6 ), Sa (p=1.9x10-6 ), MON (p=0.002), DON (p=0.04) and ZEA (p=0.003) on N28 cells between 5-250µM. The cytotoxic effects of FA did not differ significantly from controls (p=0.1). The ApoGlow™ assay revealed that in N28 cells, FB1 at 8µg.ml-1, FA at 128µg.ml-1, and (FBI+FA) combined induced growth arrest at 2 and 4µg·ml-1. Assessment of the effects of FBI and FA on the Jurkat leukaemic suspension cell line revealed that FB1 induced apoptosis at 1.56,12.5 and 50µ.ml-1, growth arrest at 100, 200 and 800µg.ml-1 and proliferation at 400µg.mg-1. Fusaric acid induced proliferation at 1. 56µg.ml-1, apoptosis at 3.15µg.mrl, growth arrest at 100 and 200µg.mrl, and necrosis at 800µg.ml-1. Combined, (FB1+FA) induced apoptosis at 1.56, 3.15,12.5 and 800µg.ml-1. Flow cytometry and fluorescence microscopy revealed that mycotoxins, Sa and So induced varying levels of apoptosis and necrosis in N28 cells. Acridine orange and ethidium bromide staining facilitated discrimination between viable, apoptotic and necrotic cells. Transition of the mitochondrial transmembrane potential was measured using Rhodamine 123 with propidium iodide, and the dual emission potential sensitive stain JC-1. Changes in mitochondrial membrane potential and plasma membrane integrity were expressed as increases or decreases in fluorescence intensity. An increase in mycotoxin concentration from 50 to 200µM was usually paralleled by a decrease in J-aggregate formation, suggesting a decrease in the ?¦¥m. Staining with Rh 123/PI indicated at specific concentrations whether N28 cells were either late apoptotic or necrotic reflected by the levels of PI uptake. No dose dependant mechanism of cell death was established using either method, as fluctuations were evident. Immunolocalisation of T2, ZEA and FB1 within cellular organelles that exhibited ultrastructural pathology provided correlation between mycotoxin exposure and effects. Multinucleate giant cells and retraction of cellular processes were observed. At the electron microscope (EM) level, FB1 was immunolocalised within microsegregated and peripherally condensed nucleoli, the nucleoplasm, distorted mitochondria and dilated endoplasmic reticulum (ER). The capacity of cells to incorporate mycotoxins and effect cytological changes represents a major factor in the potential for initiation of malignant transformation. Exposure of N2a cells to FB1 for 72 hours increased intracellular free Sa and depleted complex sphingolipids. Using High Performance Liquid chromatography (HPLC), acid hydrolysis revealed reduction in Sa from a level of O.6±0.12µM in control cells, to 02±0.lµM in cells exposed to 50µM and lOOµM FB1. Base hydrolyses revealed increase in free Sa: So ratios from 0.52±0.2 in control cells, to 1.14±0.2 and 1.4±0.3 in cells exposed to 50 and l00µM FB1 respectively. The Sa: So ratio in the complete culture media (CCM) increased from 1. 7±0. 3 for control cells to 2.0±0.2 and 2.50±0.4 for cells exposed to 50 and lOOµM FB1 respectively. Correlation coefficients between Sa: So ratios to FB1 exposure in CCM (R=0.75) and within cells (R=0.85), imply that the free Sa: So ratio within cells appears to be a better biomarker for FB1-induced disruption of sphingolipid metabolism in vitro, than the Sa: So ratio in CCM. Optimisation of HPLC analytical procedures improved recovery of FB I from spiked human sera to 95.8% (n=15) and detection limits to -5ng.ml-1 at a signal to noise ratio of 5:1. Optimisation of methods for recovery of Sa and So from spiked sera, led to recoveries of 77.9% and 85.0%, for So and Sa respectively at levels of spiking with lOng per 500µl of serum. Matched sera Sa:So ratios and FB1 levels in brain cancer and non-cancer subjects in KwaZulu-Natal were determined using these optimised methods. Fumonisin B1 was detected in sera of non-cancer (76.7±62.2nM) and brain cancer subjects (l07.38±116nM). Mean serum Sa:So ratios of 21 non-cancer subjects was 1.7±0.7. There was no correlation (R=0.26) between these variables in non-cancer subjects. The mean serum FB1 level in brain cancer subjects was 107.4±116nM (range 10.5-298nM) (n=50) and the mean Sa:So ratio (n=50) was 1.9±1.7 (range 0.40-8.16). No correlation was found between these variables in the brain cancer subjects either (R = -0.23). Fumonisin B1 was irnmunolocalised in 49 of 76 brain tumour tissue samples analysed using immunohistochemistry (IHC). Thirty-eight of the 76 specimens had matched serum FBI levels and Sa: So ratios, and 23 of these were positive for FB1 presence. Although not significantly different (p=0.ll), the FBI sera levels in the cancer group with FBI within the tumour tissue had higher levels of FB1 in sera than the IHC FB1 negative group. Fumonisin B1 was localised within irregular profiles of nuclei, elongated and swollen mitochondria and ER. Immunolocalisation of FB1 within organelles in the brain showing ultrastructural cellular pathology suggests FBI may be implicated in the aetiology of human brain carcinogenesis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.

The role of vitamin K in the blood clotting cascade has been well documented. Vitamin K has recently been implicated in improving bone health. The current studies were conducted to determine the effects of vitamin K in diets with and without mycotoxin contaminated corn on growth performance, bone characteristics, and related blood metabolites in pigs from weaning to market. Menadione sodium bisulfite complex (MSBC, 33% vitamin K) was chosen as the source of supplemental vitamin K because it is the most common form fed to swine. Vitamin K was tested at 0, 0.5, and 2.0 ppm in a corn-soybean meal based diets on two generations of pigs to evaluate any time and dose responses. The first generation of pigs was subjected to mycotoxin contaminated corn in the nursery phase to test for any interactions between the toxins and vitamin K. The addition of 0.5 ppm vitamin K reduced (P < 0.0001) prothrombin time. No additional decrease in prothrombin time was detected when increasing vitamin K inclusion from 0.5 to 2.0 ppm. With regard to growth performance, daily gain, feed intake, and feed efficiency were unaffected (P > 0.10) by supplemental vitamin K. However, pigs fed mycotoxin contaminated corn ate less (P = 0.005) and grew slower (P = 0.015) compared to those receiving good corn. The addition of vitamin K did not alleviate the negative growth effects in response to corn type. Vitamin K did not affect bone characteristics (P > 0.10), blood Ca (P > 0.05) or OC (P > 0.10). Other than blood clotting it does not appear that dietary vitamin K provides any additional benefits at these levels of inclusion and stages of swine production.

Two experiments were conducted to determine the effect of feeding diets with a 2009 and 2010 naturally-contaminated corn to weaning pigs. For both experiments three diets were blended to contain 100% 2010 naturally-contaminated corn (control), 50-50% blend of the 2009 naturally-contaminated corn and 2010 corn (Diet 2), and 100% 2009 naturally-contaminated corn (Diet 3). In Exp. 1, 24 crossbred pigs with an average body weight of 7.64 ± 0.70 kg were allotted to 4 replicates of 3 treatments with 2 pigs per pen, on the basis of gender, litter mate, and BW in a randomized complete block design. Fecal and urine samples were collected and dry matter, energy, and nitrogen apparent digestibility were determined. Dry matter, energy, and nitrogen digestibility were not affected by either Diet 3 or Diet 2 compared to the control diet. In Exp. 2, 30 crossbred pigs with an average body weight of 7.98 ± 1.15 kg were allotted to 3 replicates of 2 comparisons with 5 pigs per pen. Comparisons consisted of: 1) Control vs Diet 3, and 2) Control vs Diet 2. Two feeders were located in each pen containing one of the two diets. Feed preference and growth performance were determined. A preference for the feed containing 2010 corn feed was observed; pigs showed the ability to discriminate mycotoxin-contaminated feed (95.34 vs. 4.66%; P< 0.01). Nutrient digestibility was not affected by these diets, but a clear decrease in feed intake was observed in the pigs.

Three experiments were conducted to assess the efficacy of including selected feed additives in the diet of weaning and grow-finish pigs. Experiment 1 utilized 24 crossbred grow-finish pigs and measured the effect of added EHY on DM, N, and energy digestibility. There were no differences in DM, Energy, and N digestibility between diets 1 through 4. Experiment 2 utilized a total of 36 crossbred pigs [18 barrows, 18 gilts] in order to determine if preference would be shown when presented with naturally-contaminated corn. There were three dietary comparisons, Control vs Diet 2 (Comparison 1), Control vs Diet 4 (Comparison 2), and Diet 2 vs Diet 4 (Comparison 3). A preference was shown for the control diet over Diet 2, as well as for the control diet over Diet 4. Experiment 3 utilized a total of 24 crossbred pigs [12 barrows, 12 gilts] in order to measure the effect of contaminated corn on performance and DM, energy, and N digestibility. DM, energy, and N digestibility were affected by corn quality.

The use of near ambient air drying for the preservation of wheat stored in granaries is common in Western Canada. Guidelines have been developed to assist farmers in selecting appropriate drying methods. During this process the top layer of wheat can remain at moisture contents (m.c.) greater than the safe storage limit, 14.5% wet bulb (wb), for up to 12 weeks. This study tested the effects of this drying procedure on the development of ochratoxin A (OTA) using 1 m3 bulks of durum wheat at 18% m.c. (wb) contained within steel bins inside a Weather Simulation Lab. In a second study using 20 L volumes of wheat at a m.c. of 20% (wb) within an environmental growth chamber potential development of OTA was also evaluated. The wheat was exposed to two treatments, airflow and no airflow, for a period of 12 weeks under conditions of high relative humidity (greater than 80%) and typical Manitoba fall temperatures. The storage quality parameters of germination, fat acidity value, and presence of OTA were measured weekly. It was found that high moisture wheat stored under all treatment conditions showed a rapid decrease in germination and increase in fat acidity value over time, with no significant difference between the treatments. Under the tested conditions the development of ochratoxin A was not detected in significant quantities in the 1 m3 bulks of grain but was detected in the smaller 20 L bulks.