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ABSTRACT [ILLUSTRATION OMITTED] Aflatoxins are secondary metabolites produced by Aspergillus flavus and A. parasiticus species of fungi. They are highly toxic and have been designated by the International Agency for [...]

Malnutrition is a major problem in East Africa and animal source foods could provide essential protein and micronutrients to help alleviate this. The livestock sector is rapidly growing and intensifying in response to increased consumer demand for animal source foods. However, the ability of the livestock sector to contribute to improving nutritional security is constrained by a number of factors, including contaminants in animal source foods and feeds. Globally, mycotoxins (especially aflatoxins) are the most important contaminants of livestock feed. Aflatoxins are produced by the fungus Aspergillus flavus and related species, which occur naturally in soils throughout sub-Saharan Africa. They are toxic to humans, fish and many other animals, even in low concentrations. Susceptibility to aflatoxins varies by age, health status, species and other factors. Most research has focused on aflatoxins in maize or groundnuts and their impacts on human health. However, aflatoxins are found in other foods and can also impair livestock productivity, reducing the availability of nutritious foods and the income of smallholder farmers. Aflatoxins are also transferred into animal source foods, which could harm consumers. The objective of this review was to synthesize information on the impact of aflatoxins on livestock health and productivity, with a special focus on reports from East Africa. A systematic literature review identified 2700 abstracts in 23 databases. Of these, 46 articles were relevant to the review objective and available. This review summarises key data on impacts of aflatoxins in animal health and levels of aflatoxins in animal source foods. The studies and surveys suggest that aflatoxins may be a significant risk to livestock productivity and food safety in East Africa. Impacts are likely to worsen as livestock industries intensify in response to the growing demand for animal source foods. Climate change may also aggravate aflatoxin problems. In light of this challenge, this review identifies major research gaps and discuss the way forward. Key words: mycotoxins, livestock, animal source foods, food security, food safety, East Africa, aflatoxins, fish, INTRODUCTION Malnutrition, including micronutrient deficiencies, is a major problem in East Africa. Animal source foods are essential sources of protein and micronutrients and could contribute to improving nutritional security. Indeed [...]

Maize, a staple food in most African countries, is prone to contamination by aflatoxins, toxic secondary metabolites mainly produced by Aspergillus flavus and A. parasiticus. Aflatoxins are known to cause liver cancer, and chronic exposure has been linked to other adverse health outcomes including growth faltering in children. To mitigate exposure in maize-dependent populations, there is need to identify the factors associated with aflatoxin contamination. This is difficult, however, because of high sampling cost and lack of affordable and accurate analytical methods. Publicly available, remotely-sensed data on vegetation, precipitation, and soil properties could be useful in predicting locations at risk for aflatoxin contamination in maize. This study investigates the utility of publicly available remotely-sensed data on rainfall, vegetation cover (indicated by normalized difference vegetation index or NDVI), and soil characteristics as potential predictors of aflatoxin contamination in Kenyan maize. Aflatoxin was analyzed in maize samples (n=2466) that were collected in 2009 and 2010 at 243 local hammer mills in eastern and western Kenya. Overall, 60% of maize samples had detectable aflatoxin. Global positioning system coordinates of each mill location were linked to remotely-sensed, spatially explicit indicators of average monthly NDVI, total monthly rainfall, and soil properties. Higher rainfall and vegetation cover during the maize pre-flowering period were significantly associated with higher prevalence of aflatoxin contamination. Conversely, higher rainfall and vegetation cover during the maize flowering and post-flowering periods (not including harvest) were associated with lower prevalence of aflatoxin contamination. Water stress throughout the growing season may cause increased plant susceptibility to fungal colonization and aflatoxin accumulation. Soil organic carbon content, pH, total exchangeable bases, salinity, texture, and soil type were significantly associated with aflatoxin. In conclusion, this study shows that remotely-sensed data can be regressed on available aflatoxin data highlighting important potential predictors that could reduce the cost of data collection and the cost of aflatoxin risk forecasting models. Key words: Aflatoxin, GIS, NDVI, soil characteristics, rainfall, mycotoxins, East Africa, Kenya, INTRODUCTION It is estimated that approximately 4.5 billion people, predominantly those living in developing countries, are at risk of being exposed to dietary aflatoxins, with many chronically exposed at high [...]

Aflatoxins are toxic by-products of fungi contaminating maize and other crops; they can be carried over into milk, meat and eggs when livestock eat aflatoxin-contaminated feed or fodder. People who consume such animal products are exposed to the toxins. To assess the aflatoxin contamination status in marketed raw milk and associated risk factors in peri-urban Nairobi, we conducted a census of raw milk retailers in Dagoretti Division, Nairobi, Kenya. Structured questionnaires were filled in by face-to-face interviews with all retailers who agreed to participate in this study. Small portions of milk were purchased from each respondent and tested for aflatoxin M1 (AFM1) using competitive enzyme-linked immunosorbent assay (cELISA) tests. Geographic coordinates of each eligible retailer were recorded. A total of 350 milk retailers were visited and 344 were plotted on a map. Of these, 250 retailers answered the questionnaire and a milk sample from each of 200 retailers underwent cELISA analysis for AFM1. Four types of businesses were found: kiosks (71%), dairy shops (21%), street or mobile vendors (3%) and grocery stands (1%); for 4% the business type was not identified. Milk was mainly sourced directly from dairy farms (59%) or from intermediate distributors (35%). Most retailers sold less than 20 litres per day of raw milk. The mean daily milk consumption of the milk retailers' households was 940 ml for adults and 729 ml for children. Although 58% of retailers had heard about aflatoxins and the majority of them agreed that aflatoxins could be present in milk, only 29% believed that 'milk safety cannot be solely judged by sight or taste' and only 6% that 'milk is not completely safe even after boiling'. The cELISA test found the mean concentration of AFM1 was 128.7 parts per trillion (ppt) (median=49.9; 95% confidence interval=3.0-822.8) with a maximum of 1675 ppt. Overall, 55% of samples exceeded the European Union maximum level of 50 ppt and 6% exceeded the recommended maximum level of the United States Food and Drug Administration of 500 ppt. Compared to milk from street vendors, a significantly higher amount of AFM1 was detected in milk from kiosks and dairy shops, especially when the milk was sourced from farms without an intermediate distributor. Our findings indicate the need to better understand and manage aflatoxin in milk in Nairobi. Key words: Aflatoxin M1, Kenya, milk, dairy value chain, milk retailers, Dagoretti, mycotoxin, informal milk marketing, INTRODUCTION Aflatoxins are toxic by-products of fungi, mainly Aspergillus flavus and A. parasiticus, which contaminate maize and other crops in areas with hot and humid climates. Aflatoxin contamination is a [...]

Mycotoxin-producing fungi contaminate food and feeds before, during and after harvest. Aflatoxins are important mycotoxins and aflatoxin B1 (AFB1) is a class 1 human carcinogen (definitely carcinogenic). Aflatoxin M1 (AFM1) is a class 2B (possible) human carcinogen. Aflatoxin B1 in feeds can decrease milk production, reduce fertility and increase susceptibility to infections. A cross-sectional study of aflatoxin contamination of milk and dairy feeds was carried out in five counties in Kenya representing different agro-ecological zones: Kwale, Isiolo, Tharaka-Nithi, Kisii and Bungoma. Dairy feed concentrates and cattle milk were collected twice (dry season and rainy season) from 285 dairy farmers in the five counties and analysed for AFB1 and AFM1, using competitive enzyme-linked immunosorbent assay (ELISA). In the five counties, the proportion of farmers who fed cattle with dairy concentrates varied from zero to 68%. The dairy feed concentrates from farmers had AFB1 levels ranging from less than one part per billion (ppb) to 9661 ppb and the positive samples ranged from 47.8 to 90.3%. The percentages of dairy feeds from farmers with AFB1 above the World Health Organization/Food and Agriculture Organization of the United Nations (WHO/FAO) limit of 5 ppb varied from 33.3% to 87.5% while 83.3% to 100% of the feeds from retailers and 28.6% to 100% of the feeds from manufacturers exceeded the WHO/FAO limit. Aflatoxin M1 prevalence in milk was lowest in Kwale (13.6%) and highest in Tharaka-Nithi (65.1%). The proportion of milk samples with AFM1 above the WHO/FAO standard of 50 parts per trillion (ppt) varied from 3.4% (Kwale) to 26.2% (Tharaka-Nithi); the highest was 6999ppt. This study shows that aflatoxin contamination is common in dairy feeds and in milk and concentrations may be high. This may contribute to ill health effects in both humans and animals and, therefore, there is need for better understanding of the impacts of aflatoxins in the feed-dairy value chain and appropriate interventions to control aflatoxin contamination in animal feeds. Key words: aflatoxins, feeds, dairy cattle, milk, Kenya, dairy value chain, mycotoxins, food safety, INTRODUCTION Kenya's dairy industry is a major source of livelihood for farmers, milk processors, milk traders, feed manufacturers and feed retailers. The industry contributes 14% of the agricultural gross domestic [...]

Aflatoxins, especially aflatoxin B1 (AFB1), can be greater in dried distillers grains with solubles (DDGS) because it can be concentrated during the ethanol production process. Increased use of DDGS in swine diets could potentially lead to an increased incidence of aflatoxicosis, a disease associated with decreased feed intake, reduced BW gain, and impaired liver function. The objective of this study was to determine the effects of AFB1 on the health, performance, and serum profile of growing barrows. Ninety Duroc x Yorkshire crossbred barrows were purchased (age = 35 [+ or -] 5 d; BW = 14.2 [+ or -] 3.0 kg), allocated to 9 pens with 10 pigs per pen, and randomly assigned to receive diets containing 0 [micro]g/kg of AFB1 (CON), 250 [micro]g/kg of AFB1 (LO), or 500 [micro]g/kg of AFB1 (HI) for 7, 28, or 70 d in a 3 x 3 factorial arrangement of treatments. Feed intake was measured daily, and pigs were weighed and blood samples collected weekly. Serum was analyzed for concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (BILI), and blood urea nitrogen (BUN). Both ADFI and ADG were negatively affected (P [less than or equal to] 0.001) by AFB1 treatment. Average daily feed intake was less (P < 0.05) in HI barrows than in CON barrows from wk 5 to 10 and was less (P < 0.05) in LO barrows than in CON barrows in wk 5 and again from wk 8 to 10. Also, ADFI was less (P = 0.022) in HI barrows than LO barrows in wk 10. Decreased ADG (P < 0.05) was observed in HI barrows than in CON barrows in wk 8 and 10; no differences (P [greater than or equal to] 0.665) in ADG were noted between CON and LO barrows. There was no effect (P > 0.080) of AFB1 treatment on ALT or BILI concentrations. However, both AST and BUN were affected (P < 0.05) by AFB1 treatment. Serum AST was greater (P = 0.010) in LO barrows than CON barrows in wk 5, and serum BUN was greater (P = 0.004) in CON barrows than LO barrows in wk 3. Results from this study demonstrate that the performance and health of young growing barrows were affected by consumption of an AFB1-contaminated diet, especially when fed for a more extended period. Key words: aflatoxin, health, performance, swine doi: 10.2527/jas.2009-2663

Invasion of maize grain by fungi leads to losses in quantity and quality but also to contamination by mycotoxins, fungal metabolites, which are harmful to human and animal health. The problem of mycotoxins is higher in developing countries because of poorly managed environmental factors and due to the gap in supply and demand of food that forces people to consume what they might have otherwise rejected. The objective of this study was to determine the fungi and major mycotoxins that are naturally occurring in maize from Ethiopia where little information existed so far. Maize grain samples of 2004/2005 harvest were collected from small scale farmers' storage in Adama, Ambo and Dire Dawa representing three agroecological zones of Ethiopia. Surface and internal fungi of samples were determined employing dilution plate method on DG-18 agar. Samples were also analyzed for aflatoxins (AFT) and fumonisins (FUM) using enzyme linked immunosorbent assay (ELISA), and for deoxynivalenol (DON) and nivalenol (NIV) using high performance liquid chromatography (HPLC). Species of Aspergillus, Fusarium and Penicillium occurred in 94%, 76.5% and 64% of the samples, respectively. Fusarium verticillioides and Aspergillus glaucus were among the dominant species. A. flavus was less frequent. The mean levels of total fungal density ranged from 2.9x[10.sup.3] cfu [g.sup.-1] in maize samples from Dire Dawa, 1.9x[10.sup.4] [cfug.sup.-1] for samples from Adama to 3.8x[10.sup.5] cfu [g.sup.-1] for samples from Ambo. Aflatoxins were detected in 88% of the samples at 27 µg [kg.sup.-1] in one sample and less than 5 µg [kg.sup.-1] in others. Fumonisins occurred in two samples from Dire Dawa at 700 and 2400 µg [kg.sup.-1], and at 300 µg [kg.sup.-1] in one sample each from Adama and Ambo. Five samples contained DON at 50 - 700 µg [kg.sup.-1]. NIV was detected at 50, 130 and 210 µg [kg.sup.-1]. The mycotoxins analyzed played a minor role in the contamination of maize of 2004/05 harvest in Ethiopia. Further monitoring of mycotoxins in maize from different regions of the country is justified in order to conclusively determine the actual risks from mycotoxins and possibly low mycotoxinrisk maize production areas. Key words: Ethiopia, maize, aflatoxins, fusariotoxins, mycobiota, INTRODUCTION It is well established that maize grain can be invaded by fungi, which generally cause losses in weight and quality. Moreover, some of the fungi associated with maize are [...]

Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C--C ring closure reactions as opposed to thioester hydrolysis or O--C/N-C macrocyclization observed in previously reported TE structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B1, a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 [Angstrom] crystal structure displays an a/[beta]-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C-C cyclization. The biochemical studies, the 1.7 [Angstrom] crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C-C bond-forming class of TEs. biosynthesis | fungal metabolism | mutagenesis doi/10.1073/pnas.0913531107

BACKGROUND: Cirrhosis of the liver is thought to be a major cause of morbidity and mortality in sub-Saharan Africa, but few controlled studies on the etiology of cirrhosis have been [...]

Dioctatin A (DotA), a metabolite of Streptomyces, is known to be an inhibitor of human dipeptidyl aminopeptidase II. Here, it was found that DotA strongly inhibited aflatoxin production by Aspergillus parasiticus, with an [IC.sub.50] value of 4.0 [micro]M. The mycelial growth of the fungus was not affected by the addition of DotA at a concentration of 50 [micro]M, but inhibition of conidiation was observed at the same concentration. DotA inhibited production of norsolorinic acid, an early biosynthetic intermediate of aflatoxin, and it strongly reduced the mRNA levels of genes encoding aflatoxin biosynthetic enzymes, and significantly decreased the mRNA level of aflR, which encodes a key regulatory protein for aflatoxin biosynthesis. In addition to these genes, the mRNA level of brlA, which encodes a conidiation-specific transcription factor, was also reduced by the addition of DotA. It was also found that DotA dramatically enhanced kojic acid production by the fungus. Furthermore, DotA inhibited production of sterigmatocystin, which is a toxic aflatoxin biosynthetic intermediate, and it also inhibited conidiation in Aspergillus nidulans. These results indicate that DotA has pleiotropic effects on regulatory mechanisms of fungal secondary metabolite production and differentiation, leading to inhibition of aflatoxin production.

The potential of Lactobacillus rhamnosus GG to reduce aflatoxin [B.sub.1] (AF[B.sub.1]) availability in vitro in Caco-2 cells was investigated. Results suggested that (AF[B.sub.1]) transport can be reduced by GG binding in the Caco-2 model, while these Caco-2 cells can be used to estimate the intestinal metabolism and toxicity of (AF[B.sub.1]) by use of TER measurements.

Drought stress and aflatoxin contamination continue to challenge peanut (Arachis hypogaea L.) producers across the USA. Thus, the continued development of drought- and aflatoxin-resistant peanut cultivars is essential to maintain productivity under less than ideal growing conditions. Remote sensing of canopy reflectance is a well-established method of evaluating crop condition and shows promise as a tool for rapid selection of drought- and aflatoxin-resistant peanut genotypes. The objective of this study was to evaluate ground-based reflectance measurements to more accurately quantify differences in genotype response to drought conditions. In April 2004 and 2005 several small plots (4 m x 2 m) were established at the Gibbs Farm research facilities in Tifton, GA. Treatments consisted of five peanut genotypes encompassing a range of drought tolerance and yield characteristics. Drought conditions were simulated beginning 90 d after planting and maintained through harvest. Once drought conditions were established, a handheld radiometer was used to acquire twice-weekly reflectance measurements in the visible and near-infrared. Benchmark indices were developed based on the change in remotely sensed vegetation indices as a measure of the change in crop response between nonstressed and drought-stressed conditions. Significant treatment differences in benchmark indices were observed between drought-tolerant, moderately drought-tolerant and drought-intolerant varieties. Benchmark indices were also highly correlated with yield (r = -0.41 to -0.75, [alpha] = 0.05) in all three planting environments. However, the relationship between aflatoxin contamination and benchmark indices was less consistent, having a strong correlation with aflatoxin contamination in the second and third planting environments only (r = 0.38-0.73, [alpha] = 0.05). These indices could aid plant breeders in more accurately assessing genetic differences, which would accelerate breeding progress and the development of peanut cultivars with resistance to drought and aflatoxin contamination.

The structural studies of aflatoxin [B.sub.1]-formamidopyrimidine (AFB-FAPY) derivatives conducted with the adducted base, nucleoside and oligodeoxynucleotides are described. The results have helped in understanding the structural basis of the differential processing that occurs during replication of DNA containing the two forms of the AFB-FAPY lesion.

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius and a few other species. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. Although Aspergillus oryzae and Aspergillus sojae, which are used in fermented foods and in ingredient manufacture, have no record of producing aflatoxin, they have been shown to possess an aflR gene. This study examined 34 strains of Aspergillus section Flavi. The aflR gene of 23 of these strains was successfully amplified and sequenced. No aflR PCR products were found in five A. sojae strains or six strains of A. oryzae. These PCR results suggested that the aflR gene is absent or significantly different in some A. sojae and A. oryzae strains. The sequenced aflR genes from the 23 positive strains had greater than 96.6% similarity, which was particularly conserved in the zinc-finger DNA-binding domain. The aflR gene of A. sojae has two obvious characteristics: an extra CTCATG sequence fragment and a C to T transition that causes premature termination of AFLR protein synthesis. Differences between A. parasiticus/A. sojae and A. flavus/ A. oryzae aflR genes were also identified. Some strains of A. flavus as well as A. flavus var. viridis, A. oryzae var. viridis and A. oryzae var. effuses have an A. oryzae-type aflR gene. For all strains with the A. oryzae-type aflR gene, there was no evidence of aflatoxin production. It is suggested that for safety reasons, the aflR gene could be examined to assess possible aflatoxin production by Aspergillus section Flavi strains.

The function of the cypX and moxY genes in aflatoxin biosynthesis in Aspergillus parasiticus is studied. A simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined, was devised in this work.

Aflatoxins are toxic, carcinogenic, and mutagenic secondary metabolics mainly produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. The work is the first report of an enzyme that is involved in two distinct and disconnected reactions in aflatoxin biosynthesis.

The role of AflR and AflR1 in expression of nor-1 in Aspergillus parasiticus is defined and the additional cis-acting sites that are required for maximum nor-1 transcriptional activation are identified. The results show that the NorL site appears only once in the aflatoxin gene cluster and hence its association with the nor-1 promoter might have important implications for the overall regulatory scheme for the aflatoxin pathway.

Aflatoxin is a carcinogenic and toxic compound produced by the fungus Aspergillus flavus (Link:fr) that can be found at detrimentally high concentrations in maize (Zea mays L.) grain. Screening has led to the discovery of sources of resistance to aflatoxin accummulation in maize, but associated poor agronomic characteristics and complex inheritance have limited transfer of resistance to elite inbreds. A set of 210 [F.sub.2:3] families derived from a cross between inbred lines Mp313E (resistant) and B73 (susceptible) was evaluated in replicated trials in four environments for resistance to aflatoxin accumulation. Families were also genotyped using simple sequence repeal (SSR) markers to develop a genetic map for quantitative trait loci (QTL) analysis. Composite interval mapping (CIM) was used to identify 2, 3, 5, and 3 QTL within the tests at Stoneville (2000) and Mississippi Stale (2000, 2001, 2002), respectively. The QTL were primarily additive in nature, with Mp313E contributing to reduced aflatoxin concentration in all but one case. Two QTL regions were significant in at least three environments. The afl3 locus, represented by marker bnlg371, was located on chromosome two and accounted for 7 to 18% of variation in aflatoxin levels depending on environment. The afl5 locus, represented by marker bnlg2291, was located on chromosome four, with explained variance ranging from 8 to 18%. This QTL has been noted in earlier studies whereas afl3 is new. Identified QTL confirm important regions influencing aflatoxin accumulation previously identified and present new ones of equal effect., AFLATOXINS ARE TOXIC secondary metabolites produced by the fungal pathogen Aspergillus flavus. These toxins have carcinogenic, immunosuppressive, and hepatotoxic properties in animals (Castegnaro and McGregor, 1998). Aflatoxin B1 is the [...]

The characterization of a veA ortholog in the aflatoxin-producing fungus Aspergillus parasiticus is reported. As veA is required for both mycotoxin production and morphological development, it makes a very good potential target gene to control aflatoxin biosynthesis or fungal development.

A study investigating the potential of 'Tulare' walnut to resist the production of aflatoxins by Aspergillus flavus is presented.

A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin [B.sub.1] (AF[B.sub.1]). Hypotheses have been put forth that AF[B.sub.1], in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AF[B.sub.1] is 8,9-dihydro-8-([N.sup.7]-guanyl)-9-hydroxyaflatoxin [B.sub.1] (AF[B.sub.1]-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AF[B.sub.1]-formamidopyrimidine (AF[B.sub.1]-FAPY) adduct. AF[B.sub.1]-FAPY is detected at near maximal levels in rat DNA days to weeks after AF[B.sub.1] exposure, underscoring its high persistence in vivo. The present study reveals two striking properties of this DNA adduct: (i) AF[B.sub.1]-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AF[B.sub.1]-N7-Gua, and (ii) one proposed rotamer of AF[B.sub.1]-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.

A feeding trial was conducted to evaluate the effect of aflatoxin (AF)-contaminated diets on growth and hematological and immunological parameters. Low doses of aflatoxins (140 and 280 ppb) were included in a corn-soybean diet provided for ad libitum consumption to 36 weanling piglets for a period of 4 wk. A 'dose-related' decrease in weight gain was observed in treated animals. This effect was significant (P < 0.05) in the 280 ppb-treated group compared to the control group. Ingestion of AF-contaminated feed at either level had no effect on total red blood cell numbers or on their relative number of lymphocytes, monocytes, neutrophils, basophils, and eosinophils in blood. Likewise, AF did not alter globulin, albumins, or total protein concentrations in serum, nor did AF alter the expression of regulatory cytokines produced by either Th1 (IL-2) or Th2 (IL-4) lymphocyte subsets in phytohemagglutinin-stimulated blood samples. By contrast, AF had a biphasic effect on total white blood cell number; the low dose of AF (140 ppb) decreased the total number of white blood cells, whereas the high dose (280 ppb) had the opposite effect. Consumption of AF also increased the concentration of [gamma]-globulin in the serum. A reduced immune response induced by Mycoplasma agalactiae in the 280-ppb-treated group was also observed. Cytokine mRNA expression in phytohemagglutinin-stimulated blood cells indicated that AF decreased proinflammatory (IL-1[beta], TNF-[alpha]) and increased anti-inflammatory (IL-10) cytokine mRNA expression. These results demonstrate that low doses of AF depress growth and alter many aspects of humoral and cellular immunity in pigs. Key Words: Aflatoxins, Antibodies, Blood, Body Weight, Cytokines, Pigs

A technique has been developed for the preparation of aflatoxin [B.sub.1] (AF[B.sub.1])-tagged liposomes encapsulating a visible dye. These liposomes have several useful potential analytical applications, one of which is demonstrated. A simple plastic-backed nitrocellulose strip is the basis for an assay for detecting AF[B.sub.1]. Samples containing aflatoxin [B.sub.1] are allowed to migrate by capillary action along the strip into a zone containing immobilized antibodies; then aflatoxin [B.sub.1]-tagged, dye-containing liposomes are allowed to migrate into the same area, filling any remaining antibody sites. The liposomes that bound to the antibody zone exhibit an intense purplish pink color whose optical density is inversely proportional to the aflatoxin concentration in the sample. The device is capable of detecting aflatoxin [B.sub.1] at levels down to 20 ng and could serve as a rapid procedure for visual screening of agricultural and food samples for AF[B.sub.1] or, with densitometry, as an inexpensive quantitative assay.

A reporter plasmid is described that can be used to study the expression of the nor-1 gene, which produces a ketoreductase involved in aflatoxin synthesis in Aspergillus spp. The resulting knowledge could be used to reduce aflatoxin contamination of food crops.

Some lactic acid bacteria can remove aflatoxin B(sub1) from solutions. Lactobacillus rhamnosus strains GG (ATCC 53103) and LC-705 (DSM 7061) were most efficient at removing aflatoxin B(sub 1) and non-viable bacteria retained more aflatoxin B(sub 1) than viable bacteria.

Researchers have isolated a gene in Aspergillus parasiticus whose protein product dehydrogenates 5'-hydroxyaverantin to averufin. This is one of the steps in the synthesis of aflatoxin B(sub 1).

Research has been conducted on the aflastatin A that has a tetramic acid derivative structure. Stereochemistry of flastatin has been determined.

Research was conducted to examine the enzymatic formation of G-group aflatoxins and biosynthetic relationship between G- and B-group aflatoxins. Enzyme activity for G-group flatoxin formation was detected by using a cell extract and combined cytosol and microsome fractions of Aspergillus parasiticus NIAH-26. Results suggest that G-group aflatoxin biosynthesis may be regulated at the transcriptional level by mechanisms common to the other enzymes.

Aflatoxin pathway regulatory protein (AFLR), a DNA-binding protein of 444 amino acids, transactivates the expression of aflatoxin biosynthesis genes in Aspergillus parasiticus and Aspergillus flavus, as well as the sterigmatocystin synthesis genes in Aspergillus nidulans. By fusion of various aflR gene coding regions to the GAL4 DNA-binding coding region, it was found that the AFLR carboxyl terminus contained a region that activated GAL1::lacZ gene expression in Saccharomyces cerevisiae and that the AFLR internal region was required for the activation activity.

There are significant differences in production of aflatoxin B1 and cyclopiazonic acid (CPA) by the L-strain soil isolates from Aspergillus flavus among regions along a transect extending from eastern New Mexico through Georgia to eastern Virginia. This was gleaned from an examination of soil isolates from A flavus to determine whether A flavus populations from different geographic regions differ in production of aflatoxin B1 and CPA.Results affirm the geographic differences found in production of aflatoxin B1 by A flavus in surveys conducted in Japan and in the US' cotton-growing areas.

A study was conducted to clone the ordA and ordA1 genes of the aflatoxigenic strain Aspergillus parasiticus SRRC 143 and SRRC 2043, respectively, and to evaluate the function of the ordA gene in the formation of aflatoxins. Point mutations were introduced into the ordA gene using polymerase chain reaction-based site-directed mutagenesis while mycelia were obtained by filtration. Results indicated that cytochrome P-450 type enzymes were involved in the synthesis of different primary and secondary metabolites in filamentous fungi.

The refined structure of a thermally stable formamidopyrimidine (FAPY) adduct in a DNA duplex was investigated using nuclear magnetic resonance spectroscopy. The FAPY adduct arose from imidazole ring opening of the initially formed trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct and was positioned in the 5'-d(CTATFAPYGATTCA)-3'*5'-d(TGAATCATAG)-3' oligodeoxynucleotide. Results show that stacking of the aflatoxin B1 moiety above the 5'-face of the modified guanine causes the unwinding of the DNA by almost 15 degrees.

A study was conducted on the purification of O-methyltransferase I to apparent homogeneity from the cytosol fraction of the mycelia of Aspergillus parasiticus NIAH-26. Phenyl-Sepharose, DEAE-Sepharose, phenyl-Sepharose, Sephacryl S-300 and Matrex gel Green A chromatography series was used. Results using the purified enxyme indicate that the formation of sterigmatocystin from demethylsterigmatocystin was suppressed when dihydrodemethylsterigmatocystin was added to the reaction mixture and dihydrosterigmatocystin was created.

Aflatoxin Bsub1 is produced by Aspergillus parasiticus, A. flavus and A. nominus, and its mutagenic and carcinogenic effects in humans and other vertebrates have been extensively studied. Biosynthesis of aflatoxins is lengthy and complex, and is distinguished by diverse oxidative rearrangement processes. An intact incorporation of hexanoic acid provided a significant early insight into the first stages of precursor assembly in aflatoxin biosynthesis.

A study was conducted on the influence of the overexpression of the aflatoxin biosynthetic pathway regulatory gene, aflR, on Aspergillus flavus aflatoxin biosynthesis, temporal gene expression, aflatoxin production and nitrate inhibition. Results suggest that aflatoxin biosynthesis nitrate inhibition does not only occur at the aflR transcription level. Results also reveal that higher pathway gene transcript accumulation and aflatoxin production is achieved through the constitutive overexpression of aflR.

The exo isomer of aflatoxin [B.sub.1] (AF[B.sub.1]) 8,9-epoxide appears to be the only product of AF[B.sub.1] involved in reaction with DNA and reacts with the [N.sup.7] atom of guanine via an [S.sub.N]2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in [H.sub.2]O (0.6 [s.sup.-1] at 25 [degrees] C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA ([K.sub.d] = 0.43 mg [ml.sup.-1], or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 [s.sup.-1]. Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 [s.sup.-1] and half-maximal velocity at a DNA concentration of 1.8 mg [ml.sup.-1] (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 [s.sup.-1] at pH 6.2 with 0.17 mg DNA [ml.sup.-1]). The same rate enhancement effect was observed with poly[dA-dT][center dot]poly[dA-dT], in which the epoxide can intercalate but not form significant levels of [N.sup.7] purine adducts, and with single-stranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.

Aspergillus parasiticus SU-1 has two ver-1 genes, ver-1A and ver-1B, of which only ver-1A is functional during aflatoxin (AF) biosynthesis. The gene product of ver-1A is associated with the conversion of versicolorin A into sterigmatocystin in AFB(sub 1) biosynthesis. A linearized plasmid used to disrupt the ver-1A and ver-1B gene by gene replacement disrupts only the ver-1A gene. The aflR gene, located in a duplicated chromosomal region upstream from ver-1A and ver-1B, also regulates AFB(sub 1) synthesis.

Functional expression of aflatoxin biosynthesis genes in Aspergillus flavus 649 WAF2 transformants failed to exhibit aflatoxin production. Since strain 649 lacks a 90-kb genomic region, it was suggested that the gene cluster in the 90-kb region is involved in aflatoxin expression. Cloning and sequencing of the genes in the 90 kb of A. flavus genomic DNA detected avf1 and the oxidoreductase gene, ord1, indicating these these two genes are involved in aflatoxin biosynthesis.

The kinetics and dynamics underlying the hydrolysis of the mycotoxin aflatoxin B1 (AFB1) were studied. Specifically, the hydrolysis rate of AFB1 exo-8,9-epoxide was determined with respect to the pH of H2O. The results, which indicated the genotoxic isomer's fast and spontaneous reaction, revealed the reversible conversion of the hydrolysis product, AFB diol, to the oxyanionic AFB1 alpha-hydroxydialdehyde under moderately basic conditions.

The role of versicolorin B synthase (VBS), an enzyme from Aspergillus parasiticus, in biosynthesis of a potent mycotoxin, aflatoxin B1, was investigated by radiochemical assay and optical measurements. The results revealed that VBS cyclize the enzyme involved in catalysis of aflatoxin B1. Analysis of the pH-rate profile of VBS and chemical labeling studies identified an acidic residue of the enzyme that plays an important role in catalysis.

The use of selected genes, present in the aflatoxin biosynthesis pathway, in the PCR is effective in the detection of aflatoxigenic molds inhabiting stored grains. The ver-1, omt-1 and apa-2 are the genes involved in the aflatoxin biosynthetic pathway. The amplification occurs only in the DNA of the Aspergillus parasiticus and Aspergillus flavus in all the primer pairs of the three genes. The inoculation of sterile corn with other molds fails to amplify the DNA even at the highest inoculum level.

Whole-body autoradiography of 3H-labeled aflatoxin B1 in young pigs showed a localization of bound label in the nasal olfactory and respiratory mucosa, in the tracheo-laryngeal mucosa, and in the conjunctiva, in addition to the liver. Whole-body and microautoradiography also showed a labeling of pigmented tissues, which can be ascribed to a melanin binding of [AFB.sub.1]. In vitro experiments with microsomal preparations of various tissues from sows revealed that the nasal respiratory and olfactory mucosa had the highest capacity to form DNA-bound aflatoxin [B.sub.1]-metabolites. The tracheal mucosa and the liver, in order, had lesser binding capacity. The lung was found to be devoid of aflatoxin [B.sub.1]-bioactivating capacity. In vitro microautoradiography revealed bound label in specific cell types in the nose and trachea and in some cells of the conjunctiva. A drastic decrease in the aflatoxin [B.sub.1]-DNA binding was observed when microsomal preparations of the nasal respiratory and olfactory mucosa were incubated in the presence of reduced glutathione, but without any addition of cytosolic glutathione-S-transferases. In incubations of liver microsomes under these conditions a somewhat lower inhibition of the aflatoxin [B.sub.1]-DNA binding was seen. Our results demonstrate that the nasal olfactory and respiratory mucosa and the tracheal mucosa have a higher capacity than the liver to bioactivate aflatoxin [B.sub.1] in swine. Our data further show that microsomal-associated glutathione-S-transferases with a high capacity to catalyze the conjugation of the reactive aflatoxin [B.sub.1]-epoxide to reduced glutathione are present in the nasal olfactory and respiratory mucosa of swine. Key Words: Aflatokins, Pigs, Conjunctiva, Melanins, Glutathione Transferase

The rate of destruction of pure aflatoxins and aflatoxins deliberately added to yellow corn and peanuts increases with an increase in the heat power setting of the microwave and the duration of exposure to microwaves. There is a greater percent decrease in the toxicity index of the yellow corn and peanuts deliberately infected with aflatoxins, as compared to pure aflatoxins, at similar exposure durations and low, moderate and high power settings. This indicates that wet food systems have a higher toxicity index than the dry model system, particularly during low microwave heating.