Integration of enzyme-linked immunosorbent assay with conventional chromatographic procedures for quantitation of aflatoxin in individual cotton bolls, seeds, and seed sections.

Integration of an enzyme-linked immunosorbent assay (ELISA) with conventional chromatographic methods proved the versatility of ELISA as a research tool and allowed for rapid assessment of aflaxtoxin in individual cottonseeds, parts of cottonseed, and composite samples of seeds taken from individual cotton bolls. Aqueous acetone was substituted for methanol in the extraction procedure prescribed by ELISA. The substitution allowed the use of a common extract for all analytical methods. An aliquot of the extract was used to screen samples by ELISA. Negative samples were identified, and toxin levels between 1 and 70 ng/g were quantitated by ELISA. Samples with toxin levels beyond the upper limit of detection by ELISA were then subjected to more time-consuming conventional cleanup prior to quantitation by liquid chromatography (LC) or thin-layer chromatography (TLC). Toxin levels detected by LC or TLC ranged from 100 to 845,000 ng/g sample. The screen by ELISA detected large numbers of toxin-negative cotton bolls or individual seeds in minimum analysis time. The combination of techniques verified the presence of seed with no toxin adjacent to toxin-containing seed in the same lock. Toxin-negative portions of individual seed with high toxin in another portion were identified for the first time. Integration of techniques provided needed information on distribution patterns of aflatoxin in cotton so that preventive measures can be developed.

Metabolic behaviour of aflatoxin producing strain and non-toxigenic strain of Aspergillus flavus to different sources of nitrogen and glucose concentration.

The effect of different nitrogen sources and varying glucose concentration on aflatoxin production by a toxigenic and non-toxigenic strain of Aspergillus flavus was studied. Greatest production (3.8 ppm) of aflatoxin B1 was produced in a synthetic medium when casamino acids were supplied as the nitrogen source. Optimum sugar concentration for aflatoxin B1 production ranged between 3 and 10 g/100 ml. There was no appreciable difference in the metabolic behaviour between toxigenic and non-toxigenic strains of A. flavus when dry mycelial weight, total proteins, non-protein nitrogen and reducing sugar were the criteria.

Saprophytic fungi isolated from animal and bird pens in Egypt.

Forty-four samples collected from animal and bird pens were screened for their content of saprophytic fungi by using the dilution plate method. 76 species in addition to one variety of Aspergillus flavus belonging to 33 genera were recovered on three types of media: 20 genera and 49 species on Littman-oxgall agar, 19 genera and 41 species on cellulose- and 19 genera and 43 species on glucose-Czapek's agar. The most frequent genera were Aspergillus (21 species), Scopulariopsis (4 species) and Penicillium (10 species). Among the isolated fungi A. fumigatus, A. flavus, A. terreus, A. niger, A. sydowi, A. nidulans, S. brevicaulis, S. brumptii, P. chrysogenum and P. funiculosum were the most common species.

Isolation of DNA from fungal mycelia and sclerotia without use of density gradient ultracentrifugation.

A rapid method for extracting total DNA from Aspergillus flavus and Aspergillus parasiticus has been developed. The procedure can be completed in 2 h and yields 200 to 350 micrograms of DNA from 0.5 to 1.0 g wet wt of mycelia and 150 micrograms from 0.5 g of sclerotia. DNA samples had an OD260/OD280 of 1.6 to 1.8. Most of the DNA was at least 50 kb pairs in size and showed little degradation. DNA prepared by this method was used for restriction endonuclease digestion and Southern blotting. A DNA fragment containing the repeat unit of the ribosomal RNA genes of A. flavus has been identified.

Occurrence of mycotoxins in cereals and animal feedstuffs in Natal, South Africa.

During the period 1982-1983, just under 800 samples of agricultural commodities, comprising cereals, compound feeds, hay, and silage, were examined for molds and mycotoxins. Aflatoxin B1 showed the highest incidence rate; it occurred in over 27% of all samples analyzed, the highest levels being found in peanut meal at 1500 ppb. Other mycotoxins detected were patulin and a number of trichothecene toxins at incidence rates in all commodities of 5.6 and 3.1%, respectively. The commodities at highest risk were oil seeds, excluding soya bean; the latter was found to be fairly free from contamination with mycotoxins. The most prevalent fungi were Aspergillus flavus and parasiticus, which were found in over 22% of all samples, whereas Penicillium spp. showed the lowest incidence of genera, specifically identified in 8.3% of all samples examined. This latter finding explains in part the low incidence of Penicillium mycotoxins.

[Fatty acid composition of the lipids in fungi of the genus Aspergillus developing on mineral media with different carbon sources]. / Zhirnokislotnyi sostav lipidov gribov roda Aspergillus pri razvitii ikh na mineral'nykh sredakh s razlichnymi istochnikami ugleroda.

This work was aimed at studying the effect of different carbon sources in the composition of mineral media on the growth of fungi belonging to the Aspergillus genus and on the fatty acid composition of their lipids. A chemically-defined medium with glucose was shown to be optimal for the growth of 18 Aspergillus strains and for the synthesis of lipids by them. The fatty acid composition of lipids was studied when the fungi grew in media with different carbon sources. The lipids were shown to contain saturated and unsaturated fatty acids with the prevalence of oleic, linoleic and linolenic acids.

Rapid, economical qualitative method for separation of aflatoxins B-1, B-2 & G-1, G-2 by dry column chromatography.

A good correlation of four components of aflatoxins was accomplished by using the dry column chromatography method. The decolorization process of interfering substances, by 0.01 N KOH and defatting the extract with petroleum ether yields a clean residue for DCC separation. It is clear that the dry column chromatography is a very simple and time-saving procedure for separation of aflatoxins. DCC columns are more economical than precoated 'thick layer' preparative plates and, in DCC, no large developing tanks need to be used. Hazards associated with the use of large volumes of flammable solvents are greatly reduced.

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus.

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.

Comparative mutagenicity of palmotoxin Bo and aflatoxins B1 and M1.

The mutagenicity of palmotoxin Bo and of aflatoxin M1 relative to that of aflatoxin B1, the potent mutagen, was studied in five Ames' Tester Strains of Salmonella typhimurium (TA-98, TA-100, TA-1535, TA-1537, TA-1538). Aflatoxins B1 and M1 are both highly mutagenic in a microsome-mediated system in TA-100. The prediction of the relative carcinogenicity of aflatoxin M1 to aflatoxin B1 posed by the mutation of TA-100 is probably more authentic than TA-87. The mutagenic potency of palmotoxin Bo is less than that of either aflatoxin B1 or M1.

Regulation of aflatoxin biosynthesis. 1 Comparative study of mycelial composition and glycolysis in aflatoxigen and nonaflatoxigenic strains.

A comparative biochemical study of an aflatoxigenic strain Aspergillus parasiticus NRRL 3240 and a nonaflatoxigenic strain A. flavus NRRL 3237 was carried out in order to have a better idea of regulation of aflatoxin biosynthesis. The results obtained revealed continuous primary metabolic activity (protein synthesis) in the nonaflatoxigenic strain while the aflatoxigenic stain showed inhibition of protein and nucleic acid synthesis. The aflatoxigenic strain showed higher levels of oxygen uptake, RNA, NAD, FMN and activities of glycolytic enzymes. Furthermore, it had lower of lipids and reduced activity of glucose-6-phosphate dehydrogenase, which is a source for NADPH. The differences observed have been discussed in relation to aflatoxin biosynthesis and its regulation.

Inhibition of aflatoxin production and tentative identification of an aflatoxin intermediate "versiconal acetate" from treatment with dichlorvos.

In general, aflatoxin production by Aspergillus flavus and A. parasiticus was greatly reduced in vitro in the presence of the insecticide dichlorvos. Reduction in yield of the toxins was accompanied by the apperance of a previously unidentified orange pigment. Spectral analyses of the pigment and of its methylated and acetylated derivatives indicated the compound to be versiconal acetate (IV). The data suggest that IV is an intermediate in the metabolic cycle that may terminate in the production of aflatoxin or of the versicolorins, or both. Dichlorvos apparently inhibits biosynthesis of the difurano ring structure common to the aflatoxins and the versicolorins.