Low Levels Of Aflatoxin Research Articles

Coupled-Column System for Quantitating Low Levels of Aflatoxins

The combination of Styragel and Florisil columns allows recovery of aflatoxins at low levels. Two switching valves between the columns reduce the analysis time and exposure to solvents. Florisil is modified with oxalic acid, allowing recovery of aflatoxins at levels below 500 pg. The method is useful for contaminated corn and peanut meal samples.

Effect of dietary aflatoxin concentration on the assessment of genetic variability of response in a randombred population of chickens.

The effect of graded levels of dietary aflatoxin on the assessment of genetic variability of body weight and gain and plasma protein response was tested utilizing the Athens-Canadian randombred population of chickens. Dietary aflatoxin was administered at levels of either 0, 1.25, 2.50 or 5.0 microg/g of diet ad libitum from 7 to 21 days of age to progeny from 58 sire families. Twenty-one-day body weights, gain and plasma protein concentration were used to assess the variation in response.-The administration of increasing levels of aflatoxin resulted in a dose-related decrease of gains and plasma protein concentrations. Plasma protein concentrations were significantly higher among males than females within the control group; however, this difference was reversed as the severity of the aflatoxin challenge increased. Heritability estimates for all responses increased as the level of aflatoxin administered increased. This change was most notable for total plasma protein concentration. Phenotypic correlations for plasma protein concentration and growth measurements tended to diminish with increasing levels of aflatoxin. A similar trend was noted for the genetic correlations; however, a moderate correlation between growth responses and plasma protein response was detected in the 5.0-microg/g aflatoxin treatment group. Genetic correlations were calculated for the same characters between the different levels of aflatoxin. Regardless of which aflatoxin challenges were compared, a very high genetic correlation for 21-day body weight and 7- to 21-day gain was estimated. This variation in growth potential in the toxic environment paralleled that observed in the control environment but at a lower plane. Genetic correlations for plasma protein response across aflatoxin levels diminished as the difference between the levels of aflatoxin administered increased. Plasma protein concentration in the control environment was positively correlated with plasma protein response in groups fed a low level of aflatoxin, but negatively correlated when an aflatoxin challenge of 2.5 microg/g or more was given, suggesting that selection for aflatoxin resistance using plasma protein response as a selection criterion should be made under an aflatoxin stress environment.

Effect of Dietary Aflatoxins on Protein and Energy Utilization by Two Indonesian Breeds of Ducklings (Alabio and Tegal)

The effect of graded levels of dietary aflatoxins produced by the fungus Aspergillus flavus on protein and energy utilization by two breeds of growing ducks (Alabio and Tegal) was measured. Assays used were protein efficiency ratio (PER), net protein utilization (NPU), nitrogen digestibility, nitrogen and energy retention, and histopathology of livers in ducklings fed diets containing either soybean meal, peanut meal, or coconut meal free of aflatoxins or contaminated with aflatoxin extract. The discriminant computed PER (DC-PER) derived from dietary amino acid profiles was also determined.Alabio and Tegal ducklings had similar responses to dietary aflatoxins. NPU was affected more than PER by low levels of aflatoxins in tested feeds. NPU values were significantly lower at 30 μg aflatoxins/kg diet than at 0 μg, and significantly more urinary nitrogen was excreted by ducklings on diets containing aflatoxins. However, these effects of aflatoxins were accompanied by an increase in metabolizability of dietary energy. Thus, when liveweight gain was used as the measure of protein utilization as in PER or when amino acid composition was used as in DC-PER, the deleterious effect of aflatoxin on protein utilization in growing ducklings was not detected.The results indicate that the deleterious effect of aflatoxins on growth and on the gross histological appearance of the liver are magnified by either low quality or lack of protein in the daily ration.

Field Performance of Two Peanut Cultivars Relative to Aflatoxin Contamination

Abstract Two runner-type peanut cultivars, “Sunbelt Runner” and “Florunner,” were compared under differing field conditions for contamination of the seed by Aspergillus flavus Link and aflatoxin. Laboratory tests had shown marked differences in resistance between the two cultivars. Peanuts were grown on three nonirrigated farms during 1980 using two planting dates and three harvest dates for each cultivar. Peanuts grown on two farms experienced moderate to severe drought stress and both cultivars contained high levels of aflatoxin. Peanuts on the third farm received adequate rainfall and contained very low levels of aflatoxin. Microflora, grade and aflatoxin data showed that Sunbelt Runner (reported to be resistant to A. flavus infection) had no advantage over Florunner (reported to have moderate resistance to A. flavus) in reducing levels of A. flavus and subsequent aflatoxin contamination under field conditions. Levels of infection and contamination were related primarily to environmental conditions, (especially drought stress), during pod maturation. These and prior results show that the current laboratory assay method for selecting resistant lines should be carefully reassessed.

Evaluation of Sampling Methods for Detecting Aflatoxin Contamination in Small Test Plots of Maize Inoculated With Aspergillus flavus

AbstractTwo commercial maize (Zea mays L.) hybrids were grown in 1978 and 1980 to evaluate 13 sampling methods (grain‐sampling‐inoculation combinations) for formation of aflatoxin by Aspergillus flavus Link. The effectiveness of each method in producing uniform, consistent, genotype‐differentiating levels of aflatoxin was determined. Differences in level of aflatoxin contamination did not occur between years, but were highly significant between hybrids and among evaluation methods. Interactions among years, hybrids, and evaluation methods were nonsignificant. Mid‐ear inoculations with a knife at 20 d after full silk were more effective than were needle inoculations at the ear‐tip in eliciting high and consistent aflatoxin levels. Kernels from the immediate inoculated area of the ear or from inoculated segments of ears had the highest levels of aflatoxin contamination. Kernels from entire ears, or portions of ears, that appeared clean of fungus infection contained lower levels of aflatoxin contamination. Kernels from entire ears contained low to intermediate levels of aflatoxin, but the differences in levels seem adequate for detecting differences between genotypes. Treatments with low aflatoxin levels were generally associated with the highest coefficients of variation, although variances per se could not be positively associated with aflatoxin concentrations of the various methods.

Association of Low Levels of Aflatoxin in Feed with Productivity Losses in Commercial Broiler Operations , ,

Six growers from each of five broiler companies were classified into equal numbers of good, mediocre, and poor growers based on a productivity index. Feed samples were collected weekly during the growout period of a flock from each grower and from the mill where the feed was produced. Samples of corn from which the feed was made were also collected. Aflatoxin, moisture contents, and bacterial, coliform, and fungal counts of the samples were determined. On the same weekly basis, the temperature and relative humidity of the chicken houses were measured and the age of the feed samples ascertained. Aflatoxin concentration in feed of good growers was 6.1 ppb with an 18.0% frequency of contamination while the values for poor growers were 14.0 ppb and 31.3%. Mean aflatoxin concentration in corn of 1.2 ppb increased to 8.8 ppb in feed from the farms. Optimum conditions for aflatoxin formation were 19 to 27 C, 79 to 89% relative humidity, and 10 to 13% moisture. Increasing age of feed was associated with low moisture and high aflatoxin. High aflatoxin was associated with high relative humidity but not with fungal, bacterial, or coliform counts. These data suggest that aflatoxin is produced during and after feed manufacture in apparently normal operations, that low levels of aflatoxin are associated with productivity losses in apparently healthy broilers, and that the aflatoxin formation is associated with high relative humidity in houses and with long residence time of the feed in the house. Because relative humidity and residence time in houses can be altered, aflatoxin formation might be limited by controlling these two factors.

Aflatoxin Production on Various Popcorn Genotypes1,2

AbstractAflatoxin, a metabolite of the ubiquitous fungus, Aspergillus flavus Link, is a chronic contaminant of preharvest corn (Zea mays L.) produced in some areas of the USA. Growing hybrids that sustain relatively low levels of toxin has been suggested as a method of minimizing this potentially serious threat to animal and human health. The purpose of this 3‐year study was to evaluate ears of selected popcorn genotypes for corn earworm, Heliothis zea (Boddie), damage, incidence of greenish‐yellow fungus (GYF) sporulation characteristic of A. flavus Link infections, and aflatoxin contamination levels in the grain. Evaluations were conducted under field conditions in Tifton loamy sand (Thermic, Plinthic Paleudult) and involved 17 popcorn genotypes. Results demonstrated significant differences among popcorns for levels of aflatoxin contamination, fungal infection, and corn earworm damage. A significant interaction between genotypes and years indicated that information from a single year cannot be relied upon to accurately identify host plant genotypes with consistent low aflatoxin levels.

Isolation and characterization of two nonaflatoxigenic classes of morphological variants ofaspergillus parasiticus

AbstractSerial transfer of mycelial macerates of aflatoxigenic strains ofAspergillus parasiticus produced 2 morphological variants. The “fan” variant type showed flat growth, a gradient of sporulation density, absence of mycelial pigmentation and deep furrows on the back of the colony. The “fluff” variants displayed abundant aerial mycelium, sparse sporulation, absence of mycelial pigmentation and furrows on the reverse of the colony. Neither “fan” nor “fluff” produced detectable aflatoxin. In a marked mutant strain, the appearance of “fan” and “fluff” and their concomitant loss of aflatoxin production occurred whereas spore color and auxotrophic markers were retained. A parasexual diploid synthesized from the mutant strain with the “fan” phenotype produced high levels of aflatoxin whereas a parasexual diploid containing the “fluff” pheno‐type produced low levels of aflatoxin. The unusual mode of isolation, the differential retention of aflatoxin and other genetic markers in a mutant strain, and the different levels of aflatoxin production in parasexual diploids with “fan” and “fluff” imply an unusual genetic mechanism for the control of the aflatoxin pathway.

Loss of Norsolorinic Acid and Aflatoxin Production by a Mutant of Aspergillus parasiticus

SUMMARY: A mutant strain of Aspergillus parasiticus that accumulated the red mycelial pigment norsolorinic acid and low levels of aflatoxin was subjected to serial transfer in defined media. The isolation of unpigmented, non-aflatoxigenic forms was associated with conditions in which the inocula for serial transfers contained hyphae rather than conidia, but was not associated with either zinc deficiency or the presence of barium in the medium. The unpigmented, non-aflatoxigenic variants were stable.

A comparison of the effects of pretreatment with phenobarbitone and 3-methylcholanthrene on the metabolism of aflatoxin B 1 by rat liver microsomes and isolated hepatocytes in vitro

In agreement with previous reports (Neal and Colley, Biochem. J., 174 (1978) 839; Gurtoo and Dave, Cancer Res., 35 (1975) 382) the capacity of isolated hepatic microsomes to metabolise aflatoxin B 1 (AFB 1) is enhanced by pretreatment in vivo with phenobarbitone. Microsomes isolated from rats pretreated with 3-methylcholanthrene also exhibit increased metabolism of AFB 1. The levels of metabolism are increased in proportion to the increased levels of cytochromes P-450 and P-448 indicating similar overall efficiencies of the two cytochromes to metabolise AFB 1. However, the qualitative aspects of the metabolism induced by the two compounds are very different. In agreement with the previous data (Neal and Colley, Biochem J., 174 (1978) 839) the formation of aflatoxins Q 1, M 1 and B 1-dihydrodiol are enhanced by pretreatment with phenobarbitone whereas pretreatment with 3-methylcholanthrene, while not affecting the production of aflatoxin Q 1 and slightly lowering AFB 1-dihydrodiol formation, increases the production of aflatoxin M 1 > 10-fold. Covalent binding of AFB 1 to microsomal protein in vitro is increased by pretreatment in vivo with phenobarbitone, but decreased by pretreatment with 3-methylcholanthrene. The changes in the level of protein binding in vitro brought about by these pretreatmens are quantitatively similar to the relative changes in AFB 1-dihydrodiol tris complex production induced by the two compounds indicating a role for this metabolite in protein binding. Covalent binding of AFB 1 to DNA in vitro is also increased by pretreatment in vivo with phenobarbitone, but not by 3-methylcholanthrene and the total level of binding of AFB 1 to DNA in the in vitro system is greater than binding to microsomal protein. The possible mechanisms for binding AFB 1 to microsomal protein and DNA are discussed. In contrast to the microsomal incubations, only low levels of aflatoxins Q 1 and M 1 are present after 2 h incubation AFB 1 with intact hepatocytes isolated from control, methylcholanthrene or phenobarbitone pretreated rats. Pretreatment with either phenobarbitone or 3-methylcholanthrene induces the formation of polar metabolites, presumably conjugates, emphasising the predominant role of phase 2 metabolism in intact cellular systems.

Effect of Caffeine on Aflatoxin Production on Cocoa Beans

ABSTRACTThe effects of caffeine on aflatoxin production by Aspergillus parasiticus NRRL 2999 were studied on 13 cocoa bean types. Caffeine levels from bean types ranged from 0.30‐3.6 mg/g and only very low levels of aflatoxin were produced on bean types having > 1.80 mg caffeine/g. These data provide additional evidence that caffeine is an effective inhibitor of aflatoxin production and help explain why aflatoxin does not accumulate in cocoa beans under natural storage conditions.

Refractory Phagocytosis by Chicken Thrombocytes during Aflatoxicosis ,

The percentage phagocytosis of chicken thrombocytes was insensitive to dietary aflatoxin (10 μg/g diet) while the mean phagocytosis was slightly but significantly (P<.05) reduced by 5 or 10 μg/g but not lower levels. Thrombocytic phagocytosis was dependent on a heat sensitive serum factor which is not affected during aflatoxicosis. These findings contrast with reports that other phagocytic types such as heterophils, macrophages, and monocytes are sensitive to low levels (e.g., .625 μg/g) of dietary aflatoxin.

Pre-neoplastic changes in the liver: Neal, G. E. & Butler, W. H. (1978). A comparison of the changes induced in rat liver by feeding low levels of aflatoxin B 1 or an azo dye. Br. J. Cancer37, 55

Pre-neoplastic changes in the liver: Neal, G. E. & Butler, W. H. (1978). A comparison of the changes induced in rat liver by feeding low levels of aflatoxin B 1 or an azo dye. Br. J. Cancer37, 55

Aspergillus flavus and aflatoxin production in acid-treated maize

Abstract Isobutyric acid (IBA) and propionic-acetic acid (PA) were applied to comparable 52.8 m 3 lots of freshly harvested yellow dent maize containing 27% moisture. After 6 months storage, 30% Aspergillus flavus infection and low levels of aflatoxin were detected in adjacent bins of IBA-treated and PA-treated maize. Extensive samples were taken after 7 months from moldy spots in each bin and evaluated for aflatoxin, zearalenone, ochratoxin and microorganisms. Aspergillus flavus (10 6 propagules/g) was detected in 40% of the PA samples, but no aflatoxin was found. Also, counts of Aspergillus fumigatus, Absidia and Penicillia were high. In addition to the molds found on PA maize, Aspergillus niger was identified on IBA-treated maize. Aspergillus flavus (10 4 –10 7 propagules/g) was present in 79% of the IBA samples; aflatoxin (from 2 to 857 ng/g) was detected in 57%. Aflatoxin contamination varied between locations within a moldy area. Among 20 individual kernels picked at random from each location, aflatoxin contamination ranged from 150 to 21.800 ng/g in positive kernels. Evidently, bulk quantities of maize must be appraised on the basis of individual kernels because toxin-free kernels often are adjacent to highly contaminated kernels.

Effect of varying levels of dietary protein on tumor development and lipid metabolism in rats exposed to aflatoxin

AbstractReports in the literature concerning the relationship of protein nutrition to aflatoxicosis are contradictory. In an attempt to elucidate this relationship more clearly, we have examined the effects of low, normal, and high protein‐containing diets on tumor incidence and development, as well as on several biochemical indices, in rats which have been exposed to low levels of aflatoxin in a “chronic” rather than “acute” situation. In our study, male weanling rats were place for 3 months on otherwise adequate diets containing either 8, 22, or 30% casein with and without aflatoxin B1 at 1.7 ppm. Half of the animals in each group received diets which were further supplemented with the amino acid, cystine, at 0.6% of the diet. (Sulfur‐containing amino acids are the most limiting amino acids in casein, and the addition of cystine to the diet serves to improve the biological quality of the protein source.) After 3 months the animals were fed control diets without aflatoxin until they were killed at 1 year. Weight gain was markedly decreased and liver weights increased in response to aflatoxin in all groups except those on the low protein diets, where aflatoxin had no effect on these protein diets, where aflatoxin had no effect on these indices. No tumors were found in the livers of rats fed the low protein, aflatoxin‐supplemented diet. In the other groups, the severity of the liver involvement increased progressively with increased protein levels in the diet. When cystine was included in the diet, tumors were observed also in the animals fed the low protein diet; furthermore, the livers of those animals on “normal” and high protein diets were much more severely involved than were the livers of animals on non‐cystine supplemented diets. Plasma cholesterol levels were increased in response to aflatoxin when the diets containing 22 and 30% protein were fed and when cystine was included in the 8% protein diet. Liver cholesterol levels were increased in response to aflatoxin in all groups except in those receiving the low protein diets. Among these latter animals, aflatoxin administration had no effect on liver cholesterol values. Changes as a result of aflatoxin administration were also observed in the fatty acid composition of sterol esters, triglycerides, and phospholipids of liver and tumor tissue.

AFLATOXIN CONTENT AND SKIN REMOVAL OF SPANISH PEANUTS AS AFFECTED BY TREATMENTS WITH CHEMICALS, WATER SPRAY, HEATED AIR, AND LIQUID NITROGEN

ABSTRACTUnshelled Spanish peanuts contaminated with aflatoxin were shelled and treated with forced heated air, liquid nitrogen, H2 O2, HCl, sodium oleate, and water spray. After passing through a whole nut blancher the aflatoxin content and percentages of blanched (skin removed) and whole kernels were determined Blanching percentages were significantly higher for sodium oleate, water spray, liquid nitrogen, and H2 O2 than for HCl or heat treatments. The heat treatment produced the highest percentage of whole kernels. In most tests, the unblanched kernels contained higher levels of aflatoxin than those that blanched fully. The treatments which were most effective in producing low levels of aflatoxin in the blanched kernels were H2O2, water spray and HCl.

Estudo da aflatoxina no amendoim, da colheita à industrialização, na região de Matão, S.P.

Neste trabalho foi investigada, na região de Matão, a incidência de aflatoxina no amendoim (Arachis hypogaea L.) em três estágios de seu ciclo de industrialização: a) ao ser entregue à fábrica: Épocas I e II; b) durante seu armazenamento: Épocas III e IV, e c) após a extração do óleo (farelo): Épocas V e VI. Em cada Estágio foram feitas duas coletas de 10 amostras cada, num total de 40 amostras de amendoim e 20 de farelo. Dos resultados concluiu-se que: a) a maioria das amostras, representando 85% do total, continha aflatoxina; b) o nível nas amostras, em termos de aflatoxina B1, elevou-se continuamente da Época I para a Época VI: médias de 0,06 até 1,22 ppm com média geral de 0,44 ppm; c) as boas práticas de secagem e adequado armazenamento concorrem decisivamente para manter um nível baixo de aflatoxina, e d) o lavrador entrega amendoim já tóxico à fábrica.

Some interactions of light, riboflavin, and aflatoxin B1 in vivo and in vitro.

In previous studies, artificial sunlight and riboflavin synergistically increased acute aflatoxin toxicity in rats. Three new experiments were designed to provide information on the interaction of riboflavin, aflatoxin, and light. In a study of carcinogenesis, rats received low levels of aflatoxin 5 days/wk for 3 wk; 30 min after each dosing, half of them were irradiated for 2 hr. In some, levels of glucose-6-phosphatase and acid phosphatase were determined 5 days after completion of treatment. Remaining rats were killed at 30 or 53 wk. All underwent complete necropsies and histopathologic examination. In the second experiment, rats were dosed with riboflavin and divided into four groups: no further treatment; aflatoxin (LD50); irradiation (1-2 hr); or aflatoxin plus irradiation. Blood riboflavin levels were determined at intervals following these treatments. In the third experiment, the chemical reactions of irradated aflatoxin and/or riboflavin were studied by uv spectroscopy and TLC. The 53-wk study showed clearly that light decreased the incidence of aflatoxin-induced cancer. The other results may provide an explanation. Aflatoxin caused blood riboflavin levels to decrease-an effect enhanced by irradiation, suggesting that photosensitized riboflavin and aflatoxin form a complex. This interpretation gains support from studies in vitro that showed that riboflavin quenched aflatoxin photodegradation, perhaps by complexing with aflatoxin. Thus, low, carcinogenic doses of aflatoxin may complex with endogenous, photosensitized riboflavin, inhibiting its degradation into carcinogenic metabolites.

Dietary factors and aflatoxin toxicity: I. comparison of the effect of two diets supplemented with aflatoxin B1 upon two different strains of rats

AbstractPrevious studies in this laboratory with rats fed low levels of aflatoxin as a component of peanut discards, suggested either a strain tolerance for aflatoxin or a possible protective factor present in the diets used. In this study, two strains of rats, the Charles‐River strain and the former USC strain, were used to test the effect of 1.7 ppm purified aflatoxin B1 included for 3 months in two different diets; one previously used in this laboratory and one used by other investigators in aflatoxin studies. After an experimental period of either 12 or 18 months, growth, mortality, gross pathology, and organ wt were measured, and histopathological examination and biochemical analyses were performed. Plasma and liver cholesterol levels, total liver lipids, and fatty acids in the various lipid fractions of plasma, liver, and liver tumor lipids were measured. Both strains of rats proved to be susceptible to aflatoxin toxicity at this level as manifested by the appearance of hepatomas; however, liver involvement was more extensive in the Charles‐River rats. The diet used by other investigators produced symptoms similar to those observed as a result of essential fatty acid deficiency and also affected the response to aflatoxin through an aggravation of symptoms, i.e. an inhibition of growth and increased size and severity of the liver tumors.

TISSUE DISTRIBUTION AND METABOLISM OF AFLATOXIN B1‐14C IN LAYER CHICKENS

The effects of administering low levels of aflatoxin B1‐14C by crop intubation daily for 14 days to layer chickens were determined. Studies on the distribution of 14C in the blood, selected organs, tissues, eggs and excreta were conducted. No toxic effects were observed in layer chickens during the course of the experiment. The layer chickens excreted 92.15% of the 14C administered. Of the 14C retained, 19.5, 16.1, 3.9, 7.2, 26.4 and 26.9% were detected in the blood, liver, heart, gizzard, breast and leg, respectively. No radioactivity was detected in egg samples collected. Chemical assay of those samples demonstrating radioactivity revealed that 81.2% of the radioactivity in these substrates was soluble in aqueous extracts while approximately 10% was ex tractable by classical extraction procedures. Treatment of the aqueous extracts for conjugated steroids by treatment with Beta‐glucuronidase revealed that 31.5% of the 14C detected in the aqueous extract was a liberated glucuronide conjugate of aflatoxin M1−14C