Pat B. Hamilton

Altered lipid transport during aflatoxicosis.

The effects of graded doses of dietary aflatoxin (0, 0.625, 1.25, 2.5, 5.0 and 10.0 μg/g) on serum triglyceride fatty acids, serum total phospholipids and serum free and esterified cholesterol were measured in chickens. These classes of lipid were decreased significantly (P < 0.05) by a dose of 0.625 μg/g, and a dose of 2.5 μg/g was required to decrease the growth rate and the ribonucleic acid content of liver. The deoxyribonucleic acid content of liver was unaffected even by 10 μg/g. These data suggest that lipid transport is a primary lesion during aflatoxicosis in the chicken and is not secondary to the general effects on nucleic acid metabolism.

Concentrations of serum proteins during aflatoxicosis

The concentrations of different classes of serum proteins of chickens to graded doses of dietary aflatoxin (0, 0.625, 1.25, 2.5, 5.0, and 10.0 μg/g of diet) were measured using disc gel electrophoresis. Total serum proteins were reduced significantly (p < 0.05) by a dose of 1.25 μg/g or greater. The α-globulins and β-globulins were reduced at levels of 2.5 and 1.25 μg/g, respectively. The IgG component was reduced at 2.5 μg/g while the IgM component was not affected significantly at any level. The prealbumin fraction was reduced at 5 μg/g. The most sensitive component was serum albumin that was decreased significantly at the smallest level and was decreased to the greatest extent at the highest level. Its response curve roughly paralleled that of total serum lipids. Serum lipoproteins were decreased at 1.25 and 2.5 μg/g but not at lower or higher levels. These data can be explained by a hypothesis that aflatoxin or an active metabolite binds randomly to template deoxyribonucleic acid and inhibits the larger transcribing units such as those for serum albumin and lipid before the smaller transcribing units are inhibited.

Mycotoxins – Their Biosynthesis in Fungi: Ochratoxins – Metabolites of Combined Pathways

Little work has been done on the biosynthesis of ochratoxins. Nevertheless. a scheme based on known chemical and enzymatic reactions and incorporating known facts about ochratoxin biosynthesis is proposed as a working model. The scheme calls for formation of an unbranched pentaketide from acetyl and malonyl CoA, its cyclization and aromatization to an is ocoumarin, methylation and oxidation to the carboxy derivative which is chlorinated by chloroperoxidase before acyl activation for reaction with an ester of phenylalanine which arises via the shikimic acid pathway. The amide, ochratoxin A, is considered the final product of this biosynthetic pathway.

Determining Safe Levels of Mycotoxins

The establishing of safe levels of mycotoxins to date has been a legal rather than scientific exercise. This has resulted in levels which have varied in response to economic and political pressures. The data base for rationally determining safe levels is very small. This has resulted in subjective evaluations of the worth of different studies in attempts to deduce safe levels from experiments designed to demonstrate effects, and in assumed safe levels which vary from field experiences. Using physiological parameters other than growth as criteria of safety, known deleterious interactions of mycotoxins with other factors, and statistical corrections for inadequate numbers of animals tested, permit better agreement between safe levels determined from laboratory data and from field data. However, the number of animals required makes impractical the laboratory determination of truly safe levels. Well-conceived and executed epidemiological studies coupled with laboratory studies designed to elaborate underlying principles appear to be the best approach to determining safe levels of mycotoxins. Until safe levels are based on sound animal experimentation, the prudent person would assume there is no truly safe level and that increasing levels of mycotoxins carry increasing risk.

Impaired phagocytosis by heterophils from chickens during aflatoxicosis.

Abstract The effects of graded concentrations of dietary aflatoxin (0, 0.625, 1.25, 2.5, 5.0, and 10.0 ppm) on the in vitro phagocytic and bactericidal abilities of heterophils from chickens were investigated. The mean, percentage, and rate of phagocytosis were reduced. There were several populations of heterophils in our experimental system; 10% were unable to phagocytize (0 ppm), 15% were very sensitive to aflatoxin (0.625 ppm), 30% were resistant to aflatoxin (5 and 10 ppm), and about 45% were of intermediate sensitivity (1.25 and 2.5 ppm). Crossover experiments demonstrated that both cellular and serum factors required for optimal phagocytosis were impaired during aflatoxicosis and that the serum factor had the heat sensitivity of complement. Both spontaneous and chemotactic locomotion by heterophils were impaired. The heterophils from birds with aflatoxicosis which phagocytized bacteria had a reduced ability to kill the bacteria. Thus, a representative of the circulating phagocytes responsible in part for nonspecific host resistance has impaired function during aflatoxicosis.

T-2 Toxicosis and blood coagulation in young chickens

Recent reports have suggested that T-2 toxicosis may be a contributing factor in hemorrhagic manifestations such as alimentary toxic aleukia in man and hemorrhagic anemia syndrome and general clotting dysfunction in animals. The present communication describes the effect of graded levels of dietary T-2 toxin (0, 1, 2, 4, 8, and 16 μg/g) on the activities of tissue thromboplastin, coagulation Factors VII, X, and V, prothrombin, and fibrinogen in young chickens. In the extrinsic clotting pathway a qualitative change was demonstrated in the lipid fraction of thromboplastin, and Factor VII activity was significantly (p > 0.05) reduced by the growth inhibitory dose of 4 μg/g. Of the common pathway functions, Factor X, prothrombin, and fibrinogen were depressed by 16 μg/g T-2 toxin, with the latter two parameters being reduced to below 60% of normal. Factor V was unaffected. These data show that T-2 toxin can produce a distinct coagulopathy in chickens characterized by a primary defect in Factor VII activity and secondary effects on prothrombin and fibrinogen. Additionally, the effects of T-2 toxicosis on clotting competence provide further evidence of a possible role of T-2 toxin in alimentary toxic aleukia.

Possible outbreaks of fusariotoxicosis in avians

A disease syndrome characterized by raised yellowish-white lesions in the oral cavity was observed in several commercial broiler flocks. Growth rates were depressed, and 10% died. Fowl pox was ruled out by infectivity tests in susceptible birds with lesion material. A similar outbreak occurred in fancy pigeons fed visibly moldy feed. The oral lesions in pigeons contained large numbers of avirulent Staphylococcus epidermidis and Escherichia coli. Identical oral lesions were produced in the laboratory by feeding chickens small concentrations of fusariotoxin T-2 produced by the fungus Fusarium tricinctum.

Impairment of coagulation function during aflatoxicosis in young chickens

Abstract Reports of hemorrhagic episodes and high incidence of bruising in poultry during aflatoxicosis prompted an investigation of the effect of aflatoxin on parameters relating to hemostatic competence in chickens. Three-week-old broiler chickens fed dietary aflatoxin (0, 0.625, 1.25, 2.5, 5.0 and 10 μg/g of feed) were tested for specific coagulation responses. Tissue thromboplastin was found to possess two active components, one of which was lost during aflatoxicosis. Thromboplastin protein content was unaltered but lipid content was depressed at 10 μg/g. Factors VII and V activities and fibrinogen were reduced at 2.5 μg/g and above. Factor X activity was depressed at 5.0 and 10 μg/g. Prothrombin, the most sensitive factor tested, was reduced by 0.625 μg/g, the lowest dose fed. In addition, clot retraction in whole blood was measured, and it was found to increase with growth-inhibitory levels of aflatoxin. These data suggest that severe impairment of extrinsic and common clotting pathway functions occurs during aflatoxicosis in the chicken and that prothrombin is primarily affected during this coagulopathy.

DESTRUCTION OF AFLATOXIN IN CORN WITH SODIUM BISULFATE

The ability of sodium bisulfite to destroy aflatoxins B1 and B2 in naturally contaminated corn containing about 2350 ppb of B1 and 450 ppb of B2 was investigated. Under certain conditions, complete destruction of aflatoxin B1 was achieved. Aflatoxin B2, on the other hand, was resistant to sodium bisulfite and never over about 50% was destroyed. Moisture, sodium bisulfite level, time, as well as temperature had significant effects on aflatoxin degradation. Moisture levels of over 50% (wet weight basis) had a strongly adverse effect on the aflatoxin-bisulfite reaction. The most effective treatment involved soaking whole-kernel corn in a 10% sodium bisulfite solution for 72 h, removing the solution and incubating the corn in sealed plastic bags at 50°C. Complete destruction of aflatoxin B1 was achieved by 21 d. Sodium bisulfite exhibited antimicrobial activity in corn comparable to that of propionic acid, indicating possible utility as an effective mold inhibitor in stored corn at up to 40% moisture. Feed consumption by young chickens was unaffected until feed containing over 20 g of sodium bisulfite/kg was presented.

Destruction of Aflatoxin B1 with Sodium Bisulfite: Isolation of the Major Product Aflatoxin B1S

The reaction between aflatoxin B1 and sodium bisulfite yielded almost quantitatively a light yellow, highly fluorescent water-soluble product, aflatoxin B1S. Reverse-phase (C18) high pressure liquid chromatography (with and without the paired-ion technique), and normal- and reverse-phase thin layer chromatography revealed the ionic product at greater than 98% yield. The products intense fluorescence, identical in color to that of aflatoxins B1 and B2, supported the conclusion that the lactone of aflatoxin B1 was not opened by bisulfite and that the reaction took place at some other location on the aflatoxin B1 molecule. The UV spectrum of aflatoxin B1S exhibited identical absorbance maxima to aflatoxin B1 and these maxima exhibited no bathochromic shift in alkaline solution. Aflatoxin B1S exhibited change to bright yellow fluorescence under 365 nm UV light after being sprayed with 20% sulfuric acid in methanol. The IR spectrum of aflatoxin B1S indicated the vinylene group of the furofuran ring system of aflatoxin B1 was missing from aflatoxin B1S, whereas the coumarin-cyclopentenone ring system present in aflatoxin B1 was intact in aflatoxin B1S. Furthermore, the IR spectrum suggested the presence of a sulfonate moiety in B1S. Proton NMR spectra of aflatoxin B1S also indicated that the cyclopentenone region was intact and that the furofuran had undergone reaction with bisulfite. Integration of the NMR spectrum of aflatoxin B1S revealed 12.75 to 13.25 protons, consistent with a bisulfite addition across the double bond of aflatoxin B1 to form aflatoxin B1S. Aflatoxins B2 and G2 were not susceptible to the action of bisulfite, again supporting the conclusion that bisulfite acts at the unsaturation in the furofuran ring system of aflatoxin B1. There was no evidence that aflatoxin B2 was produced in the degradation of aflatoxin B1 by bisulfite under these conditions. The fact that previously reported chemical modifications of this furofuran ring system resulted in reduced toxicity support the potential utility of bisulfite as a means of detoxification of aflatoxin-contaminated commodities.