Production of a novel steroid sulfate metabolite [4,4,24-trimethylcholesta-8,14,24(28)-trien-2 alpha,3 beta,11 alpha,12 beta-tetrol 12-acetate, 3-sulfate] by Fusarium species and its biological activity.

A novel steroid sulfate, 4,4,24-trimethylcholesta-8,14-24(28)-trien-2 alpha,3 beta,11 alpha,12 beta- tetrol 12-acetate, 3-sulfate, was discovered in Fusarium spp. Forty Fusarium strains belonging to F. sporotrichioides, F. chlamydosporum, E. equiseti, F. acuminatum, F. sambucinum, F. culmorum, and F. graminearum produced the steroid on white corn grits at 25 degrees C for 20 days. This steroid sulfate is one of the more abundant and easily attainable microbial steroids. At a concentration of 160 micrograms/ml, it inhibited the growth of six fungi, two gram-positive bacteria, and an alga, as well as the germination of both wheat and tomato seeds.

Pharmacokinetics of the trichothecene mycotoxin, T-2 toxin, in swine and cattle.

The pharmacokinetics of the trichothecene mycotoxin, T-2 toxin, were determined in growing gilts and heifers. Following intra-aortal administration in swine and intravenous administration in calves, the disappearance of the parent T-2 toxin followed a 2-compartment open model. Mean elimination phase half-lives were 13.8 and 17.4 min and mean apparent specific volumes of distribution were 0.366 and 0.376 l/kg in swine and calves, respectively. The fraction of T-2 toxin eliminated as parent compound in the urine was negligible. In spite of administration of a lethal oral dose in swine (2.4 mg/kg) and toxic oral doses (up to 3.6 mg/kg) in calves, no parent T-2 toxin was detected in plasma or urine. After intra-aortal administration in swine, tissue concentrations of T-2 toxin were consistently highest in lymphoid organs. Tissue residues of T-2 toxin were rapidly depleted such that, in spite of administration of a potentially lethal intra-aortal dose, no quantifiable T-2 toxin was present in any of the tissues collected at 4 hr after dosing. No T-2 toxin could be detected in liver, even at 1 hr after dosing.

Isolation and characterization of two new fusaric acid analogs from Fusarium moniliforme NRRL 13,163.

Fusarium moniliforme NRRL 13,163 produced two new fusaric acid analogs, a 10,11-dihydroxyfusaric acid and a diacid of fusaric acid in which the C-11 methyl was oxidized to a carboxyl. Several hundred milligrams of the 10,11-dihydroxyfusaric acid were routinely recovered from a kilogram of corn grit medium. It crystallized as white, irregularly shaped rectangles that melted at 153 to 154 degrees C. The diacid analog of fusaric acid crystallized as white rods that melted at 210 to 211 degrees C. Unlike the consistent recovery experienced with the 10,11-dihydroxyfusaric acid, the diacid analog proved difficult to purify after the initial discovery and was detectable in subsequent fermentations only by mass spectrometry.

Production and purification of a peptide of Fusarium tricinctum that causes conidia of Penicillium to swell.

A process is described for production of a cyclodepsipeptide complex (CDPC) from autoclaved white corn grits fermented with Fusarium tricinctum NRRL 3510. Yields of more than 2.5 g CDPC/kg of medium were obtained. Previously, we described the conspicuous swelling of Penicillium digitatum conidia and hyphal tips incubating in a medium supplemented with the CDPC, a trio of metabolites produced by strains of F. tricinctum and F. roseum. Analysis of the CDPC mass spectra and the amino acid composition indicated one major and two minor cyclodepsipeptides. The two major products differed from the major cyclodepsipeptide only in the substitution of either isoleucine or valine for leucine in the threonyl-alanyl-alanyl-glutaminyl-tyrosyl-leucine peptide portion of the molecule.

Comparison of ochratoxin, aflatoxin, and T-2 toxin for their effects on selected parameters related to digestion and evidence for specific metabolism of carotenoids in chickens.

Aflatoxicosis, ochratoxicosis, and T-2 toxicosis were produced by feeding diets containing graded concentration of the appropriate toxin to broiler chicks from hatching unit 3 weeks of age. Aflatoxin, even at levels not growth inhibitory, produced a malabsorption syndrome characterized by steatorrhea, hypocarotenoidemia, and decreased concentrations of bile salts and pancreatic lipase, trypsin, amylase, and RNase. The T-2 toxin at concentrations higher than required to inhibit growth produced a mild malabsorption syndrome characterized by steatorrhea and decreased levels of pancreatic lipase, trypsin, amylase, and RNase. The only suggestion of malabsorption during ochratoxicosis was a severe hypocarotenoidemia. The following observations indicated a lack of correlation between lipid malabsorption and hypocarotenoidemia. The T-2 toxicosis exhibited lipid malabsorption in the absence of hypocarotenoidemia, ochratoxicosis exhibited hypocarotenoidemia in the absence of lipid malabsorption, and aflatoxicosis exhibited both symptoms. These findings imply that carotenoids are physiologically active compounds with specific metabolic processes and are not inert substances swept along with lipids as is commonly assumed from the ability to grow apparently healthy birds free of carotenoids. The current findings also indicate that great specificities exist in mycotoxicoses despite superficial similarities.

Toxicity of dietary and intravenously administered moniliformin to broiler chickens.

Moniliformin (1-hydroxy-cyclobut-1-ene-3,4-dione), either purified (0, 8, and 16 mg/kg of diet) or from culture of Fusarium moniliforme strain NRRL 6322 on corn grits (8, 16, and 64 mg/kg of diet) was fed to growing broiler chicks from 1 to 21 days of age. Up to 16 mg moniliformin/kg of diet from either source was without effect on chick weight gain, feed consumption, and mortality. Chicks fed 64 mg moniliformin/kg of diet from culture had reduced weight gain and feed consumption. Total daily moniliformin consumption by these chicks was nearly twice the reported single oral 50% lethal dose. Three of 10 chicks fed 64 mg/kg of moniliformin in the diet died. No lesions were found upon necropsy. The LD50 of purified moniliformin upon intravenous injection of 7-week-old female broiler chickens was 1.38 +/- .035 mg/kg body weight. Average time to death was 65 minutes. Progressive symptoms noted included lack of muscular coordination, tachypnea from moderate to severe followed by slow labored respiration, coma, terminal agonal struggle, and death.

Moniliformin and butenolide: effect on mice of high-level, long-term oral intake.

Mice survived 21 days without apparent illness while consuming a daily amount of either moniliformin or butenolide near or exceeding their oral 50% lethal dose.

Mouse bioassay for Fusarium metabolites: rejection or acceptance when dissolved in drinking water.

Ten metabolites of Fusarium species (butenolide, diacetoxyscirpenol, equisetin, fusaric acid, gibberellic acid, moniliformin, NRRL 6227 peptide, T-2 toxin, vomitoxin, and zearalenone) were added to the drinking water of mice to determine whether they were consumed or refused. Of the 10, only the trichothecenes--diacetoxyscirpenol, T-2 toxin, and vomitoxin--were refused. Refusal of 2 mg of the trichothecenes per liter was not enhanced by adding 100 mg of zearalenone per liter.

Acceptance by swine and rats of corn amended with trichothecenes.

Swine and rats demonstrated the same response factor (i.e., the average amount of corn amended with trichothecenes consumed by animals per the average amount of uncontaminated corn consumed by animals) for consumption of corn amended with 40 ppm of either T-2 toxin or diacetoxyscirpenol Rat response factor for corn containing 40 ppm of vomitoxin was 1.8 times more than corn containing either T-2 toxin or diacetoxyscirpenol at 40 ppm. For the corn containing 40 ppm of vomitoxin, swine response factor was 1.8 times greater than rat response factor.

Moniliformin, a metabolite of Fusarium moniliforme NRRL 6322: purification and toxicity.

Fusarium moniliforme NRRL 6322 produced about 600 mg of recoverable moniliformin, a mycotoxic metabolite, per kg of corn grit medium. The moniliformin was extracted from the grits with methanol, purified by preparative thin-layer chromatography, and crystallized from ether. The 50% lethal dose for chicken embryos was 2.8 microgram per egg. For 1-day-old chicks dosed with moniliformin by crop intubation and for female and male mice injected intraperitoneally, the 50% lethal doses were 5.4, 20.9, and 29.1 mg per kg of body weight, respectively. The toxin did not cause a reaction on mouse skin.

Swelling of Penicillium digitatum conidia by a Fusarium acuminatum NRRL 6227 metabolite.

Fusarium acuminatum NRRL 6227 produces an antifungal metabolite that causes incubating conidia of several Penicillium species to swell 5-10 diameters while inhibiting germination. The swollen conidia are spherical, translucent, nonviable and easily shattered with a slight physical pressure; however, they remain resistant to osmotic shock. This antibiotic has been identified as a cyclic peptide composed of alanine, glutamic acid, leucine, threonine and tyrosine.

Interaction of T-2 toxin with Salmonella infections of chickens.

A significant (P less than 0.05) interaction resulting in increased mortality occurred in chickens fed T-2 toxin (16 mug./g. of diet) and infected with either Salmonella worthington, S. thompson, S. derby, or S. typhimurium var. copenhagen, all species that cause paratyphoid. No interaction on growth rate or relative size of the bursa of Fabricus occurred, although T-2 toxin alone caused a significant (P less than 0.05) regression of that organ. The spleen size relative to the body weight was decreased (P less than 0.05) by T-2 toxin and increased (P less than 0.05) by the Salmonella infections. Interactions were observed on spleen size between the toxin and S. thompson (P less than 0.05) and S. derby (P less than 0.10). Total serum proteins were not affected by T-2 toxin or Salmonella infections. Agglutinins were formed in response to the infections, but the titers were unaltered by T-2 toxin.

Altered feathering of chicks caused by T-2 toxin.

Dietary T-2 toxin (0, 1, 2, 4, 8, and 16 mug./g.) was fed to 4 groups of 10 chickens at each treatment level from hatching until 3 weeks of age. Growth inhibitory levels (4, 8, and 16 mug./g.) caused abnormal feathering which appeared dose related. The chickens were sparsely covered with short feathers protruding at odd angles in comparison to controls. There were few feathers on the base of the neck, on the anterior dorsal surface of the wing, and on the side and back adjacent to the tail. Feather tips frequently were constricted and bent downward while the quill could have a reverse curve. A literature survey suggests that T-2 toxin may cause this effect through a nutritional imbalance.

Egg production, shell thickness, and other physiological parameters of laying hens affected by T-2 toxin.

T-2 toxin has been reported to cause severe oral lesions and neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 mug per g of feed, caused oral lesions but no abnormal neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 mug per g of feed, caused oral lesions but no abnormal neural symptoms in laying hens. T-2 toxin had no effect on either hemoglobin, hematocrit values, erythrocyte count, plasma glucose, prothrombin times, or the sizes of the liver, spleen, pancreas, and heart. Lipid content of the liver was not altered. Feed consumption, however, was reduced, as were the total plasma protein and lipid concentrations and the total leukocyte count. Most important economically was the lowered egg production and a thinner egg shell. The timing and severity of the symptoms suggest that T-2 toxin causes primary oral lesions that reduce feed consumption with a consequent reduction in serum proteins and lipids, which culminate in decreased egg production. The leucopenia and thinner egg shell may be independent systemic effects of T-2 toxin in laying hens.

Antibiotic produced by Fusarium equiseti NRRL 5537.

Fusarium equiseti NRRL 5537 grown on an autoclaved white corn grit medium for 3 to 4 weeks at room temperature produced a substance in excess of 5 g/kg of substrate that inhibited some gram-positive bacteria including mycobacteria. Most Bacillus subtilis, Mycobacterium phlei, and Staphylococcus aureus strains were inhibited when 1 mug of the antibiotic per ml was incorporated into the culture medium. Except for Neisseria perflava, gram-negative bacteria, yeasts, and molds were not inhibited by 128 mug/ml. The antibiotic was recovered as a white powder, had a melting point of 65 to 66 C, and had an intraperitoneal mean lethal dose in white mice of 63 mg/kg of body weight. In thin-layer chromatographic analysis the compound appeared as a single spot in two different solvent systems. Mass spectrometry determined that the molecular weight of the antibiotic was 373 with a molecular formula of C(22)H(31)NO(4). Chemical microanalysis was in accord with the formula.

Neural disturbances in chickens caused by dietary T-2 toxin.

Graded concentrations of dietary T-2 toxin (0, 1, 2, 4, 8, and 16 mug/g) were fed to groups of 40 chickens. T-2 toxin was found to cause an abnormal positioning of the wings, hysteroid seizures, and impaired righting reflex in young chickens. The abnormal wing positioning occurred spontaneously or as the result of dropping from a height of 1 meter. The seizures could be elicited by rough handling or loud noises. The seizures and the abnormal wing posture would not occur again when the stimulus was repeated unless a rest period of 3 to 6 h was allowed. The loss of righting reflex could be demonstrated at any time. The total incidence of neural symptoms was dependent on the length of exposure to T-2 toxin and to its concentration. Neural toxicity occurred at dosages of 4, 8, and 16 mug per g of diet, which are the same doses that retard growth. This neural toxicity of T-2 toxin in chickens is similar to the neural disturbances associated with alimentary toxic aleukia, a nutritional toxicosis of humans produced by eating moldy grain. T-2 toxin has been implicated also in moldy corn toxicosis which has neural manifestations in horses and swine.