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Mycotoxicology deals with diseases in man and animals caused by or resulting from ingestion of foods made toxic by metabolic products of fungi. Of the numerous reviews devoted to this subject over the last ten years, the foll owing are selected: Brook & White (14), Christensen (18 ), Ciegler & Lilleho j (26), Goldblatt (54), Hesseltine (60), Detroy et al (35), Purchase (115), Scott (13 2), Wogan (17 7, 179), Mateles & Wogan (94), and Ciegler et al (25). This review is restricted to those mycotoxins found in feedstuffs (excluding ergot) or in the ingredients of foods and feedstuffs in the field and is little concerned with the host of toxic metabolic products of fungi produced in pure culture in the laboratory. It often is tacitly assumed that if a fungus in pure culture in the laboratory under some conditions produces a compound or compounds in some way toxic to test animals when ingested or otherwise admin istered (including intraperitoneal injection, which by no stretch of the imagination could occur in the feedlot or poultry house) the presence of this same fungus in food or feedstuffs is cause for alarm. This is by no means true; even when a potentia l toxin-p roducing strain of As per gillusfiavus is added to moist grain and allowed to grow, along with the other microflora normally present, it may produce little or no toxin.

A derivative of resorcinylic acid, produced by the fungus Fusarium graminearum , has been found to be responsible for the estrogenic signs in swine and laboratory rats. An estrogenic response in rats can be incited by injecting intramuscularly as little as 20 μg of the estrogen (F-2). Stimulation in growth of rats was noted at the lower concentrations (20 to 40 μg) of a series. Up to 3,500 ppm of the estrogen was produced on a solid corn medium. The compound is relatively stable to heat and ultraviolet irradiation. Methods of analysis have been developed and include: extraction procedures, evaluations by ultraviolet absorption spectrophotometry, thin-layer chromatography, and gas-liquid chromatography.

The development of Plodia interpunctella, in shelled corn (maize) discouraged the growth of stored grain fungi, and slightly raised the moisture content of the infested corn. Mouldy corn, on the other hand, hindered the development of this insect. Fewer larvae reached the adult stage on mouldy corn and only after a prolonged developmental period; the adults produced were smaller in size and produced fewer eggs in their ovaries than those on mould-free corn. The more mouldy the corn the more it became unfavourable for the insect but this depended in part on the fungus species present. The relationship between this insect and the majority of stored grain fungi appeared to be antagonistic. An exceptional relationship was found with Aspergillus halophilicus, in plated cultures, which attracted the adult insect to lay its eggs and on this fungus larvae developed almost as normally as on mould-free corn.

1.1. The mold colonies isolated from 76 samples of house dust collected in homes in and near St. Paul and Minneapolis, Minnesota, in the winters of 1950 and 1951 to 1952 varied from less than 10,000 per gram to more than 3,000,000 per gram. with an average of 179,966 mold colonies per gram. 2.2. The number of bacteria from 14 samples of house dust ranged from 1,144,000 to 20,000,000 per gram, and averaged 10,700,000 per gram. 3.3. Molds and bacteria were present in some numbers in the samples of new furniture stuffing, and were present in considerably greater numbers in some used materials. 4.4. Foam rubber appears to be somewhat susceptible to invasion by molds, though to a lesser degree than certain fibers. Bacteria do not appear to be present in any considerable numbers in either new or used foam rubber. 5.5. The molds present in the air within homes may differ in both kind and numbers from those in the outdoor air at the same time. The number of viable mold spores (or other inoculum) in the air within homes may fluctuate sharply during a single day, apparently increasing when the amount of physical activity within the home increases.

Numbers and kinds of microflora were determined in 160 samples of barley grown in different regions of the United States; microflora were more abundant in the grains grown in the central states than in those grown in the western states. During steeping and germination in micromalting equipment, the number of colonies of filamentous fungi increased from two to five times, colonies of yeasts from five to ten times, and bacteria from 50 to more than 100 times the numbers present in the grain before malting. Kiln drying according to a commercial schedule reduced the number of all types of microflora below the number present before kilning, but all were present in larger numbers in the kilned malt than in the original grain. In barley stored at room temperature and at a moisture content of 15 to 18%, members of the Aspergillus glaucus group increased with increasing time and increasing moisture content, and germination percentage of the seeds decreased. Stored free of storage fungi at room temperature, barley with a moisture content just over 15% retained a high germination percentage for 5 months, but at a moisture content of 16% the germination decreased to zero.

The mycotoxin potential of a plant product can be gauged by seeking answers to two of three questions basic to the mycotoxin problem; namely, ‘Which of the fungi invading the product can be toxicogenic?’, and ‘How can they become established in the product?’ Both questions relate to the quite new science of microbial synecology. In contrast, the third basic question (‘What factors are necessary for toxin production by a given toxicogenic fungus?’) relates to the much older but more complex science of microbial autecology. Fruits of Arachis hypogaea L., called ‘peanuts’ (U.S.A.) or ‘groundnuts’, are completely hypogeic in their development, and there seems to be a successional invasion of them by soil-borne or ‘field’ fungi with the result that sound mature fruits have a dormant or quiescent endogeocarpic microfloral community. Results of co-operative research in six peanut producing states (U.S.A.) and at the University of Minnesota, interpreted in the light of present-day peanut harvesting practices in the U.S.A., justify the following conclusions: (1) Aspergillus flavus can be toxicogenic under field conditions and is a serious problem. (2) Many other of the fungi from the peanut endogeocarpic community are toxicogenic under laboratory conditions. (3) Until the conditions under which such fungi may become toxicogenic are better known, their presence in lifted groundnuts constitutes a real mycotoxin potential. (4) The genera posing the greatest threat are, in estimated order of importance, Penicillium, Aspergillus (primarily because of A. flavus), Alternaria, and Fusarium. (5) Much care must be exercised with peanuts after lifting, to minimise opportunity for proliferation of such fungi. This care must be exercised at least until we determine extent to which such fungi might be expected to produce toxins in peanut fruits on the farm and during subsequent handling.

There were large differences in the number of Plodia interpunctella completing the life cycle from egg to adult on nine hybrid varieties of corn (maize) conditioned to 13·5 and 15·7 per cent m/c and held at 70 and 80 per cent r.h., respectively. These differences were significant statistically at the 1 per cent level, the poorest survival being only 12 per cent at 70 per cent r.h. and the highest 74·5 per cent. The mean developmental periods also varied significantly, from about 31 to 35 days at 70 per cent r.h. The percentage survival and the developmental period were negatively correlated. These differences were removed by milling the corn and supplying it as meal. Differences of size and fecundity of moths emerging from different corn varieties were not then significant. More moths emerged in a shorter period at the higher m/c on every variety. As a rule males tended to predominate but not to a significant extent.

Dilution cultures of 30 samples of ground black pepper yielded an average of 39,000 colonies of fungi per g, with a range of 1,700 to 310,000 per g. Total numbers of colonies of bacteria from 11 samples averaged 194,000,000 per g, with a range from 8,300,000 to 704,000,000 per g. A variety of fungi grew from nearly all surface-disinfected whole peppercorns that were cultured. Thirteen samples of ground red pepper from the United States yielded an average of 1,600 colonies of storage fungi per g and an equal number of other fungi; five samples from India yielded an average of 78,900 colonies of storage fungi per g and 169,400 colonies of other fungi per g. Among the fungi from both black and red pepper were Aspergillus flavus and A. ochraceus , some isolates of which, when grown for 8 to 10 days on moist autoclaved corn and fed to white rats or to 2-day-old Pekin ducklings, were rapidly lethal to them. Aflatoxin B 1 was isolated from one of the samples of corn on which A. flavus from black pepper was grown. Among the bacteria isolated from ground black pepper were Escherichia coli, E. freudii, Serratia sp., Klebsiella sp., Bacillus sp., Staphylococcus sp., and Streptococcus sp. No cultures of Shigella or Salmonella were found.

1. 1. The experimental results to date indicate that Mehrlich's medium with 75 Gm. of sodium chloride per 1000 c.c. of H 2 O will give a significantly higher mold count per plate than the casein hydrolysate medium now employed in the air-borne mold survey. This medium supports the growth of a large number of the air-borne saprophytic fungi common to this region. 2. 2. There is evidence, however, that this medium is still not the best possible, since readings from the modified Smith-Humfeld salt agar show a mold count almost double the mold count obtained on Mehrlich's salt agar and almost triple the mold count from the casein hydrolysate salt medium. 3. 3. As a result, therefore, experiments with a further modified Smith-Humfeld salt medium, a purified malt salt agar, and reduced strengths of the casein hydrolysate medium are being continued. 4. 4. None of these media will disclose the presence of air-borne spores of obligate parasites or spores of some other fungi as mushrooms and wood-rotting fungi which may often be present in considerable numbers, but they may considerably increase the value of the surveys now being conducted.

The predominant fungi isolated from damaged kernels of corn (maize) plated on agar media were species of Aspergillus (mainly A. glaucus) and Penicillium. Masses of spores and mycelium of Aspergillus, Penicillium, Cladosporium and Fusarium were present in the cavities of the embryo of many kernels from which no fungi grew. Some samples had a high percentage of blue-eye kernels, evidently caused mainly by A. restrictus and A. glaucus. The embryos of all damaged kernels were decayed to some extent by fungi. Weight averaged 51 · 7 lb/bu, not much below that of sound corn. Fat acidity values ranged from 90–324. No aflatoxin was found in any of the 24 samples tested.

Fifty-seven of 87 isolates of Alternaria alternata (Fr) Keissler grown on autoclaved, moist corn-rice substrate and fed to rats were lethal. The major toxin produced was isolated and characterized as tenuazonic acid. Twenty of 23 toxigenic Alternaria isolates examined produced tenuazonic acid. No tenuazonic acid could be detected in either of the field samples of sorghum or blackeyed peas, which were heavily invaded by Alternaria .

Those who are familiar with fleshy Agaricales know that the geotropic response which results in an orientation of pores, gills, or other spore bearing surfaces perpendicular to the surface of the earth, sometimes goes awry, especially if fruit bodies are diseased or mechanically injured. Fruit bodies produced in unnatural environments, as on agar cultures or on wood in jars, sometimes exhibit a capricious orientation of pores or gills, indicating that forces other than gravity are involved in such orientation. Following will be described two interesting cases of such nongeotropic development. The first case involves an agaric (Russula sp., tentatively identified as R. atropurpurea Peck) observed at Itasca Park, Minnesota, in September, 1937. More than a decade before that time several pits about 5 feet square and 4 to 5 feet deep had been dug for experimental purposes in the level ground in a Jack pine stand. During a rainy spell in September, 1937, when fleshy fungi were rather abundant, the writer observed 2 fruit bodies of this species of Russula growing out of the vertical walls of one of the pits. One appeared about a foot from the surface of the ground, the second about 2 feet from the surface and on an adjoining wall. The stem of each, although short, extended straight out from the vertical wall, no upward curve being visible. The cap in each case was parellel to the wall. The gills were normal, although the free edges of those on the upper side bent over as they lost their turgidity with age, as can be seen in figure 1. In other words, the fruit bodies were oriented in the same way to the perpendicular surface from which they grew, as fruit bodies growing on approximately

The mycotoxins diacetoxyscirpenol, deoxynivalenol, and zearalenone, produced by Fusarium roseum, were found naturally occuring in mixed feed samples. In all cases analyzed, deoxynivalenol occurred together with zearalenone. The natural occurrence of zearalenone in sesame seed is reported for the first time. Strains of F. roseum isolated in various parts of the world form feed implicated in animal mycotoxicosis produced monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and zearalenone.

Balanced rations containing 2.5 and 5.0% of corn invaded by Fusarium tricinctum, (with 8 and 16 p.p.m. of T-2 toxin) were fed to White Leghorn laying hens. The ration containing 5% of the fungus-invaded corn resulted in reduced feed intake and reduction in weight gain and in egg production. Rations containing 2.5 and 5.0% of corn invaded by F. roseum ‘Gibbosum’ (with 25 and 50 p.p.m., respectively, of monoacetoxyscirpenol) resulted in an abrupt decrease in feed intake to 10–20% of normal, subsequent loss in weight, and cessation of egg production. Purified T-2 toxin consumed at the rate of 16 p.p.m. in the ration resulted in loss of body weight and decreased egg production; lesser amounts of T-2 toxin resulted in lessened but still detectable injurious effects. Mouth lesions developed in the birds fed these rations, their severity being proportional to the amount of toxin present.

Consumption of an otherwise balanced ration containing 1% of corn invaded by Fusarium tricinctum isolate 2061-C resulted in the death of 13% of turkey poults within 35 days, in decreased feed efficiency and weight gain, and moderate development of bilateral necrotic lesions at angles of the mouth, especially in those that succumbed. Consumption of a ration with 2% of corn invaded by F. tricinctum resulted in death of 60 to 83% of the birds, in greatly reduced growth and feed efficiency in the survivors, and in development of severe mouth lesions. Consumption of rations containing 5, 10, and 20% of corn invaded by the fungus resulted in death of all birds in 5 to 15 days.

Of 53 isolates of Chaetomium globosum Kunze isolated from corn, manufactured feed, and fiberboard, grown on autoclaved corn, and fed to rats, 25 were lethal within 4 to 6 days. Thirty-six single ascospore cultures of one isolate were uniformly lethal when similarly grown and fed to rats. Incubation of the fungus on corn for 6 to 8 weeks resulted in greater toxicity than shorter incubation times. Premortem symptoms indicated damage to the central nervous system of the affected animals, and postmortem lesions included hemoglobinuria, hemorrhagic enteritis, and subdural hemorrhaging. Corn invaded by C. globosum and toxic to rats had no detectable effects on swine when fed as their sole ration for 6 weeks, and appeared to be consumed with relish. The toxic substance was best obtained by defatting the fungus-invaded corn with petroleum ether (bp, 60 to 70 C), extraction with acetone, and further purification by elution from a silica gel column with acetone and 5% acetone in chloroform.

Filamentous fungi, especially Aspergillus flavus-oryzae , and bacteria were present in all samples of macaroni and spaghetti products tested. No aflatoxin was detected in the three samples, all relatively high in A. flavus-oryzae , that were tested for it. Five of the nine isolates of A. flavus-oryzae from macaroni and sphaghetti grown in corn, incorporated into an otherwise balanced ration, and given to ducklings resulted in death of one to five ducklings within six days.

Eighty-five fungi isolated from prepared feed and from corn collected on farms were grown separately in moist autoclaved corn. The corn was fed to virgin weanling rats for 5 to 12 days; the rats were then killed, and their uteri were removed and weighed. Twelve isolates of Fusarium from corn and one from poinsettias caused increases of five to eight times in weight of the uterus as compared with controls that were fed sound corn. The greatest increase in weight of the uterus was caused by corn inoculated with Fusarium No. 5 incubated for 21 days at 20 to 25 C followed by 14 days at 12 C. Extraction of this corn with methylene chloride, separation into fractions by means of a silicic acid column, and further purification by thin-layer chromatography yielded a compound with ultraviolet-absorption maxima at 314, 274, and 236 mμ.

The mycotoxins zearalenone (2.8 micrograms/g), deoxynivalenol (1.5 microgram/g), and T-2 toxin (110 ng/g) have been found in the pith of corn stalks standing in the field. Such contaminated stalks may contribute to mycotoxicoses of farm animals.

Zearalenol, the reduction product of zearalenone produced by Fusarium roseum growing in cereals, was found for the first time naturally occurring in oats and corn. This metabolite is three to four times more active estrogenically than zearalenone.

The fungal metabolites, F-2 and F-3, associated with estrogenism in swine, are produced by some races of Fusarium moniliforme isolated from toxic feeds.

A gilled fungus identified according to descriptions by Smith and Singer (2) and by Kauffman (1) as Galerina hypnorum (Schrank ex Fr.) Kiihner fruited on the leaf sheaths of cattails (Typha latifolia L. and T. angustifolia L.) that had been transplanted into water tanks in the greenhouse. Eight fruit bodies developed on five different cattails during a 4-wk period in late June and early July. All of them developed on the shady side of the stem and on a portion of the leaf sheath that appeared to be senescent; typical examples are shown in FIGS. 1, 2. According to Smith and Singer (2), many species of Galerina require a special habitat such as mosses, conifer needles, burned ground, and hardwood logs. Kauffman (1) recorded that G. hypnorum usually occurs on mosses, but, so far as we are aware, this is the first report of its occurrence on Typha. Cultures of the fungus were obtained readily from cast spores placed on sterile agar, and were subsequently transferred to a number of media -potato dextrose, malt, cornmeal, Czapek Dox, and water agar with and without pieces of Typha leaves. Fruit bodies (FIG. 3) developed on the water agar containing portions of Typha leaves, but not on the other media tested, usually only one fruit body per dish or flask. 879

Both male and female pigs given a ration containing corn invaded by Fusarium roseum , in amounts sufficient to provide 500 to 600 ppm of F-2, for the first 64 days of the test and which for another 60 days were given a commercial pig ration were much lower in weight than the controls which were given a commercial pig ration throughout. In relation to the weight of the animals, in the pigs receiving the ration containing F-2, the weight of the uterine horn of the gilts was much greater and the weight of the testes of the males was much less than the weight of the same organs of the controls.