CIRCULAR ANR-767 (02/93)

A L A B A M A    A & M    A N D    A U B U R N    U N I V E R S I T I E S MYCOTOXINS AND MYCOTOXICOSES

     Aspergillus flavus and A. parasiticus are common in most soils and are usually involved in decay of plant materials. They commonly cause stored grams to heat and decay and, under certain conditions, invade grain in the field. The problem is serious in subtropical and tropical regions of the world where cereals, peanuts, corn, and copra are important in the human diet.
     Aflatoxins B₁, B₂, G₁, and G₂ are produced by A. flavus and A. parasiticus in grains in both field and storage. Infection is most common after the kernels have been damaged by insects, birds, mites, hail, early frost, heat and drought stress, windstorms, and other unfavorable weather. Aflatoxins M_l and M₂ are found in milk from animals fed aflatoxin-contaminated feeds. The presence of A. flavus or A. parasiticus in a given feed sample does not imply that the feed is unwholesome and will contain high levels of aflatoxin. Aflatoxin persists under extreme environmental conditions and is even relatively heat stable at temperatures above 212°F, the boiling point of water. Roasting, ammoniation at ambient temperatures, and some microbial treatments may sharply reduce but not eliminate the aflatoxin content. Ammoniation has been shown to be most effective in reducing aflatoxin levels. Currently, these treatments have limited application, with roasting being the least effective. Pelletizing feeds may eliminate fungi present in the stock but not reduce or eliminate aflatoxin present in any of the ingredients.
     Recently, the addition of binding agents such as hydrated sodium calcium aluminosilicate (HSCAS) and bentonite clays to corn has been shown to decrease the effects of aflatoxin when fed to swine. These compounds probably work by nonspecific binding to the mycotoxin and reducing the passage time through the gut. Although not specifically approved for this purpose, various products that have this ability are approved as binding or anti-caking agents.
     One HSCAS product, NovaSil¹, was experimentally demonstrated in Texas and Virginia studies to improve the performance of swine when Novrasil was mixed with aflatoxin-contaminated corn. Control swine fed aflatoxin-contaminated corn had significantly poorer average daily gain and feed efficiency than those fed the same contaminated corn mixed with HSCAS. NovaSil was shown to be effective at either 5 or 10 pounds of compound per ton of aflatoxin-contaminated feed. The current cost of including this product in feed at this level would be $3 to $4 per ton of feed.
     Another binding agent that has been shown to be effective in reducing the effects of aflatoxin in corn are the clays, Volclay or FD-181.² In a Virginia study these products, added to aflatoxin-contaminated corn, increased average daily gain and feed efficiency to levels similar to those of control pigs. The products were included in the feed at 10 pounds per ton. At that level the cost of including these products would be approximately $1 per ton of feed.
     All animal species are susceptible to aflatoxicosis, although sensitivity varies considerably from species to species. For example, birds, fish, dogs, and swine appear to be more susceptible than mature cattle. In poultry, besides fatty liver and kidney disorders, leg and bone problems can develop as well as outbreaks of coccidiosis. Aflatoxins may cause vaccines to fail, increase the birds' susceptibility to disease, and result in suppression of the natural immunity to infection. The animals become susceptible to infection by bacteria such as Salmonella and to various viruses and other infectious agents commonly found around the farm yard, feedlot, or poultry house that normal healthy animals ward off. Decreased blood clotting results in a greater downgrading and condemnation of the birds because of massive bleeding and bruises. .Less carcass pigmentation is exhibited and egg yolks are paler. The hatchability of eggs can drop, and reduced production may be noted as well as smaller eggs with shell problems. Growth is restricted and mortality increases, especially during the growing period.
     Regular or occasional consumption by farm animals of feed containing aflatoxin in the range of less than 100 ppb to a few hundred parts per million (ppm) results in decreased feed consumption, poor feed conversion, stunting, and decreased flesh growth. Decreased productivity may be accompanied by damage to the liver, hemorrhaging into the muscles or body cavities, and suppression of natural immunity to parasites and pathogens always present in the environment. Once the damage has been done, the animals will not fully recover, even if returned to a toxin-free ration.
     Aflatoxin is present in the spores of A. flavus, which sometimes are produced in great abundance on the ears of fungus-infected corn. When corn is combined and unloaded at elevators or other transfer points, it generates considerable dust, and some of this dust may contain aflatoxin. Dust collected near a combine in Georgia in 1980 contained from 2,030 to 41,200 ppb of aflatoxin. The aflatoxin content of the dust at the elevator receiving this corn ranged from 621 to 1,480 ppb.
     Dust masks should always be worn when handling obviously moldy grain. Inhaling aflatoxin-contaminated dust is presumed to be a health hazard. Grain handlers have more respiratory problems than the general population. "Farmer's lung" is a disease that afflicts grain handlers and is frequently associated with skin irritation, fever, wheezing, breathlessness, cough, and ulcers. Farmer's lung is thought to be caused by an allergic reaction to fungal spores and other material in grain dust. Pulmonary mycotoxicosis is a disease that occurs in farmers when they inhale large amounts of grain dust containing fungal hyphae and spores. This latter disease is a direct effect of the fungal toxins, not an allergic reaction.
     Grain invaded by Aspergillus species is highly friable, therefore great care should be taken when feeding grain screenings. Broken grains often have very high levels of aflatoxin concentration.

     Zearalenone and zearalenol are produced almost exclusively by Fusarium species that contribute to the ear and stalk rot that occurs in the ears of corn and on the heads of cereal grains (scab) standing in the field or in stored ear corn in the Corn Belt. However, in 1986, these mycotoxins were detected in delayed harvest soybeans at up to 5 ppm. When consumed by swine at more than 0. 1 to 5 parts per million (ppm) (mg toxin per kg body weight), these compounds cause the estrogenic syndrome, which is characterized in females by a swollen and edematous vulva with enlarged mammary glands and in young males by a shrinking of the testes. Young gilts may show uterine prolapse. The financial loss to farmers comes about primarily through poor reproductive performance.
     Estrogenism in swine and dairy cows is usually more prevalent in the winter and early spring because, once the fungus is established in the grain, it generally requires a period of relatively low temperatures to produce biologically significant amounts of zearalenone. When some strains of Fusarium graminearum grow in corn they produce a mixture of toxins along with zearalenone. One or more of these can cause severe stunting and other deleterious effects in swine.
     Decreased fertility, prolonged estrus, and swelling of the vulva are signs that dairy cows have fed on rations containing zearalenone as well as other natural toxicants produced through natural infection of feed ingredients (corn, hay, barley). Animals vary as to their response, but some will show standing estrus at mid-cycle.
     Broiler chicks and laying hens, unlike swine and dairy cows, are affected very little by dietary zearalenone even when fed massive doses. Pure zearalenone fed to broiler chicks and finishing broilers at rates from 10 to 800 ppm produced no effect on weight gain, feed consumption, and feed-to-gain ratio. The weights of the liver, heart, spleen, testicles, oviduct, comb, and bursa were similar to those in the controls that received no zearalenone. In laying hens, zearalenone had no effect on egg production, egg size, feed consumption, body weight, fertility, hatchability of fertile eggs, or reproductive performance. When turkeys ate feed containing 300 ppm of zearalenone (a massive dose), they developed greatly enlarged vents within 4 days, but there were no other gross effects.
     The effects of zearalenol are similar to zearalenone, but zearalenol is generally considered to produce estrogenic effects five to ten times greater than those of zearalenone.
     Fusarium graminearum requires a minimum of 22 to 25 percent moisture to grow in cereal grains. Generally, shelled corn stored at these moistures is likely to be colonized by a mixture of other fungi, yeasts, and bacteria with which F. graminearum competes poorly. F. graminearum ear rot is primarily a problem in stored corn in cribs exposed to low temperatures.

     Fusarium graminearum (sexual state Gibberella zeae) growing in the ears of corn and on the heads of cereal grains before harvest may produce other toxins besides zearalenone. These include deoxynivalenol (DON), which makes the grain unpalatable to swine. Field-infected corn with visibly more than 5 percent damaged kernels is refused by pigs. Feed refusal may be accompanied by swollen vulvas and reproductive problems from zearalenone and DON in the same ration, and sometimes a complex of effects can occur. Swine producers often encounter serious problems when they attempt to make such corn palatable by applying molasses or other similar materials.
     Wet, rainy, warm, and humid weather from flowering time on promotes infection of corn and cereals by Fusarium species, resulting in ear rot in corn and scab or head blight in wheat, barley, oats, and rye. Low temperatures following infection may increase the production of DON. The toxin already present in corn at harvest may increase in ear corn stored in cribs, as does zearalenone. Shelled grains free of the toxin at harvest have not been observed to develop either DON or zearalenone mycotoxins in storage.
     Feeds that contain 1 ppm of DON may result in significant reductions in swine feed consumption and weight gain. Vomiting is rather uncommon in field cases because pigs usually will not eat enough of the contaminated feed.
     In Illinois, cool, wet weather before and during the 1981 harvest of corn and small grains was followed by reports in late 1981 and 1982 of feed refusal and clinical signs of ill health in farrowing operations, feeder pigs, and breeding sows. DON was found in 80 percent of the nearly 400 samples taken, in concentrations of 0. 1 to 41.6 ppm. Zearalenone was found in 12 percent of the samples, at concentrations of 0. 1 to 8 ppm. Some samples contained both toxins. Clinical signs and lesions in affected swine included feed refusal, a few instances of vomiting, lack of weight gain, poor feed efficiency, failure of mature sows to return to estrus, reduced efficiency, high mortality of nursing pigs, intestinal tract inflammation, and acute diarrhea in young pigs. Autopsies of young pigs revealed hemorrhaging into the abdominal cavities and pale, friable livers. In all cases investigated in detail, the problems were reduced or disappeared when the pigs were given sound feed. Dairy cattle and poultry are relatively insensitive to the dietary concentrations of DON likely to be found in feeds.

     Fusarium tricinctum and some strains of F. graminearurm, F. equiseti, F. sporotrichioides, F. poae, and F. lateritium produce T-2 and other toxic trichothecenes. These fungi commonly attack grains and can grow at temperatures from slightly above freezing to about 86°F. T-2 and HT-2 toxins are produced over a temperature range of 46° to 77°F, with the maximum production at temperatures below 59°F. This group of toxins was associated with ATA, which killed thousands of human beings in the USSR in 1913 and after World War II.
     Apparently all domestic animals are susceptible to injury by dietary intake of T-2, HT-2, and diacetoxyscirpenol (DAS) in the range of a few ppm. In poultry, T-2 toxin in feed contaminated with 1 to 3.5 ppm of T-2 and 0.7 ppm of HT-2 (a closely related toxicant) may produce lesions at the edges of the beaks, abnormal feathering in chicks, a drastic and sudden drop in egg production, eggs with thin shells, reduced weight gains, and mortality. The same feed given to turkeys results in reduced growth, beak lesions, and less immunity to infection.
     T-2 and DAS in cattle feed results in unthriftiness, decreased feed consumption, slow growth, lowered milk production, and sterility. An outbreak of the hemorrhagic bowel syndrome and death of some animals can occur in herds of cattle and swine.
     In swine, infertility with some lesions in the uteri and ovaries result from consumption of feed contaminated with 1 to 2 ppm of T-2 toxin. When sound feed was provided to all domestic animals, the troubles quickly disappeared. T-2 toxin and DAS in amounts sufficient to cause to cause toxicoses have been found in corn still in the field, in silage, and in prepared feeds made in part from corn. These toxins have also been identified in weather-delayed-harvest soybeans. Feed contaminated with these toxins must be handled carefully because these toxins can cause severe skin irritation. As with most other mycotoxins, the only control is to avoid contaminated feeds.

     Tibial dyschondroplasia (TDP) is a common and economically important bone deformation in growing broiler chickens and turkeys. The lesion appears in a cone of cartilage extending distally from the proximal tibiotarsalphysis. The most likely cause of this deformation is a toxin called fusarochromanone produced by Fusarium equiseti. When added to the diet of broiler chicks at 75 ppm, 100 percent of the chicks showed symptoms of TDP. This toxin may be largely responsible for the TDP syndrome in poultry; it also kills chick embryos in fertilized eggs.

     Blind staggers (technically known as equine leucoencephalomalacia) occasionally occurs in horses, mules, or donkeys foraging corn left standing in the field after harvest or fed grain or screenings heavily infected with F. moniliforme. The toxins fumonisin B₁ and B₂ are produced only by certain strains of F. moniliforme. This toxicant is also carcinogenic in laboratory tests. Fumonisin B₁ and B₂ have been extracted from corn infected with F. moniliforme. Fumonisin B₁ was administered to horses and, within 8 days, the horses exhibited signs of blind staggers. F. moniliforme is common even in food-grade corn and is often abundant in ground feeds and in silage. Growing pigs fed a ration containing 78 to 82 percent corn heavily colonized by F. moniliforme grew as well as the control pigs fed a ration of sound corn. It is therefore likely that fumonisin is not always present when the fungus is, or that pigs are not sensitive to fumonisin B₁. Research on the fumonisin toxins began only recently, and current thought is that concentrations of more than 5 to 10 ppm are necessary for mycotoxicosis in horses and more than 10 to 20 ppm for swine. As with other mycotoxins, various strains of this fungus vary greatly in their toxin producing ability.

     Ochratoxin A, produced primarily by members of the Aspergillus ochraceus group and a number of species of Penicillium, especially P. viridicatum have been found in some samples of food and feed grains. Frequently, citrinin or PA is produced by these same fungi simultaneously. In the field, however, injury from ochratoxin poisoning has occurred chiefly (or only) in poultry and swine. Listlessness, huddling, diarrhea, tremors, and other neural abnormalities are often encountered in broiler poultry production in the southeastern United States. Ochratoxin damage to the kidneys of swine is characteristic enough to be called "porcine nephropathy," which is recognizable and recognizable in commercial slaughtering.
     All kinds of laboratory animals tested have been sensitive to injury by ingested ochratoxins. Regular consumption of a ration containing several hundred ppb of ochratoxin results in poor feed conversion, reduced growth rate, and general unthriftiness, accompanied by reduced immunity to infection by bacteria and viruses. Other prominent features of ochratoxin poisoning are increased water consumption and increased urine production because of kidney damage. The increased urine production in pigs results in the floor of the pig pen being constantly wet and needing to be cleaned daily. Toxicosis from citrinin and ochratoxin A occurs most often in Denmark and other Scandinavian countries and is associated with P. viridicatum in barley. At slaughter, the kidneys may be found to be enlarged and pale, with an uneven cortical surface and cortical fibrosis. Lesions may also be evident in the liver.
     A toxin produced by Penicillium icelandicum in high moisture barley destroyed a purebreed Ayrshire dairy herd in Nova Scotia, Canada.

     The fungus Rhizictonia leguminicola growing in red clover produces a compound that, when consumed by cattle, results in profuse salivation (hence the name "slobber syndrome"), which is relatively common throughout the Midwest. The compound itself is not to toxic before being consumed but is transformed by the animal into a toxin compound.

     Ergot toxicity, caused by the fungus Claviceps purpurea, differs from other mycotoxicoses, since it results from the consumption of considerable amounts of fungal tissue. In other mycotoxicoses the toxins are secreted into plant tissues in which the fungus is growing, and very little fungal material is consumed. The ergot fungus infects the flowers of cereals and many grasses when flowering occurs during predominantly cool, moist weather Infected florets show characteristic black, spur-like sclerotia that replace the seed. The sclerotia or ergot bodies contain a variety of ergopeptine and clavine alkaloids that, when consumed regularly in small amounts, result in a complex of signs collectively called ergotism. Symptoms of ergotism are poor hair condition, gangrene or loss of extremities, and poor performance.

     Many tall fescue pastures in Alabama and most of the United States are infected with a systemic fungus, Acremonium coenophialum. This fungus is harmless to the host plant, but it is responsible for a variety of symptoms known as fescue toxicosis, summer syndrome, and summer slump when infected plants are consumed by cattle. The fungus is endophytic, meaning it grows within the tillers, culms, and inflorescence of the grass without invading the host, as do most of the other saprophytic or pathogenic fungi producing mycotoxins. In fact, there is evidence that the endophyte has evolved a mutualistic relationship with the grass, conferring a survival advantage in some situations. In cattle, symptoms of fescue toxicosis include reduced average daily gains, lower milk production, reduced reproductive potential, elevated body temperature, rough hair coat, and reduced prolactin levels. "Fescue foot" is like ergotism in that feet or other extremities may become gangrenous and drop off. Horses typically show only reproductive disorders when eating infected fescue. Agalactia, abortions, thickened placenta, prolonged gestation, and large foals resulting in dystocia are typical. The ergopeptine alkaloids similar to those produced by ergot (Claviceps) have been identified in endophyte-infected fescue, but other alkaloids may be involved as well.

     Evidence that a mycotoxin is responsible for illness in animals that consumed feed requires that the toxin or toxins be isolated, purified, and quantified. Procedures have been developed for the extraction, purification, and quantification of the major mycotoxins such as aflatoxins, zearalenone, T-2, DAS, DON, ochratoxin A, citrinin, fumonisins, ergot alkaloids, and some of the other trichothecene toxins. Table 2 provides information on methods for detecting mycotoxins. To achieve reliable results, these procedures require considerable expertise in the performance and interpretation of the tests plus sophisticated and relatively expensive equipment. However, preliminary screening for toxins can be done with commercially available kits³.
     Routine handling of contaminated grain - particularly heavily contaminated grain - may present a significant health hazard to technical personnel. Therefore, samples should be handled only by trained individuals working in appropriate facilities and within the guidelines of an acceptable safety protocol.

     An adequate sample of suspect grain must be obtained to use any assay method. Proper sampling is essential because one aflatoxin-contaminated kernel in 1,000 kernels of grain may be a source of significant contamination. Occasionally a biased sample may be more revealing than a truly representative one. For example, in studying stored grain or feed that shows evidence of moisture damage, heating, or "caking", a sample of damaged grain may be more appropriate than a composite one from an entire lot. A 10-pound sample is usually collected by using a probe or continuously taken from a stream or flow of grain. The sample must then be finely ground so that it will pass through a 15- to 20-mesh screen and be thoroughly blended to obtain a subsample appropriate for analysis. The objective of any sampling procedure is to acquire a representative sample. A representative sample may require random sampling of plants in all areas of a production field, whereas, in freshly mixed grain (after harvest or following handling), a representative sample may be easily acquired by a few subsamples. Samples stored for analysis should be placed in a paper bag or cardboard box and kept under cool, dry conditions that will not permit fungal growth or the possible continued production of mycotoxins. Care must be taken to keep samples in the same condition as at the time of sampling. For example, moist grain samples stored in plastic bags under warm, humid conditions may have significant aflatoxin contamination occur during sample storage. When probing a ship hold, grain bin, vehicle, or hopper car, numerous random probes may be required and site-selective probing should be done if signs of moisture leakage, insect activity, or hot spots are identified.

     The methods of aflatoxin analysis fall into three categories (Table 2):

     The estrogenic syndrome, feed refusal, or vomiting may be a first sign of a Fusarium toxin. If analysis is required, thin-layer chromatography, gas-liquid chromatography, high-pressure liquid chromatography, or mass spectroscopy can be utilized (Table 2).

     In animals, few mycotoxins produce clinical signs so characteristic that they permit unequivocal diagnosis. For example, the estrogenic syndrome in swine can be caused by diethylstilbestrol (DES) in the feed as well as by zearalenone and zearalenol. Refusal of feed containing corn or cereal grains usually indicates Fusarium toxins. At times, other contaminants may lead to refusal. Some mycotoxins, including the trichothecenes and aflatoxins, may bring about reduced productivity or depressed growth, but certain environmental factors may cause similar effects. Lowered resistance to infections by microorganisms or opportunistic parasites and reduced protection from immunization may be the result of ingesting mycotoxins. Such effects, however, may have other causes.

1. Harvest at maturity and as soon as the moisture content allows minimum grain damage. For shelled corn (23 to 25 percent moisture), ear corn (25 to 30 percent), small grains, including sorghum (12 to 17 percent), and soybeans (11 to 15 percent). Unfortunately, exact timing is not always possible because of unfavorable harvesting conditions. Grain from fields with severe insect, drought, or frost damage should be stored separately from other grain.

2. Adjust the harvesting equipment for minimum seed or kernel damage and maximum cleaning.

3. Dry all grain to at least 15-percent moisture as rapidly as possible, not to exceed a 24- to 48-hour period after harvest. Safe, long-term storage can be achieved at a uniform moisture level of 13 percent or somewhat below. Slow drying (accomplished by low heat or natural air drying) is being used increasingly, but the grain can contain no more than 20 or 21 percent moisture in full-bin drying. Another possibility is high-temperature drying until the grain reaches 20- to 21-percent moisture, followed by low-heat drying to 13-percent moisture.

4. Cool the grain after drying and maintain dry storage conditions. When possible, continue cooling until the grain temperature reaches 36° to 41°F.

5. Thoroughly clean the grain and all bins before storage to remove dirt, dust, and other foreign matter, crop debris, chaff, and cracked or broken seeds and kernels. Remember, mold-infected kernels are friable and easily broken. Broken or damaged kernels are more likely to be mycotoxin contaminated. The use of seed- or grain-cleaning equipment can significantly reduce the mycotoxin content (particularly aflatoxin) of a grain lot.

6. Store in water-, insect-, and rodent-tight structures.

7. Continue periodic aeration and probing for "hot spots" at intervals of 1 to 4 weeks throughout the storage period. The analytical tests now in use can detect levels of aflatoxin lower than those of toxicological significance in raw materials and finished food products and feeds.

8. Use propionic acid or a mixture with ammonium isobutyrate, as registered on high-moisture grain in storage. Propionic acid is sold under various trade names. Although this acid will not remove any aflatoxins already present in the grain, it will prevent the growth of fungi if properly applied. Grains treated with propionic acid can be used only for livestock and poultry feeds.

9.Where feasible, choose varieties of grain that are resistant to insects, diseases, and mechanical damage. Any damage to the grain provides a route of entry for A. flavus and other toxin-forming fungi. Once the fungus or fungi has invaded the plant, then the appropriate environmental conditions will lead to toxin formation.

     1. NovaSil, Englehard Corporation, Cleveland, Ohio (216-292-9200). NovaSil is available in Alabama through Fuller Supply Company, Birmingham (800-292-8567).
     2. Volclay and FD-181, American Colloid Company, Arlington Heights, Illinois. Volclay and FD-181 are available in Alabama through Agri Products, Incorporated, Birmingham (205-979-2474).
     3. Aflatoxins (B₁ and M₁), zearalenone, DON, and T₂ toxin test kits are available from Neogen Corporation, Lansing, Michigan (800-234-5333). Other companies who have kits for aflatoxins and others are: Environmental Diagnostics, Burlington, North Carolina (800-334-1116); Vicam, Somerville, Maine (800-338-4381); Romer Labs, Washington, Missouri (314-239-3009), IDEXX Corporation, Portland, Maine (800-548-6733); and Rialdon Diagnostics, Bryan, Texas (409-846-6202). Mycotoxin analysis is available on a fee basis from the Department of Plant Pathology, Auburn University, AL 36849-5409 (334-844-5003).

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