Stored Grain

Mycotoxins

Barry J. Jacobsen, Robert W. Coppock, and Michelle Mostrom

Identification and Life Cycle

Toxigenic Fungi

Mycotoxins are toxic fungal metabolites that cause intoxication when consumed by animals, including humans. Fungi that produce mycotoxins are called toxigenic fungi.  The toxigenic fungi do not produce mycotoxins after they have been ingested by animals and humans.  Toxigenic fungi grow in corn, cereals, soybeans, sorghum, peanuts, silage and other food, feed crops or hay in the field, and in grain during transportation. Mycotoxins can be produced in storage under conditions favorable for the growth of the toxin-producing fungus or fungi.  Mycotoxins can be found in any animal feedstuff or human foodstuff that has previously supported growth of toxigenic fungi.  It is estimated that there may be 20,000 to 300,000 unique mycotoxins and relatively few (<50) have been well characterized. These toxins can be found in processed foods and feeds produced from contaminated feedstocks.  The most common mycotoxins are produced by fungi in the genera Aspergillus, Penicillium and Fusarium.  However, fungi in the genera Alternaria, Stachybotrys, Claviceps and Epichloe produce common and important mycotoxins.

Mycotoxicoses

Mycotoxicoses is the disease caused by animals and humans consuming feedstuffs and foodstuffs contaminated with mycotoxins. Mycotoxins can cause death or chronic ill health resulting from damage to the kidneys and liver.  Mycotoxins can also damage the immune, cardiovascular, endocrine, reproductive and nervous systems.  Mycotoxins cause hemorrhage, abortion, reproductive disorders, tremors, convulsions, immune system dysfunctions, skin disorders and gangrene of appendages.  In addition, mycotoxins cause economic losses from unthriftiness, reduced growth rate, poor feed conversion, feed refusal, increased restlessness, agalactia, and lameness (Table 1).  Historic records exist of mycotoxins causing disease in humans and animals. Alimentary toxic [1]aleukia (ATA) a serious gastrointestinal syndrome was linked with consumption of  overwintered wheat, barley and prosomillet infected by Fusarium species that are potent producers of trichothecene mycotoxins.  Alimentary toxic aleukia was observed in tens of thousands of people in Russia and central Asia from 1941-1947.  In 1934 a malady called "moldy corn disease" occurred in the Midwest.  More than 5,000 horses died because of mycotoxin-contaminated feed. The mycotoxins eventually linked to “Moldy Corn Disease” were the fumonisins.  In 1972, Gibberella ear rot caused extensive feed-refusal problems in swine in the Corn Belt. Aflatoxin has caused problems in several animal species in the southeastern USA for many years, and fescue toxicosis has been a common problem with fescue pastures in the Mid-southern and Southern regions. 

Human suffering from mycotoxicoses also includes “Holy Fire” or “St. Anthony's Fire” which is linked to consumption of ergot alkaloids in rye and wheat flour. A disease called “Yellow Rice Disease” was described in Asian countries when humans consumed rice colonized with Penicillium molds. A disease called “Acute Cardiac Beriberi” was also associated with yellow rice. This disease is linked to the neuro- cardiotoxic mycotoxin citreoviridin produced by Penicillium species. Several mycotoxins have been linked to an increased incidence of cancer in humans.  Aflatoxins are linked to liver cancer in humans and esophageal cancer has been linked to consumption of fumonisins in grain infected with Fusarium moniliforme. Mycotoxins have also been linked to other pathology in humans. Zearalenone has been associated with precocious breast development in girls and ochratoxin A is suspected as a cause of the Balkan endemic nephropathy.  Gliotoxin, a mycotoxin, is suspect in the development of multiple sclerosis. 

The adverse effects of feeding moldly grains and other feedstuffs have long been known by livestock and poultry producers.  The specific implication of a mycotoxin did not occur until 1960 after the outbreak of “Turkey X Disease" in Great Britain.  The contaminated feed was traced to peanut meal imported from Brazil. The chemicals that became known as aflatoxins were identified, and the mycotoxicosis was reproduced by dosing poultry and livestock with aflatoxins.  Thus, aflatoxins were discovered and identified as the etiology of death in >100,000 young turkeys, ~20,000 ducklings, pheasants, partridge poults and numerous other livestock.  Identification of aflatoxins stimulated research on toxigenic fungi and the mycotoxins they produce (Table 1).

Table 1. Major Mycotoxins and Toxin-Producing Fungi from Corn, Cereal, Soybeans,  Peanuts, and Other Products and Some of their Effects on Animals.
Toxin or Syndrome	Fungal source	Feeds or foods affected		Possible effects on animals
Aspergillus Toxins- (primarily) Aflatoxins B1, B2, G1, and G2  (B2a, G2a, M1, and M2 are metabolites and seldom present in grain; M1 and M2 are important contaminants in milk)	Aspergillus flavus and A. parasiticus	Cereal Grains, peanuts, soybeans, and other foods		Hepatotoxin; carcinogenic; reduced growth rate; hemorrhagic enteritis; suppression of natural immunity to infection; decreased production of meat, milk and eggs, pulmonary mycotoxicosis
Ochraoxins  (nephrotoxins)	Aspergillus alutaceaus var. alutaceus ( ochraceus) and Penicillium viridicatum	Cereal grains		Toxic to kidneys and liver; abortion; poor feed conversion, reduced growth rate, general unthriftiness; reduced immunity to infection
Sterigmatocystin	Aspergillus nidulellus, A. glaucus, A. sydowii A. versicolor and Bipolaris sorokiniiana	Cereal grains		Toxemia; carcinogenic, hepatotoxic
Termorgenic toxin	Aspergillus flavus, Aspergillus terrus, Penicillium cyclopium, and P. palitans	Cereal grains, soybeans, peanuts, and other food feeds, etc.		Tremors and convulsions, death
Penicillum Toxins   (primarily) Luteoshyrin	Penicillium islandicum	Rice		Hepatotoxic, tremors and convulsions
Patulin	Penicillium urticae, P. expansum, P. clavirome, and Aspergillus clavatus	Cereal grains, apple products		Hemorrhages of lung and brain; edema toxic to kidneys; possibly carcinogenic
Rubratoxin	Penicillium rubrum			Liver damage, nephrotoxic and hemorrhage
Citrinin	Penicillium citrinum			Kidney damage
Penicillic Acid	Penicillium viridicatum and several other Penicillium sp.	Cereal grains		Similar to ochratoxin
Ergot Toxins  Ergopeptines	Claviceps purpurea	Cereal Grains		Vasoconstriction, loss of extremities (ears, tail, fee, etc.), skin necrosis, agalactia
Ergovaline	Neotyphodium (Acremonium) and Epichloe sp.	Fescue		Reduced weight gain, abortion,  poor survivability of offspring, fescue foot
Fusarium Toxins
Zearalenone  (Estrogenic syndrome)  Zearalenol	Fusarium graminearum, F. colmorum, F.equiseti		Cereal grains, soybeans	Hyperestrogenism, infertility, stunting, and even death
Emetic or feed refusal  Factor, (Vomitoxin)  Deoxynivalenol or DON	Fusarium graminearum (sexual state), Gibberella zeae), F. culmorum		Cereal Grains	Food refusal by swine, cats, dogs; reduction in weight gain
Other trichothecenes  (T-2, HT-2,  Monoacetoxyscripenol  or MAS,  Diactoxyscripenol  or DAS)	Fusarium graminearum, F. equiseti, F. poae, F. acuminatum, F. sambucinum and F. sporotrichoides		Cereal grains, soybeans, potato	Severe inflammation of gastrointestinal tract and possible hemorrhage; edema; vomiting And diarrhea; infertility; degeneration of bone marrow; death; reduced weight gain; slow growth; sterility, abortion
Fumonishin B1, B2	F. verticillioides, F. proliferatum		Corn	Leukoencephalomalacia “moldy corn disease” in horses, pulmonary edema in swine, neural tube defects and esophageal cancer in humans

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mixtures of Mycotoxins

Feed grains can be infected with multiple toxigenic fungi with production of multiple mycotoxins.  When one mycotoxin group is present, there is a distinct possibility that mycotoxins from a different group may be present.  The toxic effects observed for multiple mycotoxins can be unique in that they do not mimic the toxicology reported for any particular mycotoxin. .  The predominant interaction between mycotoxins is an additive effect. There is also evidence that synergism and potentiation can also occur. 

When horses and pigs consume DON with other trichothecene mycotoxins that are not generally included in mycotoxin analytical screens, the toxic effects of DON also increases. Fusaric acid is considered to be synergistic with DON.  A source of fusaric acid can be moldy hay or silage.  The combined effects of fumonisins and aflatoxins are considered to be additive.    It is important to consider the presence of multiple mycotoxins when estimating the safe levels of mycotoxin-contaminated feed that can be fed to animals

 

Laboratory Testing for Mycotoxins

Evidence that mycotoxins are responsible for illness in humans or animals is generally based on:

The occurrence(s) of the disease observed is linked with feeding a particular feed or consuming a particular food.

Examination of the suspect feed shows evidence of fungal activity. 

The disease is not transmissible from animal to animal.

Laboratory testing of the affected animal or person does not clearly identify an infectious agent.

Young, old and pregnant animals are generally the most susceptible and are the first to show symptoms.

 When one or more of these criteria are met, the suspect food or feed should be tested for mycotoxins in the laboratory.

For chemical analyses, the mycotoxins in the feed need to be extracted, compounds that interfere with the assay removed, and the mycotoxin identified and quantified.  Procedures have been developed for the extraction, purification, and quantification of the common mycotoxins such as aflatoxins, zearalenone, T-2, DAS, DON, ochratoxin A, citrinin, fumonisins, ergot alkaloids, sterigmatocystin, patulin and some of the other trichothecene toxins. Table 4 provides information on methods for identification and quantification of mycotoxins. To achieve reliable results, these procedures require considerable expertise in the performance and interpretation of the analytical results plus sophisticated and relatively expensive laboratory equipment. Preliminary screening for toxins can be done with commercially available kits. Kits are available for aflatoxins (B₁, B₂, G₁,G₂, and M₁), zearalenone, DON, T-2, fumonisin (B₁, B₂, B₃) , ochratoxin A, sterigmatocystin, citrinin, and patulin. Sources of test kits can be found in Table 5.  The majority of these kits are immunoassays, and false negative and false positive results can occur more commonly than those observed using other analytical chemistry techniques for identification. . 

 Routine handling of contaminated grain, particularly heavily contaminated grain or hay, may present a significant health hazard to technical personnel. Therefore, samples should be handled only by trained individuals working in appropriate facilities and within guidelines developed by state and federal agencies. Mycotoxin analysis is available on a fee basis from Romer Labs (www.romerlabs.com) and veterinary toxicology centers at North Dakota State University, Iowa State University and several other state land grant universities.

 

Sampling for Mycotoxins and Sample Preparation

An adequate and representative sample of suspect feed grain or other feed should be obtained.  This can be difficult in livestock feeding operations because the majority of the suspect feed has been consumed.  It may be necessary to remove feed from the corners of the feeders or retrieve feed from the corners of the storage unit.   

Proper sampling is essential because one kernel in 1,000 kernels of grain may be a source of significant mycotoxin contamination and contamination may occur only in pockets (hot spots) in the feed mass. 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. Typically a 10-pound (5-kilogram) sample is collected using a probe from random sites in the feed mass or continuously taken from a stream or flow of grain. This sample can be subdivided such that a representative but smaller sample is submitted for chemical analysis. The sample must then be finely ground so that it will pass through a screen of 15 to 20 mesh and be thoroughly blended to obtain an aliquot appropriate for chemical analysis. The objective of any sampling procedure (protocol) is to acquire a representative sample. A representative sample may require random sampling of feed from all areas of a feed mass whereas in freshly mixed feed (after harvest or following handling), a representative sample may be easily acquired by taking a few aliquots.

            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 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.

            It is often practical to determine which fungi are present and then test for the mycotoxins produced by the fungi present because testing for mycotoxins can be expensive.  Samples submitted for fungal identification should be chosen and stored as suggested above.  Services for fungal identification are available from extension plant pathologists in most states.  In Montana, samples for fungal identification should be sent to the Schutter Diagnostic Lab, 121 Plant BioScience Building, Montana State University, Bozeman, MT 59717-3150 and marked attention Barry Jacobsen.

 

Detecting Mycotoxins

Some methods of mycotoxin analysis are presented in Table 4. These methods differ in their sensitivities and are appropriate for only certain commodities.  Typically aflatoxin, ochratoxin and fumonisin tests should be sensitive in the parts per billion (ppb) range, while tests for DON, T-2 and zearalenone are sensitive in the parts per million (ppm) range.  It should be remembered that detection of a mycotoxin is just part of the story.  It is the dose that makes the poison and it is critical to understand what portion of the diet is from the mycotoxin-contaminated feed as well as sensitivity of the animal species and animal age exposed. It is also important to recognize that there can be significant variability in test results.  There are published results of aflatoxin tests from 10 subsamples of a single lot of peanuts that show results varying from 0 to 230 ppb. Ingestion of multiple mycotoxins can change the dose response and it is important to remember that mycotoxins other than those identified can be present in the feed. 

 

Regulatory Issues

Many countries including the USA have established advisory guidelines or action limits that restrict the amount of mycotoxin permitted in food and feed.  Table 2 provides information on advisory guidelines for mycotoxins in food and feed in the USA.  Only levels of aflatoxins B₁,B₂, G₁, G₂ and M₁ and DON are currently regulated by the FDA, and there are proposed limits for fumonisins.  It is important to understand that knowingly mixing lots of grains or feeds that exceed the regulatory limit with uncontaminated grain to achieve lower levels of mycotoxin concentration is considered adulteration and is subject to civil and criminal penalties.

 

Diagnosis

In animals, few mycotoxins produce clinical signs so characteristic that they permit unequivocal diagnosis. For example, the estrogenic syndrome in cattle can be caused by phytoestrogens in forage as well as by zearalenone and zearalenol in grain.  Refusal of feed containing corn or cereal grains usually indicates mycotoxins produced by Fusarium spp.  Other substances in feed can also cause feed refusal. Some mycotoxins, including the trichothecenes and aflatoxins, may bring about reduced productivity or depressed growth, but certain environmental factors and nutrient deficiencies 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 or it could be due to a deficiency in selenium or other factors.  Therefore, diagnosis should be made based on symptoms observed, tissue pathology, and the presence of the mycotoxin and the estimated dose of the mycotoxin.  Information of the microbial colonization of the suspect grain or feed is very helpful to determine the risk for multiple mycotoxins being present.    Factors such as animal age and condition, duration of exposure and presence of more than one mycotoxin should be considered.  The affects of exposure to multiple mycotoxins is largely unknown. Bioassays can be used to assess the toxicity of the suspect feed but are not commonly performed.  Animals used in a bioassay should have a detailed pathologic examination.

 

Minimizing Mycotoxin Problems

1.  Harvest grains at maturity whenever possible and adjust harvesting equipment to minimize physical damage to the grain. Once harvested, grain or seed should be stored at moistures that preclude the growth of mycotoxin-producing fungi.  If drying is not possible, the producer should  consider the use of propionic or other acid storage aids.  Such “acid” treated grain can only be used for livestock feed. Acid treated grains must be stored under the conditions and for the duration recommended by the manufacturer.  Such storage aids can also be used for hay.  Hay and grain treated with storage aid and stored beyond the recommended interval or under conditions different than those recommended by the manufacturer can allow rapid growth of mycotoxigenic fungi.   When harvesting grains where harvest has been delayed by wet conditions extra care should be taken to assure the grain is dry enough for safe storage.  Where scab is a problem in cereal grains, extra air should be used during combining so that much of the light weight “scabby grain” is left in the field.  Screening out broken grain may reduce aflatoxin, fumonisin and ergot contamination. Use proper techniques to ensure proper ensilement of grain silages and haylage.  Once conditions are anerobic and sufficient lactic acid is produced, the mycotoxin producing fungi cannot grow in silage or haylage.  However, any mycotoxin present in grain or forage prior to ensiling will not be destroyed by the ensilement process.

 

2.  Store cereal grains and oil seeds in weatherproof structures that have been cleaned and treated for storage insects.  Cleaning grain to remove light weight “scabby grain”, broken grain and ergot sclerotia is helpful.  Remember that mold-infected kernels are very friable and easily broken.  Commonly, cereals with high DON or aflatoxin levels can be reconditioned such that the heavily contaminated grain is cleaned out and levels of DON or aflatoxin are below advisory levels. Controlling storage insects and rodents is critical.

 

3.  Control mycotoxin-producing molds in the field.  Corn varieties with the Bt gene will typically have lower levels of fumonisin and aflatoxin because ear-damaging insects are controlled.  If scab is of concern in wheat, choose varieties that are more tolerant to Fusarium head blight and use an approved foliar fungicide at heading to anthesis.  Fescue endophyte free seed is available and should be used for new seedings.

 

4.  If aflatoxin is of concern, anhydrous ammonia treatment of contaminated grain will reduce aflatoxin levels by 30-50%.  Once treated, the grain can only be used for animal feed.

 

5. Avoid feeding grain screenings (unless tested for mycotoxins), moldy silage, or moldy hay.

 

6. If grain is purchased for feeding from an area with known mycotoxin problems, have the grain tested before shipping or have this as a contract specification.

Table 2.  Regulatory limits for mycotoxins in the USA.

 

 

Table 5. Commercial kits available for mycotoxin analysis

 

Categories: Stored Grain, Mycotoxicoses, Toxigenic fungi

Date: 11/11/2007