Mycotoxins in Grain and Feed Industries I. Mycotoxin Development Erin Bowers, Iowa State University, Agricultural Engineering Charles Hurburgh, Iowa State University, Agricultural Engineering Alison Robertson, Iowa State University, Plant Pathology Welcome to the FDA safety training for grain and feed industries. This and another module will discuss mycotoxins and their significance for grain and feed industries. These presentations are a joint effort of Iowa State University and Kansas State University. This module covers mycotoxin production by various fungal species and the impact of mycotoxin contamination in animal feed.

Learning Objectives Module Objectives This learning module will focus on the development of fungi in the field and the production of mycotoxins under specific environmental conditions Module Objectives Understand the relationship of fungi and the environment to mycotoxin production Recognize harmful levels and effects of certain mycotoxins on humans and animals This module will discuss the development of fungi in the field and the production of mycotoxins by those fungi under specific environmental conditions. The first learning objective for this module is to understand the relationship of fungi and the environment to mycotoxin production. This includes recognizing environmental conditions that promote the development of fungi as well as conditions leading to the subsequent production of mycotoxins. The second objective for this module is to recognize harmful levels and effects of certain mycotoxins on humans and animals. At the end of the module you should be able to recognize what contamination levels might be dangerous to a particular animal species, and what environmental conditions might create higher mycotoxin risks.

Basics about Mycotoxins Gibberella ear rot caused by Fusarium graminearum (Gibberella zeae ) Source: Pioneer Hi-Bred, Intl Chemical compounds produced by some fungi Contaminate grains, food, and feed worldwide Aflatoxins were the first mycotoxins discovered Hundreds now known worldwide 30 are significant health hazards 5 principle mycotoxins affect cereal grains (corn, wheat, rye, barley, oats) aflatoxins, fumonisins, ochratoxin A, deoxynivalenol (vomitoxin), and zearalenone Fusarium head blight http://www.ars.usda.gov/Main/docs.htm?docid=9765 Mycotoxins are secondary metabolites that are produced by fungi under specific environmental conditions. They are not living organisms; rather, they are chemical compounds capable of causing an array of diseases and, in some cases death, in both humans and animals. These compounds, and the fungi that produce them, contaminate grains, food, and feed worldwide. Aflatoxins were the first mycotoxins discovered, as a result of their role in a new disease in turkeys-“Turkey X” disease. In 1960 in the United Kingdom, thousands of turkeys died after eating peanut meal. The contaminated peanut meal contained what we now know as aflatoxin. Since this discovery, hundreds of mycotoxins have been detected, but only 30 are significant concerns for human or animal health. Of these 30, 5 affect cereal grains like corn, wheat, rye, barley and oats, and are monitored under the FDA surveillance program. These five include structurally similar compounds that are classified as aflatoxins and fumonisins, plus ochratoxin A, deoxynivalenol (also known as vomitoxin), and zearalenone. Mycotoxin contamination is less common in oilseeds than cereal grains. This is because soybeans and other oil seeds are typically not subject to fungal contamination in the field. This also typically holds true for soybean meal and other feed ingredients that are derived from oil seeds. The pictures on the slide display two examples of field molds on crops. The top picture is an example of a corn ear suffering from Gibberella ear rot and the bottom shows wheat affected by Fusarium head blight (also known as head scab). Both of these crop diseases are caused primarily by the fungus Fusarium graminearum. Both diseases may result in the grain being contaminated with mycotoxins.

Fungal Disease Cycle Fungi infect plants and cause disease through various pathways. Spores are the reproductive units of fungi. Fungal spores generally overwinter in the soil or on crop residues and, when these microscopic spores come into contact with above-ground plant tissues, they can infect the plant. Some examples of how this contact may occur are precipitation, wind, and insects. Plants can also be infected by spores in the soil through the plant roots. Seeds that are infected when they are planted can also result in subsequent grain infection. Host plant contact with the fungal pathogen must coincide with the proper environmental conditions in order for the fungus to grow and contaminate grain with mycotoxins. Fungal infection and development can occur at multiple stages of crop growth and maturity, but grain development is a particularly vulnerable time. Damaged or stressed plants have an increased susceptibility to fungal growth and subsequent mycotoxin production. Fungi that colonize crops and produce mycotoxins before harvest are often referred to as “field fungi” while those that are capable of fungal spread and toxin production under storage conditions are called “storage fungi”.

Developing Fungus is Dependent on the Environmental Conditions During Pollination and Early Grain Development Mycotoxin(s) Fungi Favorable Conditions Primary Grains Aflatoxins Aspergillus flavus Aspergillus parasiticus Hot and dry, drought Corn, Durum (in EU) Deoxynivalenol (Vomitoxin) Zearalenone Fusarium graminearum Fusarium culmorum Cool, wet, humid at grain fill. Corn, Wheat, Oats, Rye, Barley, Durum Fumonisins Fusarium verticillioides Fusarium proliferatum Warm to hot, dry at and after flowering Corn Ochratoxin A Penicillium verrucosum Harvest conditions determine Fungi can colonize grain over a wide temperature and pH range, but each fungal species has a unique set of optimal growth conditions. Each also has optimal conditions for mycotoxin production, which are typically narrower than those for growth. Fungal growth is favored above 65% relative humidity. Not all fungi produce mycotoxins, and those fungi capable of producing mycotoxins do not always do so after they infect a plant. Climate, weather, plant health and development stage, and the timing of these interacting factors govern the risk for both fungal and mycotoxin contamination. Corn is susceptible to all of the mycotoxins that will be discussed in this presentation, so corn is the primary example we will use. It is not the only crop affected, though. You can see wheat, oats, rye, barley, and durum can also be impacted by mycotoxins, and this list is not comprehensive. Fungal activity is heavily influenced by environmental stresses, such as lack of moisture, insect feeding, and hail and wind damage. This table gives, as best we know it, the weather conditions that are favorable for the growth of mycotoxin-producing fungi in cereal grains.

Mycotoxins in Grain and Feed Significant food/feed safety hazard Unavoidable contaminants Stable and persistent Once present, they are hard to get out Remain intact after cooking, drying, freezing or storage conditions. Low levels (ppm or ppb) cause serious health problems for humans and animals There is usually no treatment for mycotoxin poisoning (mycotoxicosis) Mycotoxins are the primary food safety hazard in raw grain and first-process products, like corn distillers grains or wheat middlings. They generally are the highest in terms of both risk and prevalence. Even when growers use good agricultural practices and handlers use good manufacturing practices, mycotoxin contamination is still a risk as the fungi that produce them are unavoidable soil contaminants. There’s no way to sanitize fields to destroy these fungi and other potential crop contaminants. Mycotoxins are also stable compounds, so once they are in a product they are hard to get out. Most mycotoxins remain intact through processing conditions like heating, cooking, freezing, drying, or long periods of storage. The persistence of mycotoxins in the food and feed supply is a risk; many mycotoxins are capable of causing serious health problems at relatively low dosages - on the scale of parts-per-million (ppm) or parts-per-billion (ppb). Treating mycotoxin poisoning in animals is generally not successful. ppm: parts per million ppb: parts per billion

Economic Impact of Mycotoxins Worldwide, ~25% of crops are affected by mycotoxins Annual economic burden of mycotoxins to U.S. agricultural estimated at $1.4 billion Product recalls (pet food is especially sensitive) 2005 dog food 2008–2009 dog food 2012 dog food and other feed Approximately 25% of crops produced worldwide are affected by mycotoxins. Costs associated with protecting grain from mycotoxin contamination, diverting contaminated grain to appropriate markets, and managing recalls of contaminated foods and feeds are in the billions of dollars annually. Annual costs and related control efforts vary with climate and weather conditions. Mycotoxin risks can be location-dependent. In the hot, humid climates of Southeast Asia, mold and mycotoxins are a constant issue. On the other hand, the climate of the United States is more variable and contains colder regions which are less conducive to fungal growth. Recent advances in corn genetics have aided the U.S. in its ability to control some risk factors for mycotoxin contamination. Another expense incurred due to mycotoxins is product recalls. Pet food is often subject to mycotoxin-related recalls. Dogs and cats are sensitive to mycotoxins, particularly aflatoxins, and their owners are sensitive to the health of their pets. Pet food production uses a number of raw agricultural commodities, resulting in an industry that is sensitive to mycotoxins in their ingredients. Listed on the slide are recent pet food recalls attributable to mycotoxins.

Poisonous Ingredients in Food Federal Food, Drug, and Cosmetic Act §402 [21 U.S.C. 342] A food shall be deemed adulterated—(a)(1) If it bears or contains any poisonous of deleterious substance which may render it injurious to health; but in case the substance is not an added substance such food shall not be considered adulterated under this clause if the quantity of such substance does not ordinarily render it injurious to health. Mycotoxins are poisonous ingredients that cannot be entirely avoided by good agricultural and manufacturing practices. As such, they are regulated to varying degrees under the Federal Food, Drug, and Cosmetic Act. The consumption of mycotoxins in contaminated food and feed can lead to a variety of negative health effects in both humans and animals. In animals, these health effects vary depending on the species as well as the toxin. There are different levels of regulation and concern for different toxins but in general, their poisonous nature creates a need for monitoring.

Action Levels, Advisory Levels, and Guidance Levels Action Level: A level above which poisonous substances are believed to be harmful to humans or animals. FDA is prepared to intervene at these levels if necessary. Advisory Level: A level below which adverse health effects are not expected. FDA may intervene but often will not. Guidance Level: A level that prudent practice should not exceed; there is insufficient scientific data to establish an action or advisory level. The FDA has not yet adopted regulations that establish tolerances for mycotoxins, but there are critical levels for some mycotoxins at which the FDA will intervene, if necessary, to protect public health. At this time, there is a regulatory action level for aflatoxins in grain and in grain-based food and feed. Grain contaminated above this action level is limited in its use. Action levels may be enforced through regulatory intervention, when it’s clear that contaminated product is being directed towards a market which will not tolerate its use. For example, the action level for aflatoxin in pet food (and ingredients used in its production) is 20 parts-per-billion. This means grain being used for pet food production should not contain aflatoxin contamination above this level. If grain being directed towards a pet food manufacturer is found to contain aflatoxin at a concentration higher than 20 parts-per-billion, then regulatory intervention is possible. There are appropriate uses for moderately-contaminated grain. For example, beef cattle can tolerate much higher levels of aflatoxin in their feed without health effects. It is recommended that aflatoxin in feed intended for finishing beef cattle not exceed 300 parts-per-billion. Diverting moderately-contaminated grain towards beef cattle feed is an example of an appropriate way to utilize it without inciting regulatory intervention. Advisory levels exist for deoxynivalenol (also known as vomitoxin) and guidance levels have been established for fumonisins. These state the maximum level in human food and animal feed that is sufficient to protect health, based on the best available knowledge. Fumonisin and deoxynivalenol are roughly 1000 times less potent than aflatoxin. Their contamination of food and feed is concerning at the parts-per-million level rather than parts-per-billion, as was the case with aflatoxin. By establishing these recommended levels, the FDA acknowledges that there is a need for monitoring and restricting their presence in food and feed. Scientific strength of evidence

Aspergillus Ear Rot-Aflatoxins Now we will examine the individual toxins and the fungi responsible for producing them. This picture shows a corn ear affected by Aspergillus fungus. The grain likely contains the class of mycotoxins known as aflatoxins. The two primary fungal species which produce aflatoxins are Aspergillus flavus and Aspergillus parasiticus. These species typically appear as a grey- to olive-green powdery mold that grows between rows of kernels. Affected kernels are often lightweight and brown. Most often the fungus is seen at the tip of the ear or in association with physically damaged kernels, such as those damaged by insect or bird feeding, or hail. Aspergillus ear rot Source: photo © Gary Munkvold

Aspergillus Ear Rot Disease Cycle and Symptoms Aspergillus flavus fungal spores survive between growing seasons in soil and crop residue Spores are carried by air movement and insects Infects corn at pollination and during grain development Initial infection favored by heat and drought Late season and post-harvest growth (after grain maturity) and mycotoxin production favored by Heat (80°F – 100°F) High humidity (≥ 85%) FDA has established action levels for aflatoxin. As with most fungi, Aspergillus fungal spores survive in soil and on crop residues. Spores can be carried by air movement, insects, birds, or animals. Anything that moves material will move fungal spores. When spores are deposited on the corn plant, the right environmental conditions can lead to fungal growth and aflatoxin production in the grain. Typically, the fungus infects ears at pollination, when the silk is coming out and receives pollen from the tassel. Hot dry conditions at pollination and during subsequent kernel fill favor infection and fungal colonization by aflatoxin-producing fungi. These fungi may also infect damaged kernels which have enhanced susceptibility as a result of their exposure to the elements. After the grain is mature, when the kernels have accumulated their maximum dry matter content, high temperatures (around 80°F – 100°F) with high humidity (about 85% or higher) can intensify the growth of existing mold, which increases the likelihood that mycotoxins are produced. This can also result in aflatoxin production in post-harvest stored grains in hot and/or tropical climates. Because it is quite drought-tolerant, in these climates slight increases in storage moisture or temperature favor Aspergillus growth and aflatoxin production over other contaminants in storage that require higher moisture. In regions where storage conditions are cooler, like most years in the Corn Belt region, for example, Aspergillus fungi generally do not produce aflatoxins in storage. If there is a problem of fungal growth in stored grains in cooler regions it is often that of more aggressive (but less toxic) storage fungi that crowd out Aspergillus fungi. Because of their high carcinogenic potential, the FDA has established action levels for aflatoxins.

Action Level and Recommended Limits for Total Aflatoxins in Livestock Feed Class of Animals Feed Aflatoxin level Finishing beef cattle Corn and peanut products 300 ppb Beef cattle, swine or poultry Cottonseed meal Finishing swine over 100 lb. 200 ppb Breeding cattle and swine, mature poultry 100 ppb Immature animals Animal feeds and ingredients (excluding cottonseed meal) 20 ppb Dairy animals, animals not listed elsewhere, or unknown use (general market) Animal feeds and ingredients The FDA has set an action level of 20 parts-per-billion for aflatoxin in general commerce (unknown final use), or grain with known end-use in human, dairy animal, immature animal, or pet food. FDA has also recommended maximum levels for aflatoxin contamination in other livestock feed ranging up to 300 parts per billion. Aflatoxin contamination is a problem in feed components other than corn, for example peanut and cottonseed and their associated feed products. Ensuring that the individual as well as the sum of aflatoxins in all feed components does not exceed the action level or recommended feeding limits is necessary for both animal health and human food safety. Finishing beef cattle are most tolerant to aflatoxin, with a recommended maximum level of 300 parts per billion in their finished feed. Swine are more sensitive, and feed levels should not exceed 200 parts-per-billion. Poultry, and breeding cattle and breeding swine feed should not exceed 100 parts-per-billion. These species-specific recommendations are enforced at the discretion of FDA when necessary to protect public health. The action level of twenty parts per billion equals the weight of seven kernels in a rail car full of corn. Sampling and testing for mycotoxins at these low concentrations is a significant issue that is covered in another module.

Impact of Aflatoxins on Animals Aflatoxins are potent liver toxins and potent carcinogens Negative effects seen in animals include: Dairy Cattle: Decreased breeding efficiency, lower birth weights, respiratory disorders, kidney damage Toxin passes to milk Swine: Decreased growth rate, liver and kidney damage, system hemorrhages Poultry: Decreased egg production, embryo loss, decreased weight gain Horses: Lack of muscular control, lethargy, rapid weight loss Aflatoxins affect different species of livestock in different ways. Aflatoxins are also recognized as the most potent naturally-occurring liver toxins known, and cause a variety of other health effects in production animals. Some of these effects endanger the human food supply; like the transmittance of aflatoxin metabolites into cow’s milk. This poses danger to all humans, but is especially concerning for children and the elderly who tend to be more susceptible to toxicity. No animals are entirely immune to the negative impact of aflatoxins. The slide shows an example of the array of aflatoxin-induced health implications that arise in animals. Aside from the previously mentioned transmission into the milk of dairy cattle, aflatoxins also decrease breeding efficiency, lower birth weights, create respiratory disorders, and promote kidney damage in cattle. Swine can experience reduced growth rates, liver and kidney damage, and bleeding. Poultry experience reduced egg production, embryo losses, and reduced weight gain. Horses can lose muscle control and experience lethargy and rapid weight loss.

Impact of Aflatoxins on the Grain Industry Aflatoxin is a food/feed adulterant Grain >20 ppb cannot be deliberately blended FDA blending dispensations have been allowed in rare instances when a large portion of the U.S. corn supply is affected State by state basis Resultant grain used only for specific livestock feed >500 ppb aflatoxin grain has never been allowed to be blended Aflatoxin is considered a food and feed adulterant. Grain which tests above 20 parts-per-billion aflatoxin in an analysis performed by a third-party analytical laboratory with competency recognized by FDA cannot be deliberately blended with uncontaminated grain to achieve overall reductions in the lot aflatoxin concentration. Over the years, when we’ve had aflatoxin outbreaks, the FDA has allows dispensations for blending, as long as the resultant grain is used by specific livestock. For example, in 2012, severe drought stretched across a large portion of the central United States resulting in a concern for large-scale contamination of the corn supply with aflatoxin. In a rare instance like this, the FDA allowed temporary blending policies for some states, for corn contaminated above 20 parts per billion with corn containing low or no aflatoxin. This temporary blending policy  required compliance agreements from grain dealers and state departments of agriculture, and the blended corn could only be used for animal feed. The purchaser was also required to provide written assurance of the intended use of the blended grain. The policy also mandated confirmatory testing of every blended lot with test results being reported to the purchaser, and labeling of all blended corn with the designation “for animal feed use only”. Grain contaminated above 500 parts per billion has never been allowed to be blended.  http://www.iowaagriculture.gov/press/2012press/press09182012.asp- link for information on the temporary blending policy

Gibberella Ear Rot/Fusarium Head Blight-Deoxynivalenol http://www.ars.usda.gov/Main/docs.htm?docid=9765 Gibberella ear rot Source: Photo Courtesy of A. Robertson Fusarium graminearum is the primary fungus responsible for producing the mycotoxin deoxynivalenol (also known as vomitoxin or DON). This fungus is also known as Gibberella zeae. It causes the diseases Gibberella ear rot in corn and Fusarium head blight or head scab in wheat and barley. The pictures on the slide show what both of these diseases look like. The fungus typically appears as a pale pink to red mold on corn ears, and invades the ear at the tip working its way down the ear from there. On wheat, individual diseased spikelets will appear bleached. Kernels will often be shriveled and discolored pink, gray, or light tan.

Gibberella Ear Rot/Fusarium Head Blight Disease Cycle and Symptoms Overwinters on crop residue Predominantly occur in the northern Corn Belt Optimal conditions for colonization are Cool with high humidity Frequent precipitation during early grain fill Typically infects through the silk (corn) or head (wheat) two to six days after emergence Fungal infection preceeds mycotoxin production Deoxynivalenol (also known as DON or vomitoxin) Zearalenone FDA has established advisory levels for deoxynivalenol Fusarium graminearum overwinters in crop residues and typically infects during pollination in corn or, in the case of wheat, near anthesis. Gibberella ear rot of corn and Fusarium head blight of wheat occur mostly in the northern United States and Canada because this fungus is favored by cool temperatures, high levels of humidity, and precipitation during the growing season, especially during grain fill. Infection typically occurs through the silk of corn or into the wheat head. In the case of corn, physically damaged kernels can promote grain infection, but silk infection is the primary infection pathway for this fungus. Like all of the mycotoxins, deoxynivalenol is not automatically present in grain when the fungus is present; but once the fungus has invaded the grain, if conditions are favorable, the fungus will produce it. Fusarium graminearum is also responsible for the production of another mycotoxin, zearalenone, which will be addressed shortly. The FDA has established advisory levels for deoxynivalenol.

Advisory Levels for Deoxynivalenol in Livestock Feed Class of Animals Feed Ingredients & portion of the diet DON level in ingredients and (finished feed) Ruminating beef and feedlot cattle older than 4 months 10 ppm (10 ppm) Ruminating dairy cattle older than 4 months Grain and grain by-products not to exceed 50% of the diet 10 ppm (5 ppm) Chickens Swine Grain and grain by-products not to exceed 20% of the diet 5 ppm (1 ppm) All other animals Grain and grain by-products not to exceed 40% of the diet 5 ppm (2 ppm) There are advisory levels for deoxynivalenol. Deoxynivalenol is not recognized as a carcinogen. However, there is evidence that it causes negative health effects in exposed humans and animals. Beef cattle can tolerate the highest exposure down to swine which are currently recognized as being most sensitive. Advisory levels can be confusing as they are given as the maximum concentration in grain or feed product, plus a maximum percentage of the diet which can contain contaminated grain or feed. This shifts the burden to the user not to over-feed or over-include grain that contains deoxynivalenol in the diets of susceptible livestock.

Deoxynivalenol (DON, Vomitoxin) Most commonly encountered mycotoxin in food and feed. Swine are particularly sensitive, compared to other livestock. Negative effects on swine and young animals: Feed suppression Feed refusal Reduced weight gain Impaired organ function Mycotoxin deoxynivalenol may induce vomiting in swine. Deoxynivalenol is the most commonly encountered mycotoxin in food and feed. Swine are particularly sensitive to deoxynivalenol. If they eat feed contaminated with deoxynivalenol they experience a number of negative gastrointestinal effects. Its nick-name, vomitoxin, comes from the vomiting it induces in pigs. Often pigs will refuse to eat feed contaminated with deoxynivalenol, which results in reduced weight gain. They also can experience impaired organ function.

Zearalenone is often found in association with Deoxynivalenol Zearalenone may occur simultaneously with deoxynivalenol, which is not surprising as it is produced by the same fungus, Fusarium graminearum. The figure displays data obtained by Iowa State University researchers after a severe hail storm. Hail damaged corn from the 2009 harvest was examined for the presence and quantity of multiple mycotoxins. This data demonstrates an association between deoxynivalenol production and zearalenone production. As an aside, this graphic also shows a number of samples exceeding the lowest guidance level for deoxynivalenol content in finished feed.

Zearalenone Estrogenic activity in swine and dairy that manifests as reproductive effects Negative effects on cattle: Infertility Reduced milk production Hyper-estrogenism Negative effects on swine: Enlarge mammae Swelling of uterus and vulva Atrophy of the ovaries Withered testes There are no FDA action, advisory, or guidance levels Zearalenone is not particularly common, especially compared to deoxynivalenol, but when it is found it can impose relatively severe economic consequences for the swine and dairy industry. Zearalenone is primarily an estrogenic mycotoxin, and many of the health impacts are related to reproductive functions. Some examples in cattle are infertility, reduced milk yield, and hyper-estrogenism. In swine, symptoms include enlarged mammae, swelling of the uterus and vulva, and atrophy of the ovaries in females, and withered testes in males. At this point, the FDA has not established action, advisory, or guidance levels for zearalenone in agricultural crops. Monitoring and establishing safe feeding levels for grain contaminated with this mycotoxin is left to veterinarians working with nutritionists.

Fusarium Ear Rot-Fumonisins Scattered or groups of infected kernels are typical of Fusarium ear rot Source: Pioneer Hi-Bred Intl, Inc. Fusarium ear rot Source: ©Gary Munkvold Fusarium ear rot in corn is a disease caused primarily by Fusarium verticillioides or Fusarium proliferatum. These fungi produce the class of mycotoxins known as fumonisins. Diseased corn ears typically have visible mold that appears white to pale pink. The infected or moldy kernels tend to be randomly scattered across the ear as shown on the image on the left. They will also often turn brown. This differs from Gibberella ear rot, caused by Fusarium graminearum, which typically originates from the ear tip and grows down the ear, affecting large blocks of kernels together. The white or pink mold areas are the most obvious indication that Fusarium ear rot is present, but individual kernels may also display a white streak or starburst pattern which can be seen on the image on the right.

Fusarium Ear Rot Disease Cycle and Symptoms Most common corn ear disease in the Midwest Fusarium verticillioides usually overwinters in crop residue Favored by warm-hot, dry weather during grain fill Also favoring infection are: Optimum growth temperature ~86°F Drought stress before and after silking Damage to kernels by insects, birds, hail *Prevention of insect feeding results in lower levels of fumonisins (e.g. Bt corn) The FDA has provided guidance levels Fusarium fungi are probably the most common molds in corn in the Midwest. There are a variety of Fusarium fungi, many of which are capable of producing fumonisins. The fungus overwinters in crop residues. Spores are dispersed by weather or insects throughout the growing season. The environmental conditions favoring Fusarium ear rot are warm to hot, dry weather during the grain maturation period, with an optimum temperature of about 86°F. Most recently these weather conditions occurred in the Corn Belt in 2012 in the weeks during and after silking. This is a particularly high-risk time for fungal infection in corn. These conditions resulted in a high risk for fumonisin contamination. Kernels damaged by insects, hail or birds compound the issue. This fungus is often associated with physically damaged kernels, like those caused by insect feeding. For this reason, corn hybrids which resist insect feeding have reduced levels of some mycotoxins as compared to hybrids that lack insect resistance. On the picture of the corn ear shown on the slide, you can see evidence of Fusarium ear rot in the patches scattered across the corn ear. At the tip of the ear you can also see evidence of Aspergillus. This is not surprising as the conditions which favor the development of these two types of fungi are relatively similar – warm to hot, dry weather, especially with drought around the silking and grain development period. And so, in 2012, the Corn Belt had a risk for contamination of corn with both fungi. Once fungal contamination is present, weather conditions during the grain fill period determine the risk of mycotoxin production. So, while both toxins may not occur, both types of fungus can be present on the same ear. The FDA has established guidance levels for fumonisins in grain, food, and feed. Fusarium ear rot Source: ©Gary Munkvold

Guidance Levels for Total Fumonisins in Livestock Feed Class of Animal Feed Ingredients & portion of the diet Fumonisin level in ingredients and (finished feed) Equids and rabbits Corn and corn by-products not to exceed 20% of the diet 5 ppm (1 ppm) Swine and catfish Corn and corn by-products not to exceed 50% of the diet 20 ppm (10 ppm) Ruminants, Poultry, and mink (all breeding) 30 ppm (15 ppm) Ruminants ≥3 months old being raised for slaughter and mink for pelt production 60 ppm (30 ppm) Poultry being raised for slaughter 100 ppm (50 ppm) All other species or classes of livestock and pet animals 10 ppm (5 ppm) The table shows the current FDA guidance levels for fumonisins in grain, food, and feed. Like the advisory levels for deoxynivalenol, guidance levels for fumonisins are specified as a maximum level of contamination and then percentage of the diet which can contain this contaminated grain, so that there is a lower total fumonisin level in the finished feeds than in the individual ingredients. The guidance level has been set at one part-per-million in finished feed for the most sensitive species which are equids (this includes horses) and rabbits and for pet animals. The guidance level is set at 10 parts-per-million in finished feed intended for swine. Non-breeding ruminants, like cattle and sheep, should not exceed 30 parts-per-million in finished feed. Poultry are quite resistant to fumonisins and can tolerate up to 50 parts-per-million fumonisins in finished feed. These recommended levels are based on the current best available knowledge and are considered sufficient to protect consumer and animal health. The FDA has deemed these levels achievable with the use of good agricultural and manufacturing practices.

Fumonisin Symptoms of Exposure Adverse effects in animals Horses: Leukoencephalomalacia Swine: Liver damage, pulmonary edema Cattle and Sheep: Mild liver damage, moderate feed refusal Fumonisins have been associated with a variety of adverse effects in animals. Horses are most sensitive of all livestock animals to the effects of fumonisins. Even low levels of contamination in horse feed can cause a fatal brain disorder, leukoencephalomalacia. Swine can develop liver damage or fluid on their lungs, a condition known as pulmonary edema. Ruminants, like cattle and sheep, may display moderate feed refusal and suffer mild amounts of liver damage. Infected kernels scattered or clustered are typical of Fusarium ear rot Source: Photo Courtesy of Pioneer HiBred Intl, Inc.

Penicillium Ear Rot-Ochratoxin A Penicillium ear rot and affected kernels Source: Photo Courtesy of Don White, University of Illinois Penicillium species produce ochratoxin A Source: Photo Courtesy of Don White, University of Illinois The last class of mycotoxins we will discuss are ochratoxin A. In cereal grains, ochratoxin A is produced by Penicillium verrucosum fungal species. This fungus is often misdiagnosed as aflatoxin-producing Aspergillus fungal species in corn. It’s difficult to tell the difference, but Penicillium is typically more blue-green than olive-green. Like Aspergillus, Penicillium also tends to develop in streaks between kernels. It takes a trained crop scout to identify the difference in the field, but that difference is important in terms of what mycotoxin may be present in grain.

Penicillium Ear Rot Disease Cycle and Symptoms Primarily caused by Penicillium verrucosum Fungus invades at maturity, not during grain development Optimal growth conditions for fungus: Temperatures between 68-77°F Grain moisture content ≥ 16% Harvested corn stored at moisture levels >18% can increase disease severity Humidity > 80% during and after maturity Most commonly found in fields infested with stalk boring insects Typically, Penicillium fungi do not invade corn during reproductive development of the seed. This is different from the other toxigenic fungi discussed in this presentation. It generally invades the field at maturity which, for corn plants, is identifiable when the kernels are at ‘black layer’. At that point, the corn has reached its maximum dry matter content. Penicillium fungi can grow in a fairly wide temperature range, but ideally in the range of 68-77 degrees Fahrenheit. It prefers reasonably high moistures (grain moisture greater than or equal to 16% and relative humidity above 80%) but can grow slowly even under less favorable conditions. Humid weather at or just before harvest can predispose grain to having Pencillium problems throughout its storage life. Penicillium fungi, like Aspergillus, is considered a storage fungus. Penicillium requires little moisture to grow; small changes in water availability in stored grain can instigate growth and mycotoxin production. This can pose a problem, particularly in the spring and summer following harvest when temperatures are warming up. This fungus is most commonly found in fields infested with stalk-boring insects. If this fungus is found in the field, it is crucial that handlers dry the grain to low moisture and keep the grain at low temperatures to deter further fungal activity.

Ochratoxin A Symptoms of Exposure Ochratoxin A is primarily a kidney toxin Swine and poultry are the primary livestock affected Adverse effects in swine and poultry Reduced feed intake, dehydration Growth retardation Kidney dysfunction Diarrhea and excessive urine production Reduced egg production (poults) Vomiting (swine) In many mammals, ochratoxin A is a kidney toxin. Swine and poultry are the livestock most susceptible to the toxic effects of ochratoxin A, while ruminants like sheep and cattle are significantly less susceptible. Some of the detrimental effects of dietary ochratoxin A on swine and poultry are listed on the slide. These include reduced feed intake and dehydration, growth retardation, kidney disfunction, diarrhea and excessive urine production, reduced egg production in poultry, and vomiting in swine. The picture on the slide shows a chicken with a lesion that developed as a result of eating ochratoxin A-contaminated feed. The FDA does not currently have action, advisory, or guidance levels in place for ochratoxin A in grains.

Many fungi in one place! Cladosporium Fusarium Sac County, Iowa August 9, 2009 Gibberella Fusarium Under unusual situations, you can get a variety of fungi all in one place. These pictures show damage from a very large hail storm that went across central Iowa in 2009, taking out almost a million acres of corn and soybeans. This particular image is from an affected corn field in Sac County, Iowa. Cool, wet weather through the subsequent harvest period resulted in these contaminated ears having significant deoxynivalenol and zearalenone contamination. This was a problem throughout Iowa, Illinois, Ohio, Southern Minnesota and was worse in the areas where this hail storm damaged the kernels. All of the corn ears pictured came from that field and they contain many fungal diseases including Fusarium, Gibberella, and Penicillium ear rots. These ears also contain species of fungi which don’t produce mycotoxins, like Cladosporium and Trichoderma. The presence of even these fungi detracts from the value of the grain, as moldy kernels classify as total damage in the grades, even if no mycotoxins are produced. Penicillium Trichoderma

Summary Mycotoxins are: Chemical compounds Produced by specific fungi Contaminants of crops and other commodities worldwide 5 mycotoxins monitored under FDA surveillance programs Aflatoxins Deoxynivalenol (Vomitoxin) Zearalenone Fumonisins Ochratoxin A Toxic to humans and animals at very low levels Mycotoxin-producing fungi survive in soil/on crop residue In summary, mycotoxins are a group of chemical compounds which are produced by some fungal species that contaminate crops and other commodities worldwide. There are 5 primary mycotoxins that are frequently found in grains and feed and are monitored under FDA surveillance programs. These are aflatoxins, deoxynivalenol (also known as vomitoxin), zearalenone, fumonisins, and ochratoxin A. These mycotoxins are capable of causing illness, disease and, in extreme instances, death in humans and animals at very low levels. The fungi that produce these mycotoxins are natural environmental contaminants that survive in soil and on crop residues.

Summary The FDA has established Action levels for aflatoxin Advisory levels for deoxynivalenol Guidance levels for fumonisins Growing-region climate and weather determine type and severity of mycotoxin risk Especially conditions during pollination, grain fill, anthesis (flowering) Climate and weather monitoring can be used as tools to 1) predict mycotoxin risk 2) direct testing efforts appropriately 3) protect the industry from large-scale contamination The FDA has esetablished action levels for aflatoxin, advisory levels for deoxynivalenol, and guidance levels for fumonisins. The FDA does not currently have advisory, action, or guidance levels for zearalenone or ochratoxin A. Climate and weather are significant determinants for the type of mycotoxin risk in a given area, as these factors largely determine which fungi in a given field will grow and prevail over other fungal competitors for the host plants. Conditions around pollination and grain fill in corn and at anthesis for wheat are especially important, as these are periods during plant development when disease susceptibility is high. The toxin risk at an individual processing plant level, then, is highly dependent on the growing season weather in the specific trade area. Weather is also a very important diagnostic piece of information if you, as an inspector, are attempting to predict what mycotoxin or mycotoxins might be an issue at a facility, or if a grain buyer is proactively protecting their industry against mycotoxins. Monitoring and knowing the environmental conditions in a particular area is probably the best predictive and preventative tool that a grain buyer or grain user can employ to control or limit the entry of mycotoxin-contaminated grain into their facilities and to employ mycotoxin testing in a cost-effective way. This, and working with the producer or seller to divert moderately-contaminated grain to its appropriate animal feed use before it enters the general market is probably the best, and certainly the cheapest, mycotoxin preventative control measure that we have. There are other, more complex control measures which will be discussed in the second mycotoxins module, along with considerations for the prevention and management of mycotoxins in stored grains.

This training was a joint effort of *Funding for this Grain and Feed Mill Operations course was made possible, in part, by the Food and Drug Administration through grant (1U54FD004333-01), views expressed in written materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.* This training course is a joint effort, between Iowa State, Kansas State, and North Carolina State Universities. Some of you listening to this right now will be taking the onsite training, or maybe have taken the onsite training, at Kansas State or North Carolina State. We hope that this has been helpful. The next module on mycotoxins will cover the testing and handling and storage considerations, which are essentially the diligence that grain dealers and grain users should use to identify and divert potential mycotoxin-containing grain as it arrives in their facility.