Mycotoxins and their Toxicity in Livestock

Mycotoxins are toxic secondary metabolites produced by fungi (molds) that cause an undesirable effect (mycotoxicosis) when animals are exposed. Exposure is usually by consumption of contaminated feeds but may also be by contact or inhalation. Biological effects include liver and kidney toxicity, central nervous system effects, and estrogenic effects, to name a few. Only some molds produce mycotoxins, and they are referred to as toxigenic. The fungal toxins are chemically diverse—representing a variety of chemical families—and range in molecular weight from about 200 to 500. There are hundreds of mycotoxins known, but few have been extensively researched, and even fewer have good methods of analysis available. The primary classes of mycotoxins are aflatoxins, zearalenone, trichothecenes, fumonisins, ochratoxin A, and the ergot alkaloids.

Molds Can Cause Disease

A mold (fungal) infection resulting in disease is referred to as a mycosis. Fungal pathogens include Aspergillus fumigatus, Candida albicans, Candida vaginitis, certain species of Fusarium, and others. Aspergillus fumigatus is known to cause mycotic pneumonia, mastitis, and abortions and has been recently proposed as the pathogenic agent associated with mycotic hemorrhagic bowel syndrome (HBS) in dairy cattle (Puntenney et al., 2003). It is thought that Aspergillus fumigatus is a fairly common mold in both hay (Shadmi et al., 1974) and silage (Cole et al., 1977). While healthy cows with an active immune system are more resistant to mycotic infections, dairy cows in early lactation are immune suppressed, and HBS is more likely in fresh cows (Puntenney et al., 2003). It is theorized that with a mycosis, mycotoxins produced by the invading fungi can suppress immunity, therefore increasing the infectivity of the fungus. A. fumigatus produces several mycotoxins, including gliotoxin and tremorgens that are toxic to cattle. A. fumigatus-contaminated silage was found to contain fumigaclavine A and C and several fumitremorgens (Cole et al., 1977). Cattle consuming this silage demonstrated symptoms including generalized deterioration typical of protein deficiency, malnutrition, diarrhea, irritability, abnormal behavior, and occasional death. The hay was fed to goats and rats and resulted in retarded growth and histopathological changes in the livers and kidneys. Gliotoxin, an immune suppressant, has been found to be present in animals infected with A. fumigatus (Bauer et al., 1989). Gliotoxin is also shown to be produced in mice associated with A. fumigatus (Eichner et al., 1988). Gliotoxin produced by A. flavus has immunosuppressive, antibacterial, and apoptotic effects. Gliotoxin is shown to affect rumen fermentation, reducing digestibility and VFA production in vitro (Morgavi et al., 2004). Reeves et al. (2004) using an insect model demonstrated the significance of gliotoxin in increasing the virulence of A. fumigatus. Niyo et al. (1988a, b) demonstrated that in rabbits, T-2 toxin decreased phagocytosis of A. fumigatus conidia by alveolar macrophages and increased severity of experimental aspergillosis. It is possible that gliotoxin, T-2 toxin, or other mycotoxins suppress immunity and may be a trigger to increased infectivity by the fungus, ultimately resulting in HBS or other fungal infections. Perhaps reducing animal exposure to mycotoxins and moldy feeds may be a key to control of mycoses such as HBS. A commercial feed additive with anti-fungal and adsorbent properties appears to reduce HBS (Puntenney et al., 2003).

Mold Growth, Mycotoxin Formation

Many species of fungi produce mycotoxins in feedstuffs. Molds can grow and mycotoxins can be produced pre-harvest or during storage, transport, processing, or feeding. Mold growth and mycotoxin production are related to plant stress caused by weather extremes, insect damage, inadequate storage practices, and faulty feeding conditions. In general, environmental conditions—heat, water, and insect damage—cause plant stress and predispose plants in the field to mycotoxin contamination. Computer models to predict mycotoxin concentrations in corn prior to harvest are based on temperature, rainfall, and insect pressure (Dowd, 2004).

Because feedstuffs can be contaminated pre-harvest, control of additional mold growth and mycotoxin formation is dependent on storage management. After harvest, temperature, water activity, and insect activity are the major factors influencing mycotoxin contamination of feedstuffs (Coulombe, 1993). Molds grow over a temperature range of 10° to 40°C (50° to 104°F), a pH range of 4 to 8, and above 0.7 aw (equilibrium relative humidity expressed as a decimal instead of a percentage).

Molds can grow on feeds containing more than 12 to 15% moisture. In wet feeds such as silage, higher moisture levels allow mold growth if oxygen is available. Because most molds are aerobic, high moisture concentrations can exclude air and help prevent mold growth. The conditions most suitable for mold growth and for mycotoxin formation are not necessarily the same. For example, the Fusarium molds associated with alimentary toxic aleukia have been reported to grow prolifically at 25° to 30°C without producing much mycotoxin, but at near-freezing temperatures, they produce large quantities of mycotoxins with minimal mold growth (Joffe, 1986). Field applications of fungicides may reduce mold growth, thus reducing production of mycotoxins. However, the stress or shock of the fungicide to the mold organism may reduce mold growth and yet not reduce the production of mycotoxins (Boyacioglu et al., 1992; Gareis and Ceynowa, 1994; Simpson et al., 2001).

Aspergillus species normally grow at lower water activities and at higher temperatures than the Fusarium species. Therefore, Aspergillus flavus and aflatoxin in corn are favored by the heat and drought stress associated with warmer climates. Aflatoxin contamination is enhanced by insect damage before and after harvest.

The individual Penicillium species have variable requirements for temperature and moisture but are more likely to grow under post-harvest conditions, in cooler climates, in wet conditions, and at a lower pH. Penicillium molds are a major contaminant of silage, probably because they are acid tolerant.

The Fusarium species are important plant pathogens that can proliferate pre-harvest but continue to grow post-harvest. In corn, Fusarium molds are associated with ear rot and stalk rot, and in small grains, they are associated with diseases such as head blight (scab). In wheat, Fusarium is associated with excessive moisture at flowering and early grain-fill stages. In corn, Fusarium graminearum is referred to as a red ear rot and is more commonly associated with a cool, wet growing season and with insect damage. Fusarium ear rots that produce fumonisins are referred to as pink ear rots and vary in their environmental requirements. They are generally associated with dry conditions in mid-season followed by wet weather (CAST, 2003).

Mycotoxin Occurrence

Worldwide, approximately 25% of crops are affected by mycotoxins annually (CAST, 1989), which would extrapolate to billions of dollars (Trail et al., 1995). Annual economic costs of mycotoxins to the U.S. agricultural economy is estimated to average $1.4 billion (CAST, 2003). Economic losses are due to effects on livestock productivity, losses in crops, and the costs and effects of regulatory programs directed toward mycotoxins. Table 1 provides mycotoxin analyses of feed samples submitted by North Carolina farmers over a nine-year period indicating that mycotoxins in feeds including corn silage and corn grain occur commonly at unsuitable concentrations (Whitlow et al., 1998).

Table 1. Occurrence of five mycotoxins in corn silage and corn grain and in all feed samples submitted for analysis by producers in North Carolina over a nine-year period.

Mycotoxin occurrence and concentrations are variable by year, which is expected because of the annual variation in weather conditions and plant stresses known to affect mycotoxin formation (Coulombe, 1993). It was concluded that mycotoxins occur frequently in a variety of feedstuffs and are routinely fed to animals. Sometimes, mycotoxins occur at concentrations high enough to cause major losses in health and performance of animals. However, a more likely scenario is to find mycotoxins at lower levels interacting with other stressors to cause subclinical losses in performance, increases in incidence of disease, and reduced reproductive performance. To the animal producer, these subclinical losses are of greater economic importance than losses from acute effects but even more difficult to diagnose.

Mycotoxin Effects

Although the potentially harmful effects of feeding moldy grain and foods has been known for many years (Matossian, 1989), mycotoxicology, the study of mycotoxins, really began in 1960 with the outbreak of Turkey-X disease in the United Kingdom. This outbreak was linked to peanut meal imported from Brazil (Sargeant et al., 1961). Because of an intensive multidisciplinary research effort, a blue-fluorescent toxin was isolated and mycelia of A. flavus were observed. A. flavus was soon shown to produce the same toxic compound(s) found in the toxic peanut meal. The toxin was characterized chemically and biologically and was given the trivial name aflatoxin. Aflatoxin was shown to be very toxic and carcinogenic in some of the test animal species used, and it resulted in a toxic metabolite in milk of dairy cows (Allcroft and Carnaghan, 1962; 1963). The discovery of aflatoxin and elucidation of some of its effects led to research on other livestock health and production problems linked with moldy feedstuffs and to the discovery of additional mycotoxins.

Mycotoxins, in large doses, can be the primary agent causing acute health or production problems in a dairy herd. But more likely, mycotoxins are a factor contributing to chronic problems, including a higher incidence of disease, poor reproductive performance, or suboptimal milk production. Ruminal degradation of mycotoxins helps to protect the cow against acute toxicity but may contribute to chronic problems, associated with long-term consumption of low levels of mycotoxins. Ruminal degradation of mycotoxins may have helped mask mycotoxin effects in dairy cows which were recognized in recent years as production stresses have increased and as the industry has paid more attention to management details.

Mycotoxins exert their effects through several means:

1. reduced intake or feed refusal;
2. reduced nutrient absorption and impaired metabolism;
3. altered endocrine and exocrine systems;
4. suppressed immune function;
5. altered microbial growth.

Recognition of the impact of mycotoxins on animal production has been limited by the difficulty of diagnosis. The progression and diversity of symptoms are confusing, making diagnosis difficult (Hesseltine, 1986; Schiefer, 1990). The difficulty of diagnosis is increased due to limited research, occurrence of multiple mycotoxins, non-uniform distribution, interactions with other factors, and problems of sampling and analysis. Because of the difficulty of diagnosis, the determination of a mycotoxin problem becomes a process of elimination and association. Certain basics can be helpful (Schiefer, 1990):

1. Mycotoxins should be considered as a possible primary factor resulting in production losses and increased incidence of disease.
2. Documented symptoms in ruminants or other species can be used as a general guide to symptoms observed in the field.
3. Systemic effects as well as specific damage to target tissues can be used as a guide to possible causes.
4. Postmortem examinations may indicate no more than gut irritation, edema, or generalized tissue inflammation.
5. Because of the immune suppressing effects of mycotoxins, increased incidence of disease or atypical diseases may be observed.
6. Responses to added dietary adsorbents or dilution of the contaminated feed may help in diagnosis.
7. Feed analyses should be performed, but accurate sampling is a major problem.

Symptoms are often nonspecific and may be wide-ranging. Symptoms result from a progression of effects or of opportunistic diseases, making a diagnosis difficult or impossible because of the complex clinical results with a wide diversity of symptoms. Symptoms vary depending on the mycotoxins involved and their interactions with other stress factors, and animals may exhibit few or many of a variety of symptoms. The more stressed cows, such as fresh cows, are most affected, perhaps because their immune systems are already suppressed. Symptoms may include reduced production, reduced feed consumption, intermittent diarrhea (sometimes with bloody or dark manure), reduced feed intake, unthriftiness, rough hair coat, and reduced reproductive performance including irregular estrous cycles, embryonic mortalities, pregnant cows showing estrus, and decreased conception rates. There generally is an increase in incidence of disease, such as displaced abomasum, ketosis, retained placenta, metritis, mastitis, and fatty livers. Cows do not respond well to veterinary therapy. The FDA regulatory control program for mycotoxins is discussed by Wood and Trucksess (1999).

Safe Levels of Mycotoxins

Some of the same factors that make diagnosis difficult also contribute to the difficulty of establishing levels of safety. These include lack of research, sensitivity differences by animal species, imprecision in sampling and analysis, the large number of potential mycotoxins, and interactions with stress factors or other mycotoxins (Hamilton, 1984; Schaeffer and Hamilton, 1991). The FDA established action, guidance, and advisory levels, in part, to protect public health. Grains with mycotoxin(s) that exceed the appropriate action, advisory, or guidance levels may be considered by CVM as adulterated. Grains with mycotoxin(s) exceeding the highest action, advisory, or guidance levels may be considered by CVM as unfit for use in animal feed (Henry, 2006).

A mycotoxin provided by a naturally contaminated feed appears more toxic than the same level of a pure mycotoxin supplemented into a clean diet. Aflatoxin produced from culture was more toxic to dairy cattle than pure aflatoxin added to diets (Applebaum et al., 1982). In swine, Foster et al. (1986) demonstrated that a diet containing pure added deoxynivalenol (DON) was less toxic than diets with similar concentrations of DON supplied from naturally contaminated feeds. Smith and MacDonald (1991) have suggested that fusaric acid, produced by many species of Fusarium, occurs along with DON to produce more severe symptoms. Lillehoj and Ceigler (1975) gave an example where penicillic acid and citrinin were innocuous in laboratory animals when administered alone but were 100% lethal when given in combination. These studies strongly suggest the presence of other unidentified mycotoxins in naturally contaminated feeds and that mycotoxin interactions are important. It is well documented that several mycotoxins may be found in the same feed (Hagler et al., 1984). Abbas et al. (1989) demonstrated Fusarium species isolated from Minnesota corn produced multiple mycotoxins. Also, because animals are fed a blend of feedstuffs and because molds produce an array of mycotoxins, many mycotoxin interactions are possible. Speijers and Speijers (2004) have discussed the combined toxicity of mycotoxins and, therefore, suggest daily tolerable intake limits for groups of mycotoxins. Interactions of multiple mycotoxins are discussed in the CAST (2003) report.

Mycotoxin interactions with other factors make it difficult to determine safe levels of individual mycotoxins. Mycotoxin effects are affected by factors such as animal species, gender, age, duration of exposure, and stresses of the environment and production. Animals under environmental or production stress may show the more pronounced symptoms. With fescue toxicity, more pronounced symptoms are expressed during heat stress (Bacon, 1995). Fumonisin at 100 parts per million (ppm) has been shown to reduce milk production in dairy cattle (Diaz et al., 2000) and in a separate study did not affect average daily gain in beef cattle fed 148 ppm (Osweiler et al., 1993). While not a direct comparison, this difference in response may suggest a difference in stress levels of early lactation dairy cattle compared with growing beef cattle.

Jones et al. (1982) demonstrated that productivity losses in commercial broiler operations can occur when aflatoxin concentrations were below those levels of concern established by controlled research in laboratory situations. This suggests that conditions in commercial operations are different from laboratory conditions and that the toxicity of mycotoxins may be influenced by interactions with those conditions. The known dietary factors that interact with mycotoxins include nutrients such as fat, protein, fiber, vitamins, and minerals (Brucato et al., 1986; Galvano et al., 2001; Smith et al., 1971). Dietary ingredients such as clay pellet binders adsorb some mycotoxins, reducing exposure of the animal. Thus, many factors and interactions make it difficult to relate field observations to those from controlled research.

Because of partial degradation in the rumen, mycotoxins are generally less toxic to ruminants than to most other animals. However, most mycotoxins are not completely degraded, and some of the degradation products remain toxic (Kiessling et al., 1984). Extent of ruminal degradation of mycotoxins appears to be variable and may be reduced in feeding situations where ruminal turnover rate is high or when rumen microbial population is reduced. Ruminal degradation of mycotoxins appears to be more dependent on protozoal than bacterial activity (Kiessling et al., 1984; Hussein and Brasel, 2001). Effects of mycotoxins in ruminants are reviewed by Jouany and Diaz (2005).
Conjugated mycotoxins in which a mycotoxin is bound to another substance, such as sugars, may be “masked” during laboratory analysis and yet toxic to animals. Both zearalenone (Gareis et al., 1990) and deoxynivalenol (Berthiller et al., 2005) are known to occur in “masked” forms. Therefore, mycotoxicoses cases may have occurred in situations where “masked” mycotoxins resulted in only low concentrations of mycotoxins detected in the laboratory, yet higher levels existed in the feed.

Toxicity of Individual Mycotoxins

Aflatoxin

Aflatoxins are extremely toxic, mutagenic, and carcinogenic compounds produced by Aspergillus flavus and A. parasiticus. Aflatoxin B1 is excreted in milk in the form of aflatoxin M1. The FDA limits aflatoxin to no more than 20 parts per billion (ppb) in lactating dairy feeds and to 0.5 ppb in milk. A rule of thumb is that milk aflatoxin concentrations equal about 1.7% (range from 0.8 to 2.0%) of the aflatoxin concentration in the total ration dry matter. Cows consuming diets containing 30 ppb aflatoxin can produce milk containing aflatoxin residues above the FDA action level of 0.5 ppb. Aflatoxin appears in the milk rapidly and clears within three to four days (Diaz et al., 2004 and Frobish et al., 1986).

Symptoms of acute aflatoxicosis in mammals include inappetence, lethargy, ataxia, rough hair coat, and pale, enlarged fatty livers. Symptoms of chronic aflatoxin exposure include reduced feed efficiency and milk production, jaundice, and decreased appetite. Aflatoxin lowers resistance to diseases and interferes with vaccine-induced immunity (Diekman and Green, 1992). In beef cattle, Garrett et al. (1968) showed an effect on weight gain and intake with diets containing 700 ppb aflatoxin, but if increases in liver weights are used as the criteria for toxicity, 100 ppb would be considered toxic to beef cattle. Production and health of dairy herds may be affected at dietary aflatoxin levels above 100 ppb, which is considerably higher than the amount that produces illegal milk residues (Patterson and Anderson 1982, and Masri et al., 1969). Guthrie (1979) showed when lactating dairy cattle in a field situation were consuming 120 ppb aflatoxin, reproductive efficiency declined, and when cows were changed to an aflatoxin-free diet, milk production increased over 25%. Applebaum et al. (1982) showed milk production was reduced in cows consuming impure aflatoxin produced by culture, but production was not significantly affected by equal amounts of pure aflatoxin.

Aflatoxin is more often found in corn, peanuts, and cottonseed grown in warm and humid climates. Aflatoxin can be found in more temperate areas, as was seen in the drought year of 1988 when aflatoxin was seen in 5% of corn grain in the midwestern United States (Russell et al., 1991). The U.S. General Accounting Office has concluded that industry, federal, and state programs are effective in detecting and controlling aflatoxin and that it is doubtful that additional programs or limits would reduce the risk of aflatoxin in the food supply. The FDA action levels for aflatoxin are presented in Table 2 (Henry, 2006). Aflatoxin regulations worldwide have been reviewed by Van Egmond and Jonker (2005).

Table 2. Action levels for total aflatoxins in livestock feed (Henry, 2006).

Deoxynivalenol (DON) or Vomitoxin

Deoxynivalenol is a Fusarium-produced mycotoxin, commonly detected in feed. It is sometimes called vomitoxin because it was associated with vomiting in swine. Surveys have shown DON to be associated with swine disorders including feed refusals, diarrhea, emesis, reproductive failure, and deaths. The impact of DON on dairy cattle is not established, but clinical data show an association between DON and poor performance in dairy herds (Whitlow et al., 1994). Dairy cattle consuming diets contaminated primarily with DON (2.5 ppm) have responded favorably (1.5 kg milk, P<.05) to the dietary inclusion of a mycotoxin binder, providing circumstantial evidence that DON may reduce milk production (Diaz et al., 2001). Field reports help substantiate this association (Gotlieb, 1997 and Seglar, 1997). Results from a Canadian study using six first-lactation cows per treatment during mid-lactation (average 19.5 kg milk) showed that cows consuming DON-contaminated diets (2.6 to 6.5 ppm) tended (P<0.16) to produce less milk (13% or 1.4 kg) than did cows consuming clean feed (Charmley et al., 1993). DON had no effect on milk production in eight cows fed over a 21-day period (Ingalls, 1996). DON has been associated with altered rumen fermentation (Seeling et al., 2006) and reduced flow of utilizable protein to the duodenum (Danicke et al., 2005). Beef cattle and sheep have tolerated up to 21 ppm of dietary DON without obvious effects (DiCostanzo et al., 1995).

Like other mycotoxins, pure DON added to diets does not have as much toxicity as does DON supplied from naturally contaminated feeds, perhaps due to the presence of multiple mycotoxins in naturally contaminated feeds. These mycotoxins can interact to produce symptoms that are different or more severe than expected. For example, it is now known that fusaric acid interacts with DON to cause the vomiting effects, which earlier was attributed to DON alone and resulted in use of the trivial name of vomitoxin for DON (Smith and MacDonald, 1991). It is believed that DON serves as a marker, indicating that feed was exposed to a situation conducive for mold growth and possible formation of several mycotoxins. The FDA has set advisory levels for DON shown in Table 3 (Henry, 2006).

Table 3. Advisory levels for deoxynivalenol (vomitoxin) in livestock feed (Henry, 2006).

*Includes lactating dairy cattle and hens laying eggs for human consumption.
**Dry weight basis.

Ergot alkaloids, including fescue toxicity

One of the earliest recognized mycotoxicoses is ergotism caused by a group of ergot alkaloids. They are produced by several species of Claviceps that infect the plant and produce toxins in fungal bodies called sclerotia or ergots, which are small black bodies similar in size to the grain. Ergotism primarily causes a gangrenous or nervous condition in animals. Symptoms are directly related to dietary concentrations and include reduced weight gains, lameness, lower milk production, agalactia, and immune suppression (Robbins et al., 1986). Sclerotia concentrations above 0.3% are related to reproductive disorders.

Fescue infected with Neotyphodium or Epichloe may contain toxic alkaloids associated with “fescue toxicity” (CAST, 2003). Symptoms include lower weight gains, rough hair coat, increased body temperature, agalactia, reduced conception, and gangrenous necrosis of the extremities such as the feet, tail, and ears. Fescue is a major pasture grass in the United States, growing widely throughout the lower Midwest and upper South. More than 20% of U.S. beef cattle graze fescue, and more than half of the fescue is endophyte infected, making this a serious problem for cattle producers. Endophyte-free varieties are available, but they are not as hardy as infected varieties. Fescue infected with a nonpathogenic endophyte appears to be more field hardy and less toxic.

Ochratoxin A

Ochratoxin A (OTA) is produced by species of Penicillium and Aspergillus and is a causative agent of kidney disease in pigs that has been referred to as mycotoxin porcine nephropathy (Krogh, 1979). The primary toxic effect is inhibition of protein synthesis (Creppy et al., 1984). In cattle, OTA is rapidly degraded in the rumen and thus thought to be of little consequence unless consumed by young pre-ruminant calves (Sreemannarayana et al., 1988). With high-grain diets, less of the dietary ochratoxin may be degraded in the rumen and thus be more toxic in those situations (Hohler, et al., 1999). Moldy alfalfa hay containing A. ochraceus was implicated as producing OTA associated with abortions in cattle (Still et al., 1971). OTA in moldy forage has also been implicated in cattle deaths (Vough and Glick, 1993). The FDA has established no guidelines for ochratoxin in feed, so any contamination issue is dealt with on a case-by-case basis (Henry, 2006).

PR toxin

PR toxin is one of the several mycotoxins produced by Penicillium molds. Penicillium grows at a low pH and in cool, damp conditions and has been found to be a major contaminant of silage. PR toxin, produced by P. roqueforti, has been suggested as the causative agent associated with moldy corn silage problems (Seglar 1997 and Sumarah et al., 2005). Surveys of grass and corn silage in Europe have found an occurrence of P. roqueforti in up to 40% of samples (Auerbach, 2003) and associated with cattle disorders (Boysen et al., 2000). PR toxin caused acute toxicity in mice, rats, and cats by increasing capillary permeability resulting in direct damage to the lungs, heart, liver, and kidneys (Chen et al., 1982) and was the suspected vector in a case study with symptoms of abortion and retained placenta (Still et al., 1972). Other Penicillium-produced mycotoxins in silages, such as roquefortine C, and mycophenolic acid, have been associated with herd health problems (Auerbach, 1998; Scudamore and Livesay, 1998, and Sumarah et al., 2005).

Patulin

Patulin is produced by Penicillium, Aspergillus and Byssochlamys (Dutton et al., 1984; Hacking and Rosser, 1981). Patulin is most likely to occur in moldy fruits such as apples but may also be found in grains, especially wet grains, and silage. Patulin is antibiotic against gram-positive bacteria. Added to rumen continuous cultures at 0, 20, 40, or 80 mg per day, patulin reduced VFA production, fiber digestion, and bacterial yield (Tapia et al., 2005). The potential for patulin toxicity of livestock is thought to be low, but there are reported case studies of toxicity (Sabater-Vilar et al., 2004).

Citrinin

Citrinin can co-occur with OTA and is produced by both Penicillium and Aspergillus. Like OTA, citrinin targets the kidney (Kitchen et al., 1977). The toxicity of citrinin was reviewed, indicating that it is a parasympathomimetic agent, and causes necrosis of tubular epithelial cells in the kidney, and in some cases, hepatotoxicity (Hanika and Carlton, 1994).

Forage Mycotoxins

Many other mycotoxins may affect ruminants, but there is less information about them, or they are of less consequence. There is much less information available about mycotoxins in forages. The array of mycotoxins found in forages may be different from those found in grains and are of major importance in mycotoxicoses of ruminants. Mycotoxins in forages and associated mycotoxicoses in cattle have been reviewed (Lacey, 1991; Gotlieb, 1997; Scudamore and Livesay, 1998; Seglar, 1997; Whitlow, 1997). El-Shanwany et al. (2005) isolated 43 fungal species belonging to 17 genera from 40 silage samples collected in Egypt. The most prevalent genera were Aspergillus and Penicillium followed by Fusarium and Gibberella. Molds were found in 206 of 233 grass or corn silage samples collected in Germany during 1997-98 (Schneweis et al., 2000). Penicillium was the dominant genus followed by Mucoraceae, Monascus and Aspergillus. Mycophenolic acid was present in 32% of samples. In 25 hay and silage samples collected in Minnesota, Wisconsin, and Illinois, there was a high incidence of cyclopiazonic acid, DON, FB, PR toxin, and alternaria TA toxin (Yu et al., 1999). It appears that A. flavus does not grow well in hay or silage; however, aflatoxin concentrations of up to 5 ppm have been reported (Kalac and Woolford, 1982). The most important pasture-induced toxicosis in the United States is tall-fescue toxicosis caused by endophytic alkaloids (Bacon, 1995). Other forage toxicosis of fungal origin include ergotism, perennial ryegrass staggers, slobbers syndrome, a hemorrhagic disease associated with dicoumarol produced in fungal-infected sweet clover and sweet vernal grass, and syndromes of unthriftiness and impaired reproduction associated with Fusarium (Cheeke, 1995).

Mycotoxin Testing

Analytical techniques for mycotoxins are improving. Several commercial laboratories are available and provide screens for an array of mycotoxins. Cost of analyses has been a constraint but can be insignificant compared with the economic consequences of production and health losses related to mycotoxin contamination. Newer immunoassays have reduced the cost of analyses.

Collection of representative feed samples is a problem because molds can produce large amounts of mycotoxins in small areas, making the mycotoxin concentrations highly variable within the lot of feed (Whittaker, 2003). Variability of mycotoxins from core samples of horizontal silos confirms that mycotoxins can be highly variable throughout the silo. Because mycotoxins can form in the collected sample, samples should be preserved and delivered to the lab quickly. Samples can be dried, frozen, or treated with a mold inhibitor before shipping.

The accurate determination of mycotoxin concentrations in feedstuffs depends on a number of factors. First, a statistically valid sample must be drawn from the lot (Whittaker, 2003). Because mycotoxins are not evenly distributed in grains and other feedstuffs, most of the error in a single analysis is due to sampling; as much as 90% of the error is associated with the taking of the initial sample. Proper collection and handling of representative feed samples is essential. Since molds grow in “hot” spots, mycotoxins are not uniformly distributed within a feed, making it difficult to obtain a representative sample, especially from whole seed, coarse feeds, or feeds not adequately mixed. Once collected, samples should be handled properly to prevent further mold growth. Wet samples may be frozen or dried before shipment, and transit time should be minimized.

The collected sample must should be finely ground and subsampled for analysis; this step is the second-largest source of error in an analysis. Finally, the subsample is extracted, the extract purified using one of several techniques, and then the mycotoxins are measured. Toxin determination may be by thin-layer chromatography plates, high-performance liquid chromatography, gas-liquid chromatography, enzyme-linked immunosorbent assays, and spectrophotometer, or by other techniques.

Mold spore counts may not be very useful, but their presence is a gross indication of the potential for toxicity. Mold identification can be useful to suggest which mycotoxins may be present. Because tests for some potentially important mycotoxins such as PR toxin are not generally available, it is currently recommended to analyze silages for mold spore count and mold identification to provide some insight to possible problems.

Blacklighting for bright-greenish-yellow fluorescence (BGYF) is often used as a screening technique for aflatoxin in corn grain, but it is very inaccurate. Newer and better methods should be used. As far as we are aware, blacklighting is completely inappropriate for other mycotoxins.

Generally, laboratories provide analysis for only a limited number of mycotoxins, perhaps including aflatoxin, ochratoxin, DON, ZEA, fumonisin, and T-2 toxin. Minimum detection levels may be limited because the purpose of the laboratory is often directed at finding high levels that cause acute toxicity and serious animal disease rather than low levels associated with chronic effects such as production losses, impaired immunity, and significant economic losses. Analytical techniques for mycotoxins are improving, costs are decreasing, and several commercial laboratories are available that provide screens for an array of mycotoxins. The Federal Grain Inspection Service (USDA-GIPSA) provides a list on the Internet of approved mycotoxin tests for grains and provides excellent background materials for the feed industry. Laboratory methods can be found in “Official Methods of Analysis of AOAC International” (Horwitz, 2000). Analytical protocols for mycotoxins are published (Trucksess and Pohland, 2000).

Standards for acceptable concentrations of mycotoxins should be conservatively low due to non-uniform distribution, uncertainties in sampling and analysis, the potential for multiple sources of mycotoxins in the diet, and interacting factors affecting toxicity (Hamilton, 1984).

Prevention and Treatment

Prevention of mycotoxin formation is essential since there are few ways to completely overcome problems once mycotoxins are present. Drought and insect damage are most important in instigating mold growth and mycotoxin formation in the field. Therefore, varieties with resistance to fungal disease or to insect damage (Bt hybrids) have fewer field-produced mycotoxins. Varieties should be adapted to the growing area. Irrigation can reduce mycotoxin formation in the field. When harvesting, avoid lodged or fallen material because contact with soil can increase mycotoxins. Mycotoxins increase with delayed harvest and with late season rain and cool periods. Damaged grains have increased mycotoxin levels; thus, for dry grain storage, harvesting equipment should be maintained to avoid kernel damage. Mycotoxin concentrations are greatest in the fines and in broken and damaged kernels; thus, cleaning can greatly reduce mycotoxin concentrations in the feedstuff.

After harvest, grains should not be allowed to remain at moisture levels greater than 15 to 18%. While there is little mold growth in grain below 15% moisture, drying to levels below 14% and preferably to <13% help to compensate for non-uniform moisture concentrations throughout the grain mass. High temperatures increase the amount of free moisture (water activity) in the grain which is the primary cause of mold growth in storage. Storage should be sufficient to eliminate moisture migration, moisture condensation, or leaks. Grain stored for more than two weeks should be kept aerated and cool. Aeration is critical because as molds start to grow in isolated spots, the moisture produced by metabolism is sufficient to stimulate spread of the mold growth. Aeration reduces moisture migration and non-uniform moisture concentrations. Commodity sheds should protect feedstuffs from rain or other water sources. They should be constructed with a vapor barrier in the floor to reduce moisture. If wet feeds are stored in commodity sheds near dry feeds, a method must be devised to prevent moisture contamination of the dry feed. Bins, silos, and other storage facilities should be cleaned to eliminate source of inoculation. Check stored feed at intervals to determine if heating and molding are occurring. Organic acids can be used as preservatives for feeds too high in moisture for proper storage.

It can be difficult to make hay at moisture levels low enough to prevent mold growth. Mold will grow in hay at moisture levels above 12 to 15%. Hay harvested at high moistures will tend to equilibrate to moisture contents of 12 to 14%, but the rate of moisture loss is dependent on moisture at harvest, air movement, humidity, air temperature, bale density, and the storage facility. The rate of dry down is enhanced by ventilation, creation of air spaces between bales, reduced size of stacks, alternated direction of stacking, and avoidance of other wet products in the same area. As molds and other microorganisms grow, they produce heat and cause deterioration. Heating can be great enough to cause spontaneous combustion and hay fires.