titrating mycotoxins out of the diet

As we learn more about mycotoxins and make new discoveries in this area, we begin to realize that there are many unanswered questions involving these toxins. Mycotoxins are produced by molds, which are aerobic unicellular organisms. Mold growth can occur in environments that contain lower available water than would be needed to support bacterial growth. For example, bread will support mold growth but not bacterial growth under normal conditions. Just as there are beneficial bacteria and pathogenic bacteria, not all molds produce mycotoxins. Mycotoxins are produced by certain molds and are considered a secondary metabolite of mold growth. This means that mycotoxin production occurs late in the growth phase of a mold and is usually associated with some aspect of mold strain survival. Controlling mold growth is an important first step in controlling mycotoxins.

This can be best accomplished by:

1) Low feed moisture. Ideally moisture levels should not exceed 12%.

2) Keeping feed fresh. Mold growth takes time; and storage time before mold growth occurs depends on ambient temperature and oxygen levels in feed.

3) Keeping equipment clean. Hazard Analysis of Critical Control Points(HACCP) not only controls pathogenic bacteria but is a major step in reducing mold and mycotoxin growth in hard-to-reach areas of feed preparation equipment.

4) Keeping the grain intact until adequately dried. Mold growth is more prevalent in damaged or processed grains.

5) Using mold inhibitors. Propionic acid-based products are very effective for mold inhibition, but will do nothing to mycotoxins. Buffered propionic acid has the advantage of being less caustic to equipment and more stable and effective for a longer period of time than acid salts or free acids. The reason for this is that when properly buffered, the acid dissociates when exposed to moisture in the feed. This prevents volatilization of the propionic acid in storage before being applied. It also allows more consistent and longer lasting mold inhibition.

Moisture in feed comes from several sources; improperly dried feed ingredients, feed manufacturing processing, and environmental storage conditions. Controlling moisture in feed manufacturing is a critical step. Heat from grinding can cause the migration and external concentration of moisture. This is important since, although it does not affect the total moisture of the feed, it causes a ‘dry inside and wet outside’ phenomenon that can contribute to mold growth. Pelleting also involves heat and moisture addition to the feed. In the pelleting process 3-5% moisture is added to the feed. If cooling conditions are improper, moisture is not allowed to dissipate leading to favorable conditions for molds. When done properly, pelleting is effective in reducing mold concentrations in the feed. Reductions of near 10,000-fold have been observed in pelleted feed compared to the same feed in mash form.

After the feed is manufactured, it is necessary to maintain conditions antagonistic to mold growth. It is necessary to eliminate sources of moisture in the handling and storage of feed. Low moisture feed can pick up moisture in humid conditions unless stored in sealed packaging. Equipment cleanliness is paramount to controlling mold growth since contaminated surfaces can inoculate otherwise ‘clean’ feed.


Mycotoxins in feed

Although the global nature of the feed market limits the ability to pinpoint ingredient region of origin, mycotoxin prevalence is somewhat regional. Aflatoxin and certain ochratoxins are primarily found in tropical and subtropical regions. Zearalenone, deoxynivalenol (DON or vomitoxin), ochratoxin A, T-2, and fumonisin are most notable in temperate regions.


AFLATOXIN

Aflatoxin can cause liver damage, decreased reproductive performance, reduced milk production, embryonic death, birth defects, tumors and suppressed immune function. Most food and feedstuff are free of aflatoxin at time of harvest. The exceptions to this rule are cotton, corn, and groundnuts such as peanuts. Corn is infected in the field where ears are subjected to insect attack followed by invasion and infection with Aspergillus flavus and the subsequent formation of aflatoxins. Cereal grains harvested at high moisture content such as rice and corn must be dried before they can be safely stored. In climates where humidity is high and drying and storage facilities are poor, aflatoxin risk may be high. Many species of Aspergillus grow slowly at moisture contents below 18% (fresh weight basis) making it critical to protect stored products by obtaining and keeping the moisture below 15%.

Overcoming the adverse effects of aflatoxin in feed has been extensively researched and a number of compounds have been identified that aid the animal consuming unavoidably contaminated diets. Biochemically, the presence of lactone in the molecule makes it susceptible to alkaline hydrolysis (Figure 1). If the alkaline treatment is mild, acidification may retrograde to original toxicity.




Figure 1. Structure of aflatoxin.


Activated charcoal, a porous material with a high surface area, has been used to aid in the control of aflatoxin. The efficacy of adsorbing aflatoxin with activated charcoal is unaffected by pH and retained material is usually retained throughout the gastrointestinal tract (GIT). The addition of 200 ppm activated charcoal to broiler diets containing 500 ppb aflatoxin B1 (AFB1) was found to be somewhat effective in reducing the adverse effects of the toxin (Jindal et al., 1994). Other studies have found charcoal of minimal benefit when added to aflatoxin-containing poultry diets (Dalvi and Ademoyero, 1984).

Furthermore, a management problem is encountered with the use of charcoal in the diet as it blackens the feed, feces, birds and their environment. Some manufacturers have overcome some of these problems by producing a product that contains up to 65% water (Buck and Bratich, 1986). The anti-caking agent, hydrated sodium calcium aluminosilicate (HSCAS) has also been used to adsorb aflatoxins from the diet. In poultry, 0.5% HSCAS added to diets contaminated with 7.5 mg/kg AFB1 reduced the growth inhibitory effects and hepatic changes associated with AFB1 (Phillips et al., 1988). Other studies have examined HSCAS with combinations of aflatoxin and T-2 or ochratoxin. In trials with aflatoxin and T-2, HSCAS provided almost complete protection against aflatoxin alone, limited protection against the combination and no protection against T-2 (Kubena et al., 1990). Similar tests with diacetoxyscripenol (DAS) and ochratoxin A revealed limited or no improvements of HSCAS with these toxins (Kubena et al., 1993; Huff et al., 1992).

Sodium bentonite has been used to aid mycotoxin control with promising results. However, Schell et al. (1993) found that the addition of this clay had slight affects on calcium, phosphorus, iron and zinc absorption and lowered magnesium and sodium absorption in control and contaminated feeds. Similar problems have been associated with zeolites because the adsorbent could interfere with tissue mineral distribution (Watkins and Southern, 1991) and negatively affect, for example, the utilization of dietary phosphorus by chickens (Mostaghian et al., 1991).


Esterified glucomannans (Mycosorb) demonstrate a high binding affinity for aflatoxin and other toxins when compared to either activated charcoal or HSCAS. In contrast to many of the clay-based adsorbents, interference with mineral metabolism has not been noted due to more specific binding characteristics of Mycosorb. In vitro data have demonstrated a strong binding affinity of Mycosorb to aflatoxin. In vivo work in a number of species confirms these findings. Data in dairy cows indicate reductions in milk aflatoxin levels with Mycosorb added at 500 g/tonne of complete feed and sodium bentonite at 12.5 kg/tonne of feed (Figure 2, Diaz et al., 1999).




Figure 2. Effects of different binding agents on milk aflatoxin residues.



Similar results have been noted in poultry (Khararern, personal communication). In vitro titration data indicate that at levels of aflatoxin below 500 ppb there is no additional benefit in terms of mycotoxin binding by adding more than 1 kg of Mycosorb per tonne of feed (Figure 3, Evans, personal communication).




Figure 3. Titration of Mycosorb and aflatoxin levels.



OCHRATOXIN

Ochratoxin was first detected by van der Merwe et al. (1965) in laboratory tests to detect toxic feedstuffs in South Africa. Ochratoxin is a potent nephrotoxin and teratogen. Adverse effects have been noted in pigs and poultry at levels at or near 2 ppm. Cattle effects can include decreased performance, reduced milk production, kidney failure and death (at levels greater than 800 ppm). Studies on horses are lacking to document adverse effects. The fungus responsible for the production of ochratoxin can invade starchy cereal grains such as corn and wheat with a moisture content of 15.5-16%. Ochratoxin A is only slightly soluble in water and is absorbed in the upper sections of the GIT in a passive manner in the non-ionized form and is subjected to secretion and reabsorption via enterohepatic recycling (Leeson et al., 1995). In mammals, ochratoxin A is absorbed primarily in the stomach and proximal jejunum although absorption through the lungs into the systemic circulation has also been documented (Di Paolo et al., 1993). Absorption is faster in areas of the GIT where pH is low. Ochratoxin A toxicity in ruminants is thought to be relatively low due to the rumen microflora. Studies postulate that levels up to 12 mg of ochratoxin A per kg of contaminated feed are tolerated by ruminants (Hult et al., 1976).

However, Hohler and coworkers (1999) found that even at dosages of 2 mg/kg of concentrate feed, substantial amounts of ochratoxin A were detected in the serum of animals fed the toxin.

Ochratoxin A (Figure 4) enters circulation through the portal vein and the lymphatic vessels bound to plasma proteins, especially albumin. Due to the relatively high affinity of ochratoxin A for albumin, a number of studies have examined increasing dietary protein levels to help alleviate the deleterious effects of this toxin. Feed efficiency of broilers given 4 ppm ochratoxin A was improved at protein levels of 22 and 26% compared to broilers fed 14 and 18% protein (Bailey et al., 1989; Gibson et al., 1989). Vitamin C has also been shown to be beneficial in layers exposed to ochratoxin A (Haazele et al., 1993). Dietary adsorbents such as charcoal and sodium calcium aluminosilicate did not alleviate ochratoxin A toxicity (Huff et al., 1992; Rotter et al., 1989). To date no in vivo data on the ability of esterified glucomannans to alleviate ochratoxin A toxicity are available. In vitro ochratoxin A binding by Mycosorb ranges from 8-15%. Due to the biochemical affinity of ochratoxin A for certain proteins, it is believed that improved binding to ochratoxin A could be accomplished by using cell wall extract with greater protein content.




Figure 4. Structure of ochratoxin A.



FUSARIUM MYCOTOXINS

The Fusarium mycotoxins consist of over 100 fungal metabolites with the same basic structure primarily produced by Fusarium spp. Mycotoxins produced from Fusarium are less well-known than the Aspergillus-produced aflatoxin, but can be more detrimental to animal health. The Fusarium mycotoxins of practical concern are listed in Table 1 and illustrated in Figure 5.


Table 1. Mycotoxins from Fusarium.

Figure 5. Fusarium mycotoxins: deoxynivalenol (vomitoxin), T-2 toxin, zearalenone, fumonisin B1 and fusaric acid.


Deoxynivalenol is the most commonly detected Fusarium mycotoxin. It is a trichothecene mycotoxin, which inhibits protein synthesis. Incremental reductions in intake have been documented in pigs above 2 ppm, with vomiting at higher concentrations. Deoxynivalenol may also cause immunosuppression and affect reproduction. Similar responses have been noted in dogs and cats. In dogs feed intake was significantly reduced by DON concentrations greater than 4.5 ppm. Cat food intake was reduced at DON levels greater than 7.7 ppm (Hughes et al., 1999).

Certain reports suggest that poultry and ruminants tolerate higher levels of DON than pigs and pets. However, Trenholm and coworkers (1984) indicated that DON levels above 5 ppm may be deleterious. In cattle, reduced feed intake and milk production have been noted (Whitlow and Hagler, 1999). In horses, DON-contaminated barley (40 ppm) had no effect on intake but reductions in serum levels of IgG and IgA were associated with the mycotoxin (Johnson et al., 1997).

T-2 is less prevalent but more toxic than DON. Levels of 1-12 ppm can cause significant reductions in pig performance and fertility. T-2 and related toxins cause irritation, hemorrhage and necrosis throughout the digestive tract. Oral lesions have also been noted in pigs and poultry and have been suspected in horses. Zearalenone imitates the female hormone estrogen and can impair reproduction in many species. At low doses, increased mammary gland and reproductive organ size have been documented in many species. Pigs appear to be the most susceptible. Swelling of the vulva leading to rectal and vaginal prolapse is an overt symptom. In addition, uterine enlargement and ovarian atrophy is common with zearalenone.

Studies conducted in Germany demonstrated that Mycosorb bound more than twice the zearalenone after 10 minutes than other adsorbents tested at pH 4.5 and 6.0. Additionally, desorption of this toxin at pH 8 was least with Mycosorb (Volkl and Karlovsky, personal communication).

Titration of zearalenone and Mycosorb indicate reactivity similar to enzyme kinetics. Increasing concentrations of toxin yield greater binding capacity of Mycosorb up to 1000 ppb zearalenone (Evans, personal communication, Figure 6).




Figure 6. Effect of Mycosorb ( 1 kg/T) on increasing concentrations of zearalenone in vitro.


Fumonisin is a recently discovered mycotoxin that can impair immune function, cause kidney and liver damage, decrease animal performance and cause death. In pigs, fumonisin has been linked with porcine pulmonary edema (PPE). Fumonisin in horses can cause equine leukoencephalomalacia (ELEM), staggers, stupor, unilateral blindness, lameness, seizure (due to brain necrosis) and death. Fumonisin levels associated with PPE and ELEM contained levels of fumonisin B1 ranging from 1 to 330 ppm (PPE) and 1 to 126 ppm (ELEM) (Ross et al., 1991). Ross et al. (1992) suggested that fumonisin B1 concentrations greater than 10 ppm in horse feeds were likely to be candidates for ELEM. Although naturally contaminated feeds containing fumonisin have been shown to elevate brain serotonin levels, from recent work by Trevor Smith in Canada, it seems clear that fumonisin is not responsible. Fusaric acid, also produced by Fusarium molds, has been shown to cause vomiting in pigs and elevate brain concentrations of tryptophan and serotonin. The difference in responses seen in scientific studies using purified Fusarium toxins and naturally contaminated feeds may be explained, in part, by the apparent synergism that exists between fusaric acid and other mycotoxins.


Mycotoxins in forages: fescue and ryegrass toxicities

In 1993, Hoveland estimated that approximately 688,000 horses in the US graze tall fescue. The presence of an endophytic fungus in tall fescue has been associated with lower weight gains, rough hair coats, reproductive problems and necrosis of the foot, tail and ears of horses and cattle. Increased gestation length, retained placentas, higher numbers of stillborn foals, and agalactic mares were associated with endophyte infected fescue consumption compared to mares consuming endophyte-free fescue.

Although endophyte-free fescue is currently available, it is less hardy, and less resistant to overgrazing, insect damage and drought than infected grass. Perennial ryegrass intoxication or ryegrass staggers is a neurotoxic syndrome characterized by ataxia, lack of coordination, head shaking, and collapse. Animals appear normal until disturbed. The neurological effects are temporary, but lack of coordination can lead to drowning, and running into and through barns and fences. The causative agents are compounds called tremorgens. A number of tremorgens have been identified with the most important being lolitrem B (Figure 7), which is produced by the endophytic fungus Acremonium lolii. Concentrations of lolitrem B are lowest in the leaf blades and highest in the leaf sheath, which tends to make ryegrass staggers most often noted in over-grazed pastures (DiMenna et al., 1992). Symptoms of ryegrass staggers appear when the lolitrem B concentrations exceed 2-2.5 ppm. As is seen with fescue, the endophyte improves the vigor of the ryegrass.





Figure 7. Lolitrem B, a tremorgen produced by Acremonium lolii, and ergovaline produced by A. coenophialum.


Ergot alkaloids have also been associated with sorghum and can have deleterious effects on poultry. Cumulative weights of broilers consuming diets containing 30 ppm sorghum ergot alkaloid with or without adsorbing agents are presented in Figure 8. Liveweight was nearly 150 g less for birds receiving sorghum ergot alkaloid compared to uncontaminated feed. In addition, these data indicate that adsorbents can help alleviate the detrimental effects of sorghum ergot alkaloid on broiler performance with the greatest numerical response seen when birds received Mycosorb (Deo et al., 1999).

As yet undiscovered mycotoxins may also hold the key in adding insight into mycotoxin effects on animal performance. It is well documented that the toxic effects of many mycotoxins are enhanced by the presence of more than one toxin. As more mycotoxins are discovered, it may come to pass that the above mentioned toxins only serve as markers or indicators of other unknown toxins.




Figure 8. Effect of sorghum ergot alkaloid (SEA) and adsorbents on broiler liveweight at 23 days.



Conclusions

Mycotoxin control begins by controlling mold growth from the point of harvest and maintaining a low mold count throughout feeding. Commercial mold inhibitors can aid in reducing mold growth at the time of harvest or shortly thereafter. Not all molds produce mycotoxins; and since mycotoxins are secondary metabolites of mold growth, high or low mold counts do not necessarily mean presence or absence of mycotoxin. Molds may have already produced mycotoxin and died, leaving a low mold count and high levels of mycotoxins. Many more mycotoxins exist than are mentioned in this text as the focus has been on the more prevalent toxins. As we learn more about mycotoxins we may be better equipped to address specific problems identified with these toxins. With this, we hope to better define the mechanisms of toxicity and identify toxic levels in feed. In many species, much more data are necessary in order to understand these toxins and the interactions of multiple toxins. In certain instances, adsorbents follow characteristics similar to enzyme kinetics with binding affinity increasing as mycotoxin concentrations increase. With clays and activated charcoal certain undesirable characteristics are associated with their use such as dust problems, blackening of the feed and animal, or interference with mineral metabolism. Biochemical adsorbents such as esterified glucomannans do not display these traits and show a greater affinity for mycotoxins due to their apparent specificity for mycotoxin binding. As we advance our knowledge in this field and learn more about the widespread nature of mycotoxins and the biochemical nature of their structure, technology exists to design adsorbents for those toxins.


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