The use of binding agents and amino acid supplements for dietary treatment of Fusarium mycotoxicoses

Introduction

Mycotoxins are metabolites produced by fungi which are toxic to livestock when consumed in biologically significant amounts. The resulting diseases are referred to as mycotoxicoses. Mold growth and mycotoxin production on feed grains are influenced by many factors but the most important of these is moisture. Stored grains should contain less than 15% moisture to minimize mold growth. Fusarium fungi are commonly found in temperate climates; and Fusarium mycotoxins are likely the most economically significant grain mycotoxins on a global basis (Wood, 1992). The numerous Fusarium mycotoxins are very diverse in chemical structure and in the characteristics of the mycotoxicoses they produce. These toxins include the trichothecenes, the fumonisins, zearalenone, moniliformin and fusaric acid. Fusarium mycotoxins differ from aflatoxin in significant ways. The feeding of blends of grains and soybean meal, however, increases the chances of aflatoxin and Fusarium mycotoxins being present in the same diet.

About 150 chemically distinct Fusarium trichothecenes have been characterized. The major effect of these toxins on livestock and poultry is a loss of appetite, and therefore these are considered to be feed refusal toxins. The most commonly reported trichothecene is deoxynivalenol (vomitoxin, DON), although others are also sometimes found. Consumption of deoxynivalenol-contaminated feeds can result in reduced feed consumption, vomiting, immunosuppression and loss of muscle coordination. Swine are the species most sensitive to dietary deoxynivalenol. Poultry are less sensitive and ruminants are the most resistant due to the action of the rumen microflora.


How hazardous are feeds contaminated with Fusarium mycotoxins?

The non-specific nature of the symptoms of trichothecene toxicoses, including reduced feed consumption, reduced growth and immunosuppression, make it difficult to confirm trichothecenes as the cause of lost performance. The cause could also be improper management practices or a wide range of health factors. It is common to observe symptoms of trichothecene toxicity in the field, but the contaminated feed is often determined to have negligible concentrations of trichothecenes (Trenholm et al., 1983). This situation has usually been attributed to inadequate sampling of feed, errors in analysis or the presence of unknown mycotoxins. The result is lost revenue and frustration for producers.

Studies in our laboratory have indicated that the presence of fusaric (5- butylpicolinic) acid, a compound synthesized from tryptophan by Fusarium molds, will increase the growth depression seen when low levels of deoxynivalenol are fed to starter pigs (Smith et al., 1997). Although fusaric acid was chemically characterized many years ago, it has not been considered to be a significant factor in Fusarium mycotoxicoses because of its relatively low toxicity. Fusaric acid is pharmacologically active, however, and alters brain neurochemistry in a wide range of animal species (Nagatsu et al., 1970) including starter pigs (Smith and MacDonald, 1991). The compound is also considered to be a phytotoxin and can cause pathology in soybean and tomato plants (Matsui and Watanabe, 1988). Bacon et al. (1996) cultured 78 different strains of Fusarium fungi and observed that all the strains produced some amount of fusaric acid. It was suggested that since the production of this compound was so common, it should be used as a marker for Fusarium contamination of feeds.

In a survey of swine producers in Ontario, Canada, who thought their reduced performance was due to mycotoxin contamination, it was found that feed grains and complete feeds contained about 10 times as much fusaric acid as deoxynivalenol (Table 1, Smith and Sousadias, 1993). The average concentrations seen in complete feeds, however, were higher than those seen in individual grains, thereby implicating soybean meal as a potential source of fusaric acid. It is very important, therefore, to determine the mycotoxin content of the entire diet, not simply suspect grains, when estimating the potential hazard posed to livestock and poultry.





Toxicological interactions between Fusarium mycotoxins

It has often been observed that the feeding of grains naturally-contaminated with mycotoxins results in a greater toxicity than the feeding of diets containing an equivalent amount of purified mycotoxin (Trenholm et al., 1994). This can be attributed to toxicological synergy among mycotoxins such as that previously described for fusaric acid and deoxynivalenol (Smith et al., 1997). Excess brain serotonin, a neurotransmitter synthesized from tryptophan, can cause loss of appetite, lethargy, sleepiness and loss of muscle coordination (Leathwood, 1987). Trichothecene mycotoxins can inhibit hepatic protein synthesis (Meloche and Smith, 1995), resulting in hyperaminoacidemia (Wannermacher and Dinterman, 1983), elevation of brain tryptophan (MacDonald et al., 1988) and increased brain concentrations of serotonin (Prelusky, 1993). Fusaric acid also increases brain serotonin concentrations, but this occurs through a different metabolic mechanism. Fusaric acid, a tryptophan analogue, competes with tryptophan for binding to blood albumin and thereby elevates blood free tryptophan (Chaouloff et al., 1986). This results in increased brain uptake of free tryptophan across the blood-brain barrier and, once again, elevated brain serotonin concentrations.


Toward a solution: development of mycotoxin binders

A useful strategy for dietary treatment of Fusarium mycotoxicoses has been the development of specialty feed additives that can be added to feeds at low levels of inclusion to bind mycotoxins in the intestinal lumen. This effectively reduces the toxicity of a given level of contamination in feeds and allows toxic grain to be fed with minimal losses of performance. The challenge is to find binding agents with a high degree of specificity for the commonly-occurring mycotoxins. A lack of specificity may result in reduction in the availability of trace nutrients and medications. It is equally important that the binding capacity be high enough that a minimal level of dietary inclusion can be achieved. Binding agents are usually non-nutritive and are considered to be diluents, thereby reducing nutrient density. Early studies in our laboratory demonstrated that non-specific mineral additives such as bentonite (Carson and Smith, 1983a) and spent bleaching clays (Smith, 1984) could reduce T-2 toxicity. Organic fibres such as those derived from alfalfa were shown to be effective both against T-2 toxin (Carson and Smith, 1983b) and zearalenone (James and Smith, 1982; Stangroom and Smith, 1984). The lack of specificity and high levels of inclusion, however, made these treatments unpractical.

Approaches to in vitro and in vivo testing of dietary binding agents have recently been reviewed (Ledoux and Rottinghaus, 1999). Although a useful guide when conducted appropriately, in vitro binding studies must be accompanied by in vivo experiments to determine the biological significance of mycotoxin binding. An example of the more sophisticated organic polymers used as an anti-mycotoxin agent is esterified glucomannan enzymatically extracted from the cell wall of Saccharomyces cereviciae1026. This material is one of the active agents in Mycosorb, produced by Alltech, Inc.


EFFECTS OF MYCOSORB IN MYCOTOXIN-CONTAMINATED DIETS FED TO TURKEY POULTS

A 21 day experiment was conducted with day-old male poults fed blends of naturally-contaminated grains with and without supplemental fusaric acid. The most highly contaminated diet was also fed supplemented with 0.2% Mycosorb. The objective of this study was to determine the effect of feeding diets contaminated with deoxynivalenol and fusaric acid to turkey poults and to observe the potential for Mycosorb to overcome these effects. Deoxynivalenol was provided by naturally-contaminated wheat and barley. The diets fed included: 1) control, 2) contaminated grains, 3) contaminated grains + 15 mg/kg fusaric acid, 4) contaminated grains + 25 mg/kg fusaric acid, and 5) diet 4 + 0.2% Mycosorb. Diets containing contaminated grains were determined to have a deoxynivalenol concentration of about 2.4 mg/ kg. The control diet was analyzed to contain 20.6 mg/kg fusaric acid. No other mycotoxins were found to be present.

Results of the trial are given in Table 2. Weight gain of poults fed contaminated grains did not significantly differ from controls. Poults fed contaminated grains + Mycosorb, however, grew significantly faster than birds fed unsupplemented contaminated grains. Feed consumption and feed efficiency were unaffected by diet. There was a trend toward increased relative gizzard weights with the feeding of contaminated grains. A clinical screen of serum metabolites was conducted at the end of the experiment. There was a significant reduction in serum cholesterol concentration in poults fed Mycosorb compared to controls.





It was concluded that there was no toxicological synergy between deoxynivalenol and fusaric acid when fed at these concentrations. Turkey poults have been shown to be quite resistant to feedborne deoxynivalenol (Morris et al., 1999). It is of interest, however, that the feeding of Mycosorb together with contaminated grains significantly increased growth rate compared to controls. It is possible that this results from the binding of fusaric acid, which was present in both the control and contaminated diets. The lowering of serum cholesterol concentrations with the feeding of Mycosorb may indicate the difficulty of providing absolute specificity in the binding of metabolites. The drop in blood cholesterol concentration is likely due to binding of bile salts in the lumen of the small intestine. This would reduce circulating levels of cholesterol that would be used for de novo synthesis of bile salts. Such synthesis would be required due to impaired recycling of bile salts through the enterohepatic circulation, since the salts were bound by Mycosorb and excreted in the feces.

It can be concluded that Mycosorb shows promise in promoting growth when turkey poults are fed diets containing Fusarium mycotoxins.


Toward asolution: expanding on the binder concept

Considering the challenge in developing very specific mycotoxin binders, some additional strategies may be employed. It is possible to reduce the Fusarium mycotoxin-induced brain uptake of tryptophan by feeding protein supplements rich in large neutral amino acids (Cavan et al., 1988). These amino acids can compete with tryptophan for active transport across the blood-brain barrier and reduce the availability of tryptophan for brain serotonin synthesis. Examples of such protein sources are corn gluten meal and blood protein supplements.


STARTER PIG TRIALS WITH PROTEIN SUPPLEMENTS

In the summer of 1999, an experiment was conducted at the University of Guelph to determine the potential for various dietary treatments to overcome the toxicity of diets containing blends of grains naturally-contaminated with deoxynivalenol and fusaric acid. Purebred Yorkshire pigs (average initial weight 8.1 kg) were fed diets formulated to contain 4.0 mg/kg deoxynivalenol and 20.0 mg/kg fusaric acid for 21 days. Diets included 1) control, 2) contaminated grains and 3) contaminated grains + 6% red blood cell protein. There was a significant reduction in weight gain of pigs fed contaminated grains compared to controls (Table 3). The difference was largely eliminated through the feeding of red blood cell protein.

At the end of the study, a subgroup of 12 pigs fed each diet was euthanized and brains were excised and dissected into frontal cortex, pons-medulla and hypothalamus. Brain regional neurochemistry was determined by high performance liquid chromatography with electrochemical detection. The largest effects of diet were seen in the pons-medulla. The feeding of contaminated grains reduced brain tryptophan concentrations. The feeding of red blood cell protein, however, significantly increased brain tryptophan compared to controls. While brain serotonin levels were significantly elevated by the feeding of contaminated grains, this was numerically reduced by the feeding of red blood cell protein. Concentrations of 5-hydroxyindoleacetic acid, a metabolite of serotonin which can be used, with caution, as an index of serotonergic neuronal activity, increased significantly with the feeding of contaminated grains. There was a numerical decline with the feeding of red blood cell protein. It was concluded that the growth depression seen when pigs were fed contaminated grains was due to neurochemical changes that could be largely overcome by the feeding of red blood cell protein.





Summary

The active component of choice in commercial preparations for overcoming mycotoxin contamination of feeds is a mycotoxin binding agent. It has been demonstrated, however, that the Fusarium mycotoxin-induced brain neurochemical changes can be largely overcome by dietary supplements of large neutral amino acids. Such supplements minimize brain uptake of tryptophan, which prevents increased behaviors such as loss of appetite. These are characteristic of stimulation of the serotonergic nervous system. The evolution of commercial anti-mycotoxin products should include combining mycotoxin binding capacity with the ability to favorably alter brain neurochemistry.


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