toxins present in endophyte-infected

Tall fescue (Festuca arundinacea) is the most important cool season perennial grass used as a forage throughout the southeastern United States.

It is able to withstand arid conditions mainly due to the fungal endophyte Acremonium coenophialum that infects most tall fescue. While the endophyte is beneficial to the plant, the toxins it produces have negative effects on the health and performance of grazing animals and result in a condition known as fescue toxicosis.

Current methods of dealing with fescue toxicosis include removing the infected fescue completely and replacing it with endophyte-free tall fescue, reducing toxin intake through grazing management, or treating the symptoms of fescue toxicosis through antagonist therapy.

These methods have had limited success. Mycosorb, a mycotoxin binder based on yeast glucan, has been shown to be effective in binding various mycotoxins produced by storage fungi in vitro and to reduce toxicity symptoms when added to livestock diets. Therefore, trials were conducted to determine its ability to bind the toxins produced by A. coenophialum.


Introduction

Tall fescue (F. arundinacea) is the major forage grass in the eastern and northwestern United States, covering 10,140,000 hectares (>25 million acres) in 21 states (Hoveland, 1993). It is the most abundant and economically important cool season perennial grass grown in the US.

The region of cultivation (Figure 1) is predominantly from Missouri to Virginia on the north, Oklahoma to the west, Virginia to the east, and across Mississippi, Alabama, and Georgia to the south (Rohrbach et al., 1995). A survey of 21 states revealed that tall fescue is mainly used for hay and pasture and estimated that 8.5 million cattle and 688,000 horses graze fescue pastures (Hoveland, 1993).



Figure 1.Range of fescue in the United States (Buckner et al., 1985).



Tall fescue is desirable because of its ease of establishment, range of adaptation, and extended grazing season (Stuedemann and Hoveland, 1988). The nutritional composition compares favorably with other cool season grasses; however, performance of cattle grazing fescue during summer months is generally less than would be expected based on the nutrient composition of the grass (Joost, 1995).

As tall fescue became a popular forage grass in the mid-20th century, reports of adverse effects on cattle grazing fescue began to accumulate (Bacon, 1995). It was not until 1977 that conclusive evidence of the presence of the endophytic fungus A. coenophialum in tall fescue and its correlation with fescue toxicosis in cattle was reported (Bacon et al., 1977). The presence of endophyte was reported in 58% of forage samples submitted from 26 states (Shelby and Dalrymple, 1987).

A more recent survey of 200 fields in 42 counties reported infection rates as high as 97%, and infection levels of individual fields greater than 67% for the majority of the fields (Strickland et al., 1993). Problems with the endophytic fungus A. coenophialum are not limited to the US. Infected tall fescue has been reported in New Zealand, Italy, Wales, France, and Poland (Strickland, 1993).

Economic losses due to endophyte infections are substantial. Beef cattle grazing endophyte-infected tall fescue have been shown to have reduced weaning weights and reduced conception rates (Hoveland, 1993). Losses to the dairy industry are mainly due to reduced milk production while infected fescue is responsible for poor reproductive performance in mares. Foals born to mares grazing infected pasture are weak and frequently stillborn. The endophyte reduces circulating progesterone and prolactin in mares (Porte and Thompson, 1992).

Researchers from a variety of disciplines have contributed to a better understanding of the problems involved in fescue toxicosis. Research to date has focused on four main areas: 1) identification and characterization of the toxins produced by A. coenophialum; 2) effects of endophyte infection on plant performance characteristics; 3) impact of fescue toxicosis on animal performance; and 4) treatment and prevention of fescue toxicosis in livestock. These studies have yielded a great deal of basic knowledge, but have failed to provide a universal technique or strategy for overcoming toxic symptoms and related performance losses.


Identification and characterization of the toxins produced by A. coenophialum

Much of the research since the identification of A. coenophialum has been dedicated to identifying the compounds responsible for toxic reactions in animals and the development of assay techniques.

Fescue toxicity symptoms appear to result from the presence of a group of toxins. The two classes of compounds receiving the most attention as causative agents are the loline alkaloids and ergot alkaloids (Strickland et al., 1993). N-acetyl and N-formyl loline account for the majority of the loline alkaloid content in endophyteinfected tall fescue (Yates et al., 1990).

The clavine alkaloids, lysergic acid amides and ergopeptines all belong to the broader group of ergot alkaloids. Several ergopeptines have been identified in endophyte-infected tall fescue including ergocornine, ergocryptine, ergocrystine, ergonine, ergosine, ergotamine and ergovaline (Yates et al., 1985; Lyons et al., 1986; Yates and Powell, 1988) (Figure 2). Ergopeptines account for 50% of the total ergot alkaloids, and ergovaline accounts for 80% of the total ergopeptines present (Lyons et al., 1986). Clavine alkaloids and lysergic acid amide alkaloids have received less attention as causative agents for fescue toxicosis than the loline alkaloids and the ergot alkaloids (Strickland et al., 1993).

A variety of procedures for the extraction, isolation, and identification of toxins produced by A. coenophialum have been developed. High pressure liquid chromatography (HPLC) is the preferred method of routine screening for ergopeptine alkaloids in endophyte-infected grasses (Yates and Powell, 1988). An immunological method capable of quantifying ergot alkaloids is also available (Hill et al., 1994) Capillary gas chromatography is used to test for the loline alkaloids (Porter, 1995).


IMPACT ON PLANT PERFORMANCE CHARACTERISTICS

The development of endophyte-free grass cultivars began after the endophyte-associated toxins were identified as causative agents of fescue toxicosis. Comparing the new cultivars to endophyte-infected tall fescue revealed that the fungi and the host exist in symbiosis (Joost, 1995).

Infected varieties show a higher germination rate, enhanced tiller formation and increased growth. Improvements in germination and tiller development promote establishment of the grass and give it a competitive advantage (Hill et al., 1991).

In addition, the endophyte seems to improve the ability of tall fescue to survive both biotic and abiotic stress. Endophyte-infected tall fescue has enhanced drought resistance and is a very valuable cool season grass from an agronomic perspective. The hardiness, other agronomic characteristics and high nutrient value of this grass make a strong case for finding a way to prevent its detrimental effects on animal health and performance.



Figure 2.Important ergopeptines indentified in endophyte-infected tall fescue.



IMPACT ON ANIMAL PERFORMANCE

Since the identification of a toxic endophyte in tall fescue, numerous research projects have examined effects on animals. A variety of different symptoms, including reduced body weight gain, increased body temperature, rough hair coat, reduced reproductive performance, fescue foot, excessive salivation, lower milk production and lower serum prolactin levels have been reported in animals consuming endophyte-infected tall fescue (Strickland et al., 1993).

Research has been conducted on horses, sheep, cattle, quail, rabbits, and rats.

The earliest symptom recognized in cattle grazing endophyte-infected tall fescue was ‘fescue foot’. Ergot alkaloids have a vasoconstrictive effect that decreases blood flow to extremities, which results in tissue death (Abney et al., 1993). Vasoconstriction also reduces blood flow to the skin thereby affecting thermoregulation and can account for increased rectal temperatures in animals fed endophyte-infected tall fescue (Browning and Leite- Browning, 1997).

The two most economically significant losses associated with fescue toxicosis are reductions in feed intake and body weight gain. Reductions in weight gain have been noted in cattle, and to a lesser extent in horses (Bond et al., 1986; Redmond et al., 1994). Strickland et al. (1993) suggested that reduced feed intake may be due to physiological mechanisms. Increased body temperature may be the primary reason for depressed feed intake. Lower feed intake in combination with reduced digestibility could be responsible for weight gain depression (Patterson et al., 1995).

Major reproductive problems in cattle and horses have been related to endophyte-infected tall fescue. Fescue toxins affect reproduction in both males and females. The majority of reproductive research in horses has focused on the pregnant mare (Porter and Thompson, 1992). Agalactia is the most commonly reported clinical sign in mares consuming infected fescue (Brendemuehl et al., 1995). Other effects in mares include prolonged gestation, thickened placentas, extremely high levels of foal mortality and dystocia. The mechanisms thought to be responsible for reproductive problems are decreased concentrations of prolactin and melatonin, vasoconstriction affecting blood flow to internal organs and hyperthermia (Porter and Thompson, 1992).

The reduction in prolactin levels has been suggested to result from the interaction of endophyte toxins with the D2 dopamine receptor on the lactotroph (Aldrich et al., 1993; Cross et al., 1995). Decreased milk production has been reported in both cattle and horses (Strickland et al., 1993). While the reduction in prolactin secretion is the most likely cause of reduced milk yields, other factors such as reduced nutrient uptake and vasoconstriction could also play a role (Strickland et al., 1993).


Yeast cell wall preparations as feed additives and toxin binders

Yeasts have been used for many years as high quality protein in animal diets. High vitamin content, enzymes, and other important cofactors make yeast attractive digestive aids in ruminant and monogastric animals (Dawson, 1994). The positive effects of live yeast cultures on animal production have mainly been associated with yeast metabolites (Girard, 1996). Recent evidence suggests that other specific positive effects such as mycotoxin binding may be associated with certain fractions of the yeast cell wall. The ability of this material to bind toxins is of particular interest and was the basis for this research.

Yeast cell wall consists almost entirely of protein and carbohydrate. The carbohydrate fraction is composed primarily of glucose, mannose, and Nacetyglucosamine.

Glucans and mannans, the two main sugars, are present in about equal concentrations in Saccharomyces cerevisiae (Figure 3). Chitin forms about 1% of the cell wall. S. cerevisiae contains glucans with mainlyß-1-3 linkages and some ß-1-6 linkages. Yeast mannan chains of various sizes are exposed on the external surface and are linked to cell wall proteins.



Figure 3.Structure of the cell wall of Saccharomyces cerevisiae.



Alltech, Inc. has been involved in the research and development of products derived from the cell wall fractions of yeast for over 10 years.

Two commercial products were developed out of this work. One is a mannan oligosaccharide-based product derived from the yeast S. cerevisiae (Bio- Mos). It has been shown to bind enteric pathogens (Spring, 1996), enhance immune function (Savage et al., 1996), and adsorb some mycotoxins present in animal feed (Trenholm et al., 1994; Devegowda et al., 1996). It has also been shown to improve animal health and performance in different monogastric species (Spring, 1996). A second-generation product shows great promise in mycotoxin binding ability (Table 1). It is a modified yeast cell wall preparation from S. cerevisiae consisting of esterified glucomannans.

During development of the commercial products, yeast cell wall preparations were screened for ability to adsorb various toxins in vitro. Binders were initially tested in a defined in vitro system; and products effective under those conditions were evaluated in a more complex system containing feed substrate and buffers to more closely simulate conditions in the gastro-intestinal tract. The in vitro tests demonstrated that the extent to which toxins were bound varied with the toxin and the cell wall preparation.

The most efficient of these preparations in vitro was an esterified glucomannan produced from the cell wall of S. cerevisiae.

The yeast-derived glucomannan product (Mycosorb) has also been tested in vivo. Stanley et al. (1997) observed that layers given diets containing aflatoxin were less affected by the toxin when the diet included the glucomannan. In a study with lactating cows, Diaz et al. (1999) found that adding Mycosorb to feed reduced the concentration of aflatoxin B1 in milk by more than 65%.

This work suggested that such preparations are stable in ruminant feeds and can decrease absorption of certain types of fungal toxins from the gut. These observations were consistent with those from a study conducted by Chandler and Newman (1994) with yeast cell wall mannanoligosaccharides that demonstrated inability of the major rumen bacteria to utilize the cell wall preparation as a carbon source.

This study showed that the cell wall preparations were fairly resistant to ruminal degradation. This suggests that the stable binding capacity of these preparations may be exploited to bind other potential toxins in the rumen. As a result of these studies, we became interested in examining the ability of the cell wall preparations to bind toxins associated with endophyte-infected fescue.


Table 1. Binding percentages of certain mycotoxins by modified yeast cell wall preparation and two other commercially-available binding agents.*





Research with modified yeast cell wall material: ability to bind endophyte toxins

In vitro toxin binding assays have been extensively used to evaluate ability of various toxin-binding agents to adsorb mycotoxins such as aflatoxin, zearalenone, T-2 toxin and vomitoxin present in stored grains. Research into the ability of a modified yeast cell wall preparation (Mycosorb) to bind toxins found in endophyte-infected was conducted in three phases:

1. Compare the ability of Mycosorb and a clay-based binder to bind toxins present in endophyte-infected fescue seed in an in vitro system.

2. Examine the ability of Mycosorb to bind toxins present in endophyteinfected fescue seed in an in vitro system containing minerals and/or organic material.

3. Test the ability of Mycosorb to bind toxins present in endophyteinfected fescue in an in vitro rumen simulation system.

The phases were completed sequentially, and the results of each group of experiments are described below.


COMPARATIVE ABILITY OF MYCOSORB AND A CLAY BINDER TO BIND TOXINS PRESENT IN ENDOPHYTE-INFECTED FESCUE SEED IN AN IN VITRO SYSTEM

Ergotamine was chosen as a representative toxin for initial studies because it was readily available in a pure form and could be easily standardized in the analytical systems used. This toxin has been used in similar in vitro trials (Chestnut et al., 1992) because of its structural similarity to ergovaline, the major ergopeptine alkaloid associated with endophyte infestation of tall fescue (Lyons et al., 1986).

The toxin binding studies were conducted in distilled water and carried out in volumes large enough to ensure a homogenous binder mixture. Initial tests demonstrated that maximum binding of ergotamine by Mycosorb was observed after a 1.5 hr incubation period (Figure 4). The amount of toxin bound was estimated by subtracting the amount of toxin measured in solution after centrifugation. This provided the basis for a rapid screening test to evaluate the relative ability of different agents to bind toxins.




Figure 4.Effects of time and Mycosorb concentration on the percentage of ergotamine (10 ppm) bound.


A comparison of Mycosorb and a commercially available clay-based product tested ability of each binder to bind ergotamine in vitro. The binders were tested at 2 mg/ml with ergotamine concentrations of 0.5, 1, 2.5, 5 and 10 ppm. The lower levels of toxin represent the minimum levels that could be quantified using this assay. The upper limits are well above the ergotamine concentrations that have been shown to be toxic in animals. These upper and lower limits provide a good range in which to determine affinity of binders for the toxin. Each binder was mixed in water with the toxin and shaken for 90 minutes. Toxin levels were measured in the supernatant fluid after centrifugation.

At the concentrations tested, Mycosorb proved more effective at binding ergotamine than the clay-based material (Figure 5). At high ergotamine concentrations, it bound more than twice the amount of toxin (47 mg/g vs. 22 mg/g) under the same conditions and application rates.




Figure 5.Comparison of the ability of Mycosorb and a clay-based binder to bind ergotamine in vitro. Binders were added at 2 mg/ml.



Mycosorb was then tested at rates of 0.5, 1 and 2 mg/ml with the ergotamine concentrations used in the previous test. The amount of toxin bound was clearly dependent on toxin concentration. The maximum binding equilibrium was reached at ergotamine concentrations above 2 ppm (Figure 6).

A series of extraction trials were then conducted to ensure that the binder was not simply masking or destroying the toxin during analysis. Sequential extractions with alcohol demonstrated that 60-70% of the toxin could be eluted from the toxin-binder complex.

These studies demonstrated that the toxin was bound to, not destroyed by, the binder; and further that the binder did not interfere with the toxin assay. These observations also suggest that the binding mechanism is reversible and can be partially explained by simple saturation kinetics. As a result, the greatest degree of toxin binding can be expected at high toxin concentrations.



Figure 6.Effects of ergotamine concentrations on binding by Mycosorb. Binding was examined at binder concentrations of 0.5, 1.0 and 2 mg/ml.



ABILITY OF MYCOSORB TO BIND TOXINS PRESENT IN ENDOPHYTEINFECTED FESCUE SEED IN AN IN VITRO SYSTEM CONTAINING MINERALS AND/OR ORGANIC MATERIAL

The gastrointestinal environment is a very complex system containing a wide range of compounds that might be expected to affect the interaction between toxins and the binding agent. A major contributor of salts to the rumen is saliva, which contains high levels of bicarbonate and phosphate that buffer the rumen environment.

To test Mycosorb in an environment similar to that found in the digestive tract, the binding assay was conducted in McDougal’s artificial saliva (McDougall, 1948). Mycosorb was able to bind significant amounts of ergotamine in the artificial saliva, however there was an apparent reduction in binding capacity when this system was compared with water (Figure 7).


ABILITY OF MYCOSORB TO BIND TOXINS PRESENT IN ENDOPHYTEINFECTED FESCUE IN RUMEN-SIMULATING BATCH CULTURES

Rumen fluid obtained from ruminally-cannulated steers was clarified for this experiment. Initial work showed interference during attempts to quantify the toxin, however, modifications to the extraction system allowed for the preparation of cleaner samples and improved toxin quantification.



Figure 7.A comparison of the amount of toxin bound when mixed with Mycosorb at 2 mg/ml in water and in artificial saliva (McDougal’s buffer).



The amount of toxin measured in the supernatant decreased as binder level increased, and was dramatically less than the samples containing no binder. Binding kinetics were similar to those seen in water and artificial saliva; but maximum binding and toxin affinity decreased when assays were run in rumen fluid (Figure 8).




Figure 8. A comparison of amount of toxin bound when mixed with Mycosorb in the presence of five different levels of ergotamine from binding trials carried out in water, artificial saliva, and rumen fluid. The binder was added at 2 mg/ml.



While binding activities were decreased in the presence of the rumen fluid and artificial saliva, these preliminary experiments demonstrate the ability of Mycosorb to bind significant amounts of one of the toxins associated with fescue toxicosis in a simulated gastrointestinal environment.


Conclusions

The results of these studies indicate that Mycosorb was able to bind significant amounts of ergotamine. In addition, this modified yeast cell wall preparation bound the toxin to a much greater extent than a clay-based mycotoxin binder.

Binding was dependent on mycotoxin concentration; which is consistent with a saturation model predicting a high affinity for the toxin at low toxin concentrations.

Mycosorb maintained a high affinity for the ergotamine in the strong mineral environment of the artificial saliva and conditions similar to those found in rumen fluid. It appears that this binding ability could be used in strategies to decrease the bioavailability of ergotamine and similar toxins. Such strategies may be useful in preventing toxicities associated with endophyte-infected fescue.


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