Molds, Mycotoxins and their Effect on Horses

Table of Contents

1. Saprophytes
   • Fusarium
   • Claviceps

2. Endophytes
3. Ergot Alkaloid Toxicity in the Late-gestation Mare
4. Sampling and Analysis
5. Mycotoxin Levels for Horses
6. Prevention and Recommendations
7. Binding Agents
8. References

Pasture grasses, hay, grain, straw and stubble can all support the growth of various fungi. The fungi can exist as saprophytes, living on the outside of the plant and obtaining nutrients from the plant with no benefit to the plant, or exist as endophytes within the plant in a symbiotic relationship, providing benefits to the plant while obtaining nutrients from the plant. The saprophytes include the more common genera Aspergillus, Claviceps, Stachybotrys, Fusarium and Penicillium. The endophytes live between the plant's cell walls and include the more common genera Balansia, Epichloe, Acremonium and Neotyphodium.

Fungi and their associated mycotoxins are present on grain crops in varying amounts each year, depending on the climatic growing conditions. A cool, wet, growing season increases the likelihood that fungi, especially Fusarium and its mycotoxins, will be present in small grains. The high moisture level in grain encourages fungal growth while the cool temperatures increase the production of mycotoxins. Mycotoxins are secondary metabolites produced by fungi and can affect various animals upon ingestion or inhalation. High concentrations of mycotoxins in wheat grain are unusual. Normally the concern is with wet falls and the invasion of corn and corn byproducts by fungi. The research involving horses and mycotoxins is limited when compared to research in other species, such as cattle and swine.

The reporting of mycotoxin concentrations may be expressed in a number of ways. Therefore, remember;
1 ppm = 1 mg/kg = 1µg/g Example: aflatoxin @ .2 ppm is 200 ppb
1 ppm = 1,000 ppb DON @ 1200 ppb is 1.2 ppm

Saprophytes

The common mycotoxin-producing saprophytic fungi are Aspergillus, Claviceps, Stachybotrys, Fusarium and Penicillium. Limited or no information is available for the effects of Aspergillus, Stachybotrys and Penicillium and their associated mycotoxins on horses.

Fusarium

Most of the research on plant fungi-livestock interactions has been done on Fusarium. The genus Fusarium contains a large number of species of fungi. They grow mainly on cereal grains, e.g., corn and wheat, and produce a number of mycotoxins that may affect various animals upon ingestion or inhalation. The fusarium species can also grow on grasses used for horse pastures and hay. In growing years, when there is high precipitation during seed-head formation, high concentrations of fusarium and its associated mycotoxins are common in wheat and wheat by-products (wheat middlings, bran), corn and corn by-products (screenings, corn stover).

The primary mycotoxins of the Fusarium spp. present in grain include T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA), and moniliformin (MON). In corn, fumonisins are the principle mycotoxins of Fusarium moniliforme. In Canada, deoxynivalenol (DON) is the most significant mycotoxin. Fortunately, compared to the other mycotoxins, it has relatively low toxicity. Often, there is more than one mycotoxin present. Therefore, the observed toxic effects may be a result of mycotoxin interactions rather than a single mycotoxin. The known effects of DON, T-2, zearalenone, aflatoxin and fumonisins on horses are summarized below.

DON, also known as vomitoxin, was the principle mycotoxin found in Ontario wheat samples in the 2000 harvest year. Its effects on horses have not been well documented. It is also called vomitoxin because it induces vomiting in pigs and dogs after ingestion of contaminated material. In various livestock species, vomitoxin will cause feed refusal, decreased weight gains, signs of gastrointestinal irritation (e.g., diarrhea, colic, rectal prolapse, and rectal bleeding), reproductive problems, skin irritation, cardiotoxicity and interference with the immune system. In mice, ingestion of DON may cause the overproduction of IgA immunoglobulins in the intestines. IgA accumulates in the kidneys and results in glomerulonephritis (kidney failure). The maximum acceptable concentration of DON in wheat intended for human consumption in flour is 1 ppm. Wheat with a concentration greater than 1 ppm will be diverted for livestock feed.

Whole grain wheat is not normally fed in large amounts to horses. However, whole wheat, wheat bran and wheat by-products can be incorporated into blended horse feeds such as sweet feed and pelleted feeds. In addition, horses are commonly bedded on wheat-straw and some horses will consume a significant amount of their straw bedding.

In wet harvest years, ingested DON concentrations on a total ration basis could be significant. In the harvest year of 2000, the following results were obtained:

• 85% (97 of 114) of wheat samples contained concentrations of DON greater than 1 ppm (mean 4.48 ppm, range 0.33 to 28.4 ppm). The zearalenone levels were of little concern, with concentrations in most cases less than 0.15 ppm.
• 48% of 50 samples of wheat shorts had concentrations of DON greater than 1 ppm. The mean concentration was 1.8 ppm and the highest detected was 4.6 ppm.
• Analysis of wheat straw revealed concentrations of DON ranging from undetectable to 16.5 ppm.

Little research about the effect of DON on horses has been published, but the following three reviews will indicate the divergence of opinion. Johnson, Casteel and Messer (1997) fed barley containing 36-44 ppm of DON (on an as-fed basis) to horses while at pasture. No detectable effects were found during the 40 days of the trial using non-pregnant mares and geldings. They suggested that the horse may not be susceptible to the adverse effects of DON and the horse's gastric microflora may be able to detoxify it (1). No work was done with stallions or pregnant mares. Clearly, the reasons for the resistance of horses to DON are not know. (Author's note: If the horses were consuming 75% of their nutrients from pasture and 25% from the grain, the DON concentration on a total ration basis would be 8-10 ppm.)

Raymond, Smith and Swamy (2003) fed a diet in which the concentrates averaged 15 ppm DON (2). The concentrates also contained 0.8 ppm 15-acetyldeoxynivalenol, 9.7 ppm fusaric acid and 0.2 ppm zearalenone. The horses were consuming 2.8 kg of concentrates and 5 kg of a mixed hay. The DON concentration on a total ration basis would be 5.4 ppm. They concluded that consumption of the contaminated feed reduced feed intake.

The Department of Animal Science, North Carolina State University, studied the relationship between mycotoxins and equine colic. They analyzed feed samples from colics and controls seen by North Carolina practitioners. They found DON in the concentrate of 100% of colic cases (n=16) (range 0.20 to 8.3 ppm) and 70% of the controls' concentrate (n=10) (range 0-2.5 ppm). T2 toxin at concentrations greater than 0.5 ppm and zearalenone greater than 0.7 ppm were present in 31% and 44% of the colic concentrate samples respectively, while neither were found in control samples (3). Hence the causal-effect relationship of DON and colic in horses is not clear.

Since these papers represent a large range in potential toxic levels, it is suggested that, until more precise research is completed, the maximum tolerable level for DON in the total feed of 2 ppm be adopted for horses.

Fumonisins are produced by Fusarium moniliforme, the fusarium that invades corn. There are three common mycotoxins designated as FB1, FB2 and FB3. FB1 is well documented as the cause of leukoencephalomalacia in horses. The consumption of fumonisins produces a multifocal neurologic disease that affects multiple horses in a herd. Once clinical signs appear, the majority of affected horses die. Corn screenings can be heavily contaminated with fumonisins and should never be fed to horses. Fumonisin B1 at concentrations of 10 ppm have been found to be associated with leukoencephalomalacia (4). Concentrations of FB1, FB2 and FB3 in equine feed should not exceed 5 ppm and should not exceed 20% of the diet on a dry-matter basis. Concentrations in excess of 5 ppm can cause colic and death (5). Leukoencephalomalacia has not been reported in Ontario despite a large volume of corn and corn products moving between the USA and Canada.

Only one report involves the feeding of T-2 toxin to horses. The feeding of 7 mg of purified T-2 toxin per os daily, to mimic a 1-ppm concentration in the feed, had no effect on the ovarian activity of mares (6).

The same authors reported that zearalenone was administered to mares for ten days at 7 mg of purified zearalenone per os daily to mimic a 1-ppm concentration in the feed. It had no adverse effect on the reproductive parameters of cyclic mares (7).

Aflatoxins are a group of closely related, highly toxic mutagenic and carcinogenic compounds produced by Aspergillus flavus. Aflatoxin is found in both field and stored feeds such as corn, cottonseed and peanuts. Ponies fed 2 mg/kg of aflatoxin B1 per day for five days demonstrated significantly elevated serum-enzyme levels of iditol dehydrogenase (LID), also known as sorbitol dehydrogenase (SDH) and gamma glutamyl transferase (GGT). These enzymes are indicators of liver damage. Other reports have indicated that concentrations as low as 0.3 mg/kg (.3 ppm ) have killed horses (8).

Claviceps

Claviceps can live on a variety of hays and pasture grasses. They produce fruiting bodies on bluegrass and cereal rye and can cause the clinical syndrome known as Ergotism. Ergotism is probably the oldest known mycotoxicosis. The ergot alkaloids of Claviceps purpurea are hallucinogenic in humans and have been associated with historical accounts of witches. Consumption of infected rye bread has been associated with human disease dating back some 2,000 years. Claviceps has a life cycle that consists of: an airborne ascospore infecting the inflorescences (flower) of the plant; followed by the formation of the honeydew and sclerotia, which replace the ovary; sclerotia, which are masses of mycelium, mature and fall to the ground forming stalked stromata which in turn produce ascospores (9). The Claviceps sclerotia contain a large array of chemicals. Many of these same chemicals are also commonly found in the endophyte-infected grasses, such as fescue, and are responsible for fescue toxicity (9). Different genera of fungi produce the ergot alkaloids in different proportions. Claviceps commonly produces ergotamine, ergostine, ergocristine, ergocryptine and ergocornine (10). Claviceps can infect many grass species, including bluegrass and ryegrass (9).

Two clinical cases involving the production of ergot alkaloids by Claviceps have been associated with fetal loss in late-gestation mares.

• From Brazil, late-gestation mares were fed oats containing ryegrass seed. They suffered fetal losses similar to the clinical condition of fescue toxicity (11). The authors indicate that the ryegrass seed was the only source of ergot alkaloids.
• In Ontario, cereal rye straw was used for bedding. Late-gestation mares were consuming a large portion of their bedding (12). Of the first 8 mares to foal, 7 had dead foals with clinical signs similar to fescue toxicity except that the mares foaled around their normal due date. High-performance liquid chromatography (HPLC) analysis of the bedding indicated the presence of two ergot alkaloids, each equivalent to 450 ppb. One was identified as ergocornine, the second peaked just ahead of 2.2 minutes retention time and adjacent to the ergonovine standard.

Endophytes

Neotyphodium coenophialum was formerly called Acremonium coenophialum. It is the endophytic fungus associated with fescue toxicity. N. coenophialum lives in a symbiotic relationship with the plant, gaining nutrients to live on while providing chemical protection against grazers, both insect and higher life forms and some resistance to drought. N. coenophialum completes its entire life cycle within the plant. It passes from one generation of the grass to the next in the seeds of the plant. N. coenophialum and Claviceps produce a similar array of ergot alkaloids (ergopeptine alkaloids). Ergovaline is the main ergot alkaloid for which analysis is available. Many other ergot alkaloids can be found. N. coenophialum, as the causal agent of fescue toxicity, is well recognized as causing dystocia in mares and deaths of perinatal foals in the United States. Therefore, when seeding pastures/paddocks, ensure that the seed does not contain endophyte-infected varieties of fescue.

Ergot Alkaloid Toxicity in the Late-gestation Mare

Mares are sensitive to ergopeptine alkaloids at concentrations as low as 300-500 ppb, while cattle do not show visible signs until 400-700 ppb (13). These alkaloids exert toxic effects on the reproductive tract and mammary gland of the mare and have been associated with depression of serum prolactin and progestagens (5 alpha-pregnanes), a prolonged gestation, a thickened edematous placenta and agalactia (14). The ergopeptine alkaloids interfere with the normal rise of progestagens (mainly 5 alpha-pregnanes) and prolactin in the last 40 days of gestation. The progestagen levels normally increase from 300 days to birth (4.8 + 1.5 to 22.7 + 2.7 ng/ml). Suppression of progestagen levels indicates ergopeptine toxicity (14). Brendemuehl suggests the use of a commercially available radioimmune assay (RIA) progesterone assay to measure total immunoreactive progestagens in pre-foaling sera of mares.

Foals born without the normal increases in maternal progestagens suffer hypoadrenocortical function and are small, weak or stillborn (14). Edema of the placenta increases the placental weight. The placental weight of the normal thoroughbred mare is reported as 5.7 + 0.08 kg or about 12.5 lbs. or 11% of the foal's body weight (15,16).

The primary clinical signs of ergot alkaloid poisoning in the late-gestation mare include:

• an extended gestation length from 11 to 12 months,
• dystocia, with mares sometimes trying to foal for many hours,
• agalactia with poor quality colostrum (low immunoglobulin levels),
• "red bag" placentas from premature separation,
• thick edematous placentas with weights exceeding 6.5 kg for a thoroughbred mare,
• weak or dead foals with aspiration pneumonia from struggling to get out through a thickened placenta.

No studies have shown the effect of ergot alkaloids in horses other than with late-gestation mares. Fescue is grown extensively in the arid areas of the USA because of its ability to withstand drought and its resistance to many insect infestations. Fescue is not commonly grown for pasture or hay production in Ontario. Endophyte-free varieties have been developed to get away from the problems caused by these mycotoxins. Endophyte-infected varieties of fescues are commonly used for erosion control and golf greens. Occasionally, endophyte-infected seed will be accidentally sold to horse owners.

Sampling and Analysis

Samples of straw and hay bales can be taken using a core sampler. Laboratory testing for Fusarium mycotoxins should be performed using HPLC (high-performance liquid chromatography) or GC (gas chromatography) methodology rather than using one of the quick ELISA tests. ELISA (enzyme-linked immunosorbent assay) assays are only validated for testing raw grains. The tolerance levels of mycotoxins suggested for feed can be used to determine the potential risk when using straw bedding.

Laboratory tests for ergovaline (the ergot alkaloid of fescue toxicity) and lolitrem B (the ergot alkaloid of perennial ryegrass) are commonly available. Dr. Morrie Craig's laboratory at Oregon State University analyzes thousands of samples annually for ergovaline. It is difficult to analyze for other ergopeptine alkaloids due to the lack of control standards.

Mares in the last 30 days of pregnancy can be monitored for placental edema by ultrasound. Low pre-foaling progestagen concentrations in the late-gestation mare are indicative of exposure to ergot alkaloids. Monitoring of progestagen concentrations is done by collecting serum samples from the mares when they enter the foaling barn (approximately 330 days gestation). A comparison with a second serum sample around day 335 of gestation would show whether there is a normal rise or peaking in progestagen concentration. Concentrations below 15 ng/ml are suspicious of ergot alkaloid toxicity. A pre-foaling serum progestagen concentration below 5 ng/ml is seen with fescue toxicity. In 50% of normal mares, progestagen concentrations drop 6-10 hours prior to foaling. Unfortunately, the RIA progesterone assay is not readily available and, from our work, there is wide variation between mares and on different days.

Mycotoxin Levels for Horses

The following table provides some guidelines for the tolerance levels of mycotoxins in the total ration dry matter (TRDM) above which they are potentially harmful.

Prevention and Recommendations

Research into the effects of mycotoxins on horses is in its infancy. As a general rule, err on the side of caution.

• Feed and bed horses with hay/grain and bedding free of mycotoxins.
• The amount of DON in cereal straw depends primarily on the presence of contaminated grain and chaff. Tedding or raking prior to baling the straw will reduce the chaff and kernels and, therefore, the concentrate of mycotoxins.
• Since the potential concern with horses is the risk of an additive effect, in harvest years with high DON concentrations, consider limiting the use of straw bedding; ensure horses are fed high quality hay and in sufficient amounts to limit the eating of bedding.
• Late-gestation mares should never be allowed to consume cereal-rye straw, endophyte-infected hay or stubble or eat sclerotia-containing grain.
• All pasture/hay seed mixes containing tall fescue and perennial ryegrass must be guaranteed endophyte-free.
• Mow grass pastures to keep them in a vegetative state rather than letting them develop seed heads. This will minimize the opportunity for proliferation of airborne ascospore and the invasion of pasture plants.
• Check with your laboratory. HPLC methodology may be more sensitive than thin layer chromatography (TLC) when trying to identify ergot alkaloid concentrations less than 1 ppm.
• Mares can be treated with domperidone should an ergot alkaloid problem be suspected.

Binding Agents

Feed manufacturers often utilize binders of various types in feeds to tie up mycotoxins. These include clay-based binders and yeast cell wall extracts (e.g., Bio-Mos-M). Raymond et al. concluded that supplementation with 0.2% yeast cell wall extract (a polymeric glucomannan mycotoxin adsorbent) improved feed intake over feeding contaminated feed alone but not when compared to control diets not containing mycotoxins (2). The long-term effects of binding agents are not clear; therefore, they should be used with caution.

References

1. Johnson PJ, Casteel ST, Messer NT. Effect of feeding deoxynivalenol (vomitoxin)-contaminated barley to horses. J. Vet Diagn Invest 1997; 9: 219-221.
2. Raymond SL, Smith TK, Swamy HVLN. Effects of feeding of grains naturally contaminated with Fusarium mycotoxins on feed intake, serum chemistry, and hematology of horses, and the efficacy of a polymeric glucomannan mycotoxin adsorbent. J. Anim. Sci. 2003; 81:2123-2130.
3. Barnett DT, Mowrey RA, Hagler WM Jr, Bristol DG, Mansmann RA. The correlation of selected mycotoxins to the incidence of colic in horses. World Equine Veterinary Review 1997; 3 (1).
4. Beasley VR. Equine leukoencephalomalacia/hepatosis and stachybotryotoxicosis. In: Current Therapy in Equine Medicine, 3rd ed., Philadelphia: WB Saunders, 1992: 377-380.
5. Russell MA, Eikenberry JG, Scott DH, Hope WD. Moldy corn outbreak: the Purdue CES response. Proceedings of the Equine Nutrition and Physiology Symposium 1993: 286-287.
6. Juhasz J, Nagy P, Huszenicza G, Szigeti G, Reiczigel J, Kulcsar M. Long term exposure to T-2 fusarium mycotoxin fails to alter luteal function, follicular activity and embryo recovery in mares. Equine Vet J. Suppl. 1997; 25: 17-21.
7. Juhasz J, Nagy P, Kulesar M, Szigeti G, Reiczigel J, Huszenicza G. Effect of low-dose zearalenone exposure on luteal function, follicular activity and uterine oedema in cycling mares. Acta Veterinaria Hungarica 2001; 49 (2): 211-222.
8. Asquith RL. Mycotoxicosis in horses. In: Smith JE, Henderson RS, eds. Mycotoxins and Animal Foods. CRC Press, 1991: 679-688.
9. Bacon CW. Fungal endophytes, other fungi, and their metabolites as extrinsic factors of grass quality. In: Forage Quality, Evaluation and Utilization. Nutrition Conference on Forage Quality, University of Nebraska, 1994: 318-366.
10. Lacey J. Natural occurrence of mycotoxins in growing and conserved forage crops. In: Mycotoxins and Animal Foods. CRC Press, Inc., Florida, 1991: 363-397.
11. Riet-Correa F, Mendez MC, Bergamo PN, Flores WN. Agalactia, reproductive problems and neonatal mortality in horses associated with the ingestion of Claviceps purpurea. Australian Vet. J. 1988; Vol 65, 6:192-193.
12. Wright RG, Boyce B, Van Dreumel T, Hazlett MJ, Cross DL. Ergot alkaloid toxicity in foaling mares associated with eating cereal rye straw. Abstract presented at Equine Nutrition and Physiology Symposium, Lexington KY May 28/01.
13. Craig M. Personal communication. Oregon State University, Corvallis, Oregon.
14. Brendemuehl JP, Williams MA, Boosinger TR, Ruffin DC. Plasma progestagen, tri-iodothyronine, and cortisol concentrations in postdate gestation foals exposed in utero to the tall fescue endophyte Acremonium coenophialum. In: Equine Reproduction VI. Biology Reprod Mono 1, 1995: 53-59.
15. Whitwell KE , Jeffcott LB. Morphological studies on the fetal membranes of the normal singleton foal at term. Res. Vet. Sc. 1975; 19: 44-55.
16. Schlafer DH. Gross examination of equine fetal membranes: What's important - What's not! Proceedings of the Equine Symposium: Society for Theriogenology/American College of Theriogenologists, San Antonio, Texas 2000.
17. Meronuck R, Concibido V. Mycotoxins in feed. Feedstuffs 1997; July 24: 142-148.
18. Action Levels of Aflatoxin in Corn. Aflatoxin Regulations. Food and Drug Administration Release, U.S. Department of Health and Human Services; October 5, 1998.
19. Adams RS, Kephart KB, Ishler VA, Hutchinson LJ, Roth GW. Mold and mycotoxin problems in livestock feeding. PennState's College of Agricultural Sciences, Cooperative Extension note DAS 93-21: 1-16.
20. Tarr B. Molds and Mycotoxins. OMAFRA Info Article # 2. Ontario Ministry of Agriculture, Food and Rural Affairs, 1996.
21. Fumonisin Levels in Human Foods and Animal Feeds. Guidance for Industry. Food and Drug Administration Release, U.S. Department of Health and Human Services; November 9, 2001.