Mycotoxins in equine diets: the difference between win, place and show?
Published: March 8, 2007
By: KYLE E. NEWMAN - Venture Laboratories Inc. (Courtesy of Alltech Inc.)
Limiting values or safe concentrations of specific mycotoxins are, for the most part, unknown for the horse. A unique challenge is presented when attempting to use non-equine data to effectively define mycotoxin effects on horses, since the horse is comparable to the ruminant in that it is a forage-grazing animal but has a gastrointestinal tract more closely similar to a pig with the addition of a hindgut fermentation process. The nature of the horse also makes the equine quite different from other livestock species. Food animal species are bred for growth and meat yield and have a relatively short lifespan, while in most cases the horse is bred for athletic performance, confirmation, temperament, beauty and/or durability.
Historically, mycotoxins were identified by their ability to produce severe, overt disease syndromes in animals as a result of relatively high intakes of toxin. However, low levels of toxin exposure over long periods can elicit chronic or subchronic toxicological manifestations. A concern is that for horses, especially elite athletes or breeding stock, exposure to low levels of mycotoxins may affect performance or breeding ability without the appearance of overt signs of toxicity. The outcome from exposure to chronic low levels of toxin may include general ill thrift, suppression of the immune system and increased risk of secondary infections.
Unlike other livestock production species, horses are generally expected to live a long lifespan and are expected to be reproductively sound in their later years. For these reasons, probably more so than for other species, the safe amount of specific mycotoxins is unknown for the horse. In addition to the degree of exposure, incidence of disease can also be influenced by the presence of multiple mycotoxins. Factors influencing susceptibility to mycotoxins include disease, heat stress, marginal nutritional profile, drug interactions, presence of multiple toxins, crowding, age and reproductive status.
Molds and mycotoxins
The occurrence of mold and mycotoxins in food and animal feed is a problem of major concern internationally (Wood, 1992). Mycotoxin production can occur when favorable conditions are met that allow fungi to grow on crops in the field, at harvest, in storage or during the processing of feed (Palmgren and Lee, 1986). Mycotoxins are the products of secondary fungal metabolism although not all fungal growth produces mycotoxins. Environmental and nutritional parameters and growth stage of the mold govern toxin production. Molds that are present in the field before harvest can proliferate if moisture content remains high as a result of inadequate processing and storage conditions. Mold and subsequent mycotoxin contamination of a feedstuff or forage can increase in extreme environmental conditions such as drought, excessive precipitation, or sudden frost. Contamination can also increase due to physical damage to crops that allows fungal penetration of the plant tissue (Gregory et al., 1963; Northolt et al., 1976; Schindler, 1977).
A large number of the predominant mycotoxins are produced by the Fusarium molds, which thrive in temperate climates worldwide and are common contaminants of feed grains. A wet growing season followed by cool weather increases the likelihood that fungi, especially Fusarium and its mycotoxins, will be present in grains. The high moisture level in grain encourages fungal growth while the cool temperatures can increase the production of mycotoxins. Fusarium spp. require a moisture content of 25% concurrent with a relative humidity of more than 90% in order to grow.
Such conditions can commonly occur when field-dried hay is not cured properly. Forage is the basis of most feeding programs for the horse, with long-stemmed, field-dried hay being the traditional source. Any horseman can recognize the dust aerosol when opening a bale of hay that has a high mold count and many individuals equate a high mold count with mycotoxin content. Nevertheless, a forage or feed with a low level of measurable mold contamination can still contain significant amounts of mycotoxins. The subjective appraisal of hay by horse owners has been shown to be unreliable in terms of mycotoxin, mold, and actinomycete contamination (Raymond et al., 2000). Mycotoxin production does not correlate to mold growth and conditions that support one may actually inhibit the other. Increased mycotoxin contamination and the potential for mixtures of mycotoxins to be cocontaminants in feeds are now even more likely due to improved global grain transportation systems and global trading of agricultural commodities.
Conditions favoring mycotoxin production
Aspergillus, Fusarium, and Penicillium are the three most important genera of fungi that have been extensively studied in relation to mycotoxicosis. The large surface area and vigorous activity of fungal mycelia ensure a close relationship of the mold with the environment. Extrinsic factors such as temperature, pH, water activity, and chemical composition have an influence on growth and mycotoxin biosynthesis. Other factors such as plant-fungal interactions, interactions among microorganisms, and the presence of certain chemical agents can influence mold growth and mycotoxin synthesis (Smith and Henderson, 1991).
In the field, specific fungal species may occur cyclically or seasonally. Usually optimal conditions for mold growth do not coincide with those for mycotoxin production. However, the production of several different mycotoxins by the same species, or even the same strain, may not occur optimally under identical conditions. For example, the production of deoxynivalenol (DON) and zearalenone (ZEN) by a single strain of Fusarium graminearum responded differently to changes in temperature. ZEN production reached a maximum at 25oC, whereas the production of DON continued to increase with increasing temperature, and at 28oC the production was more than twice that at 25oC (Greenhalgh et al., 1983). In field situations DON and ZEN have been produced simultaneously in infected corn and wheat by the above fungi (Cote et al., 1985).
Forage toxins
Many studies have shown that forage sources can contain toxins that adversely affect the health or reproductive function of the equine.
FESCUE TOXINS
In the eastern United States, tall fescue is extremely prevalent and represents a major forage source. In 1993, Hoveland estimated that approximately 688,000 horses in the US graze tall fescue. In 1977, Bacon and coworkers first reported the presence of the endophytic fungus now called Neotyphodium coenophialum in tall fescue. The presence of this fungus in consumed grasses has been associated with reduced weight gain, rough hair coats, higher body temperatures, reproductive problems, and necrosis of the foot, tail and ears of horses and cattle. Increases in the number of cycles for conception and early embryonic death have also been associated with endophyte-infected fescue (Brendemuehl et al., 1994). In addition, increased length of gestation, retained and so-called ‘red bag’ placentas, higher numbers of weak or stillborn foals, and agalactic mares were associated with endophyte-infected fescue consumption compared to mares consuming endophytefree fescue (Monroe et al., 1988).
Ergovaline is one of the primary ergot alkaloids produced by N. coenophialum and exerts its activity by acting as a dopamine agonist and suppressing prolactin secretion (Strickland et al., 1994). Decreased serum prolactin is one of the most consistently reported symptoms of fescue toxicosis (Neal and Schmidt, 1985; Elsasser and Bolt, 1987; Redmond et al, 1994).
Ergonovine and ergotamine have been shown to be arterio- and venoconstrictive, which plays a role in thermoregulation since vasoconstriction prevents dissipation of heat by slowing the transfer of internal body heat to the skin or respiratory tract (Abney et al., 1993). The effects of ergovaline and ergotamine are of primary importance in warm climates and in the equine athlete. Trials examining the effects of endophyteinfected fescue on exercising horses found that recovery heart and respiratory rates (post-exercise) were significantly greater than for horses grazing non-infected endophyte. Water consumption post-exercise was also found to be greater for horses consuming endophyte (9.45 gallons vs. 5.59 gallons P< 0.05; Vivrette et al., 2001). The minimum amount of exposure required to observe these effects and the reversibility is not clear.
Symptoms in this trial were observed after 14 days of grazing endophyte-infected fescue. In cattle, the adverse effects were reversed after 8 days of grazing endophytefree pastures (Rhodes et al., 1991). Although endophyte-free fescue is currently available, it is less hardy, and less resistant to overgrazing, insect damage and drought than infected grass. Recent studies in the United States indicate that daily supplementation of mare diets with FEB-200™ (Alltech, Inc.) reduced some of the deleterious effects of endophyte-infected fescue. In these studies, serum progesterone levels were greater in mares receiving FEB-200™ (10.74-ng/dL) compared with unsupplemented mares (9.68-ng/dL). Trials in cattle fed FEB-200™ indicate increased weight gain, and improved pregnancy rates (Akay et al., 2004).
RYEGRASS TOXINS
Perennial ryegrass intoxication or ryegrass staggers is a neurotoxic syndrome characterized by ataxia, lack of coordination, head shaking, and collapse. 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, which is produced by the endophytic fungus Neotyphodium (Acremonium) lolii. Concentrations of lolitrem B are lowest in the leaf blades and highest in the leaf sheath. Thus, ryegrass staggers occur most frequently in over-grazed pastures (DiMenna et al., 1992). Symptoms of ryegrass staggers appear when the lolitrem B concentrations exceed 1.5 to 2.5 ppm. As with fescue, the endophyte improves the vigor of the ryegrass.
LEGUME TOXINS
The ubiquitous soil fungus Rhizoctonia leguminicola can infect certain legumes after heavy rainfall or in situations of high humidity. Although mostly associated with red clover (Trifolium pratense), white clover, alsike clover, kudzu, alfalfa, Korean lespedeza, cow pea, soybean, black medic and blue lupine have also been reported to be susceptible to infection by this fungus (Hagler and Behlow, 1981). Examination of the leaves of the legumes will reveal bronze to black spots or concentric rings ranging in size from pinhead to pencil eraser in diameter. R. leguminicola produces slaframine, an alkaloid that after ingestion is activated by the liver yielding a cholinergic parasympathomimetic agent (Guengerich and Aust, 1977). Toxicosis is manifested by profuse salivation (slobbers) accompanied by increased water consumption. Bloating, piloerection, diarrhea, agalactia, feed refusal, abortions and death in severe cases are also occasionally observed (Sockett et al., 1982). Slobber-response tests in guinea pigs indicate that levels of 50-100 ppm in clover will elicit a response.
The alkaloid is somewhat stable in that it may still be present in baled hay from infected fields but will degrade over time. Studies have shown a decrease in slaframine concentrations from 50 to 100 ppm to approximately 7 ppm after 10 months of storage ( Hagler and Behlow, 1981). Removal of animals from infected pastures to non-infected areas or feeding grass hay will return animals to normal health within 3 to 4 days.
STACHYBOTRYOTOXICOSIS
One of the earliest recognized mycotoxin diseases was stachybotryotoxicosis. In eastern Europe and South Africa the mold Stachybotrys has been isolated from forage sources and straw and implicated in stachybotryotocosis in horses, cattle, swine and sheep. Stachybotrys has also been incriminated in domestic housing in the US as a source of ‘sick house syndrome’. This fungus has been shown to produce macrocyclic trichothecene mycotoxins such as satratoxins F, G and H, verruccarin J, and roidin E (Eppley, 1977; Harrach et al., 1981). Stachybotrys chartarum (formerly S. atra) is a fungus found in the soil and in moist dark indoor environments. This fungus produces black, ‘soot-like’ spores in hay and straw and has been found to grow extensively on moist drywall in houses.
In the early 1930s, the horse was vital to the economy of Russia. In the Ukraine in 1931 thousands of horses were afflicted with an unknown ailment and a team of scientists was sent to investigate. By 1938, it was discovered that the disease was caused by Stachybotrys alternans (also now called S. chartarum) growing on the hay fed to the animals (Rodricks and Eppley, 1974).
In the summer of 1983 and spring of 1984 three stables in Hungary experienced an outbreak of fungal toxicosis where nearly all of the 300 horses at these stables presented with drying and cracking of the facial mucosae, purulent nasal discharge and epistaxis (bloody nose). Post-mortem examination of two of the affected animals showed extensive petechiation of the internal tissue. Bedding from these stables was found to contain satratoxin G (20 to 35 μg/kg), satratoxin-H (75 to 107- μg/kg), and verrucarin-J (35 to 61 μg/kg). Samples of oats fed to horses during the 1984 outbreak were found to contain T-2 toxin, suggesting a synergism between toxins contributing to the intensity of the outbreak (Harrach et al., 1987).
When low levels of toxic feed are consumed, fissures at the corner of the mouth are followed by deeper lesions. Traditionally, the first cases of this disease appear in the autumn when animals are stabled and inhale or consume fodder containing the toxin. There is a subsequent increase in affected animals as the winter months continue into April when the toxicosis begins to decline as horses are returned to pasture and stall confinement is diminished (Forgacs, 1972). The toxins are nonantigenic, therefore no immunity seems to develop from stachybotryotoxicosis and the same horse can become affected several times in the same year. Foals nursing affected mares do not show signs of illness, indicating the toxins are not excreted in milk.
Stachybotryotoxicosis may present itself in either of two forms; the typical form which most commonly occurs in nature and the atypical (shocking) form. The typical form develops progressively with three stages of onset. Stage 1 begins with the aforementioned stomatitis within the first 2 to 10 days of exposure. The fissures of the mouth become necrotic. Edema of the underlying tissues leads to swelling of the lips and perhaps the entire head in what has been described as a ‘hippopotamus-like’ appearance (Forgacs, 1972). In many cases salivation, conjunctivitis, rhinitis, and enlarged lymph nodes develop in the next 120 hrs. In other cases these symptoms are absent and the only specific abnormality may be an increase of body temperature of 1-1.5oC accompanied by neutrophilia for a brief period.
Typically, Stage 1 lasts from 8-12 days but may last for as long as a month. Stage 2 is characterized by changes in blood profiles. The total leukocyte count may drop to 1000-3000 cells/mm3 of whole blood with a concomitant decrease in the relative neutrophil count. Blood clotting time also increases or clotting fails to occur. Body temperature, respiration, and cardiac function remain in the normal range, but in rare cases it may be possible for the clinician to detect atony of the intestinal tract. The second stage duration varies from 5 to 20 days, but may last as long as 50 days. In Stage 3 thrombocytopenia and leucopenia are exacerbated. Leukocyte counts may drop as low as 100/ mm3 and a complete loss of blood coagulation is observed. Hyperthermia is also a distinct characteristic of Stage 3 with the temperature rising to between 40 and 41.5oC. Affected horses may show signs of depression, poor or no appetite, problems swallowing and colic. This stage is usually quite short (1-6 days) and is almost always fatal.
An atypical form of stachybotrytoxicosis develops after ingestion of a large amount of toxin. In this form nervous disorders are the main visible symptoms. The animal will avoid movement and stand with legs in a wide gait or may cross the front legs or lean against objects for support. Swallowing is quite difficult, the pulse becomes weak and arrhythmic (80 to 100 beats/ min), pulmonary edema and cyanosis of the mucous membranes is observed. The animal may become blind. Death usually occurs due to respiratory failure within 72 hrs of ingestion.
OTHER TOXINS FROM FORAGE SOURCES
PR toxin produced by P. roquefortii has been found in forage sources and was the suspected toxic agent in a case study with symptoms of abortion in cattle (Still et al., 1972). In addition, fumigaclavine produced by Aspergillus fumigatus, and Alternaria spp. have been implicated with abortion, small intestinal dysfunction and other health problems (Cole et al., 1977; Pitt and Hocking, 1997).
Raymond et al. (2001) reported that higher levels of DON, ZEN and T-2 toxin were found in forages as compared with grain concentrates during a field study of performance horse farms in Ontario, Canada. Concentrates sampled did not seem to contain significant levels of mycotoxins. A similar survey was conducted by Buckley and coworkers (2004) in which zearalenone and DON were the major toxins found in samples of feed, forage and bedding, with 20-25% of the forage and straw samples containing detectable quantities of these mycotoxins. It is important to emphasize that in all of these studies commercial enzyme-linked immunosorbent assays (ELISA) test kits were used for the analysis. These kits are not validated for the complex matrices found in forage samples. ELISA procedures are prone to false positive results on non-validated matrices and should be confirmed using high performance liquid chromatography (HPLC) and/or thin-layer chromatography (TLC) testing. For this reason, the concentrations of mycotoxins in forage sources are not clear, but there appears to be situations when these toxins are present in relatively high concentrations.
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.
Aflatoxicosis
Aflatoxin is a mycotoxin produced primarily by Aspergillus flavus and A. parasiticus. These molds prefer warm and humid conditions. Mycotoxin production on feedstuffs seems to be most prevalent when drought, insect infestation or other stresses occur late in the growing season of cottonseed, peanuts, corn, or other grain products. In documented outbreaks of aflatoxicosis on two separate horse farms, peanut meal and corn were found to be the sources of the toxin (Angsubhakorn et al., 1981). In one case the elevated price of rice caused feed producers to use stored corn and peanut meal (comprising 70% of the feed mixture) in place of the rice in the feed.
Representative samples of the mixed ration contained approximately 0.2 mg aflatoxin B1 (AFB1) and 0.2 mg aflatoxin B2 per kg. A number of animals showed clinical signs of illness and 12 yearlings died. Clinical symptoms of aflatoxin intoxication include ataxia, tremor, elevated temperature, anorexia, loss of appetite, weight loss, icterus (yellowing of the eyes or skin), hemorrhages, bloody feces, and brown urine (Angsubhakorn et al., 1981; Bortell et al., 1983; Cysewski et al., 1982). The major target organ of aflatoxin is the liver, where the toxin induces centrilobular necrosis (Stoloff and Trucksess, 1979).
Clinical signs of toxicity were observed in ponies dosed with aflatoxin, at levels ranging from 0.075 to 0.3 mg/ kg BW/day (75 to 300 parts per billion (ppb)) with all animals either dying or declining and euthanized during a 39 day trial (Cysewski et al., 1982). A subsequent trial found that ponies given aflatoxin B1 at dosages equal to or greater than 2 mg/kg BW died within 76 hrs (Bortell et al., 1983). At the time of necropsy, ponies demonstrated petechiae of the viscera, free blood in the small intestine, pale tan to yellow livers and icterus. From the data, it appears that horses are more susceptible to aflatoxin B1 than calves. Calves had a reduced rate of gain with 0.2 mg B1/kg/day but died after 14 days given 0.5 mg B1/kg/day (Pier et al., 1976). In ponies, 0.3 mg B1/kg/day caused death at between 12 and 16 days and clinical symptoms and biochemical changes were observed even at 0.075 mg B1/kg/day (Cysewski et al., 1982). It should be noted that other studies in ponies have observed apparent resistance at levels of 0.2 mg B1/kg/day (Walter et al., 1981). Cysewski hypothesized that previous exposure to aflatoxin of the ponies used in the Walter study may have influenced this resistance.
The fact that Walter and co-workers dosed their ponies for 5 days and Cysewski and co-workers dosed for up to 39 days may also have played a role in the differences observed in these two trials. A separate case study of an Arkansas farm where three horses died of severe hepatic necrosis and others became ill found concentrations of 130 ppb total aflatoxin (114 ppb AFB1, 10 ppb AFB2 and 6 ppb AFM1) in the suspected corn (Vesonder et al., 1991). A summary of equine aflatoxicosis cases indicates a very large range from approximately 55 ppb to 6,500 ppb to be associated with clinical symptoms (Asquith, 1985) with duration of exposure and previous history of exposure playing a role in aflatoxicosis.
Recently authors of a study in Sweden have implied a possible link between chronic obstructive pulmonary disease (COPD) and inhaled mycotoxins (Larsson et al., 2003). Data from this study show that activation of AFB1 occurs, not only in the liver but also in the olfactory and respiratory tissues in the horse. AFB1 requires activation by cytochrome P450 enzymes to the reactive intermediate AFB1-8,9-epoxide, which then binds to nucleic acids among other molecules and leads to the carcinogenic and cytotoxic properties normally associated with this toxin. Glutathione (GSH) may also conjugate with AFB1-8,9-epoxide, however, this binding essentially detoxifies the compound (Quinn et al., 1990).
COPD is a disease of the equine lung that is similar to asthma in humans and caused by an allergic response. It is also called ‘heaves’ since horses typically are observed ‘heaving’ to push air out of the lungs at the end of an exhale. COPD is most often observed in older or stabled horses, and is rarely seen in situations where horses are pastured year round. As with asthma in humans, aerosolized particles of pollen, dander, microorganisms or other agents inhaled by a horse with COPD elicit an allergic response. The molds A. fumigatus and Micropolyspora faeni are two heavily implicated organisms that may evoke COPD in horses (Halliwell et al., 1993). In humans, the allergic condition known as ‘farmer’s lung’ has been well established to be caused by inhaling dust from moldy hay and straw (Gregory and Lacey, 1963; Blyth, 1973).
Ochratoxicosis
Ochratoxin was first detected by van der Merwe et al. (1965) in laboratory tests to identify 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. Effects observed in cattle include decreased performance, reduced milk production, kidney failure and death (at levels greater than 800 ppm). The outcome of this toxin in horses is not known but, because of the site of absorption, assumed to be similar to the outcome in pigs. 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 (OA) is only slightly soluble in water and when consumed is absorbed in the upper sections of the gastrointestinal tract. Maximal absorption occurs in the proximal jejunum in a passive manner in the non-ionized form and is then subjected to secretion and re-absorption via enterohepatic recycling (Kumagai and Aibara, 1982; Leeson et al., 1995). Absorption is faster in areas of the gastrointestinal tract with a low pH.
In mammals, ochratoxin A absorption through the lungs into the systemic circulation has also been documented (Di Paolo et al., 1993). Toxicity of OA in ruminants is thought to be relatively low due to the rumen microflora. Hult and co-workers (1976) postulated that levels up to 12 mg OA/kg of contaminated feed are tolerated by ruminants. However, Hohler and coworkers found that even at dosages of 2 mg/kg of concentrate feed, substantial amounts of OA were detected in the serum of cattle fed the toxin (Hohler et al., 1999). Although data in horses are lacking, it is assumed from the site of absorption of this toxin, that horses would have a sensitivity simlar to that of most monogastrics.
Ochratoxin A 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. As with most mycotoxins, ochratoxin is not present in isolation and is commonly found with citrinin, penicillic acid and other mycotoxins in field situations (Ribelin et al., 1978).
Fusarium mycotoxins
Fusarium mycotoxins of concern include trichothecenes, ZEN, fumonisins, moniliformin and fusaric acid (FA). The name trichothecene was derived from the fungus Trichothecium, and these compounds are chemically related sesquiterpenoids that all possess a tetracyclic 12,13-epoxy-trichothec-9-ene ring system.
Deoxynivalenol (also known as vomitoxin or DON) is considered the most common trichothecene but also one of the least toxic. Other prominent trichothecenes include T-2 (considered one of the least prevalent but most toxic), diacetoxyscirpenol, fusarenon-X and satratoxins. Zearalenone, fumonisins, moniliformin and fusaric acid (FA) are classified as non-trichothecene Fusarium mycotoxins. Most trichothecenes can produce clinical signs in the parts per million or parts per billion concentration range. The trichothecenes are known as feed refusal toxins because loss of appetite is one of the first observable symptoms (Trenholm et al., 1994). Loss of appetite, lethargy, sleepiness and loss of muscle coordination are associated with increased tryptophan and serotonin levels by inhibition of hepatic protein synthesis resulting in hyperaminoacidemia (Meloche and Smith, 1995; Wannemacher and Dinterman, 1983). Studies in laboratory animals and pigs have shown enhanced serotonergic activity in the brain as one of the possible mechanisms for DON and FA induced feed refusal, vomiting and other behavioral changes (Smith et al., 1997).
Impaired immune function is also associated with exposure to trichothecene mycotoxins (Rotter et al., 1994). Decreased resistance to a number of infectious organisms has been documented (reviewed by Otokawa, 1983; Venturini et al., 1996) and include Salmonella, Mycobacterium, Listeria, Candida, Cryptococcus and herpes virus. 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). This led the author to suggest that the horse may not be as susceptible to DON and the horse’s gastric microflora may act to inactivate the toxin. Raymond and co-workers advanced this study by using a diet containing naturally contaminated wheat and corn (Raymond et al., 2003a). In this study, the concentrate averaged 15 ppm DON, 0.8 ppm 15-acetyldeoxynivalenol, 9.7 ppm FA and 0.2 ppm ZEN. The horses were consuming 2.8 kg concentrates and 5 kg of mixed hay per day.
The authors concluded that consumption of the contaminated diet reduced feed intake, and serum levels of γ- glutamyltransferase (GGT) were significantly higher in horses consuming contaminated grain sampled on day 7 and 14 of supplementation but not for day 21. An increase in GGT levels indicates a possible effect on liver function without obvious signs of damage since GGT is a hepatic membrane-associated enzyme. The increase in GGT associated with mycotoxincontaminated feed was not seen when contaminated diets were supplemented with a mycotoxin adsorbent (Mycosorb®, Alltech, Inc.). In addition, Mycosorb®- supplemented animals had improved intake compared to those given unsupplemented contaminated diets (1.64 kg vs 1.00 kg, P< 0.05). A similar study was conducted by the same authors incorporating an exercise program (Raymond et al., 2003b). Results were similar although in this trial significant weight loss was observed over the 21 day supplementation period in horses fed contaminated grains as compared to control animals.
The differences observed between the Johnson study and those of Raymond and co-workers may be explained by a synergistic effect between low levels of different toxins. Toxicological synergism has been documented among Fusarium mycotoxins (Smith et al., 1997).
Studies 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 DON are fed to starter pigs (Smith et al., 1997). Although FA was chemically characterized years ago, it has not been considered a significant factor in Fusarium mycotoxicoses because of its relatively low toxicity.
Fusaric acid is a pharmacologically active compound that alters brain neurochemistry in a wide range of animal species. The physiological effect of FA is a drop in blood pressure. Such an effect could be of significance for the horse as one of its production parameters is athletic performance. Like DON, FA is associated with increased brain serotonin levels but through a different mechanism. It has been demonstrated in pigs that fusaric acid and DON act synergistically to reduce feed consumption and trigger loss of muscle coordination and lethargy (Smith et al., 1997).
Horses can be exposed to DON and/or FA from corn or wheat products in grain concentrates and/or from forage or wheat straw bedding. Contamination of bedding material represents a risk through ingestion, inhalation and dermal contact. An unplanned DON concentration of 1.2 ppm in the straw bedding in the previously described feeding trial is an illustration of the potential for mycotoxin contamination from bedding sources (Raymond et al., 2003a). The amount of DON in straw depends primarily on the presence of contaminated grain and chaff, two sources known to have more concentrated levels of mycotoxins. Although forage represents a possible source of contamination, the forage component in the diet may provide some protection from mycotoxin exposure. High concentrations of plant fiber have been shown to reduce some of the deleterious effects of mycotoxins (Carson and Smith, 1983; James and Smith, 1982).
A published case report cited weight loss and elevated hepatic enzymes in horses with the probable cause being straw contaminated with DON. It was reported that in 2002, approximately half of 104 Warmblood-type riding horses stabled in Germany suddenly lost weight. Further examination of nine of the affected horses revealed marked elevation of liver enzyme activity of both GDH and GGT. Analysis of the feed, hay and bedding revealed DON concentration ranging from 0.5 to 2.7 ppm in the straw. Once the horses were removed from the contaminated bedding they slowly gained weight and general condition became progressively better (Zeyner et al., 2002).
FDA levels of concern for DON are 2 ppm for wheat entering the milling process for humans and 1 ppm for the finished product for humans. The concern level for DON in wheat for livestock is 4 ppm (Wood, 1992). 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 for horses be comparable to levels allowed for humans.
There are few studies on the effects of T-2 toxin in the horse, with only one report involving the feeding of T-2 toxin. Administering 7 mg of purified T-2 toxin per os daily to mimic a 1 ppm concentration in feed had no effect on the ovarian activity of mares (Juhasz et al., 1997). In other species, oral and intestinal lesions are frequently observed in animals consuming T-2 or diacetoxyscirpenol (DAS) at levels as low as 100 ppb for a period of 25 days (Sklan et al., 2001).
Zearalenone is an estrogenic non-tricothecene mycotoxin produced by Fusarium. Zearalenone binds to estrogen binding sites and causes enlargement of the uterus and rectal and vaginal prolapse, abortions and infertility with pre-pubertal animals appearing to be more sensitive than older animals and swine being the most susceptible livestock species (D’Mello et al., 1999; Kalleta and Ettala, 1981). Because the symptoms of ZEN toxicity are quite specific, it is more easily recognized than trichothecene toxicity.
Studies by Juhasz et al. (2001) found that ZEN administered to mares for 10 days to mimic a 1 ppm concentration in the feed found no adverse effect on the reproductive parameters of cycling mares but skin lesions were observed around the mouth in 3 of 6 horses.
An earlier study by Gimeno and Quintanilla (1983) reported a natural outbreak of ZEN mycotoxicosis on a commercial horse farm. In this case, corn screenings fed to the horses were found to contain approximately 2.7 ppm ZEN. No other common mycotoxins were found. Corn screenings fed to a herd of horses on a commercial farm produced strong estrogenic symptoms after a feeding period of 30 days. Symptoms in the mares included feed refusal, prolapsed uterus and internal hemorrhage. In male horses, severe flaccidity of the genitals was observed. Reports of feeding ZENcontaminated diets to pigs indicated that 1 ppm is the minimum concentration required to produce hyperestrogenism (James and Smith, 1982) and from the above data it can be inferred that pigs are more sensitive to ZEN than horses. In cattle, traditionally considered more resistant to the effects of ZEN, abortions have been attributed to ZEN-contaminated hay (Kalleta and Ettala, 1981).
Fumonisins are produced primarily by Fusarium moniliforme and F. proliferatum, the Fusarium species that invade and are prevalent in corn. Fumonisins are recently discovered mycotoxins 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) (Harrison et al., 1990). Fumonisin in horses can cause equine leukoencephalomalacia (ELEM), which is typified by staggers, stupor, lameness, seizure (due to brain necrosis) and death. Feed associated with PPE and ELEM contained levels of fumonisin B1 (FB1) ranging from 1 to 330 ppm (PPE) and 1 to 126 ppm (ELEM) (Ross et al., 1991).
Additional studies (Ross et al., 1992) suggested that fumonisin B1 concentrations greater than 10 ppm in horse feeds made those animals consuming the feed likely candidates for ELEM. Although naturally contaminated feeds containing fumonisin have been shown to elevate brain serotonin levels, it appears that fumonisin is not the source of this activity. Smith and MacDonald (1991) demonstrated that FA increased tryptophan, serotonin and 5- hydroxyindoleacetic acid in pigs. Previous work by Porter and coworkers (1990) examined corn associated with ELEM on tryptophan levels in rats and found elevated levels, however, these investigators did not measure FA levels in the corn. Subsequent work by Porter did not show similar effects with purified FB1 (Porter et al., 1993). It seems probable that fumonisin does not affect brain serotonin levels, other toxins produced along with fumonisin seem to be responsible for this activity.
Fumonisins interfere with sphingolipid metabolism, disrupting endothelial cell walls and basal membranes (Van der Westhuizen et al., 2001). Sphingolipids are one of the primary groups of lipids in cell membranes. They have multiple functions, including signal transduction, cell growth, cell-to-cell communication, immuno-recognition, and aid in defining the physical nature of lipoproteins. There are three common fumonisin mycotoxins designated as FB1, FB2 and FB3. Fumonisin B1 is well documented as the primary cause of leukoencephalomalacia in horses. Fumonisin B2 has also been implicated as a possible cause of ELEM, however, the only trial examining this toxin (75 ppm FB2) also had dietary levels of FB1 of 3 ppm (Ross et al., 1994). Two of three ponies consuming this diet for 136 days demonstrated symptoms of ELEM. Ponies receiving a diet containing 75 ppm FB3 and less than 1 ppm of both FB1 and FB2 for 56 days were clinically normal with no differences from control ponies at the time of necropsy. From this trial it is clear that FB3 is not as toxic as FB1 and FB2. The effects of FB2 are not as obvious due to the somewhat high concentrations of FB1 in the diet containing 75 ppm FB2.
In the equine, the consumption of fumonisins produces a brain neurological disease identified by multi-focal liquefactive necrosis of the white matter affecting multiple horses in a herd. Once clinical signs appear, the majority of affected horses die. Horses that survive typically have some degree of permanent neurological disorder. Corn screenings can be heavily contaminated with fumonisins and should never be fed to horses. 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.
Mycotoxins and colic
Barnett and coworkers (1995) studied the relationship between mycotoxins and equine colic. They analyzed feed samples from farms experiencing possible feedrelated colics (n=16) and control farms (n=10) as indicated by the veterinarian. They found DON in the concentrate of 100% of colic cases (range 0.20 to 8.3 ppm) and in 70% of the control group concentrate (n=10) (range 0–2.5 ppm). T-2 toxin at levels greater than 0.5 ppm and ZEN greater than 0.7 ppm were present in 31% and 44% of the colic concentrate samples respectively, while neither were found in control samples. Forage samples were not provided by all farms. Hence, a causal relationship between mycotoxins and colic in horses is not clear, but certainly should be considered a possibility especially when a series of colic cases are observed on a single farm.
Summary
Historically, mycotoxins were identified by their ability to produce severe, overt disease syndromes in animals as a result of relatively high intakes of toxin. However, low levels of toxin exposure over long periods can elicit chronic or subchronic toxicological manifestations. A concern is that for horses, especially elite athletes or breeding stock, exposure to low levels of mycotoxins may affect performance or breeding ability without the appearance of overt signs of toxicity. The outcome from exposure to chronic low levels of toxin may include general ill thrift, suppression of the immune system and increased risk of secondary infections.
Unlike other livestock production species, horses are generally expected to live a long lifespan and are expected to be reproductively sound in their later years. For these reasons, probably more so than for other species, the safe amount of specific mycotoxins is unknown for the horse. In addition to the degree of exposure, incidence of disease can also be influenced by the presence of multiple mycotoxins. Factors influencing susceptibility to mycotoxins include disease, heat stress, marginal nutritional profile, drug interactions, presence of multiple toxins, crowding, age and reproductive status.
Molds and mycotoxins
The occurrence of mold and mycotoxins in food and animal feed is a problem of major concern internationally (Wood, 1992). Mycotoxin production can occur when favorable conditions are met that allow fungi to grow on crops in the field, at harvest, in storage or during the processing of feed (Palmgren and Lee, 1986). Mycotoxins are the products of secondary fungal metabolism although not all fungal growth produces mycotoxins. Environmental and nutritional parameters and growth stage of the mold govern toxin production. Molds that are present in the field before harvest can proliferate if moisture content remains high as a result of inadequate processing and storage conditions. Mold and subsequent mycotoxin contamination of a feedstuff or forage can increase in extreme environmental conditions such as drought, excessive precipitation, or sudden frost. Contamination can also increase due to physical damage to crops that allows fungal penetration of the plant tissue (Gregory et al., 1963; Northolt et al., 1976; Schindler, 1977).
A large number of the predominant mycotoxins are produced by the Fusarium molds, which thrive in temperate climates worldwide and are common contaminants of feed grains. A wet growing season followed by cool weather increases the likelihood that fungi, especially Fusarium and its mycotoxins, will be present in grains. The high moisture level in grain encourages fungal growth while the cool temperatures can increase the production of mycotoxins. Fusarium spp. require a moisture content of 25% concurrent with a relative humidity of more than 90% in order to grow.
Such conditions can commonly occur when field-dried hay is not cured properly. Forage is the basis of most feeding programs for the horse, with long-stemmed, field-dried hay being the traditional source. Any horseman can recognize the dust aerosol when opening a bale of hay that has a high mold count and many individuals equate a high mold count with mycotoxin content. Nevertheless, a forage or feed with a low level of measurable mold contamination can still contain significant amounts of mycotoxins. The subjective appraisal of hay by horse owners has been shown to be unreliable in terms of mycotoxin, mold, and actinomycete contamination (Raymond et al., 2000). Mycotoxin production does not correlate to mold growth and conditions that support one may actually inhibit the other. Increased mycotoxin contamination and the potential for mixtures of mycotoxins to be cocontaminants in feeds are now even more likely due to improved global grain transportation systems and global trading of agricultural commodities.
Conditions favoring mycotoxin production
Aspergillus, Fusarium, and Penicillium are the three most important genera of fungi that have been extensively studied in relation to mycotoxicosis. The large surface area and vigorous activity of fungal mycelia ensure a close relationship of the mold with the environment. Extrinsic factors such as temperature, pH, water activity, and chemical composition have an influence on growth and mycotoxin biosynthesis. Other factors such as plant-fungal interactions, interactions among microorganisms, and the presence of certain chemical agents can influence mold growth and mycotoxin synthesis (Smith and Henderson, 1991).
In the field, specific fungal species may occur cyclically or seasonally. Usually optimal conditions for mold growth do not coincide with those for mycotoxin production. However, the production of several different mycotoxins by the same species, or even the same strain, may not occur optimally under identical conditions. For example, the production of deoxynivalenol (DON) and zearalenone (ZEN) by a single strain of Fusarium graminearum responded differently to changes in temperature. ZEN production reached a maximum at 25oC, whereas the production of DON continued to increase with increasing temperature, and at 28oC the production was more than twice that at 25oC (Greenhalgh et al., 1983). In field situations DON and ZEN have been produced simultaneously in infected corn and wheat by the above fungi (Cote et al., 1985).
Forage toxins
Many studies have shown that forage sources can contain toxins that adversely affect the health or reproductive function of the equine.
FESCUE TOXINS
In the eastern United States, tall fescue is extremely prevalent and represents a major forage source. In 1993, Hoveland estimated that approximately 688,000 horses in the US graze tall fescue. In 1977, Bacon and coworkers first reported the presence of the endophytic fungus now called Neotyphodium coenophialum in tall fescue. The presence of this fungus in consumed grasses has been associated with reduced weight gain, rough hair coats, higher body temperatures, reproductive problems, and necrosis of the foot, tail and ears of horses and cattle. Increases in the number of cycles for conception and early embryonic death have also been associated with endophyte-infected fescue (Brendemuehl et al., 1994). In addition, increased length of gestation, retained and so-called ‘red bag’ placentas, higher numbers of weak or stillborn foals, and agalactic mares were associated with endophyte-infected fescue consumption compared to mares consuming endophytefree fescue (Monroe et al., 1988).
Ergovaline is one of the primary ergot alkaloids produced by N. coenophialum and exerts its activity by acting as a dopamine agonist and suppressing prolactin secretion (Strickland et al., 1994). Decreased serum prolactin is one of the most consistently reported symptoms of fescue toxicosis (Neal and Schmidt, 1985; Elsasser and Bolt, 1987; Redmond et al, 1994).
Ergonovine and ergotamine have been shown to be arterio- and venoconstrictive, which plays a role in thermoregulation since vasoconstriction prevents dissipation of heat by slowing the transfer of internal body heat to the skin or respiratory tract (Abney et al., 1993). The effects of ergovaline and ergotamine are of primary importance in warm climates and in the equine athlete. Trials examining the effects of endophyteinfected fescue on exercising horses found that recovery heart and respiratory rates (post-exercise) were significantly greater than for horses grazing non-infected endophyte. Water consumption post-exercise was also found to be greater for horses consuming endophyte (9.45 gallons vs. 5.59 gallons P< 0.05; Vivrette et al., 2001). The minimum amount of exposure required to observe these effects and the reversibility is not clear.
Symptoms in this trial were observed after 14 days of grazing endophyte-infected fescue. In cattle, the adverse effects were reversed after 8 days of grazing endophytefree pastures (Rhodes et al., 1991). Although endophyte-free fescue is currently available, it is less hardy, and less resistant to overgrazing, insect damage and drought than infected grass. Recent studies in the United States indicate that daily supplementation of mare diets with FEB-200™ (Alltech, Inc.) reduced some of the deleterious effects of endophyte-infected fescue. In these studies, serum progesterone levels were greater in mares receiving FEB-200™ (10.74-ng/dL) compared with unsupplemented mares (9.68-ng/dL). Trials in cattle fed FEB-200™ indicate increased weight gain, and improved pregnancy rates (Akay et al., 2004).
RYEGRASS TOXINS
Perennial ryegrass intoxication or ryegrass staggers is a neurotoxic syndrome characterized by ataxia, lack of coordination, head shaking, and collapse. 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, which is produced by the endophytic fungus Neotyphodium (Acremonium) lolii. Concentrations of lolitrem B are lowest in the leaf blades and highest in the leaf sheath. Thus, ryegrass staggers occur most frequently in over-grazed pastures (DiMenna et al., 1992). Symptoms of ryegrass staggers appear when the lolitrem B concentrations exceed 1.5 to 2.5 ppm. As with fescue, the endophyte improves the vigor of the ryegrass.
LEGUME TOXINS
The ubiquitous soil fungus Rhizoctonia leguminicola can infect certain legumes after heavy rainfall or in situations of high humidity. Although mostly associated with red clover (Trifolium pratense), white clover, alsike clover, kudzu, alfalfa, Korean lespedeza, cow pea, soybean, black medic and blue lupine have also been reported to be susceptible to infection by this fungus (Hagler and Behlow, 1981). Examination of the leaves of the legumes will reveal bronze to black spots or concentric rings ranging in size from pinhead to pencil eraser in diameter. R. leguminicola produces slaframine, an alkaloid that after ingestion is activated by the liver yielding a cholinergic parasympathomimetic agent (Guengerich and Aust, 1977). Toxicosis is manifested by profuse salivation (slobbers) accompanied by increased water consumption. Bloating, piloerection, diarrhea, agalactia, feed refusal, abortions and death in severe cases are also occasionally observed (Sockett et al., 1982). Slobber-response tests in guinea pigs indicate that levels of 50-100 ppm in clover will elicit a response.
The alkaloid is somewhat stable in that it may still be present in baled hay from infected fields but will degrade over time. Studies have shown a decrease in slaframine concentrations from 50 to 100 ppm to approximately 7 ppm after 10 months of storage ( Hagler and Behlow, 1981). Removal of animals from infected pastures to non-infected areas or feeding grass hay will return animals to normal health within 3 to 4 days.
STACHYBOTRYOTOXICOSIS
One of the earliest recognized mycotoxin diseases was stachybotryotoxicosis. In eastern Europe and South Africa the mold Stachybotrys has been isolated from forage sources and straw and implicated in stachybotryotocosis in horses, cattle, swine and sheep. Stachybotrys has also been incriminated in domestic housing in the US as a source of ‘sick house syndrome’. This fungus has been shown to produce macrocyclic trichothecene mycotoxins such as satratoxins F, G and H, verruccarin J, and roidin E (Eppley, 1977; Harrach et al., 1981). Stachybotrys chartarum (formerly S. atra) is a fungus found in the soil and in moist dark indoor environments. This fungus produces black, ‘soot-like’ spores in hay and straw and has been found to grow extensively on moist drywall in houses.
In the early 1930s, the horse was vital to the economy of Russia. In the Ukraine in 1931 thousands of horses were afflicted with an unknown ailment and a team of scientists was sent to investigate. By 1938, it was discovered that the disease was caused by Stachybotrys alternans (also now called S. chartarum) growing on the hay fed to the animals (Rodricks and Eppley, 1974).
In the summer of 1983 and spring of 1984 three stables in Hungary experienced an outbreak of fungal toxicosis where nearly all of the 300 horses at these stables presented with drying and cracking of the facial mucosae, purulent nasal discharge and epistaxis (bloody nose). Post-mortem examination of two of the affected animals showed extensive petechiation of the internal tissue. Bedding from these stables was found to contain satratoxin G (20 to 35 μg/kg), satratoxin-H (75 to 107- μg/kg), and verrucarin-J (35 to 61 μg/kg). Samples of oats fed to horses during the 1984 outbreak were found to contain T-2 toxin, suggesting a synergism between toxins contributing to the intensity of the outbreak (Harrach et al., 1987).
When low levels of toxic feed are consumed, fissures at the corner of the mouth are followed by deeper lesions. Traditionally, the first cases of this disease appear in the autumn when animals are stabled and inhale or consume fodder containing the toxin. There is a subsequent increase in affected animals as the winter months continue into April when the toxicosis begins to decline as horses are returned to pasture and stall confinement is diminished (Forgacs, 1972). The toxins are nonantigenic, therefore no immunity seems to develop from stachybotryotoxicosis and the same horse can become affected several times in the same year. Foals nursing affected mares do not show signs of illness, indicating the toxins are not excreted in milk.
Stachybotryotoxicosis may present itself in either of two forms; the typical form which most commonly occurs in nature and the atypical (shocking) form. The typical form develops progressively with three stages of onset. Stage 1 begins with the aforementioned stomatitis within the first 2 to 10 days of exposure. The fissures of the mouth become necrotic. Edema of the underlying tissues leads to swelling of the lips and perhaps the entire head in what has been described as a ‘hippopotamus-like’ appearance (Forgacs, 1972). In many cases salivation, conjunctivitis, rhinitis, and enlarged lymph nodes develop in the next 120 hrs. In other cases these symptoms are absent and the only specific abnormality may be an increase of body temperature of 1-1.5oC accompanied by neutrophilia for a brief period.
Typically, Stage 1 lasts from 8-12 days but may last for as long as a month. Stage 2 is characterized by changes in blood profiles. The total leukocyte count may drop to 1000-3000 cells/mm3 of whole blood with a concomitant decrease in the relative neutrophil count. Blood clotting time also increases or clotting fails to occur. Body temperature, respiration, and cardiac function remain in the normal range, but in rare cases it may be possible for the clinician to detect atony of the intestinal tract. The second stage duration varies from 5 to 20 days, but may last as long as 50 days. In Stage 3 thrombocytopenia and leucopenia are exacerbated. Leukocyte counts may drop as low as 100/ mm3 and a complete loss of blood coagulation is observed. Hyperthermia is also a distinct characteristic of Stage 3 with the temperature rising to between 40 and 41.5oC. Affected horses may show signs of depression, poor or no appetite, problems swallowing and colic. This stage is usually quite short (1-6 days) and is almost always fatal.
An atypical form of stachybotrytoxicosis develops after ingestion of a large amount of toxin. In this form nervous disorders are the main visible symptoms. The animal will avoid movement and stand with legs in a wide gait or may cross the front legs or lean against objects for support. Swallowing is quite difficult, the pulse becomes weak and arrhythmic (80 to 100 beats/ min), pulmonary edema and cyanosis of the mucous membranes is observed. The animal may become blind. Death usually occurs due to respiratory failure within 72 hrs of ingestion.
OTHER TOXINS FROM FORAGE SOURCES
PR toxin produced by P. roquefortii has been found in forage sources and was the suspected toxic agent in a case study with symptoms of abortion in cattle (Still et al., 1972). In addition, fumigaclavine produced by Aspergillus fumigatus, and Alternaria spp. have been implicated with abortion, small intestinal dysfunction and other health problems (Cole et al., 1977; Pitt and Hocking, 1997).
Raymond et al. (2001) reported that higher levels of DON, ZEN and T-2 toxin were found in forages as compared with grain concentrates during a field study of performance horse farms in Ontario, Canada. Concentrates sampled did not seem to contain significant levels of mycotoxins. A similar survey was conducted by Buckley and coworkers (2004) in which zearalenone and DON were the major toxins found in samples of feed, forage and bedding, with 20-25% of the forage and straw samples containing detectable quantities of these mycotoxins. It is important to emphasize that in all of these studies commercial enzyme-linked immunosorbent assays (ELISA) test kits were used for the analysis. These kits are not validated for the complex matrices found in forage samples. ELISA procedures are prone to false positive results on non-validated matrices and should be confirmed using high performance liquid chromatography (HPLC) and/or thin-layer chromatography (TLC) testing. For this reason, the concentrations of mycotoxins in forage sources are not clear, but there appears to be situations when these toxins are present in relatively high concentrations.
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.
Aflatoxicosis
Aflatoxin is a mycotoxin produced primarily by Aspergillus flavus and A. parasiticus. These molds prefer warm and humid conditions. Mycotoxin production on feedstuffs seems to be most prevalent when drought, insect infestation or other stresses occur late in the growing season of cottonseed, peanuts, corn, or other grain products. In documented outbreaks of aflatoxicosis on two separate horse farms, peanut meal and corn were found to be the sources of the toxin (Angsubhakorn et al., 1981). In one case the elevated price of rice caused feed producers to use stored corn and peanut meal (comprising 70% of the feed mixture) in place of the rice in the feed.
Representative samples of the mixed ration contained approximately 0.2 mg aflatoxin B1 (AFB1) and 0.2 mg aflatoxin B2 per kg. A number of animals showed clinical signs of illness and 12 yearlings died. Clinical symptoms of aflatoxin intoxication include ataxia, tremor, elevated temperature, anorexia, loss of appetite, weight loss, icterus (yellowing of the eyes or skin), hemorrhages, bloody feces, and brown urine (Angsubhakorn et al., 1981; Bortell et al., 1983; Cysewski et al., 1982). The major target organ of aflatoxin is the liver, where the toxin induces centrilobular necrosis (Stoloff and Trucksess, 1979).
Clinical signs of toxicity were observed in ponies dosed with aflatoxin, at levels ranging from 0.075 to 0.3 mg/ kg BW/day (75 to 300 parts per billion (ppb)) with all animals either dying or declining and euthanized during a 39 day trial (Cysewski et al., 1982). A subsequent trial found that ponies given aflatoxin B1 at dosages equal to or greater than 2 mg/kg BW died within 76 hrs (Bortell et al., 1983). At the time of necropsy, ponies demonstrated petechiae of the viscera, free blood in the small intestine, pale tan to yellow livers and icterus. From the data, it appears that horses are more susceptible to aflatoxin B1 than calves. Calves had a reduced rate of gain with 0.2 mg B1/kg/day but died after 14 days given 0.5 mg B1/kg/day (Pier et al., 1976). In ponies, 0.3 mg B1/kg/day caused death at between 12 and 16 days and clinical symptoms and biochemical changes were observed even at 0.075 mg B1/kg/day (Cysewski et al., 1982). It should be noted that other studies in ponies have observed apparent resistance at levels of 0.2 mg B1/kg/day (Walter et al., 1981). Cysewski hypothesized that previous exposure to aflatoxin of the ponies used in the Walter study may have influenced this resistance.
The fact that Walter and co-workers dosed their ponies for 5 days and Cysewski and co-workers dosed for up to 39 days may also have played a role in the differences observed in these two trials. A separate case study of an Arkansas farm where three horses died of severe hepatic necrosis and others became ill found concentrations of 130 ppb total aflatoxin (114 ppb AFB1, 10 ppb AFB2 and 6 ppb AFM1) in the suspected corn (Vesonder et al., 1991). A summary of equine aflatoxicosis cases indicates a very large range from approximately 55 ppb to 6,500 ppb to be associated with clinical symptoms (Asquith, 1985) with duration of exposure and previous history of exposure playing a role in aflatoxicosis.
Recently authors of a study in Sweden have implied a possible link between chronic obstructive pulmonary disease (COPD) and inhaled mycotoxins (Larsson et al., 2003). Data from this study show that activation of AFB1 occurs, not only in the liver but also in the olfactory and respiratory tissues in the horse. AFB1 requires activation by cytochrome P450 enzymes to the reactive intermediate AFB1-8,9-epoxide, which then binds to nucleic acids among other molecules and leads to the carcinogenic and cytotoxic properties normally associated with this toxin. Glutathione (GSH) may also conjugate with AFB1-8,9-epoxide, however, this binding essentially detoxifies the compound (Quinn et al., 1990).
COPD is a disease of the equine lung that is similar to asthma in humans and caused by an allergic response. It is also called ‘heaves’ since horses typically are observed ‘heaving’ to push air out of the lungs at the end of an exhale. COPD is most often observed in older or stabled horses, and is rarely seen in situations where horses are pastured year round. As with asthma in humans, aerosolized particles of pollen, dander, microorganisms or other agents inhaled by a horse with COPD elicit an allergic response. The molds A. fumigatus and Micropolyspora faeni are two heavily implicated organisms that may evoke COPD in horses (Halliwell et al., 1993). In humans, the allergic condition known as ‘farmer’s lung’ has been well established to be caused by inhaling dust from moldy hay and straw (Gregory and Lacey, 1963; Blyth, 1973).
Ochratoxicosis
Ochratoxin was first detected by van der Merwe et al. (1965) in laboratory tests to identify 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. Effects observed in cattle include decreased performance, reduced milk production, kidney failure and death (at levels greater than 800 ppm). The outcome of this toxin in horses is not known but, because of the site of absorption, assumed to be similar to the outcome in pigs. 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 (OA) is only slightly soluble in water and when consumed is absorbed in the upper sections of the gastrointestinal tract. Maximal absorption occurs in the proximal jejunum in a passive manner in the non-ionized form and is then subjected to secretion and re-absorption via enterohepatic recycling (Kumagai and Aibara, 1982; Leeson et al., 1995). Absorption is faster in areas of the gastrointestinal tract with a low pH.
In mammals, ochratoxin A absorption through the lungs into the systemic circulation has also been documented (Di Paolo et al., 1993). Toxicity of OA in ruminants is thought to be relatively low due to the rumen microflora. Hult and co-workers (1976) postulated that levels up to 12 mg OA/kg of contaminated feed are tolerated by ruminants. However, Hohler and coworkers found that even at dosages of 2 mg/kg of concentrate feed, substantial amounts of OA were detected in the serum of cattle fed the toxin (Hohler et al., 1999). Although data in horses are lacking, it is assumed from the site of absorption of this toxin, that horses would have a sensitivity simlar to that of most monogastrics.
Ochratoxin A 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. As with most mycotoxins, ochratoxin is not present in isolation and is commonly found with citrinin, penicillic acid and other mycotoxins in field situations (Ribelin et al., 1978).
Fusarium mycotoxins
Fusarium mycotoxins of concern include trichothecenes, ZEN, fumonisins, moniliformin and fusaric acid (FA). The name trichothecene was derived from the fungus Trichothecium, and these compounds are chemically related sesquiterpenoids that all possess a tetracyclic 12,13-epoxy-trichothec-9-ene ring system.
Deoxynivalenol (also known as vomitoxin or DON) is considered the most common trichothecene but also one of the least toxic. Other prominent trichothecenes include T-2 (considered one of the least prevalent but most toxic), diacetoxyscirpenol, fusarenon-X and satratoxins. Zearalenone, fumonisins, moniliformin and fusaric acid (FA) are classified as non-trichothecene Fusarium mycotoxins. Most trichothecenes can produce clinical signs in the parts per million or parts per billion concentration range. The trichothecenes are known as feed refusal toxins because loss of appetite is one of the first observable symptoms (Trenholm et al., 1994). Loss of appetite, lethargy, sleepiness and loss of muscle coordination are associated with increased tryptophan and serotonin levels by inhibition of hepatic protein synthesis resulting in hyperaminoacidemia (Meloche and Smith, 1995; Wannemacher and Dinterman, 1983). Studies in laboratory animals and pigs have shown enhanced serotonergic activity in the brain as one of the possible mechanisms for DON and FA induced feed refusal, vomiting and other behavioral changes (Smith et al., 1997).
Impaired immune function is also associated with exposure to trichothecene mycotoxins (Rotter et al., 1994). Decreased resistance to a number of infectious organisms has been documented (reviewed by Otokawa, 1983; Venturini et al., 1996) and include Salmonella, Mycobacterium, Listeria, Candida, Cryptococcus and herpes virus. 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). This led the author to suggest that the horse may not be as susceptible to DON and the horse’s gastric microflora may act to inactivate the toxin. Raymond and co-workers advanced this study by using a diet containing naturally contaminated wheat and corn (Raymond et al., 2003a). In this study, the concentrate averaged 15 ppm DON, 0.8 ppm 15-acetyldeoxynivalenol, 9.7 ppm FA and 0.2 ppm ZEN. The horses were consuming 2.8 kg concentrates and 5 kg of mixed hay per day.
The authors concluded that consumption of the contaminated diet reduced feed intake, and serum levels of γ- glutamyltransferase (GGT) were significantly higher in horses consuming contaminated grain sampled on day 7 and 14 of supplementation but not for day 21. An increase in GGT levels indicates a possible effect on liver function without obvious signs of damage since GGT is a hepatic membrane-associated enzyme. The increase in GGT associated with mycotoxincontaminated feed was not seen when contaminated diets were supplemented with a mycotoxin adsorbent (Mycosorb®, Alltech, Inc.). In addition, Mycosorb®- supplemented animals had improved intake compared to those given unsupplemented contaminated diets (1.64 kg vs 1.00 kg, P< 0.05). A similar study was conducted by the same authors incorporating an exercise program (Raymond et al., 2003b). Results were similar although in this trial significant weight loss was observed over the 21 day supplementation period in horses fed contaminated grains as compared to control animals.
The differences observed between the Johnson study and those of Raymond and co-workers may be explained by a synergistic effect between low levels of different toxins. Toxicological synergism has been documented among Fusarium mycotoxins (Smith et al., 1997).
Studies 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 DON are fed to starter pigs (Smith et al., 1997). Although FA was chemically characterized years ago, it has not been considered a significant factor in Fusarium mycotoxicoses because of its relatively low toxicity.
Fusaric acid is a pharmacologically active compound that alters brain neurochemistry in a wide range of animal species. The physiological effect of FA is a drop in blood pressure. Such an effect could be of significance for the horse as one of its production parameters is athletic performance. Like DON, FA is associated with increased brain serotonin levels but through a different mechanism. It has been demonstrated in pigs that fusaric acid and DON act synergistically to reduce feed consumption and trigger loss of muscle coordination and lethargy (Smith et al., 1997).
Horses can be exposed to DON and/or FA from corn or wheat products in grain concentrates and/or from forage or wheat straw bedding. Contamination of bedding material represents a risk through ingestion, inhalation and dermal contact. An unplanned DON concentration of 1.2 ppm in the straw bedding in the previously described feeding trial is an illustration of the potential for mycotoxin contamination from bedding sources (Raymond et al., 2003a). The amount of DON in straw depends primarily on the presence of contaminated grain and chaff, two sources known to have more concentrated levels of mycotoxins. Although forage represents a possible source of contamination, the forage component in the diet may provide some protection from mycotoxin exposure. High concentrations of plant fiber have been shown to reduce some of the deleterious effects of mycotoxins (Carson and Smith, 1983; James and Smith, 1982).
A published case report cited weight loss and elevated hepatic enzymes in horses with the probable cause being straw contaminated with DON. It was reported that in 2002, approximately half of 104 Warmblood-type riding horses stabled in Germany suddenly lost weight. Further examination of nine of the affected horses revealed marked elevation of liver enzyme activity of both GDH and GGT. Analysis of the feed, hay and bedding revealed DON concentration ranging from 0.5 to 2.7 ppm in the straw. Once the horses were removed from the contaminated bedding they slowly gained weight and general condition became progressively better (Zeyner et al., 2002).
FDA levels of concern for DON are 2 ppm for wheat entering the milling process for humans and 1 ppm for the finished product for humans. The concern level for DON in wheat for livestock is 4 ppm (Wood, 1992). 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 for horses be comparable to levels allowed for humans.
There are few studies on the effects of T-2 toxin in the horse, with only one report involving the feeding of T-2 toxin. Administering 7 mg of purified T-2 toxin per os daily to mimic a 1 ppm concentration in feed had no effect on the ovarian activity of mares (Juhasz et al., 1997). In other species, oral and intestinal lesions are frequently observed in animals consuming T-2 or diacetoxyscirpenol (DAS) at levels as low as 100 ppb for a period of 25 days (Sklan et al., 2001).
Zearalenone is an estrogenic non-tricothecene mycotoxin produced by Fusarium. Zearalenone binds to estrogen binding sites and causes enlargement of the uterus and rectal and vaginal prolapse, abortions and infertility with pre-pubertal animals appearing to be more sensitive than older animals and swine being the most susceptible livestock species (D’Mello et al., 1999; Kalleta and Ettala, 1981). Because the symptoms of ZEN toxicity are quite specific, it is more easily recognized than trichothecene toxicity.
Studies by Juhasz et al. (2001) found that ZEN administered to mares for 10 days to mimic a 1 ppm concentration in the feed found no adverse effect on the reproductive parameters of cycling mares but skin lesions were observed around the mouth in 3 of 6 horses.
An earlier study by Gimeno and Quintanilla (1983) reported a natural outbreak of ZEN mycotoxicosis on a commercial horse farm. In this case, corn screenings fed to the horses were found to contain approximately 2.7 ppm ZEN. No other common mycotoxins were found. Corn screenings fed to a herd of horses on a commercial farm produced strong estrogenic symptoms after a feeding period of 30 days. Symptoms in the mares included feed refusal, prolapsed uterus and internal hemorrhage. In male horses, severe flaccidity of the genitals was observed. Reports of feeding ZENcontaminated diets to pigs indicated that 1 ppm is the minimum concentration required to produce hyperestrogenism (James and Smith, 1982) and from the above data it can be inferred that pigs are more sensitive to ZEN than horses. In cattle, traditionally considered more resistant to the effects of ZEN, abortions have been attributed to ZEN-contaminated hay (Kalleta and Ettala, 1981).
Fumonisins are produced primarily by Fusarium moniliforme and F. proliferatum, the Fusarium species that invade and are prevalent in corn. Fumonisins are recently discovered mycotoxins 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) (Harrison et al., 1990). Fumonisin in horses can cause equine leukoencephalomalacia (ELEM), which is typified by staggers, stupor, lameness, seizure (due to brain necrosis) and death. Feed associated with PPE and ELEM contained levels of fumonisin B1 (FB1) ranging from 1 to 330 ppm (PPE) and 1 to 126 ppm (ELEM) (Ross et al., 1991).
Additional studies (Ross et al., 1992) suggested that fumonisin B1 concentrations greater than 10 ppm in horse feeds made those animals consuming the feed likely candidates for ELEM. Although naturally contaminated feeds containing fumonisin have been shown to elevate brain serotonin levels, it appears that fumonisin is not the source of this activity. Smith and MacDonald (1991) demonstrated that FA increased tryptophan, serotonin and 5- hydroxyindoleacetic acid in pigs. Previous work by Porter and coworkers (1990) examined corn associated with ELEM on tryptophan levels in rats and found elevated levels, however, these investigators did not measure FA levels in the corn. Subsequent work by Porter did not show similar effects with purified FB1 (Porter et al., 1993). It seems probable that fumonisin does not affect brain serotonin levels, other toxins produced along with fumonisin seem to be responsible for this activity.
Fumonisins interfere with sphingolipid metabolism, disrupting endothelial cell walls and basal membranes (Van der Westhuizen et al., 2001). Sphingolipids are one of the primary groups of lipids in cell membranes. They have multiple functions, including signal transduction, cell growth, cell-to-cell communication, immuno-recognition, and aid in defining the physical nature of lipoproteins. There are three common fumonisin mycotoxins designated as FB1, FB2 and FB3. Fumonisin B1 is well documented as the primary cause of leukoencephalomalacia in horses. Fumonisin B2 has also been implicated as a possible cause of ELEM, however, the only trial examining this toxin (75 ppm FB2) also had dietary levels of FB1 of 3 ppm (Ross et al., 1994). Two of three ponies consuming this diet for 136 days demonstrated symptoms of ELEM. Ponies receiving a diet containing 75 ppm FB3 and less than 1 ppm of both FB1 and FB2 for 56 days were clinically normal with no differences from control ponies at the time of necropsy. From this trial it is clear that FB3 is not as toxic as FB1 and FB2. The effects of FB2 are not as obvious due to the somewhat high concentrations of FB1 in the diet containing 75 ppm FB2.
In the equine, the consumption of fumonisins produces a brain neurological disease identified by multi-focal liquefactive necrosis of the white matter affecting multiple horses in a herd. Once clinical signs appear, the majority of affected horses die. Horses that survive typically have some degree of permanent neurological disorder. Corn screenings can be heavily contaminated with fumonisins and should never be fed to horses. 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.
Mycotoxins and colic
Barnett and coworkers (1995) studied the relationship between mycotoxins and equine colic. They analyzed feed samples from farms experiencing possible feedrelated colics (n=16) and control farms (n=10) as indicated by the veterinarian. They found DON in the concentrate of 100% of colic cases (range 0.20 to 8.3 ppm) and in 70% of the control group concentrate (n=10) (range 0–2.5 ppm). T-2 toxin at levels greater than 0.5 ppm and ZEN greater than 0.7 ppm were present in 31% and 44% of the colic concentrate samples respectively, while neither were found in control samples. Forage samples were not provided by all farms. Hence, a causal relationship between mycotoxins and colic in horses is not clear, but certainly should be considered a possibility especially when a series of colic cases are observed on a single farm.
Summary
Mycotoxins in horse diets are well documented for their ability to cause acute, severe diseases. However, the effects of mycotoxins are much more complicated. Subclinical problems such as a decrease in appetite, loss of performance and decreased reproductive capacity are non-specific disorders that can be associated with mycotoxin ingestion. Mycotoxins may be unavoidable contaminants in feeds and forages due to the unpredictability of pre-harvest contamination of susceptible crops by fungi. Various genera of fungi can produce mycotoxins whenever optimal conditions of temperature, humidity and suitable substrate prevail in a given area. It can be argued that given the complex interactions of mycotoxins with feed ingredients, varying exposure situations and the health of the individual horse, safe levels cannot be identified and that no levels of mycotoxins can be demonstrated as safe in a field situation. Nevertheless, with increased awareness, levels of toxins can be reduced. Feeds and forages should be tested to allow for proper management. Compounds with demonstrated mycotoxin-binding capabilities can then be applied to reduce equine health risk from mycotoxin exposure.
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Author: KYLE E. NEWMAN
Venture Laboratories, Inc., Lexington, Kentucky, USA
Venture Laboratories, Inc., Lexington, Kentucky, USA
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7 de abril de 2008
A good review.
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