Mycotoxins in Forages and Their Llllpact Onanilllal Health

Mycotoxins in Forages and Their llllpact onAnilllal Health

Gary Osweiler, DVM Veterinary Diagnostic Laboratory College of Veterinary Medicine Iowa State University Ames, IA 50011

Abstract mold. Secondary metabolites are produced sporadically in response to stress. Not all secondary metabolites are Moldy and damaged forages occur commonly and are toxic. Mycotoxin occurrence is often associated with widely fed to ruminants. Major mycotoxins associated with geographic areas that support environmental condi forages include the endophyticfungi responsible for fescue toxicosis, the tremorgens, slaframine associated with red clo tions for mold growth and concurrent stress of the ver, stachybotryotoxin in straw, sporodesmin, and ergot alka fungus. Table 1 indicates regions ofNorth America most loids from the grasses. Effects from forage associated mycotox likely to support growth of fungi that produce feed and ins are seldom acutely fatal, but cause effects including re forage mycotoxins. duced gain, lowered milk production, peripheral gangrene, ataxia and tremors, excessive salivation, gastroenteritis, pho Fungi offeed grains that grow on susceptible crops tosensitization and dermal necrosis. Clinical effects can mimic prior to harvest are known as field fungi. Among these, other common disease conditions and diagnosis depends on Fusarium is the most common producer of several im thorough review of history and circumstances as well as portant mycotoxins. Fusarium generally requires rela clinical and laboratory evaluation. Currently, analytical tests are used to confirm presence of the toxins, but are not rou tive humidity above 90% and rarely grows after harvest tinely available on a routine basis. Common feed grain myc because it is usually below critical moisture needs. otoxins (aflatoxin, zearalenone, trichothecenes) are occasion Growth in storage generally requires moisture concen ally reported from forages, but generally are not a clinical or trations of 22-25%. production problem associated with forages. Most of the for 0 Storage fungi (e.g. Aspergillus and Penicillium) "'O ages related mycotoxins are associated with well defined (D climatic, weather and cultural conditions; thus often can be typically do not invade intact grain prior to harvest ~ avoided by appropriate cultural and management practices. unless stress conditions such as drought or insect dam ~ Prevention is the key to reducing losses since specific effective (") age predispose them to infection in the field. Fungal (D antidotes are not available. 00 growth requires a proper substrate (carbohydrate), mois 00 0...... ture, oxygen and adequate temperature for growth. 00 General Considerations ,-+- Formation of some toxins, such as aflatoxin may require '"i prolonged periods where the minimum daily tempera ~ The spoilage of feeds and forages by fungi depends ~ ture does not fall below a critical level. This critical ..... on both the environment and the substrate on which the 0 minimum temperature combined with stress conditions p fungus grows. Additionally, the invasion of forages by related to insects and drought or heat stress is why certain fungi growing within the plant structure itself aflatoxin is primarily a problem in tropical or subtropi may establish a symbiotic relationship beneficial to both cal regions. organisms. These fungi, known as endophytes, occur in fescue and rye grass where they induce formation of Biologic Effects of Mycotoxins toxins detrimental to livestock. In some of the well known forage mycotoxicoses, fungal infection and myc The biologic effects of mycotoxins result from their otoxin production are not apparent as a pathogenic interference with basic biochemical or hormonal pro effect on the plant. Unfortunately, compared tomycotox cesses in the cell. Some mycotoxins affect metabolic and ins of feed grains, relatively little is known about the anabolic processes by inhibiting mitochondrial function toxic animal effects of forage fungi. This discussion will and altering enzymes that promote carbohydrate me cover major effects of known mycotoxins in forages, with tabolism. Others interfere with lipid metabolism, reduc some comparisons to the more familiar grain-related ing fatty acid and cholesterol biosynthesis. A third major mycotoxins, and will suggest approaches to prevention biochemical effect ofmycotoxins is impaired nucleic acid and management of these problems as part of herd function resulting in reduced or altered nucleic acid health management. orprotein biosynthesis. This effect on protein synthesis Mycotoxins are toxic secondary metabolites pro reduces the synthesis of a multitude of enzymes, and the duced by fungal (mold) growth. A secondary metabolite effects may not be manifest until existing enzymes are is not essential to normal structure or function of the

JANUARY, 1994 55 Table 1. Quick Guide to Mycotoxins That Affect Livestock

Geographic Disease Toxin Reference Substrates Fungus Manifestations (Q) n Aflatoxin South-East and Cottonseed Aspergillus flavus Hepatotoxicosis, hepatic carcinogenesis, 0 B1, B2, G1, & G2 South Central Corn Aspergillus parasiticus cholangio-hepatitis, hemorrhage coagulopathy. '"a '-< USA; From drought Peanuts Slow growth, poor feed conversion, reduced ...... >-i (JQ and insect stress Sorghum milk production. Milk residues are regulated. ~ ..-+- Fumonisins Corn Belt and all Corn Fusarium moniliforme Cattle tolerate up to 50 ppm with no adverse > effects. Above 100 ppm may cause liver damage 8 USA where corn (D is grown and immune suppresssion ...... >-i (") ~ Ochra toxin A North Central USA Corn, Wheat Aspergillus ochraceus Nephrotoxicosis with polydipsia and polyuria. ~ and Canada Peanuts Penicillium viridicatum Weight loss and anorexia. 00> 00 Citrinin North Central USA Wheat, Rye Penicillium citrinum Similar to ochratoxin 0 and Canada Oats, Barley (")...... a...... Trichothecenes North Central Corn Fusarium sporotrichioides Feed refusal, diarrhea, weight loss, 0 (e.g. T-2 toxin, USA, Canada, Barley necrotic dermatosis. Lympholysis and ~ 0 diacetoxyscirpenol, Appalachia Wheat thymolysis. Feed refusal is most 1-i; deoxyni valenol and Mid-South Sorghum common sign in natural exposure. Some to 0 vomitoxin, DON) data show reduced milk production. <...... ~ Zearalenone (F-2) North Central Corn Fusarium roseum Estrogenic vulvovaginitis in gilts, anestrus (D ~ and Zearalenol and Central USA, Sorghum infertility, and early embryonic death in sows. >-i ~ Canada Wheat Mammary enlargement and infertility in cows. (") ..-+-...... Tremorgenic mycotoxins Pacific Coast & Corn, Peanuts Penicillum cyclopium Tremors, convulsions, low mortality, ..-+-...... 0 (Penitrems, North Central Pecans Aspergillus spp. Recovery when source removed ~ (D fumi tremorgen) USA Walnuts >-i 00 Tunicamycin and Pacific Northwest Perennial Acremonium lolii Stiffness, incoordination, tremors, 0 '"a Lolitrem B and California ryegrass hypermetria, opisthotonus, seizures, recumbency, (D high morbidity and low mortality. ~ ~ (") Penitrems South Central and Dallis grass Paspalum dilatatum Tremors, ataxia, excitement, seizures, recumbency. (") (D South-East USA Rapid recovery when source is removed. 00 00 Ergotamine and North & Central USA Rye Claviceps purpurea Gangrene of the extremities; convulsions, ataxia, 0...... 00 related alkaloids (Small Grain Area) Cereal grains and tremors, agalactia due to reduced prolactin. ..-+- ...... >-i cr' Ergovaline South-East USA Fescue forage Acremonium coenophialum Heat intolerance, infertility, reduced prolactin I= ..-+-...... Slaframine Midwest & Eastern Red Clover Rhizocotonia leguminicola "Slobbers syndrome", salivation, lacrimation, 0 to North-East USA anorexia, diarrhea p Dicoumarol Entire USA Common Penicillum sp Hemorrhages due to anti-Vitamin K Sweet clover Mucor sp effect in liver. Abortions in late gestation, Humicolor sp lameness, hematomas depleted. Thus many mycotoxicoses are not apparent Diagnosis of Mycotoxicoses until after several days or weeks of exposure. Certain mycotoxins cause profound neurochemical or endocrine Mycotoxicoses are difficult to differentiate clinically effects, altering behavior, appetite and reproductive from other common disease or management problems function. Finally, several major mycotoxins are immu and may be occurring concurrently with them. When nosuppressive and may increase susceptibility to infec mycotoxins cause acute disease or large death loss, close tious diseases or reduce response to vaccinations. evaluation of the clinical history and feed supply are Metabolism or biotransformation of a mycotoxin usually necessary to suggest tentative diagnoses. Gen can lead either to activation or detoxification, depending erally the diagnosis cannot be confirmed without some on the mycotoxin. Metabolized aflatoxin is more toxic diagnostic assistance. This is especially true when and carcinogenic than the original Aflatoxin Bl while mycotoxicoses present as chronic conditions related to metabolism of the trichothecenes (e.g. T-2 toxin) reduces poor performance, ill thrift or increased susceptibility to their toxicity and increases their excretion. infectious disease.

56 THE BOVINE PROCEEDINGS-NO. 26 Mycotoxicosis are often sporadic both seasonally storage bin. These aliquots are combined and thor and geographically, so epizootiology is often difficult to oughly mixed before submission for analysis. assess quickly. Due to uneven distribution within grain Forage samples present even greater challenges in or feed supplies, the offending agent may have been sampling because plants are relatively large units, and consumed prior to investigation. differing cultural or microenvironmental conditions in a While analysis for major mycotoxins in feed and field may affect stress and mycotoxin formation. Sam forages is commonly practiced, detection of toxins in pling pasture forages should be done by establishing a tissues is not routinely available, limiting the confirma grid throughout the pasture and randomly selecting tion ofmycotoxin induced disease. Furthermore, many several samples from each section of the grid. These can mycotoxins are rapidly excreted from animals, so lack of be analyzed as a composite, by grid section or as indi consumption for several days prevents residue detec vidual samples which appear questionable. Silage can tion even when analytical tests are available. In some be sampled by an approach similar to grains. Multiple cases, diagnosis may depend on test feeding of the samples can be collected from baled hay using a core suspected ration with reproduction of the characteristic sampler. mycotoxic disease. When test feeding is used, the best Samples for mycotoxin analysis may change greatly approach is to use animals of the type and age affected if not properly preserved and shipped. Grain or forage by the suspect mycotoxin. They should be offered sus samples of greater than 15% moisture should be oven pect forage for at least two to three weeks to account for dried at 60°C and shipped in paper or cloth bags, or high the potential delayed effects of the toxins. Test param moisture samples can be frozen and shipped to arrive eters should include feed consumption, weight gain, still frozen. Remember that some molds can grow at clinical observation and a minimal clinical chemistry refrigerator temperatures, and silage when exposed to panel for liver and kidney function. In many cases it will air in a plastic bag can mold rapidly. For long term be impossible to recreate all the conditions present at the storage, dry the samples to 12% moisture and store in time of a clinical mycotoxicosis. moisture proof containers.

Sampling and Submitting Feeds for Selection of Laboratory Analyses Laboratory Analysis 0 Mycotoxins are complex and varied organic chemi "d (ti When feeds or forages are purchased, owners should cals. Only recently have state and private laboratories ~ P:l be advised to retain an identified sample of the feed stuff developed analytical tests that are accurate, precise and 0 0 (D for up to several months after use has begun. Often sensitive. Often an effective analysis is confined to en when purchased feeds are suspected, there is no remain certain matrices such as corn or feed grains, and when en 0.. ing representative sample for testing, leaving only the it is applied to forages, the pigments and oils can inter -·rJ). recollection of the parties involved as evidence on which fere with the analysis, causing failed detection and/or s-:~ to base a decision. false positives. With the advent of radioimmunoassay g Samples of feed or forage for analysis or feeding and ELISA technologies, several commercially avail 0-· trials should reflect all sources of feed available at the able test kits have been utilized (Table 2). These are ~ time the problem occurred. Individual plants or seeds generally only for screening purposes to detect presence can be heavily contaminated with mycotoxins and be of a mycotoxin at levels above a specified detection limit. responsible for measurable levels of toxin within a large In some cases, cross reactivity can occur with similar lot offorage or grain. Represen ta tive sampling offorages compounds, and quantitation is difficult. They can serve or grain is important since contamination can vary a valuable role in providing rapid high-volume screen widely among different sites in the same field or supply ing, but should not currently be considered as supplant of stored forage. ing traditional chemistry methods. Mold activity (even molds known to produce toxins) Laboratory determination of mold counts or isola is no sure indicator ofmycotoxicosis. Temperature, sub tion of molds by genus only is generally not considered strate, moisture, and number of contaminating molds adequate diagnostic confirmation of a mycotoxicosis. all may affect toxin production. Mold spores are common in feedstuffs, and only when For grains, representative samples may be taken conditions are proper for their growth and toxin produc from storage units in two ways: (1) stream sampling, tion do they have the potential for harm. When mold where a large quantity of grain is transferred by auger populations are mixed and heterogeneous, there may be or gravity and periodic samples are taken from the less probability ofmycotoxin formation than when only moving stream, combined, mixed and an aliquot submit one toxin producing species and strain are present. ted for analysis, and (2) probe sampling, where a grain When selecting a laboratory, look for an organiza probe is used to select individual aliquots from a large tion that provides a written schedule offees and services

JANUARY, 1994 57 and that provides information about their quality control Specific Forage Mycotoxicoses program, the qualifications of their staff, and where they are accredited. For example, state veterinary diagnostic Several mycotoxins are associated with forages in laboratories are accredited by the American Association North America. In some cases they are responsible for of Veterinary Laboratory Diagnosticians. In addition, widespread disease and economic loss (e.g. fescue the laboratory should be able to provide instructions in toxicosis) while others appear to occur only rarely (e.g. collection and preservation of samples and interpreta ryegrass staggers). Numerous questions arise about tion of the results in the context of the samples being moldy forages with post-harvest mold infestation. Un analyzed and the source of the specimens. Finally, the fortunately little definitive information is available to laboratory should be willing to inform you when your evaluate the effects of post-harvest fungal infestations sample is inadequate or improperly submitted. to specific livestock problems. In such cases, attention to the general principles of evaluating and managing Table 2. Commercially AvailableMycotoxin Test Kits* mycotoxin problems (i.e. careful history and appropri ate test feeding) can aid in practical resolution of imme Mycotoxin Name Methodology Sources diate animal health issues. The mycotoxins commonly associated with feed Aflatoxin Aflatest Affinity column VICAM Afla20 ELISA International grains (e.g. aflatoxin, DON or vomitoxin, zearalenone, Diagnostic ochratoxin or citrinin, and fumonisins) are rarely found Agri Screen ELISA N eogen Corp. in forages, and when reported are generally at relatively Cite-Probe ELISA IDEXX low levels. This paper will discuss, for comparative EZ Screen ELISA Environmental purposes, the common grain mycotoxins. However, Diagnostics Total Aflatoxins Affinity column BioCode, Ltd. their presence in molded forages is not expected unless grain is part of the harvested forages. Examples are Aflatoxin M1Test ELISA Transia Target Selective Absorption TerraTek ergot infested small grains incorporated in straw, and HV Mini column Mini column Romer Labs, Inc. green chop or silage where the grain mycotoxins are part DON of the chopped forages. When TMR diets are used, the (Vomitoxin) Agri Screen ELISA N eogen Corp. separation and detection of source of mycotoxin may be 0 "'O DON Romer Labs, Inc. difficult. (D ~ Fumonisins Agri Screen ELISA Neogen Corp. ~ Fumonitest Affinity column VICAM Major forage related mycotoxicoses in North ('.") (D America include the following: 00 Ochratoxin VICAM 00 Ochratest Affinity column • Fescue toxicosis 0...... T-2 Toxin T-2 ELISA N eogen Corp. 00 • Ryegrass staggers ,-+- T-2 Mini column Romer Labs,Inc. '"i • Paspalum staggers ~ Zearalenone Zearalatest Affinity column VICAM • Sweet clover disease ~...... Agri Screen ELISA Neogen Corp. • Slobbers syndrome (Slaframine toxicosis) 0p Zearalenone Mini column Romer Labs, Inc. • Photosensitization from Pithomyces chartarum *Manufacturers and/or sources of commercial mycotoxins Recognition of Mycotoxin Related Diseases Fescue toxicosis Fescue grass (Festuca arundinaceae Schreb.), also Diseases caused by mycotoxins may be difficult to commonly known as Tall Fescue is a major forage grass recognize, since many are chronic, subtle and of delayed over approximately one-third of the USA involving an onset. Conversely, they are specific chemicals and usu estimated 35 million acres. It is readily established, and ally cause somewhat consistent effects that may be provides good yields and favorable nutrient content altered by dosage received. Mycotoxic diseases may while tolerating poor quality soils, drought, heat and sometimes be regional and correlated with local weather insects. Much of the early plantings of fescue ("Ken or cultural conditions, while in other cases poor storage tucky 31") co-existed with a fungus living within the conditions allow for contamination of the feed supply in fescue plants. This endophytic fungus, Acremonium only one herd. Careful investigation may be necessary to coeniphialum does not affect appearance of the plant establish a relationship with a specific feedstuff. This is and actually confers on the fescue some beneficial effects especially difficult, since some effects may not be appar such as increased drought resistance and insect toler ent for several days or weeks after consumption, or they ance. Acremonium coenophialum is considered a mutu may be delayed and not recognized until the contami alistic mycosymbiont and natural agent of biological nated feed is unavailable to be sampled. protection of tall fescue. (Tsai et al 1992) The endophyte

58 THE BOVINE PROCEEDINGS-NO. 26 is transmitted through infected fescue seed. Recently, Laboratory analyses have shown reduced serum endophyte-free strains of fescue have been developed prolactin levels. Endophyte toxins may reduce prolactin and made commercially available, allowing producers to synthesis and release and may alter activity of dopam develop management strategies to alleviate fescue inergic neurons (Schillo et al 1988). Imbalances of pro toxicosis. Three major livestock disorders are associated lactin and melatonin resulting in reduced blood flow to with fescue. These include fescue foot, bovine fat necro internal organs are reported as potential causes of sis and fescue toxicosis ("summer syndrome") (Ball et reduced reproduction, growth, and maturation in live al1991). stock consuming endophyte-infected tall fescue (Porter Fescue foot is a dry gangrene of the extremities, and Thompson, 1992). affecting primarily the feet, tail and ears. Severity Recently, several researchers have shown that varies depending on in take ofinfected forage and weather ergopeptide alkaloids found in endophyte-infected fes conditions. Cold weather accentuates the severity of cue are associated with the fescue toxicosis syndrome. clinical signs and may increase the incidence of periph Ample evidence exists as well that ergot alkaloids affect eral necrosis. Ergopeptide alkaloids are produced in the prolactin release. When total ergovaline consumption endophyte-infected plant. Temperature and blood flow per cow was between 4.2 and 6.0 mg/day, decreased in the extremities are reduced, leading first to lameness performance (body weight) was not fully accounted for followed by separation of the hoof at the coronary band by reduced forage intakes, leading to the conclusion and dry gangrene very similar to classical gangrenous that nutrient utilization may be reduced by endophyte ergotism. The toxin(s) associated with endophyte-in infected fescue (Thompson et al 1992a) . Prolactin is fected tall fescue reduce blood flow to peripheral and associated with the onset of milk production and in core body tissues, and the effect persists for up to 8 days creases when environmental temperature is elevated. after removing the toxin(s) from the diet (Rhodes et al Studies on laboratory animals suggest potential 1991). Lameness is commonly observed to begin in the immunosuppressive effects of endophyte-infected fes rear limbs of cattle. Currently there are no antidotes for cue. Ratshad reduced serum titers to sheep red blood fescue foot. Infected pastures are more a problem if cell (SRBC) and lowered white cell counts. In addition, stockpiled for winter grazing, and mowing to reduce the spleen cells from mice fed endophyte positive diets had accumulation of seed heads seems to reduce incidence reduced response to the mitogens Concanavalin A and 0 and severity of the lesion. Known infected fescue should lipopolysaccharide (Dew et al 1990). These potential "'O (D avoided or limited during cold weather. immunologic effects in rodents have not been demon ~ Bovine fat necrosis is a condition reported mainly strated in livestock. ~ (") from the Southeastern USA and has been associated Several treatments have been investigated which (D 00 with nearly pure stands offescue that are heavily fertil show promise of reducing the effects offescue toxicosis. 00 ized with nitrogen fertilizer or poultry litter. The lesion These are reviewed briefly, even though they are cur 0...... 00 ,-+- consists oflarge masses ofhardened fa tin the abdominal rently not approved nor in general clinical use. '"i cavity. Necrotic fat is calcified and higher than normal Animals treated with metoclopramide grazed ap ~ ~...... fat in ash, calcium, magnesium and cholesterol. These proximately four times longer between noon and 4 PM 0 may cause physical interference with intestinal motility than controls offered the same toxic fescue pastures. p or with calving, but the overall incidencem of economic Results suggest that dopaminergic processes may be loss is relatively low. important in fescue toxicosis (Lipham et al 1989). Fescue toxicosis, also known as "summer syn Metoclopramide administration also increased serum drome" or "summer slump"' is the most economically prolactin in cattle fed endophyte-infected fescue damaging of the fescue problems. Animals grazing (Thompson et al 1992a). Alkaloids in ingested endo infected pastures during periods of high ambient tem phyte-infected tall fescue appear to induce thiamin peratures commonly spend less time in daytime grazing deficiencies in cattle that result in symptoms of tall and have reduced forage in take resulting in lower weight fescue toxicosis (Dougherty et al 1991). Thiamin supple gains and reduced milk production. In severe cases, mentation appears to alleviate some of the effects of agalactia may occur. In addition, signs of heat intoler grazing on endophyte-infected tall fescue during warm ance include increased respiratory rate, higher body weather (Lauriaultet al 1990). Steers fed Ammoniated temperatures and more time spent in shade or water. toxic fescue hay had significantly higher serum prolac Although heat intolerance is a factor in fescue toxicosis, tin concentrations and lower rectal temperatures than poor weight gains can occur on endophyte infected pas steers receiving only endophyte-infested fescue hay tures during cool weather as well. Some affected animals ( Kerr et al 1990). Cimetidine ( a histamine H2 receptor show increased salivation. Cows have reduced reproduc antagonist) also lowered body temperatures to control tive performance and fertility, indicated by increased levels by day 4 in sheep fed Endophyte-infected fescue postpartum intervals and reduced pregnancy rates. hay (Zanzalari et al 1989), although this approach is not

JANUARY, 1994 59 yet clinically practicable. Recently, treatment with lar in some respects to ryegrass staggers, but these ivermectin pouron has increased gains for steers grazed chemical causes can be confirmed by analytical testing. on toxic fescue pastures (Bransby, 1993). Resolution ofryegrass staggers can be enhanced by removing affected animals from contaminated pastures Perennial Ryegrass Staggers to a quiet, protected location and supplementing with Perennial ryegrass, Lolium perenne, has recently clean feed. There is no specific antidote. Prevention of been documented in the occurrence of a tremorigenic future poisoning could include diluting the forage with condition in the Pacific Northwest. The condition, known other hay or grain, avoiding overgrazing, and pasture as ryegrass staggers has long been recognized in Austra renovation with endophyte-free strains of perennial rye. lia, New Zeland and Europe. It usually occurs on intensively grazed pastures during late summer or fall. Dallis Grass Staggers (Paspalum Staggers) An endophytic fungus,Acremonium lolii, has been asso Dallis grass staggers, also known as Paspalum ciated with the toxic grasses, and the toxins lolitrem-B staggers, is caused by an ergot (Claviceps paspali) of and tunicamycin have been implicated as the causative Dallis grass (Paspalum dilatatum). The Dallis grass agents. Increasing levels ofinfection are correlated with ergot is a pale tan cauliflower shaped sclerotium two to higher concentrations of the toxins. Concentration of the three times the size of the Dallis grass seed. Infection endophyte and toxin are greatest in the lower leaves and occurs in late summer. stems of toxic pastures. Tremorgenic toxins similar to penitrem A affect Clinical signs of ryegrass staggers occur after one mainly cattle, but horses and sheep are also susceptible. to two weeks of continuous exposure. Morbidity may be Morbidity is high, but mortality is usually less than 10 high (10-75%) in a herd, but deaths are uncommon if the percent and clinical signs occur from two to three days of problem is recognized and animals are removed to un exposure to as little as 3 grams of ergot bodies per contaminated pastures. Clinical signs appear related to kilogram body weight. Clinical signs are similar to disturbances in excitatory amino acid neurotransmit ryegrass staggers, but seizures may be more severe and ters such as GABA. The signs are typical of the affected animals may be belligerent or aggressive tremorgenic mycotoxins, ranging from mild and subtle (Nicholson, 1989). when animals are at rest to exaggerated and severe 0 Diagnosis of Dallis grass staggers is suggested by "'O when animals are excited or forced to exercise. Signs (D observation of the characteristic clinical signs and detec ~ progress from fine tremors about the head and neck to tion of the typical ergot sclerotia in pasture or hay. ~ (") stiffness, incoordination, hypermetria, tremors, No specific antidote is available for Dallis grass (D 00 opisthotonus and seizures. If animals are left undis staggers. Animals should be kept quiet and in a pro 00 turbed, signs subside in five to ten minutes. Recovery tected and secure enclosure. Affected animals recover in 0...... 00 ,-+- occurs one to three weeks after ingestion of the infected a few days if clean feed is given. Preventive measures '"i ryegrass stops. Deaths are rare, except from accidental should include clipping the pasture or close grazing to ~ events such as trauma or drowning during the acute ~...... prevent seed formation which serves as a substrate for 0 seizures. The condition is considered reversible, even the fungus. p though some reports describe permanent neurologic lesions in experimental animals (Nicholson, 1989; Galey, 1991). Sweet Clover Disease Diagnosis of ryegrass staggers has been confirmed Both yellow sweet clover (Melilotus officinalis) and by use ofmouse bioassay, identification of the endophyte white sweet clover (Melilotus alba) contain coumarin in forage plants, and chemical analysis of plants to which can be dimerized by fungal activity to form detect the tremorgenic toxins. Samples for examination dicoumarol. Dicoumarol is a potent anticoagulant that should include the lower portions of stems and leaf acts by preventing the reactivation of vitamin K which sheaths (just above the root) from suspect plants. Other is necessary to complete the synthesis of factors II, VII, sources of tremorgenic toxins include Dallis grass IX and X in the coagulation system. Several general of (Paspalum dilatatum) infected with Claviceps paspali, fungi, including Aspergillus, Penicillium, Humicolor Bermuda grass (Cynodon dactylon), ergot (Claviceps and Mucor have been associated with this activity. purpurea), white snakeroot(Eupatorium rugosum), Ohio Currently, low coumarin sweet clover varieties are buckeye (Aesculus glabra), and penitrem A from moldy available, but sweet clover is not a commonly utilized walnut hulls (Juglans spp.). Consideration of the time of forage. I tis planted as a soil-building or cover crop where year and available exposure to potential tremorgens will use for hay or pasture is not expected. Exposure to sweet usually eliminate many of the differential diagnoses. clover may occur as a spurious contaminantofpasture or Insecticides such as chlorinated hydrocarbons, organo hay, or when hay is cut from federal set-aside land or phosphates and pyrethrins can cause clinical signs simi-

60 THE BOVINE PROCEEDINGS-NO. 26 other conservation-based plantings. Silage containing week alternated with alfalfa or other sources for two moldy sweet clover is also toxic. The use of large round weeks has prevented sweet clover disease. Menadione bales stored outside for long periods of time has been has not been effective in preventing sweet clover dis suggested by some as a predisposing factor to molds that ease. Sweet clover should not be fed in late gestation or could cause sweet clover disease. Dicoumarol concentra within two weeks before surgical procedures or other tions ofless than 20 ppm are usually non-toxic, while events that cause trauma and potential bleeding. levels above 40 ppm are considered unsafe. Concentra tions greater than 60 ppm experimentally have caused Slobbers Syndrome (Slaframine toxicosis) coagulopathy within approximately three weeks of con The conditions commonly known as "Slobbers syn tinuous feeding (Casper, 1993). drome" or "salivary syndrome" is associated mainly with Clinical effects of dicoumarol toxicosis can range red clover (Trifolium pratense) infected with the fungus from sudden death due to massive hemorrhaging to Rhizoctonia leguminocola (Black patch disease). Con chronic bleeding with epistaxis, vaginal bleeding, bloody tamination is most common in wet cool years and in feces, subcutaneous hemotomata, lameness, and occa stands of nearly pure red clover, although other legumes sionally neurologic signs if bleeding is in the central (white clover, alsike alfalfa,and others). The fungus can nervous system or spinal cord. Pregnant cows may abort overwinter in infested plants or survive up to two years due to placental bleeding, and calves born to dams fed in seed. Properly dried hay will remain contaminated moldy sweet clover may have coagulopathy at birth. and fungal activity can continue development in baled Hematomas usually occur in the submandibular, bris hay. An indolizidine alkaloid known as slaframine is the ket, flanks, legs and other areas that physically contact toxic principle (Smalley and Sanderson, 1993). feed bunks, fences or buildings. Diagnosis of sweet Horses appear to be most susceptible, but clinical clover disease is usually suspected when a vitamin K effects are documented in cattle. Animals may be af responsive coagulopathy occurs in the presence of moldy fected within one hour after consuming contaminated sweet clover. Differentiation of sweet clover from hay. There is profuse salivation early and this may alfalfa can be done by examining the tri-lobed leaflets of continue for 24 to 72 hours. Other concurrent signs the plant. Sweet clover has a center leaflet that is more include mild lacrimation, frequent urination and diar elongated than alfalfa and the distal third of the leaflet rhea. Bloat can occur and most affected animals have 0 margins are serrated. Clinical laboratory findings of partial to complete anorexia. "'O (D anemia, prolonged clotting time and elevated one stage In addition to stimulation of salivation, slaframine ~ prothrombin time (OSPT) and/or activated partial throm causes increased pancreatic fluid secretion, bradycar ~ (") boplastin time (APTT) are adjuncts to diagnosis. Ana dia and bradypnea, hypothermia and increased intesti (D 00 lytical identification of dicoumarol in forages(> 30 ppm) nal and uterine motility. 00 0...... or liver(> 0.5 ppm) will provide confirmation of probable Morbidity may be high, but mortality generally is 00 ,-+- toxic exposure. Live animals may have detectable con low, and prompt removal of contaminated hay is fol '"i ~ centrations of dicoumarol or its metabolites in blood or lowed by recovery in 24 to 48 hours. Presence of other ~...... urine. alkaloids, especially swainsonine, has been suggested to 0 p Treatment of dicoumarol poisoned animals should complicate the clinical response and introduce neuro begin with immediate administration of Vitamin Kl at logic disturbances as well. The real role of swainsonine 1-3 mg/kg body weight daily, given intramuscularly in in the salivary syndrome is not clear. divided doses using a small gauge needle. Menadione Diagnosis is usually based on recognition of the (vitamin K3) is not effective. In acute emergency situa characteristic excessive salivation and other clinical tions, administration of three to five liters ofblood from effects correlated with a history of consumption of red a normal animal will provide the coagulation factors clover or other legumes. Identification of the lesions or needed to save an animal. Animals must be promptly black patch disease or isolation of the fungus further removed from the sweet clover source. Alfalfa hay is a supports a presumptive diagnosis. If needed, chemical good substitute forage since it is high in natural vitamin analysis for slaframine and swainsonine is available K. Coagulation improvement will occur in from 12 to 24 through diagnostic laboratories. hours after vitamin Kinjection and affected animals are There is no specific an ti dote to slaframine toxicosis, usually normal in 4 to 7 days. Monitoring of coagulation although atropine may control at least some of the status and administration of vitamin K for 3 to 5 days is prominent salivary and gastrointestinal signs. Removal recommended (Casper, 1993). of animals from the contaminated hay is essential. Sweet clover poisoning is best prevented by feeding Prevention of black patch disease has been diffi only properly cured hay, planting low coumarin culti cult by chemical treatment of the plants themselves. vars of sweet clover, and avoiding prolonged feeding of Seed treatment may eliminate the infection for new known contaminated hay. Feeding sweet clover for one seeding, and producers can check with seed suppliers for

JANUARY, 1994 61 varieties that are relatively resistant to black patch. Major Mycotoxicoses From Feed Grains in Avoiding use of pure red clover for forages or using it as North America: part of a forage mixture is helpful, and the low incidence • Aflatoxin of the salivary syndrome is probably due in part to the • Ergot relatively infrequent use ofred clover in modern forage • Trichothecenes systems. • Zearalenone

Pithomyces chartarum Photosensitization There is little evidence that these mycotoxins occur Pithomyces chartarum is a fungal saprophyte of a with significant incidence or at clinically relevant levels variety of grasses, legumes and weeds. Historically it on forages in North America. Their significance to has been a clinical problem in Australia, New Zealand ruminants is reviewed briefly for comparative purposes and South Africa, but is also known to occur in the and because they may be economically more important United States and Canada. Favorable conditions of than the recognized forage mycotoxins just discussed. moisture and temperature promote the growth of P. chartarum which can produce a hepatotoxic mycotoxin Afiatoxin known as sporodesmin which is concentrated in the Cattle, sheep, and other ruminants are less suscep spores. Sporodesmin is a biliary system toxin, affecting tible than monogastric animals or poultry. Calves are bile duct epithelium with swelling and obliterative more susceptible than mature animals. Dietary concen cholangitis. As a result of these cholestatic effects, plant trations as low as 200 ppb fed to calves has been associ chlorophyll metabolites (phylloerythrins) are not ex ated with reduced growth and liver damage. creted in the bile. They accumulate in blood, and where Concentrations of 2.2 ppm fed for sixteen weeks skin is exposed to sunlight their photodynamic effects results in death of weanling calves. Concentrations ofl- induce photosensitization. While sporodesmin has not 2 ppm in mature cattle for short periods of time results been definitively linked to naturally occurring photosen in reduced gain and decreased milk production. Abor sitization in North America, the presence of the fungus tion and infertility are not generally expected. and conditions that support sporodesmin growth make Major clinical effects include depression, anorexia, it a potential problem (Bagley and Shupe, 1986). reduced gain or milk production, subnormal body tem 0 "'O The clinical effects of sporodesmin are similar to perature and dry muzzle. Lesions ofliver damage range (D ~ other photosensitization reactions, but are dose depen from acute to chronic. Aflatoxin has been reported to ~ dent and at high dosages significant mortality may (") decrease cellulose digestion, reduce volatile fatty acid (D 00 occur. Clinical signs may occur until several days of formation and inhibit proteolysis in artificial rumen 00 weeks after initial exposure when representative in systems. Aflatoxin at high concentrations (1-4 ppm) can 0...... 00 ,-+- fected forage is no longer available. reduce rumen motility. '"i Diagnosis of sporodesmin toxicosis would be en Although estimates vary, Aflatoxin in milk ap ~ hanced by detection of the liver lesions that include bile pears at approximately 1 % of the dietary concentration ~...... 0 duct edema and luminal occlusion accompanied in ad (Range 0.2-3.2%). Most Aflatoxin is excreted within 72- p vanced cased by proliferation ofbile ductules and fibrous 96 hours after exposure stops. Dairy cattle should not connective tissue. Clinical laboratory determination of be fed feed containing more than 20 ppb Aflatoxin. This elevated serum bilirubin, gamma glutamyl should prevent the occurrence of milk residues where transpeptidase and aspartate aminotransferase would the current action level is 0.5 ppb. Liver and kidney be consistent with sporodesmin toxicosis. Definitive appear to retain measurable residues ofAflatoxin longer chemical testing is not routinely available, but isolation than other tissues (but less than 2 weeks). of the fungus could help in establishing a presumptive Acute disease, is characterized by depression, diagnosis, although as with other mycotic infections of anorexia, icterus, and hemorrhages. Liver function plants, the fungus does not always produce toxin. tests and microscopic evaluation of liver should be used Prevention and control of sporodesmin toxicosis is as adjuncts to support clinical diagnosis. Aflatoxin is enhanced by grazing or haying management which readily detected in feed supplied at levels far below those reduces the amount of dead plant litter. This is usually necessary to produce clinical poisoning. Urine, milk, accomplished by well controlled rotation grazing. In and blood may contain detectable residues during acute high humidity areas (e.g. New Zealand) pastures are exposure, and urine concentrations of Aflatoxin Ml may sprayed with fungicides such as Thiabendazole or Benlate persist for up to a week after exposure to high levels to suppress sporulation during periods of high risk. Zinc stops. supplementation at 20 mg/kg body weight is protective Mold inhibitors may aid in preventing further against liver damage from sporodesmin, but adminis growth and toxin formation of A. flavus in prepared tration at this level has been logistically difficult.

62 THE BOVINE PROCEEDINGS-NO. 26 feeds, but will not destroy preformed toxins. Treatment on grain overwintered in the field. Reports of occurrence with anhydrous ammonia for 10-14 days reduces Afla on forages have been in past years, and recent tech toxin in grain, but due to uncertainty of the breakdown niques have not revealed a significant contamination products, FDA has not cleared this detoxication method. problem on forages. Hydrated sodium calcium aluminosilicate (HSCAS) re Except for vomitoxin, effects described, for duced but did not eliminate residues of Aflatoxin Ml in trichothecenes are mostly from dosing experiments. milk of dairy cows fed Aflatoxin Bl. HSCAS has high Most animals refuse to consume high concentrations of affinity for Aflatoxin and can reduce absorption of Afla trichothecenes and never achieve the full range of re toxin from the gastrointestinal tract. ported toxic effects. Forced intake oftrichothecenes can cause anorexia, enteritis, lymphoid depletion, leucopenia, Action levels recently suggested by FDA are: bone marrow depression, coagulopathy, and suppres Human Food and Milk 0.5 ppb sion of cell mediated immunity. Prolonged dermal Corn of Unknown Destination 20 ppb contact with T-2 and some other trichothecenes can Young Animals 20 ppb cause dermal irritation and necrosis. Dairy Cattle 20 ppb Cattle begin to refuse vomitoxin (DON) contami Breeding Cattle 100 ppb nated feed at approximately 10 ppm. Limited studies in Finishing Cattle 300 ppb dairy cows indicate six ppm may be a refusal level. Recently, some investigators have associated presence Ergot of low levels of vomitoxin in feed with reduced milk Ergot alkaloids are produced by sclerotia of production. DON itself may not be responsible, but is Claviceps purpureaand invade the ovary (seed) of grass considered by some to be a "marker" toxin indicating flowers where they appear as dark brown to black potential adverse effects. More studies are needed, and banana shaped sclerotia. Recently Tall Fescue (Festuca so far a cause-effect relationship has not been estab arundinaceae) invaded by the endophytic fungus lished. Acremonium Coeniphialum has also been shown to Trichothecenes are rapidly metabolized both by contain the ergot alkaloid ergovaline (see Fescue). rumen microorganisms and systemic enzymes. Degra Ergot alkaloids may induce uterine contractions in dation products are rapidly eliminated and persistent 0 "'O a sensitized uterus, but solid evidence for abortion is residues are not expected. Very little DON (vomitoxin) (D limited. High concentrations of ergot alkaloids cause is excreted in milk (Beasley, 1993). ~ ~ peripheral vasoconstriction and produce classical dry ('") (D 00 gangrene of extremities (feet, tail, ears) that is exacer Zearalenone 00 bated in cold weather. This mycotoxin is an estrogen but less potent 0...... 00 ,-+- Ergot alkaloids inhibit prolactin release and this estrogen than estradiol or DES. In the USA, grain rarely '"i prevents mammary development in late gestation as exceeds 3 ppm. Most common sources are corn and ~ well as reducing or eliminating milk secretion. The ~...... wheat during wet, cool seasons that cause wheat scab or 0 result is agalactia at parturition. The agalactia is pink rot of corn. It has been very rarely reported in hay, p reversible five to seven days after ergot is removed from and usually at concentrations not known to cause toxicosis the diet. in cattle. Remember that other plants (e.g. legumes) Ergot alkaloids may also be responsible for heat contain estrogens which could mimic some effects of intolerance in cattle when consumption occurs in warm zearalenone. Zearalenone is stable to drying, but the weather. This association of ergopeptide alkaloids with effect of ensiling on zearalenone is not known. heat intolerance and prolactin suppression is well estab Dairy heifers may have increased infertility and lished with endophyte infected fescue. Such animals reduced conception at zearalenone feed concentrations have elevated rectal temperature, seek shade and/or above 12 ppm. Mature cows are affected at concentra water holes, eat less and are often depressed. tions above 25 ppm. Some reports indicate vaginitis and vaginal secretion may occur. Virgin heifers may have Trichothecene Mycotoxins mammary enlargement. Small amounts ofzearalenone These include over 60 metabolites produced by may be passed in milk, but residues are not believed to Fusarium sporotrichioides or F. rose um. Best known are persist for more than a few days once exposure is stopped T-2 toxin, diacetoxyscirpenol and vomitoxin (DON). DON (Diekman and Green, 1992). is by far the most commonly reported. They are almost Alfalfa and alfalfa meal in some species will reduce always reported from feed grains. Cool, wet weather absorption and increase fecal excretion of zearalenone. during maturation of grain or harvest favor production Whether this occurs in dairy cattle has not been deter of trichothecenes. They have commonly been reported mined.

JANUARY, 1994 63 Guidelines for Prevention and Management International Diagnostic Environmental Diagnostic of Mycotoxicoses: System Corp. Systems Corp. 1. The best management is to avoid use of known P.O. Box 799 P.O. Box 908 2990 Anthony Road moldy grain or forages in dairy cattle rations. St. Joseph, MI 49085 (616) 983-3122 Burlington, NC 27215 Mycotoxins may occur for which there are no (800) 334-1116 documented effects nor means of identification. IDEXX 2. Dilution of contaminated feedstuffs will reduce 100 Fore Street the initial mycotoxin level. Remember that Portland, ME 04101 molds present may contaminate "clean" feed or (800) 548-8733 forage if storage conditions are not optimal. 3. Many mycotoxins in grain are associated with Selected References and Suggested Reading low quality, small, cracked or broken seeds. Cleaning ofgrain will often markedly reduce the Bagley, C. V., and J. L. Shupe, "Sporidesmins Facial Eczema and mycotoxin load. Pithomycotoxicosis", Current Veterinary Therapy 2, Jimmy L.Howard, ed., W. B. Saunders Company, Philadelphia, PA, pp. 375- 4. Heavily molded feedstuffs may lack normal 377,1986. Ball, Don, Steve Schmidt, Garry Lacefield, Carl Hoveland, energy and vitamin levels. Supplementation, and William C. Young, III, "Tall Fescue/Endophyte/Animal Relation after nutritional analysis, may be helpful. ships", Oregon Tall Fescue Commission, 1991. Beasley, Val R., 5. Testing of suspect molded forages and grains "Trichothecenes", Current Veterinary Therapy 3, Jimmy L. Howard, ed., W. B. Saunders Company, Philadelphia, PA, pp. 332-333, may be helpful to eliminate presence of known 1993. Bransby, D. I., "Another tool against Fescue Toxicosis?" , Beef, mycotoxins, but will not establish complete pp. 33-34, 1993. Buck, William B., and S. J . Cysewski, "Nervous form safety. Use of mold counts or spore counts are ofErgotism", Current Veterinary Therapy 2, Jimmy L. Howard, ed., W. not a definitive means to establish safety. B. Saunders Company, Philadelphia, PA, pp. 369,1986. Buck, Will iam B., and S. J. Cysewski, "Tremorgenic Toxins", Current Veterinary 6. Storage should be at 13-15% moisture to prevent Therapy 2, Jimmy L. Howard, ed., W. B. Saunders Company, Philadel recontamination by fungal activity. phia, PA, pp. 378,1986. Casper, H. C., "Sweet Clover Poisoning", 7. Organic acids will prevent mold growth, but not Current Veterinary Therapy 3, Jimmy L. Howard, ed., W. B. Saunders destroy preformed toxins. Some adsorbents, Company, Philadelphia, PA, pp. 358-359, 1993. Coppock, R. W., and S. P. Swanson, "Aflatoxins", Current Veterinary Therapy 2, Jimmy L. such as aluminosilicates are useful to alleviate 0 Howard, ed., W. B. Saunders Company, Philadelphia, PA, pp. 363- '"O aflatoxin effects, but have not been effective for 366,1986. Dew, R. K, G. A. Boissonneault, N. Gay, J. A. Boling, R. J. (D ~ other mycotoxins. Cross, and D. A. Cohen, "The effect of the endophyte (Acremonium coenophialum) and associated toxin(s) of tall fescue on serum titer ~ 8. Storage areas and feeders should be kept dry (") response to immunization and spleen cell flow cytometry analysis and (D 00 and cleaned periodically. response to mitogens", Vet. Immunol. Immunopathol., 26(3):285-95, 00 9. For most mycotoxins, there is no specific treamen t 1990. Dougherty, C. T., L. M. Lauriault, N. W. Bradley, N. Gay, and 0...... 00 P. L. Cornelius, "Induction of tall fescue toxicosis in heat-stressed ,-+- orantidote. Good supplemental nutrition and '"i alternative feed supplies are essential. cattle and its alleviation with thiamin", J. Anim. Sci., 69(3):1008-18, ~ 1991. Diekman, M.A. and M. C. Green, "Mycotoxins and Reproduc ~...... 10. Supplementation ofvitamins and selenium may tion in Domestic Livestock",J.Anim. Sci., 70:1615-1627, 1992. Galey, 0 be helpful, and provision of adequate high qual Francis D., Mark L. Tracy, Arthur L. Cragmill, Bradd C. Barr, Gary p ity protein is advisable after animals have con Markegard, Richard Peterson, and Michael O'Connor, "Staggers in sumed mycotoxins. duced by consumption of perennial ryegrass in cattle and sheep from Northern California", JAVMA, Vol. 199, 4:466-470, 1991. Hintz, H . F., "Molds, Mycotoxins, and Mycotoxicosis", Clinical Nutrition, Veteri Manufacturers and/or sources of commercial mycotoxins. nary Clinics of North America: Equine Practice, Volume 6, 2:419-431, August 1990. Kerr, L.A., C. P. McCoy, C. R. Boyle, and H. W. Essig, BioCode, Ltd. Romer Labs, Inc. "Effects ofAmmoniation ofEndophyte Fungus-infested Fescue Hay on University Road P.O. Box 2095 Serum Prolactin concentration and rectal temperature of beef cattle", Hesslington Washington, MO 63090 Am. J. Vet. Res., 51(1):76-8, 1990. Lauriault, L. M., C. T. Dougherty, (314) 239-3009 N. W. Bradley, and P. L. Cornelius, "Thiamin supplementation and the York Y0L 5DE ingestive behavior of beef cattle grazing Endophyte-infected tall fes United Kingdom cue", J. Anim. Sci., 68(5):1245-53, 1990. Lipham, L. B., F. N. Thomp +44 904 430 616 VICAM son, J. A. Stuedemann, and J . L. Sartin, "Effects of Metoclopramide on Fax: +44 904 430 495 29 Mystic Avenue steers grazing Endophyte-infected fescue",J. Anim. Sci. 67(4):1090-7, Somerville, MA 02145 1989. Nicholson, Steven S., "Tremorgenic syndromes in livestock", Transia (800) 338-4381 Clinical Toxicology, Veterinary Clinics ofNorthAmerica: Food Animal 8, rue Saint-Jeau-de-Dien Practice, Vol. 5,2:291-300,July 1989. Olson, William G., "Evaluation 69007 Lyon, France Neogen Corp. of Heat- or Mold-Damaged Ensiled Feeds and Their Effects on Live stock", Current Veterinary Therapy 3, Jimmy L. Howard, ed., W. B. 620 Lesher Place +33 72 73 03 81 Saunders Company, Philadelphia, PA, pp. 341-342, 1993. Osweiler, Lansing, MI 48912 Gary D., "Mycotoxins and livestock: What role do fungal toxins play TerraTek (517) 372-9200 in illness and production losses?", Veterinary Medicine, pp. 89-94, 400 Wakara Way January 1990. Osweiler, Gary D., "Ergotism", Current Veterinary Salt Lake City, UT 84108 Therapy 3, Jimmy L. Howard, ed., W. B. Saunders Company, Philadel- (800) 372-2522

64 THE BOVINE PROCEEDINGS-NO. 26 phia, PA, pp. 334-335, 1993. Porter, J. K., J. A. Stuedemann, F. N. ed., W. B. Saunders Company, Philadelphia, PA, pp. 336, 1993. Thompson Jr., and L.B. Lipham, "Neuroendocrine measurements in Sullivan,JohnM., and William W. Carlton, "Citrinin", Current Veteri steers grazed on endophyte-infected fescue" ,J. Anim. Sci., 68( 10 ):3285- nary Therapy 3, Jimmy L. Howard, ed., W. B. Saunders Company, 92, 1990. Porter, J. K., and F. N. Thompson, Jr., "Effects of fescue Philadelphia, PA, pp. 337, 1993. Thompson, F. N., R. D. Jones, J . A. toxicosis on reproduction in livestock", J. Anim. Sci., 70(5): 1594-603, Stuedemann, KM. Mizinga, and C. K Smith, "EffectofMetoclopramide 1992. Rhodes, M. T., J. A. Paterson, M. S. Kerley, H. E. Garner, and on Luteinizing hormone secretion in postpartum anestrous cows" ,Am. M. H. Laughlin, "Reduced blood flow to peripheral and core body J. Vet. Res., 53(5):727-30, 1992. Thompson, F. N., R. D. Jones, J . A. tissues in sheep and cattle induced by endophyte-infected tall fescue", Stuedemann, KM. Mizinga, and C. K Smith, "EffectofMetoclopramide J. Anim. Sci., 69(5):2033-43, 1991. Schillo, K. K., L. S. Leshin, J. A. on Luteinizing hormone secretion in postpartum anestrous cows",Am. Boling, and N. Gay, "Effects ofendophyte-infected fescue on concentra J. Vet. Res., 53(5):727-30, 1992. Tsai, H.F., M. R. Siegel, and C. L. tions of prolactin in blood sera and the anterior pituitary and concen Schardl, "Transformation ofAcremonium coenophialum, a protective trations of dopamine and dopamine metabolites in brains of steers", J. fungal symbiont of the grass Festuca arundinacea", Curr. Genet., Anim. Sci., 66(3):713-8, 1988. Smalley, Eugene B., and Julie M. 22(5)399-406, 1992. Zanzalari, K. P., R. N. Heitmann,J. B. McLaren, Sanderson, "Slaframine (Slobber Factor)", Current Veterinary Therapy and H. A Fribourg, "Effects ofEndophyte-infected fescue and cimetidine 3, Jimmy L. Howard, ed., W. B. Saunders Company, Philadelphia, PA, on respiration rates, rectal temperatures and hepatic mixed function pp. 338-339, 1993. Sullivan, John M., William W. Carlton, and Palle oxidase activity as measured by hepatic antipyrine metabolism in Krogh, "Ochratoxin", Current Veterinary Therapy 3, Jimmy L. Howard, sheep", J. Anim. Sci., 6702):3370-8, 1989.

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JANUARY, 1994 65