Mycotoxins in Feedstuffs and Their Effect on Dairy Cattle

Mycotoxins in Feedstuffs and Their Effed on Dairy Cattle G. P. LYNCH Animal Science Research Division, USDA, Beltsville, Maryland 20705 Abstract

This paper is a review of mycotoxins reported in feedstuffs and their possible effect on dairy cattle and other types of livestock. Toxins of the Aspergilli discussed were the aflatoxins, ochratoxins, patulin and sterigrnatocystin. Toxins of the Fusaria included zearalenone, acetamido lactone, tricothecenes and the toxin from F. solani. Those from the PeniciUia were rubratoxins, patulin, citrinin, and the tremorgenic toxins. Other mycotoxins also mentioned were slaframine, sporidesmins, ergots and those from Stachybotrys atra. Emphasis was given to reporting methods of analysis along with a summary of physiological actions of these mycotoxins. Particular attention was given to the excretion and detection of aflatoxin M from cattle and sheep. It is pointed out that the extent of livestock production or death losses from mycotoxin contamination of feeds is not known. Introduction

Both man and animals live trader a certain degree of "biological hazard" from natural toxicants that occur in feedstuffs (7, 33). Fungal toxins have been reported in feeds consumed by both animals and man. Work in recent years on the occurrence and physiological action of a number of naturally occurring mycotoxins has been stimulated by serious outbreaks of mycotoxicoses in man and animals (1,07). Mycotoxiu contamination of feedstuffs occurs either as a result of invasion of crops by field fungi or by the growth of storage fungi in crops held under improper storage conditions. Many cereal grains show a rapid increase in respiration during storage when the moisture content is increased beyond 14 percent. Species of molds normally associated with the respiration and heating of grains be-

gin to germinate at a relative humidity of about 80 percent (81). Each fungal species has its own moisture tolerance level that will influence how well it grows in competition with other species. These fungi have the potential of producing many changes associated with the reduction of grain quality. The extent of the changes produced by fungal growth depend upon environmental storage conditions, length of storage, and predominate fungal species. These fungal-induced changes in stored grain can include loss of germination, heat damage, production of toxic metabolites, increases in nonprotein N, reducing sugars, fatty acids, and the loss of nutritive value (13, 28, 81). Exposure of livestock to moldy feeds has also shown to produce mycotic pneumonia and mycotic abortion (32, 34, 77, 131). Not all mold induced changes are detrimental, for example, Rhizopus oligosporus is grown on soybeans to produce a human food called tempeh (125), nor is the positive identification of a known toxin producing fungi in moldy feed positive evidence of toxin production. Toxin production among similar species of fungi is known to be variable (53). Accurate diagnosis of mycotoxin poisoning depends upon prompt observation of post mortern lesions along with positive identification of the toxin in the feed. Toxins of the Aspergilli

Historically, moldy feeds have been implicated with outbreaks of poisoning or poor productive performance in livestock by several workers (5, 16, 18). Both natural and experimental moldy corn poisonings were held responsible for the gross and microscopic lesions observed in swine, cattle, horses, and goats. Work at several laboratories has shown that an outbreak of turkey X disease in England was caused by aflatoxin produced by AspergilIus flavus, Link and Fries (53) and more recently by Penicillium puberulum (58). Aflatoxin was then found to contain at least four chromatographic components, Bx, G1, and their dihydro-derivatives B2 and G2. The Received for publication June 21, 1971. 1Invitational Paper Presented at the SLxty-sixth chemical structures (Fig. 1) were found to Annual Meeting of the American Dairy Science be furofuran compounds with a substituted Association, Michigan State University, East Lan- coumarin ring (53). The physiochemical assay sing. of aflatoxin depends on their fluorescent prop1243

1244

LYNCH

0 I 3 B1

B2

M2

3 G1

OH

MI

G2

B2a

O0

G2a

P1

FiG. 1. Stnlctures of the a~]atoxins. erties tinder long-wave ultraviolet light. Aflatoxins are separated by thin layer chromatography and compared to primary aflatoxin standards by visual appraisal or by the more sensitive fluorodensitometrie method. Under ideal conditions with the visual appraisal method the precision limit is not better than +__ 205, but precision of the fluorodensitometrie method is -+- 9g for average of multiple observations (53). Collaborative studies for the determination of aflatoxin in peanuts (43, 124) and cottonseed (97) have been reported. A method adapted for the determination of aflatoxin in corn has also been reported (108). Recent analytical work has indicated a number of distinct aflatoxin chromatographic fractions such as B1, B2, G1, G2, M1, M..,, B2.~, G2~ (53) and P~ (35). Comparable toxicities in day-old ducklings indicates B1 to be the most toxic fraction followed by G~, B2, and Gz (127). Aflatoxin P1 failed to exhibit toxicity in chick embryo tests (117). Hydration of the furofuran double bond or loss of the furofuran ring system resuits in a reduction or loss of toxicity (103). Aqueous solutions of aflatoxins B~ and G~ undergo decomposition in light, under treatment with ammonia (36, 51), and toxicity is reduced on prolonged exposure to long wave UV light (53). The toxicity of aflatoxin M (milk toxin) appears to be similar to that of B~ in ducklings and aflatoxin B~ and G2~ are relatively nontoxic to ducklings (53) and chick embryos (95). Methods have been reported for the determination of the afatoxins in milk powder (98), fuid milk (64), and cheese

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The aflatoxins can be regarded as highly toxic, potent hepatoearcinogens and are mutagenic. Young animals are more susceptible than older and variations occur in susceptibility among different species (53). Pigs up to 12 weeks and calves up to 6 months of age are highly susceptible (127). Studies with dairy calves have indicated a depression of feed intake and live weight gains with liver cell damage. Death losses of both pigs and calves on aflatoxin contaminated feed has been reported (27, 127). Liver lesions include fibrosis, centrolubular necrosis, bile duct proliferation, and venous occlusion. Ascities and viseral edema were also reported. Metabolic changes included an increase in serum alkaline phosphatase and a reduction in liver vitamin A (127). Recent work with young calves indicates increases in serum alkaline phosphatase with daily aflatoxin levels of .02 mg B1/kg body weight or more along with a loss of liver glycogen, accumulation of fat, fibrosis and disorganization of liver lobules (74). Increased serum levels of lactic dehydrogenase, aldolase, glutamic-oxaloacetic or glutamic-pyruvie transaminase (53) and urocanase have also been reported in chicks treated with aflatoxin (94). At the cellular level, two aflatoxin effects have been demonstrated. These are carcinogenicity and necrosis of liver cells. These events are then thought to lead to the inhibition of synthesis of certain liver proteins (53, 65, 128). Information on the mechanism of this action has been derived from studies with the antibiotic actinomycin D, a biochemically analogous compound to aflatoxin in that it also inhibits protein synthesis (101). The incorporation of 3H-cytidine into nuclear RNA, a lowered RNA/DNA ratio in rat liver nuclei and the in vitro binding of aflatoxin B1 to both native and denatured DNA has been shown. Estimates have been reported that 600 moles of native DNA-phosphorous bound 1 mole of aflatoxin B 1 (53, 76, 113). DNA-dependent RNA synthesis may be inhibited by preventing the transcription of DNA by the inhibition of RNA polymerase. Recent work indicates that the inhibition of RNA polymerase activity in rat livers may be due to the action of aflatoxln B~ on some component of chromatin in the cell rather than directly on the polymerase enzyme (42, 53, 87). Spectral shifts brought about by DNA-aflatoxin binding were greatest for B1, intermediate for G~, and smallest with G2. The extent of DNA binding is related to the reported toxicity of these ariatoxins. Aflatoxin binds to single stranded DNA and purine bases and the amino groups are

MYCOTOXINS IN F E E D S T U F F S

1245

(53). Severe illness and liver damage were active in binding. Work with single stranded calf thymus DNA and bacterial RNA polymer- reported in 2 year old fattening cattle fed ase indicate that aflatoxin binding to polynu- toxic groundnut meal for 3 months (31), Cows cleotides requires the amino group of adenine fed 1.5 mg atlatoxin daily (.30 ppm) in or possibly guanine (53). More recent work groundnut cake and nursing calves showed an suggests that a metabolite of aflatoxin B1 may increase in total blood lipids and a decrease in be the active compound binding to the DNA urine urea. The calves showed a reduction of template for inhibition of gene transcription growth, an increase in serum glutamic(42, 104). In an in vitro system, aflatoxin B 1 oxaloacetie transaminase, and death of three was shown to bind with DNA and induce non- of five calves in the treated group. Analysis of reversible mutations up to 23 times that of the milk from all the treated cows indicated background. This mutagenic potential might aflatoxin M contents ranging from .08 to .30 explain the carcinogenicity of aflatoxin (76). mg/liter. Milk from cows whose calves died Disaggregation of cytoplasmic ribosomes gave aflatoxin M contents of .20 to .30 and a decreased activity of drug metabolizing mgfliter. Traces of aflatoxin were found in enzymes of rat livers has also been reported viscera and muscle samples from one of two treated cows that were sampled (23). (53, 56, 123). Subsequent work indicated that cattle fed Aflatoxin is apparently readily transported to liver cells of most species to inhibit DNA aflatoxin excreted a toxin factor in milk, found dependent, RNA synthesis and subsequent with the casein fraction and having a biologisynthesis of some liver proteins. In some cal effect on ducklings similar to that of species, histological lesions continue to appear aflatoxin. This factor was first designated "milk in the liver well beyond the time atlatoxin can toxin" and was shown to be a chromato~aphie be detected in tissues. This suggests the possi- fraction with a lower Rf value than aflatoxin bility of a metabolite of aflatoxin remaining in Ba. Aflatoxin M was then found in A. flavus the liver to retard recovery (19). Slow re- cultures in liver, kidney, and urine of sheep. coveries from sublethal doses of aflatoxin have The general term aflatoxin M was adopted. The metabolite was crystallized from shoeo been reported in sheep (6), pigs (20), and dairy calves (75). Intakes of sublethal doses urine and the milk of cows dosed with of aflatoxin through feed contamination could aflatoxin. Aflatoxin M was then found to have result in prolonged impairment of livestock two components, M~ and M2. These were the 4-hydroxy-aflatoxin derivatives of B~ and B., performance. (Fig. 1) (53). More recently, the aflatoxin M Research with young ruminants has shown that aflatoxin-induced serum enzyme changes derivative of aflatoxin G has also been shown (40, 57). The LDs0 values of aftatoxin B~ and and histological liver lesions are similar to those of other species (53, 74). This may in- M in ducklings is similar being 12 and 16.6 dicate that, qualitatively, many of the pro- /*g, respectively. Aflatoxin M was also shown posed biological mechanisms for aflatoxin to be nearly as carcinogenic in rainbow trout toxicity shown in other species may also apply as Bj (111). After the ingestion of a single dose of aflatoxin B~, excretion of M~ in milk is to the ruminant. Even though young calves up to about 6 completed within a few hours and about .3% months of age are more susceptible, aflatoxin of the administered dose appears in this milk (78). Aflatoxin Ms appeared in the milk 12 to can produce toxic symptoms in older cattle (8, 54). Rations containing 15 to 2070 of the 24 hr after feeding an aflatoxin-contaminated toxic groundnut (peanut) meal, fed over diet and disappeared in three to four days afseveral months caused loss of condition, a re- ter the removal of the aflatoxin contaminated duction in milk yield, and liver damage in diet (120). A linear relationship between the heifers and cows. Later estimates of aflatoxin amount of aflatoxin B~ ingested and the concontent were reported to be about 2 ppm with centration of aflatoxin M1 in milk has been sugdaily intakes of 3.2 mg B 1 for dry cows and gested. A rapid disappearance of aflatoxin M up to 12.7 mg B~ for milking cows (53). was reported from cows fed graded levels of Similar gross symptoms in cattle and swine aflatoxin, however, detectable amounts were were also reported from aflatoxin con- observed on the 7th or last day of treatment (12, 79, 120). The lowest daily intake of taminated cottonseed meal (52, 72) and moldy corn (17). Groundnut meals containing aflatoxin B~ to give detectable quantities of .22 to .66 ppm BI produced weight loss when aflato~n Mx (.0003 t~g/ml) in the milk of a fed to 2 year old cattle. Mild liver lesions may" 500 kg cow was from .6 to .9 mg (3, 102). Ushave been produced at the .66 ppm B~ level ing the first data (79) and assuming that a JOURNAL OF DAIRY SCIENCE VoI.

55, NO. 9

'8'

TABLE 1. Summary of data on aflatoxin residues in tissues of cattle and sheep.

z

Species

Ref.

t.D

Aflatoxin dose

Source

Tissue

Method

Finding

Remarks

3.0(mg/kg feed) 3.75 3.75

Peanut meal

Milk Liver Blood

Ducklingtoxicity

Toxic Nontoxic Nontoxic

13

Animals

o Cow

6

~' Sheep z

7

.36(mg/kg BW)

Mixed ariatoxins

Liver Kidney Urine

TLC

Bt, G1M1, GM~ B~, G~M~, G~ B~, GM~, G,, M~

3 Animals, dosed by intubation or ip injection, sacr, at 2 hr.

Cow

9

.22(mg/kg BW)

Mixed a~atoxins

Milk

TLC

M~

1 Animal negative after 5 days

Cow

109

Mixed ariatoxins

Milk

TLC

Peanut meal

Milk

TLC, duckling

.31(mg/day) .63 .90 .875 1.75 2.63 5.0 15.0 20.0 24.5

Peanut meal

Milk

TLC

360 ( tzg/mg BW )

Mixed, ariatoxins

Milk

TLC

t~

xo

Cow

16i

Cow

8

Sheep ( ewe ) Cow Steers

122

5(mg/day) 10 20 40 80

8(mg/day )

Mx

M~ M, M~ M~, B1 MI

M1

6 Animals, Detectable for 7 Detectable for 7 Detectable for 7 Detectable for 7 Detectable for 4 ( Not lactating)

days days days days days

4 Animals, no relation between M1 and milk yield 6 Animals, Mx in milk related to B~ in feed

M~ Mt M, M~ M~ M,

1

1.6(mg/kg feed)

Peanut meal

Milk

TLC, ducks

20

to 1( mg/kg BW)

Mixed ariatoxins

Meat Blood Liver

TLC

B~, G,, M1

1 Animal, no M1 after 6 days, no B~ or G1 after 1 day Animal

Trace

Animals, i mg/kg level, none 24 hr after withdrawal

z

1247

MILK FAT GLOBULE MEMBRANE

TABLE 2. Comparative excretmn of aflatoxin in ~ e cow and ewe. Aflatoxin Dose Dose recovered,

Conversion, B~ to M~ 48 hr excretion ( goOf recovered dose )

Cow Total Milk Urine Feces

.5 nag/kg (300 nag) (%) 4.52 (N) .18 (M1) (~) 1.55 (M1 + B1) (%) 2.79 (B1 + Mx) (5g) .35 85

typical 545 kg cow consumed 16 kg of feed daily, then an average of 8 /zg aflatoxin B1/kg of feed (dry weight) would give aflatoxin M~ levels in milk at the detection limits of the newer analytical methods (.1 /zg/liter). Calculation of other data (3) indicate that average aflatoxin in feed intakes of 16 ~g B1/kg feed would give atlatoxin M1 levels in milk at the detection limits of present analytical methods (64, 117). The metabolism of atLatoxin has been studied in both the lactating cow and ewe (Table 2). When a single dose of 300 mg (.5 mg/kg) of mixed aflatoxins were administered to cows, about 857o of the total detected aflatoxin was excreted in the first 48 hr. Only 4.525g of the total dose was accounted for by excretory routes using the TLC analytical method. The urine contained 1.557o, the feces 2.79~, and milk 0.18%. Only affatoxin M~ was found in milk and this represented .357O of the administered B~ dose (4). In lactating ewes only 8.17o of the dose was recovered with 6.47O in the urine, 1.6go in the feces, and .1% in the milk. About 90% of the milk and urine aflatoxin excretion occurred within 48 hr and none was detected in the milk after 6 days, in the urine after 8 days, and in the feces after 9 days. About .25~ of the administered B1 was converted to M~ (88). The most noticeable difference between lactating cows and ewes was the greater urine and fecal excretion of B1 from the cow than from the ewe (4). It is suggested that B~ is metabolized to a greater extent by the hepatic microsomal hydroxylating enzyme system in the sheep. Early reports indicated resistance of sheep to Mtatoxin poisoning (70), however, recent work indicates that the susceptibility of wethers to acute aflatoxin poisoning may be similar to that of other species (6). Recent work with wethers indicates that the total excretion of aflatoxin B~ + M~ amounts to 9.3% of the B~ dose and is similar to that of the

Ewe 1 nag/kg (78 mg) 8.1 .1 (M1) 6.4 (M1) 1.6 (M1) .25 90

lactating ewe and cow (119). A summary (Table 1) of attatoxins in tissues of cows and ewes given various levels of aflatoxin has been prepared. Since a small portion of the ingested aflatoxin dose appears as a toxic metabolite in milk, it is of importance to know how frequently commercial milk becomes contaminated. Ducklings were used as a biological test organism for aflatoxin in milk from 11 collection centers in England, Wales, and Ireland. No toxicity was shown (2). Aflatoxin M was found in 5 out of 21 samples of milk collected from retail outlets in South Africa (100). Samples of commercial and farm bulk tank milk in this country revealed no detectable amounts of altatoxin using the thin layer chromatographic method with detection limits of about 3/xg/liter (14). A considerable amount of information is needed on the metabolism of attatoxin and its effect on livestock health and productivity. Only 4 to 87O of the aflatoxin administered to cows or ewes could be recovered. Tissue levels are not high in animals dosed with varied levels of aflatoxin (Table 1) as measured by current chromatographic methods. When either 14C methoxy-labeled or 1~C ring-labeled attatoxin B 1 was given to rats in a single intra-peritoneal dose, 70 to 80g of the activity of the dose was recovered during 24 hr after dosing (129). The fact that some toxic effects persist beyond the time that the toxin can be detected indicates a possible retention of metabolites undetected in tissues by present chromatographic methods. Support for this suggestion comes from recent work in which chromatographic methods were modified to identify the metabolite P1 from monkey urine (35) and also the report of an unidentified, nonfluorescent, "metabolite x" from liver preparations of several species metabolizing aflatoxin B 1 (93). JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 9

1248

LYNCH

volved in a number of serious outbreaks of mycotoxicoses among farm animals. F. roseum (Gibberella zeae) was reported to produce a uterotrophie substance on corn that produced vulvar and mammary hypertrophy in sows (53, 69). This toxin is usually produced in storage (22) under conditions of alternating moderate and cool periods. An estrogenic metabolite of Fusarium was isolated from corn and called F-2 toxin (53). The chemical structure was reported to be a 6-(10 hydroxy-6oxo-trans-l-undeeenyl)-B-resoreylie acid laetone (Fig. 2) o:r commonly called zearalenone (53). Methods of analysis for zearalenone have been developed (21, 44, 118) and its production has been shown by Fusarium roseum (45) and other Fusarium species (21). One milligram of zearalenone will produce the uterotrophie effect in a gilt within 48 hr, but less will promote growth in swine, sheep, and cattle (11). The F-2 toxin was found in samples of hay that had been fed to dairy cattle. This hay caused an increase in the number of artificial insemination services per conception in these cows (82). Recently, isolates of F. solani halve been reported to produce symptoms of atypical interstitial pneumonia when fed to cattle (38). Tall feseue, Fest~ca arundinacea, has been associated with rescue toxicity during cold seasons. Symptoms include lameness, loss of weight, arched back, swelling of the hind legs, cracks showing separation of the hooves from the feet, dry gangrene of the extremities, altered hoof and horn growth. Vasoconstriction of the peripheral circulatory system may be responsible for fescue foot symptoms. Animal production is affected by a reduction in daffy gain and dry matter intake of certain tall rescue varieties when symptoms of rescue foot are shown (6311. Clinical symptoms of feseue foot were produced by giving cattle an intraruminal dose of an 80% ethanol extract of toxic hay (62). Feseue foot is not contagious nor infectious and occurs sporadically by season or by regions (63). This suggests that a mold may be responsible (130). A toxin, 4acetamido-4-hydroxy-2-butenoic acid-y-lactone (acetamido laetone, Fig. 2) was shown to be produced by a strain of F. tricincturn isolated from tall fescue hay. Evidence indicates a relation between symptoms of rescue foot and administration of acetamido lactone (66). The acetamido lactone was prepared synthetically and daffy intramuscular injections Toxins of the Fusarb3 given to a heifer for 90 days. Arched back and The Fusarium species is widely distributed dry gangrene at the end of the taft, but no hoof in stored cereal grains and has shown to be in- damage, were shown. The symptoms exhibited

The Aspergilli have been responsible for the production of several other mycotoxins that have a possible significance in livestock health and production. A. ochraceus Wilh. has a widespread distribution, is found in stored grains and produces a toxic metabolite called ochratoxin A (106, 121). Ochratoxin A has also been reported to be produced by Penicillium viridicatum (107, 122). This compound is an amide formed from phenylalanine and is a substituted chloroisocoumarin (Fig. 2). Methods of detection have been reported (44, 50, 106, 115, 118), and conditions of toxin production with pure cultures described (105). Ochratoxin A has been found as a natural contaminant of corn (110). In a study of possible causes of bovine abortion, ochratoxiu A produced fetal death and resorption in pregnant rats (116). A. clavatus, Penicillium patulum, and Penicillium urticae have been shown to produce an antibiotic substance called clavaein or patulin (59, 89, 91). Its structure is an a, fi-unsaturated lactone (Fig. 2), and it has been found in soil and wheat straw residue (90). Patulin has been reported to be carcinogenic and mutagenic (80). Cattle deaths were traced to a patulin-producing fungus isolated from feed (60). Samples of feed from a farm reporting deaths of dairy cattle were found to contain large numbers of A. clavatus spores. Pure cultures of the A. clavatus taken from the ration were lethal when incorporated into mice rations (73). A method of analysis for patulin has been reported (96, 118), A naturally occurring metabolite of A. versicolor and A. ~nidulans, similar in structure to aflatoxin (Fig. 2), called sterigmatocystin is reported to be both hepatoxic and carcinogenic (71, 99). Although this fungus is widely distributed, no serious livestock losses have been attributed to this toxin. A method of analysis for sterigmatocystin in grain samples has been reported (114, 118). Bovine hyperkeratosis has been attributed to the ingestion of highly chlorinated napthalenes characterized by lacrimation, salivation inappetence, low plasma vitamin A levels, and hyperkeratosis (92). Calves fed pure cultures of A. clavatus and A. chevalieri showed symptoms similar to those of bovine hyperkeratosis (24, 46, 53). The role of myeotoxins in bovine hyperkeratosis has not been clarified.

JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 9

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MYCOTOXINS IN FEEDSTUFFS

are characteristic of fescue foot (55). The natural occurrence of this compound on rescue grass has not yet been reported. A syndrome, termed "moldy corn toxicoses", has been shown to be associated with the intake of moldy corn and has been reported in both swine and cattle (112). In the acute form, cattle show depression, loss of weight, blood-stained diarrhea, and bleeding from the nostrils. Post-mortem changes in both cattle and swine include extensive hemorrhages in many tissues, yellow livers, and edema (127). Aflatoxin and ochratoxin have been isolated from moldy corn (110). Fungi cultured from moldy corn have been shown to produce aflatoxin, rubratoxin B, cyclopiazonic acid, ochratoxin A, trichodermin, diacetoxyscirpenol, and other trichothecenes (11). Some of these toxins have been discussed individually in this paper but little is known about the specific toxins involved with moldy corn toxicoses in livestock production. It is probable that more than one toxin may be involved in field outbreaks. For example, rubratoxin B is reported to be synergistic in its toxicity with aflatoxin and A. flavus and P. rubrum are frequently found growing together (11, 41). Aflatoxin administration can produce extensive hemorrhagic changes typical of those reported in moldy corn toxicoses, but very few hemorrhages were noted in aflatoxin-treated calves (74). F. tricinctum produces a tricothecene called T-2 toxin. It is a local irritant, causes extensive hemorrhaging, necrotic loci of the liver and intestines (66). More work is needed to describe all the factors concerned with moldy corn toxicoses in livestock. Toxins of the Penicillia

In general, the Penicillium species are found commonly as a contaminant of stored corn (25). Work to date indicates the possibility of several mycotoxins. Cultures of P. rubrum from moldy corn were found to be toxic when fed to swine, a goat and horses. The toxic portion was found in the water soluble fraction (17). Two toxins with hepatoxic activity called rnbratoxin A and B have been characterized from P. rubrum (53). A ration containing A. flavus, P. cyclopium, and P. paIitans produced death in cattle (1). Aflatoxins have been reported to be produced by isolates of P. puberulum (58, 68). P. cyclopium and A. flavus reportedly produce tremorgenic toxins that were neurotoxic to sheep and horses. When small amounts of the toxin from P. cyclopium were injected into

mice it produced tremors progressing to clonic or tetanic convulsions and death. The tremors were not abolished by anticholinergic agents. Microffam quantities of the toxin stimulated contraction of smooth muscle. Abnormal urine in the test subjects also suggested some impairment of glucose and electrolyte reabsorption in kidney tubules (126). A strain of P. palitans may have been involved in the deaths of several dairy cows. Both P. cyclopium and P. palitans have shown to be capable of producing treinorgenic mycotoxin (30, 126). Recent work reports that existence of tremortin A and tremortin B. A colorimetric method for the quantitative estimation of these compounds was developed (61). Cultures of P. oxalicum and P. viridicatum were shown to be hepatoxic when fed to mice. Gross lesions in mice from P. viridicatum were icterus, green kidneys and urine, mottled and greenish livers. Histological liver lesions included typical focal areas of necrosis, bile duct hyperplasia and fibrosis. Tests for aflatoxin proved to be negative (25). It is of interest to note that P. viridicatum isolated from barley also produced a nephrotoxic compound for rats and pigs called citrinin (26, 49). It is proposed that citrinin, or citrinin plus some other synergist, may be responsible for kidney degeneration that occurs among Danish pigs (67). Other Fungal Toxins

Cattle and sheep fed red clover hay slobbered excessively, went off feed, developed diarrhea, bloat, stiff joints and often died. Samples of the red clover forage were found to be infested with the fungus Rhizoctonia leguminicola or black patch disease of red clover, A parasympathomimetie alkaloid having salivation activity was isolated from the fungus. Its structure (Fig. 2) was shown to be 1-acetoxy-8-aminooctahydroindolizidine and called slaframine (53). This compound has been shown to be a potent stimulator of exocrine glands and to stimulate pancreatic activity. Slaframine is not the active compound but is thought to be converted to an active form by the liver rnicrosomal enzymes (9, 10). A syndrome of both cattle and sheep called "facial eczema" was reported from New Zealand and parts of Australia. The fungus Pithomyces chartarum was found to be the cause. The toxic metabolites were isolated and named sporidesmins after the identification of the original fungus Sporidesmium backeri. Sporidesmin is present in high concentrations JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 9

1250

LYNCH C00g

OH 0

0

U

-OH

CI

Patulln

Ochratoxin A

°H

0 0g .

CH3 _ ! u_ _112

~0 3 II

Zearalenone

Sterlgnmtocystln

(~~NHCOCH

3

Acetamldo lactone

2 ~-2

H2N

A~;

Slaframine

Fic. 2. Structures of several myeotoxins of possible importance in livestock production. in the spores. Optimal growth requirements of the fungus are 100~ relative humidity and a minimum temperature of 13 C. The structures of sporidesmin A, B, and C have been reported (53). Symptoms include loss of weight, icterus, inflammation, and scabs on the exposed skin and photosensitivity. Histological lesions of the liver were acute inflammation of the bile ducts, local areas of necrosis around occluded bile ducts and areas of proliferation around undamaged bile duct cells. An oral dose of 1.0 mg sporidesminfkg body weight in sheep will produce severe liver lesions, icterus, photosensitization and high mortality (37, 83, 84, 85). Excretion studies with sporidesmin show that the largest concentration of a single dose in the sheep was between the serum and the bile which explains the damage found in the biliary system (86). Facial eczema is a secondary effect brought on by the failure of the damaged liver t o detoxffy phylloerythrin, a normal metabolic product of chlorophyll. The skin becomes sensitized with the accumulation of phylloerythrin so that exposure to bright sunlight causes an acute inflammatory reaction on unprotected areas of the skin. No JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 9

sporidesmin poisoning of livestock in this country has been reported. The ergot fungi, Claviceps purpurea and C. paspali have long been known to be toxic to cattle, sheep, and horses. Ergot occurs on cereal crops such as rye, barley, wheat and triticale from the Northern plains states 2. Symptoms of C. purpurea poisoning include lameness, gangrene and inflammation of the digestive tract. The ergot alkaloids cause vasoconstriction of the peripheral circulatory system along with a rise in blood pressure, injury to the capillary endothelium, loss of circulation and gangrene of the extremities develops (39). Recent work with pigs fed low levels of crude ergot showed a reduction in feed intake, weight gain and nitrogen retention (48). Most recent reports are Busch, R. H. and H. D. Wilkins. 1972. Agronomic performance of triticale in North Dakota. North Dakota Farm Res. 29:29; Gilles, K. A., L. D. Sibbitt and R. L. Kiesling. 1972. Ergot causes in wheat crop analyzed; guide in handling. The Southwestern Miller, Feb. 29, 1972; Puranik, S. B. and D. E. Mathre. 1972. Biology and control of ergot on male sterile wheat and barley. Phytopathology, 61:1075.

MYCOTOXINS IN FEEDSTUFFS Symptoms of C. paspali include muscular trembling and staggers. Some of the ergot alkaloids include elymoclavine, festuc]avine, and several amides of lysergic acid (15). Stachybotryotoxicoses is a mycotoxicoses produced b y the fungus Stachybotrys atra. This fungus has world wide distribution and is found on straw, hay, and stubble. It usually effects horses and humans but the lesions can be produced in sheep, calves, and swine (53). Most of the natural outbreaks have been reported from the USSR. Symptoms include salivation, swelling and cracking of the lips, prolonged prothrombin time, leukopenia, diarrhea and death. An atypical form occurs that produces either hyperirritability or pronounced depression and death occurs within 72 hr (47). Profuse hemorrhage and tissue necrosis are found in both forms. A detailed description of post mortem lesions has been reported (127). Conclusions Research work on the identification, analysis, physiological effects of the numerous toxic fungal metabolites on sub-cellular components, cells, tissues and complete animals is still in the early stages of development. The toxicity of many of the compounds is high so that natural contamination of foods produces the threat of possible carcinogenesis and natural contamination of livestock feeds produces death losses or a severe reduction or both in livestock productivity. Incidences of mycotoxicoses in livestock are usually characterized by a rapid onset with sudden death losses. The extent of these losses throughout this country is unknown. The risk of exposure to the occurrence of mycotoxin poisoning in livestock production could be reduced by recognizing the field conditions and farm storage conditions under which these molds develop. References (1) Albright, J. L., S. D. Aust, J. H. Byers, T. E. Fritz, B. O. Brodie, B. E. Olsen, R. P. Link, J. Simon, H. E. Roades, and R. L. Brewer. 1964. Moldy corn toxicosis in cattle. J. Amer. Vet. Med. Ass., 144:1013. (2) Allcroft, R., and R. B. A. Carnaghan. 1963. Groundnut toxicity: An examination for toxin in human food products from animals fed toxic groundnut meal. Vet. Bee., 75:259. (3) Allcroft, R., and B. A. Roberts. 1968. Toxic groundnut meal: The relationship between aflatoxin Ba intake by cows and exeretSon of attatoxin M1 in milk. Vet. Ree., 82:116. (4) Allcroft, R., B. A. Roberts, and M. K. Lloyd. 1968. Excretion of aflatoxin in a

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lactating cow. Food Cosmet. Toxicol., 6:619. (5) Alsberg, C. L., and O. F. Black. 1913. Contributions to the study of maize deterioration. U. S. Dept. of Agriculture, Bur. Plant Industry Bull., 270. (6) Armbreeht, B. H., W. T. Shalkop, L. D. Rollins, A. E. Pohtand, and L. Stotoff. 1970. Acute toxicity of aflatoxin B1 in wethers. Nature, 225:1062. (7) Armbrecht, B. H. 1971. Aflatoxin residues in food and feed derived from plant and animal sources. Residue Rev., 41:13. (8) Aust, S. D. 1964. Effects of feeding moldy corn to cattle. Illinois Vet., 7:10. (9) Aust, S. D., H. P. Broquist, and K. L. Bluehart, Jr. 1968. Slaframine: A parasympathomimetie from Rhizoctonia leguminicola. Biotechnol. Bioeng., 10:403. 10) Aust, S. D. 1969. Evidence for the bioactivation of slaframine. Biochem. Pharmacol., 18:929. 11) Bamburg, J. R., F. M. Strong, and E. B. Smalley. 1969. Toxins from moldy cereals. J. Agr. Food Chem., 17:443. 12) Booth, A. N. 1969. Review of the biological effects of aflatoxins on swine, cattle, and poultry. Abstr. J. Amer. Oil Chem. See., 46:154. (13) Bottomley, R. A., C. M. Christensen, and W. F. Geddes. 1950. The influence of various temperatures, humidifies, and oxygen concentrations on mold growth and biochemical changes in stored yellow corn. Grain Storage Studies IX. Cereal Chem., 27:271. 14) Brewington, G. R., J. L. Weihrauch, and C. L. Ogg. 1970. Survey of commercial milk samples for aflatoxin M. J. Dairy Sci., 53:1509. (15) Brook, P. J., and E. P. White. 1966. Fungus toxins affecting mammals. Ann. Rev. Phytopathol., 4:171. (16) Buckley, S. S., and W. G. MacCallum. 1901. Acute haemorrhagic encephalitis prevalent among horses in Maryland. Amer. Vet. Bey., 25:99. (17) Burnside, J. E., W. L. Sippel, J. Forgaes, W. T. Carll, M. B. Atwood, and E. R. Doll. 1957. A disease of swine and cattle caused by eating moldy com. IL Experimental production with pure cultures of molds. Amer. J. Vet. Res., 18:817. (18) Burr, A. W. A., G. R. Dunton, and D. C. Thomas. 1964. Effect of fimgal contamination of hay upon the productivity of the dairy cow. Animal Prod., 6:261. (19) Butler, W. H. 1964. Acute toxicity of ariatoxin BI in rats. British J. Cancer, 18:756. (20) Cardeilhae, P. T., E. C. Schroeder, J. T. Perdomo, G. E. Combs, and G. T. Edds. 1970. Stunted pigs from sows fed crude aflatoxin. Toxieol. Appl. Pharmacol., 17:548. (21) Caldwell, R. W., J. Tuite, M. Stob, and R. Baldwin. 1970. Zearalenone production by Fusarlum species. Appl. Microbiol., 20:31. JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 9

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