Toxicity of Corn Culture Material of Fusarium proliferatum M-7176 and Nutritional Intervention in Chicks1

ENVIRONMENT AND HEALTH Toxicity of Corn Culture Material of Fusarium proliferatum M-7176 and Nutritional Intervention in Chicks1 RAMAKRISHNAN Y. NAGARAJ and W. D. WU2 Department of Poultry Science, University of Wisconsin, Madison, Wisconsin 53706 R. F. VESONDER

ABSTRACT The toxicity of Fusarium proliferatum M-7176 cultured on corn (FPC) and nutritional intervention were investigated in baby chicks (New Hampshire x Single Comb White Leghorn) in three 2-wk feeding experiments. In Experiment 1, 30% FPC decreased weight gain (P < .05) and increased relative heart weight (RHW) (P < .01). Experiment 2 included a 2 x 2 factorial arrangement of FPC (0 or 30%) and Se (0 or 5 mg/kg) and two detached treatments of Se (2.5 mg/kg) or thiamin (Bv 25 mg/kg) supplementations to 30% FPC. Only Bj was inhibitory to the toxic effects of FPC on weight gain, feed efficiency, and RHW (P < .05). Experiment 3 included 2 x 2 factorial arrangement between FPC (0 or 30%) and Se (0 or 4 mg/kg), or Bj (0 or 50 mg/kg), or vitamin E (0 or 50 IU/kg) and additional supplementations of Se (2 mg/kg), Bj (10 or 25 mg/kg), or E (10 IU/kg) to 30% FPC. A new batch of FPC was used and it caused 36% mortality. Vitamin E did not interact with FPC, but SE interacted with FPC only on RHW (P < .01). Thiamin interacted with FPC on all measured variables with significance ranging from P < .1 to P < .01. Supplementation of Ba as low as 10 mg/kg was inhibitory to some toxic effects of FPC. However, Bi as high as 50 mg/kg did not completely negate the cardiotoxicity. Water-extractable Bt in FPC diets was 13 to 27% of the control diets. Water extract of FPC reduced Bi recovery from a standard solution by 40%. The anti-thiamin factor was heat-sensitive. Both fumonisins and moniliformin were present in FPC. However, the results indicate that the anti-thiamin factor is also a major toxic factor of F. proliferatum M-7176. (Key words: Fusarium proliferatum, mycotoxin, toxicity, anti-thiamin factor, nutritional intervention) 1994 Poultry Science 73:617-626

INTRODUCTION

Cole et al. (1973) first described the fungal metabolite moniliformin (sodium or potassium salt of 1-hydroxycyclobut-

Received for publication July 12, 1993. Accepted for publication December 22, 1993. iSupported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison. 2 To whom correspondence should be addressed.

l-ene-3,4-dione). The acute toxicity of moniliformin has been recognized. It is highly toxic to baby chicks [oral 50% lethal dose (LDso) = 4.0 mg/kg] (Cole et al, 1973), ducklings (oral LD50 = 3.7 mg/kg), and weanling rats (oral LD50 = 50 mg/kg for males and 41.6 mg/kg for females) (Kriek et al, 1977). Moniliformin administered orally causes severe myocardial lesions in cockerels, ducklings, rats, and mice (Thiel, 1978). In rats, dietary

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Mycotoxin Research, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, Peoria, Illinois 61604

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moniliformin at 3, 5, and 8 mg/g of diet would be involved in the toxicity; and 3) induces hemorrhages in the intestines and to evaluate nutritional interventions of the 100% mortality within 16 h (Abbas et al, FPC toxicity with Blr vitamin E (E), and 1990). In general, muscular weakness, Se. Oxidation of pyruvate and arespiratory distress, and cyanosis are as- ketoglutarate requires the cofactor thiamin sociated with moniliformin intoxication in pyrophosphate, whereas GSSG-Px is Seanimals. Moniliformin (<5 mM selectively dependent. Both Se and E are important in inhibits mitochondrial pyruvate and a- protecting the cell from oxidative ketoglutarate oxidations by 50% (Thiel, damages. These provide the rationales 1978). It also inhibits isolated rat my- why Bj, Se, and E are considered in the ocardial glutathione peroxidase (GSSG-Px) nutritional intervention of moniliformin(Chen et al., 1990). Moniliformin- associated toxicity. containing fusarial culture caused hydropericardium, myocardial pallor, and MATERIALS AND METHODS ascites in broiler chicks, ducklings, and turkey poults (Engelhardt et al, 1988). Culture materials from isolates of Fusar- Corn Culture of Fusarium ium moniliforme or Fusarium proliferatum proliferatum M-7176 have been shown to cause equine leucoenWhole shelled corn, after soaking in hot cephalomalacia (Marasas et al, 1988), por- running water (65 C) for 4 h, was bottled cine pulmonary edema (Harrison et al, into 3.84-L plastic containers (filled to one1990), promotion of liver tumors in rats quarter of total volume) and covered with (Gelderblom et al, 1988), and reduced cotton plugs. The corn was autoclaved at growth and feed utilization in poultry 121 C for 30 min, and the autoclaving was (Brown et al, 1992; Ledoux et al, 1992; repeated the next day. Spore suspension Weibking et al, 1993). The causal factors was prepared by inoculating sterile are fumonisins (Bezuidenhout et al, 1988). Czapek-Dox broth3 with F. proliferatum MIn addition to elaboration of toxins, F. 7176 and agitating on a shaker for 3 d at 22 moniliforme was shown to produce a C. Twenty milliliters of the spore suspenthiaminase that could destroy thiamin sion was added per container. After 5 wk of (vitamin Bj), and the Bx deficiency in incubation at 22 C, the moldy corn was affected chicks could be prevented by dried at 40 C for 3 d and then ground into a adding more B1 to the diet, by autoclaving fine powder. This powdery culture material the moldy substrate, or by injecting the was referred to as FPC and kept at 4 C until chicks with thiamin hydrochloride (Fritz et use. Two batches of FPC were prepared at different times. The first batch was used in al, 1973). Fusarium proliferatum M-7176 was iso- Experiments 1 and 2 and the second batch lated from a high moisture corn that was in Experiment 3. originally associated with indigestion and feed refusal in calves. Both the dried corn Analysis of Moniliformin and and corn cultured with the fusarial mold Fumonisin were found to cause feed refusal in Pekin ducklings in a preliminary investigation. The FPC material (100 g) was extracted This mold was also found to produce with CHC13 in a Waring blender jar.4 The moniliformin and fumonisins. The pur- CHCl3-extracted FPC material was divided pose of this study was: 1) to evaluate the into two 40-g samples. Each sample was toxicity of corn fermented with F. prolifera- extracted with 250 mL acetonitrile and tum M-7176 (FPC) on 1-d-old chicks; 2) to water (60:40) for 12 h. An aliquot of the evaluate whether an anti-thiamin factor extraction solvent equivalent to 25 g of the sample was collected and evaporated to dryness. Each residue was worked with water (20 mL) and passed through a 3 Difco Laboratories, Detroit, MI 48232. 20-g Amberlite XAD-2 resin,5 and washed 4 Waring Products Division, New Hartford, CT with water until chemical analysis indi06057. 5Sigma Chemical Co., St. Louis, MO 63178-9916. cated the absence of moniliformin (three to

TOXICITY OF FUSARIUM PROLIFERATUM

Experiment 1 This experiment was designed to find out the toxic dose of FPC. One-day-old New Hampshire x Single Comb White Leghorn chicks were wing-banded, weighed, and randomly distributed into eight groups of seven chicks in each group. The groups were placed in a thermostatically controlled electric brooder batteries. Two groups were fed a control diet (0% FPC) or diets containing 10, 20, or 30% FPC, formulated according to the NRC standards (1984). The FPC replaced a portion of the corn in the corn-soybean diet. The chicks were monitored twice a day for clinical signs, and at 14 d of age they were individually weighed, killed with C0 2 gas, and necropsied. The heart and liver weights of the chicks were recorded, and relative organ weight to 100 g of body weight calculated. The feed consumed per group of chicks for the entire 2 wk was also recorded.

were combined to form a 2 x 2 factorial arrangement of treatments. The treatment of 30% FPC + 2.5 mg/kg Se was included to observe the effect of intermediate Se supplement. The treatment of 30% FPC + 25 mg/kg Bx was also included to pilot test the effect of supplemented Bj. The housing conditions and observations were similar to Experiment 1. The FPC was the same batch used in Experiment 1. Experiment 3 Two levels of FPC (0 or 30%), two levels of supplemented Se (0 or 4 mg/kg), two levels of supplemented B\ (0 or 50 mg/kg), and two levels of supplemented vitamin E (0 or 50 IU/kg) were combined to form three 2 x 2 factorial arrangements to test the interactions between FPC and an individual nutrient. Treatments of 30% FPC + intermediate levels of nutrients were also included to study the efficacy of these nutrients. Two groups of seven chicks in a group were allocated to each treatment. The housing conditions were similar to Experiment 1. A second batch of FPC was used in Experiment 3. Measurement of Water-Extractable Thiamin

The water-extractable Bj in the experimental diets was measured following the basic principles of Association of Official Analytical Chemists (AOAC) procedure (1984), with some operational modifications (i.e., no enzyme was used to release the bound thiamin). Three grams of sample were mixed with 12 mL of water for 15 min. The sample was centrifuged at 2,500 rpm for 5 min and the supernatant filtered using a Whatman No. 42 filter paper.8 In a test tube, 1 mL of the filtrate was added to 1 mL of oxidizing solution (2 mL of 1% potassium Experiment 2 ferricyanide + 48 mL of 15% potassium hydroxide, freshly mixed). To this mixture, Two levels of FPC (0 or 30%) and two 3 mL of isobutanol was added and the levels of supplemented Se (0 or 5 mg/kg) mixture was vigorously shaken for about 1 min and set aside for the separation of the two layers. About 2.5 mL of the top layer 'Pierce, Rockford, IL 60015. was carefully removed for fluorescence 7 Alltech Associates, Inc., Deerfield, IL 60015. 8 Whatman Scientific Ltd., Maidstone, Kent, ME16 measurement. A series of Bj standard solutions was prepared fresh and oxidized 0LS, UK.

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four column volumes). The XAD-2 column was next eluted with CH3OH and this eluate was saved for fumonisin analysis. The detection, quantification, and confirmation of moniliformin in the water eluate were by methods described by Vesonder (1986), which utilized thin-layer chromatography and ultraviolet spectroscopy. The methanol eluate was taken to dryness on a rotary evaporator and residue dissolved in 20 mL of CH3OH:H20 (20:80). Ten microliters of the latter solution was derivatized with orthophthalate anhydride,6 injected into a Econosphere analytical C18 reverse phase column/ and eluated with gradient 60 to 90% CH3OH/.01 M sodium acetate buffer (pH 6) at a flow rate of 1 mL/ min. The measurement was by a fluorescence detector set at excitation of 340 ran and emission of 454 ran.

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TABLE 1. Effects of various levels of Fusarium proliferatum culture material (FPC) on the mean weight gain, feed efficiency, and relative weights of heart and liver of 2-wk-old chicks, Experiment 1 Treatment

Weight gain

Feed efficiency

Heart

(%)

(g) 96* 108* 88* 76" 5

(g=g) 3.04 2.81 3.14 3.2 .27

.83bc .8C .91b 1.06a .02

0 10 20 30 Pooled SEM Source of variation Treatment a_c

.0673

.2184

Liver - (g/100 g BW)

.0001

Means within a column with no common superscript differ significantly (P < .05).

4.54 4.86 4.93 4.93 .09 .3413

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as described for the sample extract. The Statistical Analysis thiochrome fluorescence was measured usThe percentage data (mortality and thiaing a fluorometer at excitation of 365 run min recovery) were first transformed by arc and emission of 435 nm. The Bi concentrasine square root, then all data were subtions in the different samples were measured by reading directly from the standard jected to one-way ANOVA using the General Linear Models procedure of the curve. SAS® (SAS Institute, 1985) to measure the treatment effect. Means of treatments were Recovery of Thiamin after separated by least significant difference (a = Coincubation with Water Extract of .05), after the probability of Fisher's test was Culture Material of less than 10% (marginal significance). When appropriate, two-way ANOVA was carried Fusarium proliferatum out to test effects of factors (FPC and Three grams of the sample were ex- nutrient) and interaction of factors. tracted with 180 mL water for 15 min by agitation. The sample was then centrifuged RESULTS at 2,500 rpm for 5 min and the supernatant filtered using a Whatman No. 42 filter paper. Nine milliliters of the filtrate were Mycotoxins Produced by Fusarium added to 1 mL of 10 mg/mL Bi solution and proliferatum M-7176 incubated for 45 min at 22 C. Then 1 mL of Moniliformin was found to be present at this solution was added to 1 mL of a concentration at 37 and 102 ppm in the oxidizing solution. After extraction with first and second batch of FPC, respectively. isobutanol, Bj concentration was deterThe first batch of FPC contained 14 ppm of mined as described above. Recovery was fumonisin B (FB^ and 47.5 ppm of hydrox calculated as thiamin recovered from coin- lyzed fumonisin Bj (HFBj). The second cubation with the culture extract divided by batch of FPC contained 37 ppm of FBj and Ba recovered from coincubation with water. 112 ppm of HFBj. The potential influence of moniliformin on the recovery of Bx was also investigated by coincubation of Bj with moniliformin at Experiment 1 1, 5, and 10 M concentrations equivalent to Weight gain was marginally affected by that of Bi. The purified moniliformin used FPC (P < .07), and 30% FPC resulted in a in this investigation was prepared by lowest weight gain among the treatments fermentation of corn grits with F. (Table 1). Feed efficiency was not affected moniliforme NRRL 6322 as described by by addition of FPC. Relative heart weight of Burmeister et al. (1979). chicks was affected by FPC (P < .01). Chicks

TOXICITY OF FUSARIUM PROLIFERATUM

fed 30% FPC had the heaviest absolute heart weight (data not shown) and relative heart weight. Relative liver weight was not affected by FPC. Hydropericardium was observed in more than half of the chicks fed 30% FPC, whereas no other gross pathological changes were recognizable and no mortality resulted.

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Experiment 3

TABLE 2. Effects of feeding 30% Fusarium proliferatum culture material (FFC) in combination with various levels of selenium and thiamin (Bj) on the mean weight gain, feed efficiency, and relative weights of heart and liver of 2-wk-old chicks, Experiment 2 Treatment FPC

Nutrient

Weight gain

Feed effi

(%)

(mg/kg)

(g)

0 0 30 30 30 30 Pooled SEM

0 5 Se 0 5 Se 2.5 Se 25 Bj

102* 84b 58= 60= 50= 104* 5

(g:g) 2.29= 2.97bc 3.58* 3.3" 4.29* 2.39= .23

Source of variation Treatment Factorial 1 FPC Se FPC x Se

Liver .78= .93>>

1.26* 1.24* 1.27* .93" .03

(g/100 g BW) 4.6* 4.71* 4bc 4.52*" 3.98= 4.6* .09

Probability .0001

.0108

.0001

.0192

.0001 .1809 .1328

.048 .5189 .1711

.0001 .1923 .0549

.0447 .1408 .2956

^Means within a column with no common superscript differ significantly (P < .05). iFactorial (2 x 2) arrangement of FPC (0 and 30%) and Se (0 and 5 mg/kg).

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Addition of 30% FPC (from second batch of culture) suppressed body weight gain (P < .01), decreased feed efficiency (P < .05), increased relative heart weight (P < .01), decreased relative liver weight (P < .05), and caused 36% mortality (P < .05) (Tables 3 to 5). Selenium had marginal effect on relative Experiment 2 heart weight (P < .06), and interacted with Addition of FPC decreased weight gain FPC on the relative heart weight (P < .01). (P < .01), feed efficiency (P < .05), relative Relative heart weight in chicks of 30% FPC heart weight (P < .01), and relative liver + 4 mg/kg Se was less than 30% FPC, but weight (P < .05) (Table 2). Addition of Se was still heavier than control groups (P < had no effects on these measured variables. .05) (Table 3). Thiamin affected body weight gain and There was a marginal interaction between FPC and Se on relative heart weight (P < relative heart weight (P < .01) (Table 4). The .06). Addition of 5 mg/kg of Se adversely effect of interaction between FPC and Bi affected weight gain and relative heart was marginal on body weight gain and feed weight when compared with no additions efficiency (P < .1), whereas it was significant of 0% FPC + 5 mg/kg Se (P < .05) (Table 2). on relative liver weight and mortality (P < Even though not specifically tested for .05), and highly significant on the relative interaction between FPC and B^ addition of heart weight (P < .01). Supplementation of Bi (25 mg/kg) appeared to antagonize the 10 mg/kg or more Bi improved body adverse effects of FPC on weight gain, weight gain than 30% FPC and prevented relative heart weight, and relative liver FPC-induced mortality. Supplementation weight (P < .05). Hydropericardium was of 25 mg/kg or more Bi antagonized the seen in the groups of 30% FPC and 30% FPC adverse effects of FPC on organ weights. + 2.5 or 5 mg/kg Se. The FPC again did not Vitamin E had marginal effect on weight cause mortality. gain (P < .1), and there was no interaction

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TABLE 3. Effects of feeding 30% Fusarium proliferatum culture material (FPC) in combination with various levels of selenium on the mean weight gain, feed efficiency, and relative weights of heart and liver of 2-wk-old chicks, Experiment 3 Treatment FPC

gain

Selenium

(%)

(g) 9 6 ab

99a 58= 68= 7 6 bc

4

(g:g) 2.60= 2.71= 3.32*b 3.44* 2.99abc .13

.0011

.0836

.0002 .4016 .6888

.025 .615 .9909

Heart

Liver

(g/100 g BW) 4.50* .84= 4.89a .87= 4.01 b 1.19" 1.05b 4.06b 1.16a 4.56 ab .02 .1 — Probability — .0001 .0314 .0001 .0563 .0082

Mortality •

.0055 .3411 .4791

(%)

0b 0b 35.7* 57.0^ 21.4 ab 8 .059 .0003 1 1

a_c

Means within a column with no common superscript differ significantly (P < .05). iFactorial (2 x 2) arrangement of FPC (0 and 30%) and Se (0 and 4 mg/kg).

control diets (Table 6). Thiamin recovered after coincubation with water extracts obtained from FPC or FPC diets was significantly lower than with water extract of corn or control diets (Table 7). Autoclaving of FPC or water extract of FPC prevented the inhibitory effect on Bj recovery, and addition of crystalline moniliformin to a Ba solution did not prevent Ba recovery (Table 8).

between FPC and E (Table 5). Weight gain of chicks on 30% FPC with E supplement was better than chicks on 30% FPC without E supplement (P < .05). Water-Extractable Thiamin and Thiamin Recovery The water-extractable Bj from 30% FPC diets was significantly lower than that from

TABLE 4. Effects of feeding 30% Fusarium proliferatum culture material (FPC) in combination with various levels of thiamin (Bj) on the mean weight gain, feed efficiency, and relative weights of heart and liver of 2-wk-old chicks, Experiment 3 Treatment FPC

Bi

(%)

(mg/kg) 0 50 0 50 10 25

0 0 30 30 30 30 Pooled SEM Source of variation Treatment Factorial 1 FPC Bi FPC x B t

—

Weight gain

Feed efficiency

(g)

(g«)

96* 106a 58= 90b 82b 93b 3

2.60b 2.54b 3.32a 2.70b 2.94* 2.75b .09

.0001

.0603

.0001 .0011 .0955

.0303 .1428 .098

Heart (g/100 .84=> .85d 1.19a

.98= l.l»b 1.04b= .02 — Probability — .0001 .0001 .0014 .0003

g

Liver

Mortality

BW) 4.50*= 4.26b= 4.01= 4.78* 5.02* 4.44abc .1

(%) 0b 7.1 b 35.7* 0b 7.1 b 7.1 b 4

.0396

.0345

.0456 .2108 .0197

.0498 .1092 .016

a-^Means within a column with no common superscript differ significantly (P < .05). iFactorial (2 x 2) arrangement of FPC (0 and 30% and Bj (0 and 50 mg/kg).

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(mg/kg) 0 0 0 4 30 0 30 4 30 2 Pooled SEM Source of variation Treatment Factorial 1 FPC Se FPC x Se

Feed efficiency

TOXICITY OF FUSARIUM. PROLIFERATUM

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TABLE 5. Effects of feeding 30% Fusarium proliferatum culture material (FPC) in combination with various levels of vitamin E (E) on the mean weight gain, feed efficiency, and relative weights of heart and liver of 2-wk-old chicks, Experiment 3 Treatment E

(%)

(IU/kg) 0 50 0 50 10

of variation Treatment Factorial 1 FPC E FPC x E

teg)

(g)

.84b .87b 1.18a 1.20* 1.19= .03

2.60b 2.68b 3.32a 2.93* 2.82* .11

96* 98 a 58 b 81 a 90" 4

Liver

Heart

(g/100 g BW) 4.50* 4.57* 4.01 bc 4.13*c 3.72' .09

.0032

.0789

.0001

.0096

.0005 .0792 .1579

.0353 .3692 .2102

.0001 .4386 .5798

.0240 .6296 .9075

Mortality

(%) 0b 0b 35.7* 35.7* 50.0* 9 .071 .0003 1 1

a_c

Means within a column with no common superscript differ significantly (P < .05). factorial (2 x 2) arrangement of FPC (0 and 30%) and E (0 and 50 IU/kg).

7-wk-old chicks when intravenously injected, chicks can tolerate reasonable quantity of moniliformin in their diet (up to 64 ppm) (Allen et al, 1981). The highest amount of moniliformin brought into the test diet by FPC was 30.6 ppm in Experiment 3, which was half of the amount used by Allen et al. (1981), and one fifth of

DISCUSSION

Results from studies using fumonisin and • moniliformin-producing strain M7176 of F. proliferatum confirmed the observation of Engelhardt et al. (1988) that moniliformin-containing culture of F. moniliforme var. subglutinans was cardiotoxic to poultry species. However, the level of moniliformin alone in FPC could not account for the observed toxicity. Even though moniliformin is extremely toxic to TABLE 7. Percentage of thiamin recovered day-old chicks when intubated and to from 1 pg/mL solution after coincubation with water extracts of corn, control diet, Fusarium proliferatum culture material (FPC), and FPC diets1 TABLE 6. Water-extractable thiamin in the control and FPC diets1 Sample

Thiamin content

G*g/g) Control diet (Experiment 2) FPC diet (Experiment 2) Control diet (Experiment 3) FPC diet (Experiment 3) Pooled SEM Probability

1.5a .4b 1.6a .2= .2 .0001

Sample

Recovery rate

Water Corn FPC (Batch 1) FPC (Batch 2) Control diet (Experiment 2) FPC diet (Experiment 2) Control diet (Experiment 3) FPC diet (Experiment 3) Pooled SEM Probability

100a 93.9* 57.9= 59.3= 98.3a 75.3b 97.8a 81.9b 4.8 .0006

(%)

""Means within a column with no common a_c Means within a column with no common superscripts differ significantly (P < .05). lr superscript differ significantly (P < .05). The FPC diets contained 30% Fusarium proliferatum lr rhe FPC diets contained 30% FPC. Water extracts culture material (FPC). Measurements were done 2 wk were made 2 wk after the diets were prepared. after the diets were prepared.

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0 0 30 30 30 Pooled SEM

Feed efficiency

gain

FPC

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TABLE 8. Percentages of thiamin recovered from 1 /ig/mL standard thiamin solution after coincubation with water extracts of autoclaved and nonautoclaved Fusarium proliferatum culture material (FPC) or with purified moniliformin1'2 Recovery

Water FPC (Batch 2) Autoclaved FPC Autoclaved FPC extract Moniliformin:thiamin 1:1 Moniliformin:thiamin 5:1 Moniliformin:thiamin 10:1 Pooled SEM Probability

100c 58.9d 101.6bc 97.3« 101.4bc 107.0ab 109.5a 5.2 .0001

(%)

a_d Means within a column with no common superscripts differ significantly (P < .05). 1 Second batch of FPC or FPC water extract were autoclaved at 121 C for 15 min. 2 Molar ratio of moniliformin to thiamin in the mixtures were 1:1, 5:1, and 10:1.

the amount used by Engelhardt et al. (1988) (calculated on their 12.5% diet). The mycotoxin FBX alone could not account for the toxicity of FPC either. Cardiotoxicity has not been reported in broiler chicks or turkey poults that have been fed diets with as high as 400 ppm of FBj (Brown et al, 1992; Ledoux et al, 1992; Weibking et al, 1993). When heart weights were specifically measured, FBi decreased heart weights of turkey poults (Weibking et al, 1993). The highest level of total FBt (FBX + HFBi) contributed by 30% FPC was 44.7 ppm in Experiment 3, which was less than half of the lowest amount used in broiler and turkey studies (Ledoux et al, 1992; Weibking et al, 1993). The addition of Bi prevented some toxic effects of FPC. Less than 27% of Bi was extracted from diets with 30% FPC when compared with control diets. About 40% of Bj was recovered from a standard Bi solution after coincubation with water extract of FPC. Taken together, it is indicated that an anti-thiamin factor is also a major contributor of FPC toxicity. The anti-thiamin factor in FPC is heatsensitive, because autoclaving destroyed its activity. Thiaminase was reported in F. moniliforme by Fritz et al. (1973) almost 20

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Coincubated with

yr ago, whereas our report is the first to recognize anti-thiamin factor in F. proliferatum. Recognition of anti-thiamin activity of these fusarial molds should help to explain the benefits of supplemented Bj observed in poultry nutrition, because these molds are common in corn-based rations (Wu et al, 1991). Total toxicity of FPC might result from the potential interactions among fumonisins, moniliformin, the anti-thiamin factor, and perhaps yet to be identified mycotoxins. Kriek et al. (1977) suggested that moniliformin caused acute degenerative lesions in the myocardium by the suppression of energy-driven transmembrane transport mechanism, resulting in the disturbance of intracellular osmoregulation and consequently severe intracellular edema. Thiel (1978) provided experimental evidence that moniliformin selectively inhibited mitochondrial pyruvate and aketoglutarate oxidations with a consequent reduction of oxidative phosphorylation (i.e., adenosine triphosphate production). Gathercole et al. (1986) further showed the inhibition of isolated bovine pyruvate dehydrogenase complex (PDH) by moniliformin. Thiamin pyrophosphate is a cofactor of PDH. Therefore, PDH may provide a molecular basis for the possible additive or synergistic interaction between moniliformin and the anti-thiamin factor. The presence of fumonisins could further exacerbate the toxicity of FPC. The higher amounts of both moniliformin and fumonisins in the second batch of FPC than in the first batch may explain the high mortality encountered in Experiment 3, because the activity of anti-thiamin factor was not different between the two batches of FPC. It is a common experience that a mycotoxin, when provided as purified form to chicks, is less toxic than when it is provided by the culture material that contains equal amount of the toxin. One reasonable explanation for this phenomenon is that the culture material contains additional toxic factors. Fusarium proliferatum M-7176 produces at least three toxic factors. In spite of the fact that poultry feed is frequently found to be contaminated by more than one mycotoxin, there have been few studies

TOXICITY OF FUSARIUM PROLIFERATUM

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Poultry Sci. 52:1523-1530.

In conclusion, F. proliferatum M-7176 is Gathercole, P. S., P. G. Thiel, and J.H.S. Hofmeyr, 1986. Inhibition of pyruvate dehydrogenase toxic to chicks in the forms of decreased complex by moniliformin. Biochem. J. 233: feed intake and weight gain, increased 719-723. heart weight, and mortality. The toxicity is Gelderblom, W.C.A., K. Jaskiewicz, W.F.O. Marasas, P. G. Thiel, R. M. Horak, R. Vleggaar, and N.P.J. attributable to three recognizable factors: Kriek, 1988. Fumonisins—novel mycotoxins the heat labile anti-thiamin factor, with cancer-promoting activity produced by moniliformin, and fumonisins. The main Fusarium moniliforme. Appl. Environ. Microbiol. 54:1806-1811. application of these findings is that supplementation of thiamin can be prophylac- Harrison, L. R., B. Colvin, J. T. Greene, L. E. Newman, and J. R. Cole, 1990. Pulmonary tic to some of the toxic factors of F. edema and hydrothorax in swine produced by proliferatum. fumonisin Bj, a toxic metabolite of Fusarium

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involving combinations of mycotoxins. ACKNOWLEDGMENTS Studies done with chickens have shown The authors thank R. E. Peterson for synergistic effects of aflatoxin and analytical HPLC and Terry Strand, Jim ochratoxin A on weight gain (Huff and Mason, and Bradley Bruegger for asDoerr, 1981). Interactions of additive and sistance in animal care. antagonistic nature have also been observed among mycotoxins on some biologREFERENCES ical measurements (Doerr, 1986; Huff et al, 1988). The effects of combined mycotoxins Abbas, H. K., C. J. Mirocha, R. F. Vesonder, and R. on biological systems need investigation, Gunther, 1990. Acute toxic effects of an isolate of moniliformin-producing Fusarium oxysporwn if we want to assess the impact of and purified moniliformin on rats. Arch. Envinaturally occurring mycotoxicoses. ron. Contain. Toxicol. 19:433-436. Moniliformin is thought to be an etio- Allen, N. K., H. R. Burmeister, G. A. Weaver, and C. J. Mirocha, 1981. Toxicity of dietary and inlogical factor of Keshan disease (human travenously administered moniliformin to cardiomyopathy, where myocardial necrobroiler chickens. Poultry Sci. 60:1415-1417. sis is seen) as the morbidity of the disease Association of Official Analytical Chemists, 1984. was very high wherever there was severe Methods of Analysis. 4th ed. Association of Official Analytical Chemists, Arlington, VA. contamination of the corn by F. moniliforme (more likely as F. subglutinans) Bezuidenhout, C. S., W.C.A. Gelderblom, C. P. Gorstallman, R. M. Horak, W.F.O. Marasas, G. (Zhang and Li, 1989). The blood of Keshan Spiteller, and R. Vleggar, 1988. Structure elucipatients was shown to have elevated dation of the fumonisins, mycotoxins from Fusarium moniliforme. J. Chem. Soc. Chem. levels of lipid peroxide and decreased Commun. 1988:743-745. activity of glutathione peroxidase (GSSGBrown, T. P., G. E. Rottinghaus, and M. E. Williams, Px) and unusual activity of glutathione 1992. Fumonisin mycotoxicoses in broilers: perreductase (GSSG-R) (Zhu et al, 1982). formance and pathology. Avian Dis. 36:450-454. These enzymes as well as vitamin E are Burmeister, H. R., A. Ciegler, and R. F. Vesonder, 1979. Moniliformin, a metabolite of Fusarium required for the scavenging of the free moniliforme NRRL 6322: purification and toxicradicals. The inhibition or alteration of ity. Appl. Environ. Microbiol. 37:11-13. these enzymes by moniliformin could be a Chen, L. Y., X. L. Tian, and B. Yang, 1990. A study on the inhibition of rat mycocardium glutathione possible explanation for the myocardial peroxidase and glutathione reductase by toxicity observed during Keshan disease. moniliformin. Mycopathologia 110:119-124. Moniliformin has been shown to competi- Cole, R. J., J. W. Kirksey, H. G. Cutler, B. L. Doupnik, tively inhibit the rat myocardial GSSG-Px and J. C. Peckham, 1973. Toxin from Fusarium moniliforme: effects on plants and animals. (Chen et al, 1990). The interaction between 179:1324-1326. FPC and Se on cardiotoxicity might indi- Doerr,Science J. A., 1986. The growing mycotoxin challenge. cate that chicken myocardial GSSG-Px was Poult. Digest 44:86-90. affected by moniliformin. However, the Engelhardt, J. A., W. W. Carlton, and J. F. Tuite, 1988. Toxicity of Fusarium moniliforme var. subglutinans practical utility of Se supplement is profor chicks, ducklings and turkey poults. Avian hibited by its toxic nature. The lack of Dis. 33:357-360. effects of vitamin E suggests that lipid Fritz, J. C, P. B. Mislivec, G. W. Pla, B. N. Harrison, peroxidation was not a toxic mechanism C. E. Weeks, and J. G. Dantzman, 1973. Toxigenicity of moldy feed for young chickens. of FPC.

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