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Survey of Molds and Mycotoxins for Their Ability to Cause Feed Refusal in Chickens1,2,3
Survey of Molds and Mycotoxins for Their Ability to Cause Feed Refusal in Chickens1'2'3 SUSAN J. BURDITT, WINSTON M. HAGLER, JR. and PAT B. HAMILTON Department of Poultry Science and Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27650 (Received for publication March 1, 1983) ABSTRACT The cause of feed refusal by poultry is an important problem that has not been established. T-2 toxin, a culture filtrate of Fusarium roseum NRRL 1181 containing a high concentration of diacetoxyscirpenol, and a filtrate of Penicilliiim citrinum containing an unknown factor produced refusal activity in week-old chickens. Citrinin stimulated water consumption while aflatoxin did not affect feed consumption. Detection of refusal activity was more sensitive when the refusal factor was presented in the drinking water than in the feed. The experimental model used here was sensitive, and, with appropriate controls, would appear suitable for the isolation and study of new factors capable of causing feed refusal in poultry. (Key words: T-2 toxin, diacetoxyscirpenol, citrinin, feed refusal, chickens) 1983 Poultry Science 62:2187-2191 INTRODUCTION Feed refusal in animals has been k n o w n for decades t o be a serious p r o b l e m (Curtin and Tuite, 1966). Feed refusal in this c o n t e x t has c o m e to be defined as a sudden refusal b y apparently h e a l t h y animals as a direct effect of a specific factor rather t h a n as a response secondary t o some deleterious factor or condit i o n . T h e relationship of feed refusal in swine t o m o l d - c o n t a m i n a t e d feed has been investigated by m a n y workers (Curtin and Tuite, 1966; Kotsonis et al, 1 9 7 5 ; Vesonder et al, 1976, 1 9 7 7 , 1 9 7 9 ; Mirocha et al, 1 9 7 6 ; Ueno et al, 1974) w h o have d e m o n s t r a t e d t h a t feed refusal in swine can be caused by t r i c h o t h e c e n e m y c o t o x i n s p r o d u c e d b y Fusarium growing in feed and ingredients. Feed refusal in p o u l t r y , while k n o w n for m a n y years, has n o t attracted m u c h a t t e n t i o n until recently when t h e experim e n t a l models necessary for study were developed (Hagler et al, 1 9 8 1 a ) . This developm e n t p e r m i t t e d feed refusal t o be established unequivocally as a s y m p t o m of ochratoxicosis
'Paper No. 8752 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650. 2 The use of trade names in this publication does not imply endorsement of the North Carolina Agricultural Research Service nor criticism of similar ones not mentioned. 3 A preliminary report of part of this paper was given at the annual meeting of the American Society for Microbiology, Atlanta, GA, March, 1982.
in t u r k e y s b u t n o t chickens ( H a m i l t o n et al, 1 9 8 2 ; Burditt et al, 1982). T h e only o t h e r evidence linking m y c o t o x i n s directly t o feed refusal in p o u l t r y appears to be t h e finding of Hagler et al. ( 1 9 8 1 b ) t h a t culture filtrates of Fusarium roseum containing high levels of t h e m y c o t o x i n diacetoxyscirpenol (DAS) p r o d u c e d d r a m a t i c refusal when added to feed or w h e n substituted for drinking water. T h e present c o m m u n i c a t i o n r e p o r t s o n a survey of some k n o w n m y c o t o x i n s and s o m e cultures of fungi suspected of causing feed refusal for their ability t o induce refusal in a model system u n d e r l a b o r a t o r y conditions (Hagler et al, 1 9 8 1 a ) . T h e results suggested t h a t feed refusal is a characteristic of several k n o w n m y c o t o x i n s and that t h e r e are u n k n o w n feed refusal factors awaiting isolation and identification.
MATERIALS AND METHODS Measurement of Feed Refusal. Day-old cockerels (Dekalb Sex-Sal) were o b t a i n e d from a commercial h a t c h e r y (Clay's H a t c h e r y , Blackstone, VA). T h e y were h o u s e d in electrically heated batteries u n d e r c o n t i n u o u s lighting with feed and water available ad libitum. On t h e evening of t h e sixth d a y , feed was removed if feed refusal was t o be measured or water was removed if water c o n s u m p t i o n was to b e measured. On the m o r n i n g of t h e seventh d a y , t h e birds were assigned o n a r a n d o m basis in groups of 30 birds per p e n .
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There were five pens per treatment. The initial body weight, feed weight, and water volume were determined for each pen. After 6 hours of refeeding or rehydration, the same variables were measured again and the consumption calculated for each treatment. Feed. The feed was a broiler starter mash of commercial type obtained from the university feed mill. Moisture content of the feed was determined gravimetrically after samples were dried overnight at 110 C in a forced draft oven. When culture filtrates were added to the feed, the moisture content of all control and treated feed was adjusted to a constant 35% by the combined addition of culture filtrate and deionized water. Culture Filtrates. Stagnant cultures of Fusarium roseum NRRL 1181, F. tricinctum NRRL 3299, F. roseum 'Gibbosum', and Fusarium sp. 2TRK were grown in Czapek's broth (Difco) supplemented with 2% peptone (Difco) for 14 days at 29 C in 2800 ml Fernbach flasks containing 1000 ml of medium. Penicillium citrinum, Rhizoctonia leguminicola, and Scopulariopsis brevicaulis were grown in a medium containing 2% yeast extract (Difco) and 4% sucrose. Except for the NRRL strains, the fungi were isolated as the predominant organism in suspect feed or ingredients. After incubation the cultures were filtered through three layers of cheesecloth to remove the mycelium. The filtrates for each organism were combined and placed immediately in the waterers for the birds or they were added to the feed alone or mixed with deionized water in quantities sufficient to give a final feed moisture content of 35% in all cases. Gas liquid chromatography of the filtrates by the method of Pathre and Mirocha (1977) revealed that F. roseum NRRL 1181 contained DAS (335 /ig/ml), F. tricinctum NRRL 3299 contained T-2 toxin (300 /xg/ml), F. roseum 'Gibbosum' contained trace amounts of zearalenone and monoacetoxyscirpenol (MAS), and Fusarium 2TRK was free of detectable T-2 toxin, MAS, DAS, and zearalenone. The P. citrinum filtrates were free of detectable citrinin by the method of Nelson et al. (1980). T-2 toxin, Citrinin, and Aflatoxin. Pure T-2 toxin was prepared by the method of Hagler et al. (1981b) and pure citrinin was prepared by the method of Davis et al. (1975). Aflatoxin was prepared as a rice powder concentrate by the method of Shotwell et al. (1966), analyzed with high-pressure liquid chromatography (ratios
of aflatoxins B 1 : B 2 : G 1 : G 2 were 90:3:7:1) by the CB method (Association of Official Analytical Chemists, 1975), and mixed in feed to yield 0, 5, and 10 /zg total aflatoxins/g of feed. Crystalline T-2 toxin was dissolved in a volume of ethanol equal to 1% of the feed to be treated. The solution was added to about 10% of the total feed, dried to evaporate the ethanol, and then mixed with the remainder of the feed. The concentrations of T-2 toxin tested for feed refusal were 0, .625, 1.25, 2.5, 5, 10, 20, and 40 £ig/g of feed. Refusal of T-2 toxin when presented in drinking water (0, .15, .31, .62, 2.5, 10, and 40 £ig/ml) was determined after the T-2 toxin was dissolved in ethanol equal to 1% of the drinking water to be treated and diluted in the water shortly before administration. Citrinin concentrations of 0, 50, and 100jUg/ml were prepared and tested in the same fashion. In all cases the controls received an equivalent amount of ethanol and were treated identically. Statistical Considerations. Group means were submitted to analysis of variance in which an F-ratio was calculated. If F-ratio was significant (P<.05), the treatment means were compared for the least significant difference. If only two treatments were compared, Student's t test was used (Bruning and Kintz, 1968).
RESULTS T-2 toxin added to feed or water produced a dose-related refusal of the material containing it (Table 1). Presentation in water gave a more sensitive refusal with a minimum effective dose of .31 ppm as compared with a minimum effective dose of 5.0 ppm in feed. Despite the sensitivity of chickens to T-2 toxin, a concentration as high as 40 ppm caused only 74% refusal of feed and 53% refusal of water. The ethanol used to dissolve T-2 toxin to ensure even distribution was without effect when added to water alone. Citrinin, however (Table 2), stimulated the consumption of drinking water at concentrations of 50 and 100 /ig/ml. Aflatoxin (Table 3), at concentrations which induce growth retardation and low mortality (Smith and Hamilton, 1970), had no significant effect on feed consumption. The detection of refusal of culture filtrates has the complication that the growth medium itself might influence consumption. Czapek's broth amended with 2% peptone had no significant effect on consumption when mixed
FEED REFUSAL AND MYCOTOXINS TABLE 1. Refusal of T-2 toxin incorporated into feed and drinking water Consump tion T-2 toxin
Feed
Water
(ppm)
(g/kg body wt)
(ml/kg body wt)
0(H2O) 0 ( 1 % ethanol) .15 .31 .62 1.25 2.5 5.0 10 20 40
363 ± l l a
462 + 18 a 479 ± 16 a 449 ± 1 8 a 325 ± 3 1 b 339± 2 1 b
364 + 27a 338± 2 1 a 339 ± 1 3 a 271+ 1 3 b 230± 2 2 b 142 ± 9° 94 ± 7 d
213 ± 8C 197 + 2 5 c 218 ± 3 1 c
' ' ' Values in column with different superscripts differ significantly (P<.05). Values are the mean ± SE of 5 groups of 30 birds per treatment, and consumption is expressed in terms of body weight prior to refeeding or rehydration. A dash indicates the treatment was not tested.
TABLE 2. Consumption of drinking water containing citrinin Citrinin
Consumption
(Mg/ml)
( m l H 2 0 / k g b o d y wt)
0 50 100
255 ± 3 1 a 360 + 1 0 b 339±19b
ab ' Values with different superscripts differ significantly (P<.01). Values are mean ± SE of 5 groups of 30 chickens and are expressed in terms of body weight prior to rehydration.
TABLE 3. Failure of aflatoxin to produce feed refusal Aflatoxin
Consumption
(Mg/g)
(g feed/kg body wt)
0 5 10
274 ± 32 a 269 ± 2 3 a 250± 13 a
a Values are mean + SE of 5 groups of 30 birds per treatment and are expressed in terms of body weight immediately prior to refeeding. Values did not differ significantly (P<.05).
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into feed, b u t substitution of t h e a m e n d e d broth for drinking water stimulated m a r k e d l y t h e c o n s u m p t i o n of liquid (Table 4 ) . T h e major c o m p o n e n t s alone had no effect; hence, t h e stimulation resulted from t h e c o m b i n a t i o n . Filtrates of F. roseum N R R L 1181 t h a t contained a high c o n c e n t r a t i o n of DAS (335 /ug/ml) caused a 77% decrease of feed cons u m p t i o n and a 9 3 % decrease of liquid cons u m p t i o n . Filtrate diluted 1 t o 16 (21 p p m DAS) still caused a 72% refusal of liquid cons u m p t i o n compared t o t h e water control. T h e possibility t h a t t h e fungus was inducing refusal by changing t h e initial pH of t h e m e d i u m was ruled o u t b y showing t h a t adjusting t h e p H of water or feed in t h e range from 4 t o 10 was w i t h o u t effect (data n o t s h o w n ) . A survey of t h e filtrates of six fungal cultures isolated from feed and ingredients associated w i t h o u t b r e a k s of feed refusal is shown in Table 5. Filtrates of F. tricinctum NRRL 3299 t h a t contained T-2 toxin ( 3 0 0 jug/ml) depressed liquid c o n s u m p t i o n by 88%. Filtrates of P. citrinum, which did n o t contain citrinin t h a t stimulated c o n s u m p t i o n (Table 2), reduced liquid c o n s u m p t i o n b y 7 1 % . Filtrates of t h e other Fusarium cultures t h a t had trace, if any, DAS, MAS, T-2 t o x i n , or zearalenone, had n o significant ( P < . 0 5 ) effect, and neither did t h e filtrates of S. brevicaulis and R. leguminicoli. It was essential to have suitable controls, because t h e c o n s u m p t i o n of u n t r e a t e d water varied markedly from e x p e r i m e n t to experim e n t (Table 5).
DISCUSSION In the present e x p e r i m e n t s t h e refusal of feed and water containing T-2 t o x i n or DAS suggests t h a t these t r i c h o t h e c e n e m y c o t o x i n s are possible causes of feed refusal in p o u l t r y . These findings and t h e failure of t w o Fusarium culture filtrates essentially free of d e t e c t a b l e trichothecenes to cause refusal suggest t h a t refusal may be a p r o p e r t y of t r i c h o t h e c e n e s similar t o their ability t o cause skin irritation. In swine, deoxynivalenol (vomitoxin) is t h e premier cause of refusal, b u t T-2 t o x i n and other t r i c h o t h e c e n e s have been implicated (Burmeister et al, 1 9 8 0 ; Vesonder et al, 1 9 7 7 ) . T h e observation t h a t citrinin stimulated t h e u p t a k e of water (Table 2) agrees with t h e findings of Wyatt et al. ( 1 9 7 9 ) , w h o established t h a t citrinin stimulates water u p t a k e b y acting as a diuretic. Hence, refusal of filtrates of P.
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BURDITT ET AL.
TABLE 4. Refusal by chickens of water and feed amended with culture filtrates of Fusarium roseum NRRL 1181 containing diacetoxyscirpenol Consumption Treatment
None (control) Medium Sucrose' Peptone 1 Minerals' Filtrate (undiluted) Filtrate (diluted 1:16)
Feed
Water
(g/kg body wt) 121 ± l a 125 ± 8 a
(ml/kg body wt) 181± 301 + 199 ± 204± 210± 16 ±
2 2 2
32+ l b
15 a 31b 17 a la la 5C
51 ± l l d
2
a ' ' ' Values in column with different superscripts differ significantly (P<.05). Values are the mean ± SE of 5 groups of 30 birds per treatment, and consumption is expressed in terms of body weight immediately prior to refeeding or rehydration.
'These main components of the growth medium were tested separately at the concentrations occurring in the medium. 2
Treatment was not tested.
citrinum (Table 5) from which citrinin acquired its n a m e was u n e x p e c t e d . This refusal can be reconciled o n t h e basis t h a t citrinin was n o t d e t e c t e d in t h e filtrates and t h a t P. citrinum
TABLE 5. Fefusal activity of fungal culture filtrates supplied to chickens as drinking water Consumption Fungus
Water
produces an u n k n o w n refusal factor. T h e existence of such a refusal factor would be consistent with prior observations of reduced c o n s u m p t i o n of feed infested with P. citrinum ( R o b e r t s and Mora, 1 9 8 2 ) , although a specific refusal does n o t appear to have been r e p o r t e d . T h e failure of aflatoxin (Table 3) and of t h e filtrates of four species of fungi (Table 5) to cause refusal suggest t h a t t h e model of feed refusal used in these e x p e r i m e n t s is selective and, h e n c e , useful in studies of t h e field condition. T h e finding t h a t presentation of refusal factors in water is more sensitive than presentation in feed (Tables 1 and 4) should be useful in t h e isolation and study of presently u n k n o w n refusal factors. In a d d i t i o n , fewer manipulations are involved w h e n water is used as the vehicle. One limitation of presenting culture filtrates in drinking water is t h a t t h e growth m e d i u m itself may influence cons u m p t i o n (Table 4 ) . T h e preferential c o n s u m p tion of sugar and o t h e r substances has been reported previously (Kare and Pick, I 9 6 0 ; J a c o b s and Scott, 1 9 5 7 ; T h a x t o n a n d P a r k h u r s t , 1976). Such preferences can b e corrected by t h e inclusion of a p p r o p r i a t e controls in t h e experimental design. The present findings suggest t h a t T-2 toxin, DAS, and an u n k n o w n factor from P. citrinum can be added to o c h r a t o x i n A and an u n k n o w n factor from Aspergillus ochraceus (Burditt et ah, 1982) as feed refusal factors in p o u l t r y . T h e limited n u m b e r of t o x i n s and fungi surveyed to d a t e for refusal activity suggests t h a t feed refusal in p o u l t r y has m a n y possible causes. ACKNOWLEDGMENTS We t h a n k G o r u m Whitaker and Nancy Bailey for technical assistance.
Filtrate REFERENCES
(ml/kg body w t ) — — Fusarium tricinctum NRRL 3299 Penicillium citrinum Fusarium roseum 'Gibbosum' Fusarium 2 TRK Scopulariopsis brevicaulis Rbizoctonia leguminicoli
399+ 3 a
47 ± 6 b
444 ±38 a 220 ±12 a
130±26 b 245 ± 23 a
220 ±12 a 220 + 12a
230 + 31 a 260 ± 21 a
220 ± 12a
240 ±12 a
ab ' Values in a row with different superscripts differ significantly (P<.01). Values are mean ± SE of 5 groups of 30 birds per treatment, and are expressed in terms of body weight prior to rehydration.
Association of Official Analytical Chemists, 1975. Natural poisons. Pages 477—478 in Official Methods of Analysis. 12th ed. Assoc. Offic. Anal. Chem., Washington, DC. Bruning, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott, Foresman, and Company, Glenview, IL. Burditt, S. J., W. M. Hagler, and P. B. Hamilton, 1982. Feed refusal as a symptom of ochratoxicosis in turkeys. Annu. Mtg. Am. Soc. Microbiol. 1982: 204. (Abstr.) Burmeister, H. R., R. F. Vesonder, and W. F. Kwolek, 1980. Mouse bioassay for Fusarium metabolites: Rejection or acceptance when dissolved in drinking water. Appl. Environ. Microbiol. 39: 957-961. Curtin, T. M., and J. Tuite, 1966. Emesis and refusal
FEED REFUSAL AND MYCOTOXINS of feed in swine associated with Gibberella %ea infected corn. Life Sci. 5:1937-1944. Davis, N. D„ D. K. Dalby, U. L. Diener, and G. A. Sansing, 1975. Medium-scale production of citrinin by Penicillium citrinum in a semisynthetic medium. Appl. Microbiol. 29:118—120. Hagler, W. M., P. B. Hamilton, J. Hutchins, and S. Burditt, 1981a. Nutrient refusal in chickens. Annu. Mtg. Am. Soc. Microbiol. 1981:192. (Abstr.) Hagler, W. M., C. J. Mirocha, and S. V. Pathre, 1981b. Biosynthesis of radiolabeled T-2 toxin by Fusarium tricinctum. Appl. Environ. Microbiol. 41:1049-1051. Hamilton, P. B., W. E. Huff, J. R. Harris, and R. D. Wyatt, 1982. Natural occurrences of ochratoxicosis in poultry. Poultry Sci. 61:1832—1841. Jacobs, H. L., and M. L. Scott, 1957. Factors mediating food and liquid intake in chickens. 1. Studies on the preference for sucrose or saccharine solutions. Poultry Sci. 36:8—15. Kare, M. R., and H. L. Pick, 1960. The influence of the sense of taste on feed and fluid consumption. Poultry Sci. 39:697-706. Kotsonis, F. N., E. B. Smalley, R. A. Ellison, and C. M. Gale, 1975. Feed refusal factors in pure cultures of Fusarium roseum 'Graminearum'. Appl. Microbiol. 30:362-368. Mirocha, C. J., S. V. Pathre, B. Schaverhamer, and C. M. Christensen, 1976. Natural occurrence of Fusarium toxins in feedstuffs. Appl. Environ. Microbiol. 32:553-556. Nelson, T. S., J. N. Beasley, L. K. Kirby, Z. B. Johnson, and G. C. Ballam, 1980. Isolation and identification of citrinin produced by Penicillium lanosum. Poultry Sci. 59:2055-2059. Pathre, S. V., and C. J. Mirocha, 1977. Assay methods for trichothecenes and review of their natural
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occurrence. Pages 229—253 in Mycotoxins in Human and Animal Health. J. V. Rodericks, C. W. Hesseltine, and M. A. Mehlman, eds. Pathotox Publ. Inc., Park Forest South, IL. Roberts, W. T., and E. C. Mora, 1982. Noncitrinin toxicity of Penicillium citrinum contaminated corn. Poultry Sci. 61:1637-1645. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425—428. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207—215. Thaxton, J. P., and C. R. Parkhurst, 1976. Growth, efficiency, and livability of newly hatched broilers as influenced by hydration and intake of sucrose. Poultry Sci. 55:2275-2279. Ueno, Y., K. Ishii, N. Sato, and K. Ohtsubo, 1974. Toxicological approaches to the metabolites of Fusaria. IV. Vomiting factor from moldy corn infected with Fusarium spp. Japan J. Exp. Med. 44:123-127. Vesonder, R. F., A. Ciegler, H. R. Burmeister, and A. H. Jensen, 1979. Acceptance by swine and rats of corn amended with trichothecenes. Appl. Environ. Microbiol. 38:344—346. Vesonder, R. F., A. Ciegler, and A. H. Jensen, 1977. Production of refusal factors by Fusarium strains on grains. Appl. Environ. Microbiol. 34:105— 106. Vesonder, R. F., A. Ciegler, A. H. Jensen, W. K. Rohwedder, and D. Weisleder, 1976. Co-identity of the refusal and emetic principle from Fusarium infected corn. Appl. Environ. Microbiol. 31: 280-285. Wyatt, R. D., 1979. Biological effects of mycotoxins (other than aflatoxin) on poultry. Pages 87—95 in Interactions of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC.
'Paper No. 8752 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650. 2 The use of trade names in this publication does not imply endorsement of the North Carolina Agricultural Research Service nor criticism of similar ones not mentioned. 3 A preliminary report of part of this paper was given at the annual meeting of the American Society for Microbiology, Atlanta, GA, March, 1982.
in t u r k e y s b u t n o t chickens ( H a m i l t o n et al, 1 9 8 2 ; Burditt et al, 1982). T h e only o t h e r evidence linking m y c o t o x i n s directly t o feed refusal in p o u l t r y appears to be t h e finding of Hagler et al. ( 1 9 8 1 b ) t h a t culture filtrates of Fusarium roseum containing high levels of t h e m y c o t o x i n diacetoxyscirpenol (DAS) p r o d u c e d d r a m a t i c refusal when added to feed or w h e n substituted for drinking water. T h e present c o m m u n i c a t i o n r e p o r t s o n a survey of some k n o w n m y c o t o x i n s and s o m e cultures of fungi suspected of causing feed refusal for their ability t o induce refusal in a model system u n d e r l a b o r a t o r y conditions (Hagler et al, 1 9 8 1 a ) . T h e results suggested t h a t feed refusal is a characteristic of several k n o w n m y c o t o x i n s and that t h e r e are u n k n o w n feed refusal factors awaiting isolation and identification.
MATERIALS AND METHODS Measurement of Feed Refusal. Day-old cockerels (Dekalb Sex-Sal) were o b t a i n e d from a commercial h a t c h e r y (Clay's H a t c h e r y , Blackstone, VA). T h e y were h o u s e d in electrically heated batteries u n d e r c o n t i n u o u s lighting with feed and water available ad libitum. On t h e evening of t h e sixth d a y , feed was removed if feed refusal was t o be measured or water was removed if water c o n s u m p t i o n was to b e measured. On the m o r n i n g of t h e seventh d a y , t h e birds were assigned o n a r a n d o m basis in groups of 30 birds per p e n .
2187
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BURDITT ET AL.
There were five pens per treatment. The initial body weight, feed weight, and water volume were determined for each pen. After 6 hours of refeeding or rehydration, the same variables were measured again and the consumption calculated for each treatment. Feed. The feed was a broiler starter mash of commercial type obtained from the university feed mill. Moisture content of the feed was determined gravimetrically after samples were dried overnight at 110 C in a forced draft oven. When culture filtrates were added to the feed, the moisture content of all control and treated feed was adjusted to a constant 35% by the combined addition of culture filtrate and deionized water. Culture Filtrates. Stagnant cultures of Fusarium roseum NRRL 1181, F. tricinctum NRRL 3299, F. roseum 'Gibbosum', and Fusarium sp. 2TRK were grown in Czapek's broth (Difco) supplemented with 2% peptone (Difco) for 14 days at 29 C in 2800 ml Fernbach flasks containing 1000 ml of medium. Penicillium citrinum, Rhizoctonia leguminicola, and Scopulariopsis brevicaulis were grown in a medium containing 2% yeast extract (Difco) and 4% sucrose. Except for the NRRL strains, the fungi were isolated as the predominant organism in suspect feed or ingredients. After incubation the cultures were filtered through three layers of cheesecloth to remove the mycelium. The filtrates for each organism were combined and placed immediately in the waterers for the birds or they were added to the feed alone or mixed with deionized water in quantities sufficient to give a final feed moisture content of 35% in all cases. Gas liquid chromatography of the filtrates by the method of Pathre and Mirocha (1977) revealed that F. roseum NRRL 1181 contained DAS (335 /ig/ml), F. tricinctum NRRL 3299 contained T-2 toxin (300 /xg/ml), F. roseum 'Gibbosum' contained trace amounts of zearalenone and monoacetoxyscirpenol (MAS), and Fusarium 2TRK was free of detectable T-2 toxin, MAS, DAS, and zearalenone. The P. citrinum filtrates were free of detectable citrinin by the method of Nelson et al. (1980). T-2 toxin, Citrinin, and Aflatoxin. Pure T-2 toxin was prepared by the method of Hagler et al. (1981b) and pure citrinin was prepared by the method of Davis et al. (1975). Aflatoxin was prepared as a rice powder concentrate by the method of Shotwell et al. (1966), analyzed with high-pressure liquid chromatography (ratios
of aflatoxins B 1 : B 2 : G 1 : G 2 were 90:3:7:1) by the CB method (Association of Official Analytical Chemists, 1975), and mixed in feed to yield 0, 5, and 10 /zg total aflatoxins/g of feed. Crystalline T-2 toxin was dissolved in a volume of ethanol equal to 1% of the feed to be treated. The solution was added to about 10% of the total feed, dried to evaporate the ethanol, and then mixed with the remainder of the feed. The concentrations of T-2 toxin tested for feed refusal were 0, .625, 1.25, 2.5, 5, 10, 20, and 40 £ig/g of feed. Refusal of T-2 toxin when presented in drinking water (0, .15, .31, .62, 2.5, 10, and 40 £ig/ml) was determined after the T-2 toxin was dissolved in ethanol equal to 1% of the drinking water to be treated and diluted in the water shortly before administration. Citrinin concentrations of 0, 50, and 100jUg/ml were prepared and tested in the same fashion. In all cases the controls received an equivalent amount of ethanol and were treated identically. Statistical Considerations. Group means were submitted to analysis of variance in which an F-ratio was calculated. If F-ratio was significant (P<.05), the treatment means were compared for the least significant difference. If only two treatments were compared, Student's t test was used (Bruning and Kintz, 1968).
RESULTS T-2 toxin added to feed or water produced a dose-related refusal of the material containing it (Table 1). Presentation in water gave a more sensitive refusal with a minimum effective dose of .31 ppm as compared with a minimum effective dose of 5.0 ppm in feed. Despite the sensitivity of chickens to T-2 toxin, a concentration as high as 40 ppm caused only 74% refusal of feed and 53% refusal of water. The ethanol used to dissolve T-2 toxin to ensure even distribution was without effect when added to water alone. Citrinin, however (Table 2), stimulated the consumption of drinking water at concentrations of 50 and 100 /ig/ml. Aflatoxin (Table 3), at concentrations which induce growth retardation and low mortality (Smith and Hamilton, 1970), had no significant effect on feed consumption. The detection of refusal of culture filtrates has the complication that the growth medium itself might influence consumption. Czapek's broth amended with 2% peptone had no significant effect on consumption when mixed
FEED REFUSAL AND MYCOTOXINS TABLE 1. Refusal of T-2 toxin incorporated into feed and drinking water Consump tion T-2 toxin
Feed
Water
(ppm)
(g/kg body wt)
(ml/kg body wt)
0(H2O) 0 ( 1 % ethanol) .15 .31 .62 1.25 2.5 5.0 10 20 40
363 ± l l a
462 + 18 a 479 ± 16 a 449 ± 1 8 a 325 ± 3 1 b 339± 2 1 b
364 + 27a 338± 2 1 a 339 ± 1 3 a 271+ 1 3 b 230± 2 2 b 142 ± 9° 94 ± 7 d
213 ± 8C 197 + 2 5 c 218 ± 3 1 c
' ' ' Values in column with different superscripts differ significantly (P<.05). Values are the mean ± SE of 5 groups of 30 birds per treatment, and consumption is expressed in terms of body weight prior to refeeding or rehydration. A dash indicates the treatment was not tested.
TABLE 2. Consumption of drinking water containing citrinin Citrinin
Consumption
(Mg/ml)
( m l H 2 0 / k g b o d y wt)
0 50 100
255 ± 3 1 a 360 + 1 0 b 339±19b
ab ' Values with different superscripts differ significantly (P<.01). Values are mean ± SE of 5 groups of 30 chickens and are expressed in terms of body weight prior to rehydration.
TABLE 3. Failure of aflatoxin to produce feed refusal Aflatoxin
Consumption
(Mg/g)
(g feed/kg body wt)
0 5 10
274 ± 32 a 269 ± 2 3 a 250± 13 a
a Values are mean + SE of 5 groups of 30 birds per treatment and are expressed in terms of body weight immediately prior to refeeding. Values did not differ significantly (P<.05).
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into feed, b u t substitution of t h e a m e n d e d broth for drinking water stimulated m a r k e d l y t h e c o n s u m p t i o n of liquid (Table 4 ) . T h e major c o m p o n e n t s alone had no effect; hence, t h e stimulation resulted from t h e c o m b i n a t i o n . Filtrates of F. roseum N R R L 1181 t h a t contained a high c o n c e n t r a t i o n of DAS (335 /ug/ml) caused a 77% decrease of feed cons u m p t i o n and a 9 3 % decrease of liquid cons u m p t i o n . Filtrate diluted 1 t o 16 (21 p p m DAS) still caused a 72% refusal of liquid cons u m p t i o n compared t o t h e water control. T h e possibility t h a t t h e fungus was inducing refusal by changing t h e initial pH of t h e m e d i u m was ruled o u t b y showing t h a t adjusting t h e p H of water or feed in t h e range from 4 t o 10 was w i t h o u t effect (data n o t s h o w n ) . A survey of t h e filtrates of six fungal cultures isolated from feed and ingredients associated w i t h o u t b r e a k s of feed refusal is shown in Table 5. Filtrates of F. tricinctum NRRL 3299 t h a t contained T-2 toxin ( 3 0 0 jug/ml) depressed liquid c o n s u m p t i o n by 88%. Filtrates of P. citrinum, which did n o t contain citrinin t h a t stimulated c o n s u m p t i o n (Table 2), reduced liquid c o n s u m p t i o n b y 7 1 % . Filtrates of t h e other Fusarium cultures t h a t had trace, if any, DAS, MAS, T-2 t o x i n , or zearalenone, had n o significant ( P < . 0 5 ) effect, and neither did t h e filtrates of S. brevicaulis and R. leguminicoli. It was essential to have suitable controls, because t h e c o n s u m p t i o n of u n t r e a t e d water varied markedly from e x p e r i m e n t to experim e n t (Table 5).
DISCUSSION In the present e x p e r i m e n t s t h e refusal of feed and water containing T-2 t o x i n or DAS suggests t h a t these t r i c h o t h e c e n e m y c o t o x i n s are possible causes of feed refusal in p o u l t r y . These findings and t h e failure of t w o Fusarium culture filtrates essentially free of d e t e c t a b l e trichothecenes to cause refusal suggest t h a t refusal may be a p r o p e r t y of t r i c h o t h e c e n e s similar t o their ability t o cause skin irritation. In swine, deoxynivalenol (vomitoxin) is t h e premier cause of refusal, b u t T-2 t o x i n and other t r i c h o t h e c e n e s have been implicated (Burmeister et al, 1 9 8 0 ; Vesonder et al, 1 9 7 7 ) . T h e observation t h a t citrinin stimulated t h e u p t a k e of water (Table 2) agrees with t h e findings of Wyatt et al. ( 1 9 7 9 ) , w h o established t h a t citrinin stimulates water u p t a k e b y acting as a diuretic. Hence, refusal of filtrates of P.
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BURDITT ET AL.
TABLE 4. Refusal by chickens of water and feed amended with culture filtrates of Fusarium roseum NRRL 1181 containing diacetoxyscirpenol Consumption Treatment
None (control) Medium Sucrose' Peptone 1 Minerals' Filtrate (undiluted) Filtrate (diluted 1:16)
Feed
Water
(g/kg body wt) 121 ± l a 125 ± 8 a
(ml/kg body wt) 181± 301 + 199 ± 204± 210± 16 ±
2 2 2
32+ l b
15 a 31b 17 a la la 5C
51 ± l l d
2
a ' ' ' Values in column with different superscripts differ significantly (P<.05). Values are the mean ± SE of 5 groups of 30 birds per treatment, and consumption is expressed in terms of body weight immediately prior to refeeding or rehydration.
'These main components of the growth medium were tested separately at the concentrations occurring in the medium. 2
Treatment was not tested.
citrinum (Table 5) from which citrinin acquired its n a m e was u n e x p e c t e d . This refusal can be reconciled o n t h e basis t h a t citrinin was n o t d e t e c t e d in t h e filtrates and t h a t P. citrinum
TABLE 5. Fefusal activity of fungal culture filtrates supplied to chickens as drinking water Consumption Fungus
Water
produces an u n k n o w n refusal factor. T h e existence of such a refusal factor would be consistent with prior observations of reduced c o n s u m p t i o n of feed infested with P. citrinum ( R o b e r t s and Mora, 1 9 8 2 ) , although a specific refusal does n o t appear to have been r e p o r t e d . T h e failure of aflatoxin (Table 3) and of t h e filtrates of four species of fungi (Table 5) to cause refusal suggest t h a t t h e model of feed refusal used in these e x p e r i m e n t s is selective and, h e n c e , useful in studies of t h e field condition. T h e finding t h a t presentation of refusal factors in water is more sensitive than presentation in feed (Tables 1 and 4) should be useful in t h e isolation and study of presently u n k n o w n refusal factors. In a d d i t i o n , fewer manipulations are involved w h e n water is used as the vehicle. One limitation of presenting culture filtrates in drinking water is t h a t t h e growth m e d i u m itself may influence cons u m p t i o n (Table 4 ) . T h e preferential c o n s u m p tion of sugar and o t h e r substances has been reported previously (Kare and Pick, I 9 6 0 ; J a c o b s and Scott, 1 9 5 7 ; T h a x t o n a n d P a r k h u r s t , 1976). Such preferences can b e corrected by t h e inclusion of a p p r o p r i a t e controls in t h e experimental design. The present findings suggest t h a t T-2 toxin, DAS, and an u n k n o w n factor from P. citrinum can be added to o c h r a t o x i n A and an u n k n o w n factor from Aspergillus ochraceus (Burditt et ah, 1982) as feed refusal factors in p o u l t r y . T h e limited n u m b e r of t o x i n s and fungi surveyed to d a t e for refusal activity suggests t h a t feed refusal in p o u l t r y has m a n y possible causes. ACKNOWLEDGMENTS We t h a n k G o r u m Whitaker and Nancy Bailey for technical assistance.
Filtrate REFERENCES
(ml/kg body w t ) — — Fusarium tricinctum NRRL 3299 Penicillium citrinum Fusarium roseum 'Gibbosum' Fusarium 2 TRK Scopulariopsis brevicaulis Rbizoctonia leguminicoli
399+ 3 a
47 ± 6 b
444 ±38 a 220 ±12 a
130±26 b 245 ± 23 a
220 ±12 a 220 + 12a
230 + 31 a 260 ± 21 a
220 ± 12a
240 ±12 a
ab ' Values in a row with different superscripts differ significantly (P<.01). Values are mean ± SE of 5 groups of 30 birds per treatment, and are expressed in terms of body weight prior to rehydration.
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