The Effect of Rubratoxin in Broiler Chickens1

The Effect of Rubratoxin in Broiler Chickens1

The Effect of Rubratoxin in Broiler Chickens1 R. D. WYATT AND P. B. HAMILTON Department of Poultry Science and Department of Microbiology, North Carol...

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The Effect of Rubratoxin in Broiler Chickens1 R. D. WYATT AND P. B. HAMILTON Department of Poultry Science and Department of Microbiology, North Carolina State Raleigh, North Carolina 27607

University,

(Received for publication December 10, 1971)

POULTRY SCIENCE 51: 1383-1387,

INTRODUCTION UBRATOXIN is a mycotoxin produced by the fungus, Penicillium rubrum. It was extracted by Wilson and Wilson (1962) from cultures isolated from feed implicated in moldy corn toxicosis (Burnside et al., 1957). This condition in chickens is apparently synonomous with the hemorrhagic anemia syndrome (Forgacs and Carll, 1962). Forgacs et al. (1958) produced the hemorrhagic anemia syndrome in the laboratory by feeding corn infected with P. rubrum to chickens. This experimentally-produced disease was characterized by congestion, hemorrhage, and necrosis of various organs. The toxic material extracted from P. rubrum by Wilson and Wilson (1962) produced hepatic alterations and a general hemorrhagic condition when injected into mice. Townsend et al. (1966) fractionated this material into two pure compounds which they called rubratoxins A and B. Moss et al. (1969) identified these toxins as closely related nonadrides which varied only in having one of

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Paper Number 3646 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina.

1972

the maleic anhydride residues of rubratoxin B reduced to a lactone group in rubratoxin A. Hayes and Wilson (1970) subsequently investigated in some detail the effects of rubratoxin B in mice. Many different fungi have been implicated in the hemorrhagic anemia syndrome, but the syndrome, to our knowledge, has never been produced in the laboratory by a single chemical entity isolated from these fungi. The production in the laboratory of the syndrome with a pure toxin would permit definitive studies which are now lacking. Since rubratoxin is presumably the toxic principle of P. rubrum which has been implicated in the hemorrhagic anemia syndrome, we investigated the effect of rubratoxin in broiler chickens where the hemorrhagic anemia syndrome causes considerable economic loss. MATERIALS AND METHODS

Rubratoxin was produced by growing P. rubrum NRRL 3290 on Mosseray's simplified Raulin's medium according to Hayes and Wilson (1968). The culture filtrate was concentrated to one-tenth its original volume, acidified to pH 1.5, and extracted with ethyl ether for 24 hours in a liquid-

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ABSTRACT Crystalline rubratoxin produced by PenicUlium rubrum was tested for its effects in broiler chickens. The acute oral LD50 in day-old chicks was 83.2 ± 0.67 mg./kg. of body weight. The most characte.istic symptom in birds receiving a lethal dose was red mottled livers. When graded doses of dietary rubratoxin were fed for three weeks, a dose of 500 u.g./g. of diet was required to cause a significant (P < 0.0S) decrease in body weight. A dose of 1000 Hg./g. was required for a significant hypertrophy of the liver and atrophy of the bursa of Fabricius. The relative weights of the pancreas, spleen, and heart were not affected. This dose also caused a significant anemia and proteinemia with an increase in serum cholesterol and capillary fragility. Spontaneous hemorrhaging was not observed. These data suggest that rubratoxin has low toxicity to broiler chickens and that rubratoxin, by itself, does not cause hemorrhagic anemia syndrome which P. rubrum has been reported to cause.

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al. (1970). Hemoglobin concentration was measured according to the method of Sunderman et al. (1953). Serum protein was determined by the biuret method according to Wootton (1964). Serum total cholesterol was determined according to the method of Zlatkis et al. (1953). Serum total lipid was measured by the procedure of Searcy et al. (1963). Total liver lipid was determined according to the method of Smith and Hamilton (1970). Organs from the birds were excised, trimmed of extraneous tissue, blotted, and weighed. Organ weights were expressed relative to the body weight. The data were compared statistically by analysis of variance using the replicate means. The significance of the differences among treatment means was determined by calculating the least significant difference (Bruning and Kintz, 1968). Statements of significance are based P<0.05. RESULTS

The first experiment was conducted to determine the LD50 dose of rubratoxin in day-old chicks. The LD50 dose was calculated to be 83.2 ± 0.67 mg./kg. of body weight. Mortality was first noted approximately 12 hours after toxin administration and continued for five days. Chicks receiving a lethal dose were necropsied and they exhibited mild kidney congestion, red mottled livers and occasional slight hemorrhages of other internal organs, such as the intestines and pancreas. The red mottled appearance of the liver was most pronounced at the higher doses. The effect of graded doses of dietary rubratoxin on the growth rate of chickens is shown in Fig. 1. A dose of 500 [J.g./g. caused a significant decrease in body weight at three weeks while a dose of 1000 [j.g./g. significantly decreased body weight at both two and three weeks. Although not shown in Fig. 1, doses of 125 and 250 \i.g./

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liquid extractor. The toxin accumulated in the ether phase as a crystalline mass. The crude crystals were separated by filtration and dissolved in a mixture (9:1 v./v.) of ethyl acetate and benzene. The solution was passed through a silica gel column as described by Natori et al. (1970). The eluate was concentrated to dryness and the resulting crystals were washed twice with ethyl ether. This material had a melting point of 166-168°C. (decomposition) which agrees with the value reported by Hayes and Wilson (1968) for rubratoxin B. However, thin layer chromatography (Hayes and Wilson, 1968) revealed the crystals to be a mixture of rubratoxins A and B, with B the more toxic (Townsend et al., 1966), comprising at least 80% of the total. Rubratoxin administered in the LD 50 experiment was dissolved in O.SM sodium bicarbonate with controls receiving an equal volume of solvent. The oral LD 50 dose was calculated by the procedure of Miller and Tainter (1944). Experimental rubratoxicosis was induced by adding known amounts of crystalline rubratoxin dissolved in O.SM sodium bicarbonate at levels of 0, 500, and 1000 pig./g. to a commercial broiler starter mash from which all medications had been omitted. Day-old male broiler chicks obtained from a local hatchery were used in all experiments. The birds were maintained in electrically-heated batteries with continuous artificial illumination. Feed and water were available ad libitum. Each experimental diet was given to four groups of five birds each. The dose response experiments were terminated after three weeks. Lateral shear strength of segments of the pectoralis major muscle and capillary fragility in the wing web were measured as outlined by Tung et al. (1971). The specific activity of acid phosphatase was measured in fresh liver samples according to Tung et

RUBEATOXIN I N

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CHICKENS

T A B L E 2.—The effect of rubratoxin on certain blood parameters of the broiler chicken Ojjg/g

P^f. w f / S f n ^ n («5./g.) (g./lOO ml.) 0 8.68 500 8.43 8.17" 1000

Serum P r S (£/100ml) 3.15 2.84 a 2.74

Serum Serum choKterol lipid" ( m g ./i00ml.) (g./lOO ml.) 1.38 166 1.42 166 1.51 186"

a These values aresignificantly different from the corresponding control values (P<0.05).

2 AGE (weeks)

F I G . 1. Effect of graded doses of rubratoxin on t h e g r o w t h rate of broiler chickens.

g. were found to have no effect on the growth rate at any time interval. The effect of dietary rubratoxin on the size of certain internal organs is shown in Table 1. A dose of 500 pig./g. which inhibited growth rate significantly had no effect on the relative weights of these organs. The highest dose, 1000 [xg./g., caused significant hypertrophy of the liver and atrophy of the bursa of Fabricius. Analysis of the liver tissue indicated that the total lipid content was not affected. The relative weights of the pancreas, spleen, and heart were not altered significantly by any dose. The responses of various blood parameters during rubratoxicosis are shown in Table 2. A dose of 500 pig./g. did not effect significantly any of the parameters, but a dose of 1000 |xg./g. caused a significant deT A B L E 1.—The effect of rubratoxin on relative organ weights of broiler chickens Relative organ weights (g./lOO g. body weight) Dose (cg./g-) 0 500 1000

Liver 2.53 2.58 3.06"

Bursa

Pancreas

Spleen

0.44 0.38 0.37a

0.31 0.37 0.37

0.13 0.12 0.12

Heart 0.81 0.88 0.89

a These values are significantly different from the corresponding control value (P<0.05).

DISCUSSION The data presented here suggest that rubratoxin has very low toxicity for chickens. The growth rate, which is the most sensitive indicator of rubratoxicosis, required a dose of 500 [/.g./g. before it was decreased. A dose of 1000 [Jig./g. was required to affect the other parameters. Since dose levels T A B L E 3.—The effect of rubratoxin on parameters related to tissue integrity and strength in the broiler chicken

Dose G*g-/g-)

Capillary fragility (mm. Hg)

Lateral shear strength (kg.)

L i v e r acid phosphatase ( u n i t s X lO""2 / n i g . protein)

0 500 1000

319 285 255"

1.40 1.17 1.15

6.92 6.12 6.06

a T h i s value is significantly different from its control value ( P < 0 . 0 5 ) .

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I

crease in the levels of hemoglobin and serum protein concomitant with an increase in serum cholesterol levels. Serum total lipid was not affected by either dose. Since P. rubrum has been reported to cause hemorrhaging (Burnside et al., 1957; Wilson and Wilson, 1962), some parameters relating to tissue integrity and strength were measured. The effect of dietary rubratoxin on capillary fragility, lateral shear strength of muscle, and the specific activity of acid phosphatase of liver are shown in Table 3. The dose of 1000 pig./g. increased significantly the capillary fragility but had no effect on liver acid phosphatase or lateral shear strength of muscle.

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The results of this study do not offer a positive insight into the hemorrhagic anemia syndrome which caused the loss of 250,000 birds in North Carolina from 1964 to 1967 (H. W. Garren and J. R. Harris, personal communication) and which presumably is a hazard throughout the poultry industry. This laboratory has investigated the effects of rubratoxin, aflatoxin, ochratoxin, and fusariotoxin T-2 in chickens with the aim of pinpointing the cause of the hemorrhagic anemia syndrome which was described originally by the pioneering efforts of Forgacs and Carll (1962). These four toxins are the toxic principles of four fungi reported to cause the hemorrhagic anemia syndrome, yet none of these toxins can cause the symptoms of hemorrhagic anemia by themselves in the laboratory. This suggests that other toxins are produced by these fungi, but which, as yet, are unisolated, or that several toxins act in concert to cause the hemorrhagic syndrome. There is precedent for both possibilities since Aspergillus flavus produces toxins other than aflatoxin (Wilson, 1966) and since rubratoxin has been reported to

interact with aflatoxin (Edwards and Wogan, 1968). Another possibility which logically must be considered is that the disease was improperly described. This disease reportedly was caused by many unrelated fungi (Forgacs and Carll, 1962). This implies that many unrelated fungi produce the same toxin or that many different toxins cause the same effect. Considering what is known of the specificity of toxins (Ciegler and Lillehoj, 1968), both possibilities seem unlikely. Perhaps the answer lies in interactions between toxins and other factors. At any rate, the status of the elusive hemorrhagic anemia syndrome appears unsettled and deserving of more investigation. ACKNOWLEDGMENTS We thank Sharon West, E. Y. Hickman, and Nancy Goodwin for technical assistance. REFERENCES Burnside, J. E., W. L. Sippel, F. Forgacs, W. L. Carll, M. B. Atwood and E. R. Doel, 1957. A disease in swine and cattle caused by eating moldy corn. II. Experimental production with pure cultures of molds. Am. J. Vet. Res. 18: 817-824. Bruning, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott Foresman Co., Glenview, Illinois. Ciegler, A., and E. B. Lillehoj, 1968. Mycotoxins: Advances in Applied Microbiology. Vol. 10: 115-219. Academic Press, New York. Edwards, G. S., and G. N. Wogan, 1968, Acute and chronic toxicity of rubratoxin in rats. Fed. Proc. 27: 552. Forgacs, J., H. Kock, W. L. Carll and R. H. White-Stevens, 1958. Additional studies on the relationship of mycotoxicosis to the poultry hemorrhagic syndrome. Am. J. Vet. Res. 19: 744-753. Forgacs, J. and W. L. Carll, 1962. Mycotoxicoses, Advan. Vet. Sci. 7 : 273-382. Hamilton, P. B., R. D. Wyatt and H. Burmeister, 1971. Effect of fusariotoxin T-2 in chickens. Poultry Sci. 50: 1583-1584. Hayes, A. W., and B. J. Wilson, 1968. Bioproduction and purification of rubratoxin B. Appl. Microbiol. 16:1163-1167.

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this high would probably not occur in nature, rubratoxin could scarcely be termed a toxin in chickens. For example, aflatoxin requires in this experimental system a dose of 2.5 [xg./g. to affect growth (Smith and Hamilton, 1971) and fusariotoxin T-2 requires a dose of 4 ixg./g. (Hamilton et al., 1971). These compounds are 125 to 500 times more toxic to chickens than is rubratoxin as measured by their effect on growth rate. While rubratoxin in chickens does cause a slight anemia, proteinemia, and an increase in serum cholesterol, which are also characteristic of its effects in mice (Hayes and Wilson, 1970), as well as increased capillary fragility the doses required are so high that it seems unlikely that rubratoxin by itself offers much hazard to poultry health.

RUBRATOXIN IN CHICKENS

Townsend, R. J., M. O. Moss and H. M. Peck, 1966. Isolation and characterization of hepatotoxins from Penicillium rubrum. J. Pharm. Pharmac. 18: 471^173. Tucker, T. L., and P. B. Hamilton, 1971. The effect of ochratoxin in broilers. Poultry Sci. 50: 1637. Tung, H.-T., W. E. Donaldson and P. B. Hamilton, 1970. Effects of aflatoxin on some marker enzymes of lysosomes. Biochim. Biophys. Acta, 222: 665-667. Tung, H.-T., J. W. Smith and P. B. Hamilton, 1971. Aflatoxicosis and bruising in the chicken. Poultry Sci. 50: 795-800. Wilson, B. J., and C. H. Wilson, 1962. Extraction and preliminary characterization of a hepatotoxic substance from cultures of Penicillium rubrum. J. Bacteriol. 94: 283-290. Wilson, B. J., 1966. Toxins other than anatoxins produced by Aspergillus fiavus. Bacteriol. Rev. 30: 478-484. Wootton, I. D. P., 1964. Micro-analysis in Medical Biochemistry. Grune and Stratton, Inc. New York. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.

NEWS AND NOTES (Continued from page 1365) five well-known poultry industry leaders elected to the National Poultry Hall of Fame which is sponsored by the Society and housed at the National Agricultural Library of the U. S. Department of Agriculture. Related to the writing project is a special committee of the American Poultry Historical Society, headed by Professor J. L. Skinner, Extension Poul tryman, Department of Poultry Science, University of Wisconsin, Madison, Wisconsin. Other members are: Dr. James M. Gwin, Alfred N. Schwartz and Mahlon C. Sweet. Also collaborating in the project is Roland C. Hartman, long time member of the Watt Publishing Company editorial staff, who is now publisher of Poultry Digest and an Associate Editor on the Garden State Publishing Company staff. J. Harold Florea, who joined the Watt Publishing Company editorial staff in 1929, and retired as Editorial Director in 1969, will be a Consulting Editor. The book will feature a detailed chronology of

significant events occurring in the poultry industry since 1830. More than 20 chapters, each contributed by an outstanding authority, will present an overview of developments in breeding, housing, management, disease control, marketing, and industry organizations. ALBERTA NOTES The Board of Governors of the University of Alberta have approved a name change for the Faculty of Agriculture. The faculty will now be known as the Faculty of Agriculture and Forestry. The new name was considered necessary because of the increased educational responsibility taken by the Faculty of Agriculture. Two years ago the faculty began offering courses providing for education in forestry, and recently was given approval to offer a Bachelor of Science in Forestry degree. Agriculture staff members did not feel the name Faculty of Agriculture adequately covered all courses taught.

(Continued on page 1401)

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Hayes, A. W., and B. J. Wilson, 1970. Effects of rubatoxin B on liver composition and metabolism in the mouse. Toxicol. Appl. Pharmacol. 17: 481-493. Miller, L. C , and M. L. Tainter, 1944. Estimation of the ED50 and its error by means of logarithmic-probit graph paper. Proc. Soc. Exp. Biol. Med. 57: 261-264. Moss, M. O., F. V. Robinson, A. B. Wood, H. M. Paisley and J. Feeney, 1969. Rubratoxin B, a proposed structure for a Bis-anhydride from Penicillium rubrum Stoll. Nature, 220: 767770. Natori, S., S. Sakaki, H. Kurata, S. Udagawa, M. Ichinoe, M. Saito, M. Umeda and K. Ohtsubo, 1970. Production of rubratoxin B by Penicillium purpurogenum Stoll. Appl. Microbiol. 19: 613-617. Searcy, R. L., J. L. Korotzer and L. M. Bugquist, 1963. Micro measurement of serum total lipids. Clin. Chim. Acta, 8: 376-381. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-21S. Sunderman, F. W., R. P. MacFate, D. A. McFayden, G. F. Stevenson and B. E. Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Path. 23 : 519-598.

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