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Effects of T-2 Toxin on Reproductive Performance and Health of Laying Hens1,2
Effects of T-2 Toxin on Reproductive Performance and Health of Laying Hens 12 M.
S. C H I , C. J. MIROCHA, H . J. K U R T Z , G.
W E A V E R , F . BATES AND W .
SHIMODA 3
Departments of Animal Science, Plant Pathology, Veterinary Biology and Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul, Minnesota 55108 (Received for publication August 19, 1976)
ABSTRACT Purified T-2 toxin was fed to S.C.W.L. hens at levels of 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 p.p.m. of an otherwise balanced diet. Feed consumption, egg production and shell thickness were significantly (P < 0.05) decreased in hens fed 8 p.p.m. as compared with control hens. The fertility and progeny performance were not depressed by feeding T-2 toxin, but the hatchability of fertile eggs of hens fed 2 and 8 p.p.m. was significantly (P < 0.05) lower than that of hens fed the control diet. The weights of liver, heart, gizzard and spleen were not influenced by T-2 toxin. Serum levels of alkaline phosphatase, LDH and uric acid of hens fed high concentrations of T-2 toxin were greater than those of control hens. SGPT in hens fed 8.0 p.p.m. was lower when compared with ccntrol hens. No outward changes in hematocrit, hemoglobin, erythrocyte, leukocyte and differential leukocyte counts were noted with feeding T-2 toxin. Most hens fed T-2 toxin developed oral lesions: circumscribed proliferative yellow caseous plaques at the margin of the beak, mucosa of the hard palate and angle of the mouth, and tongue. The incidence and severity of lesions were proportional to the dietary level of T-2 toxin. The only other lesion observed in necropsy examination at the end of the experiment was the small mucosal ulcer in the anterior portion of the gizzard in hens fed high levels of T-2 toxin. Microscopic examination of bone marrow and various organs (liver, heart, kidney, lung, brain, gastrointestinal tract, etc.) revealed no significant pathological change except the necrotic lesions in the gizzard and crop. POULTRY SCIENCE 56: 628-637,
INTRODUCTION
1977
correlated with moldy corn toxicosis in farm animals (Gilgan et ai, 1966; Smalley et ai, 1970; H s u et al, 1972).
T
H E contamination of corn and other cereal grains with certain fungal species and the s u b s e q u e n t development of their toxic metabolites have been recently recognized to cause disease (mycotoxicosis) in farm animals (Forgacs et a/., 1962; F o r g a c s , 1964; Gilgan et ai, 1966; Brook and White, 1966) and h u m a n s (Joffe, 1964). T h e fungal flora and resulting toxic metabolites naturally occuring in cereal grains were closely related to the prevailing environmental conditions. Fusarium tricinctum was reportedly most prominent on moldy corn in low-temperature storage and its detection in moldy corn was
A m o n g the several mycotoxins elaborated by strains of F. tricinctum a major metabolite was identified to be 3-hydroxy-4, 15-diacetoxy-8-(3-methylbutyry loxy)-12,13-epoxy-A 9 trichothecene which was designated as T-2 toxin (Bamburg et ai, 1968). An application of T-2 toxin to the shaved skin of laboratory animals resulted in an inflammatory skin response and the severity of the response was proportional to the a m o u n t of the toxin applied suggesting usefulness as a semiquantitative biological assay (Bamburg et al, 1968). Hsu et al. (1972) chemically identified T-2 toxin at a concentration of 2 p . p . m . in moldy corn being fed to a herd of dairy cattle, 20% of which died of u n k n o w n causes over a 5-month period. In poultry T-2 toxin caused a decrease of weight gain, oral lesions (Christensen et ai, 1972; Wyatt et al., 1972; Chi
1. Published as paper No. 9651, Scientific Series of the Minnesota Agricultural Experiment Station. 2. Supported by Research Grant RFP-641-4-191 from Food and Drug Administration. 3. Food and Drug Administration, Beltsville, Maryland. 628
629
T-2 TOXIN
et al., 1977) and neural disturbances (Wyatt era/., 1973). Limited information is available regarding the toxic effects of dietary T-2 toxin on reporduction of farm animals and poultry. Intraperitoneal injections of T-2 toxin at a level of 1 mg./kg. body weight in mice resulted in an increased maternal mortality and decreased prenatal survival (Stanford et al., 1975). Several types of gross malformations were noted in that study; limb and bone malformations and retarded jaw development. Choudhury et al. (1971) noticed that when ochratoxin, a toxic metabolite of Aspergillus ochraceus, was fed to laying hens, it reduced egg production and hatchability of fertile eggs, and depressed subsequent performance of the progeny for their first two weeks of life. The objective of this investigation was to study the subacute effects of T-2 toxin on reproductive performance and health of laying hens. MATERIALS AND METHODS T-2 toxin was produced by growing Fusarium tricinctum NRRL 3299 on rice or corn grit substrate, extracted and purified as described by Chi et al. (1977) and Burmeister (1971). Single Comb White Leghorn hens 4 were housed in individual cages at 22 weeks of age and fed a basal diet (Table 1) until 27 weeks of age. Egg production record during this period was kept individually. At 27 weeks of age, hens were selected by body weight and the rate of egg production and divided into groups of four hens each so that each group had similar body weight and an egg production rate. The dietary treatments consisted of 6 concentrations of T-2 toxin: 0 (control), 0.5, 1, 2, 4, and 8 p.p.m. Each
4. Shaver Starcross 288, Silver Lake Hatchery and Breeding Farm, Silver Lake, Minnesota.
TABLE 1.—Composition of the basal diet Ingredient
%
Corn, No. 1 food grade, ground Soybean meal, solv. extracted, 44% protein Meat and bone meal, 50% protein Alfalfa meal, dehydrated, 17% protein Animal fat, stabilized Dicalcium phosphate Limestone Salt, iodized Trace mineral mix 1 Vitamin mix (VMH-69) 2 Di_-methionine
59.20 22.50 4.00 2.50 4.00 1.00 6.00 0.40 0.10 0.25 0.05
1
Trace mineral mix provided in mg. per kilogram of the diet: zinc, 60.0; manganese, 60.0; iron, 20.0; copper, 2.0; iodine, 1.2; cobalt, 0.2. 2 Vitamin mix VMH-69 provided per kg. of the diet: vitamin A, 5,000 I.U.; vitamin D 3 , 1,500 I.U.; vitamin K, 1.0 mg.; riboflavin, 4.0 mg.; calcium pantothenate, 8.0 mg.; niacin, 20.0 mg.; choline chloride, 250 mg.; vitamin B 1 2 , 5.0 n-g.
treatment was randomly assigned to 6 groups of hens. T-2 toxin was prepared as a premix by dissolving it in 50% ethanol, spraying onto finely ground corn and evaporating the ethanol. This premix was then substituted into the basal diet for an equivalent amount of ground corn to provide each concentration of T-2 toxin in the final diet. The basal diet (Table 1) was based on corn and soybean meal without supplements of antibiotics or any other drugs to prevent interactions with T-2 toxin. Experimental diets and water were provided ad libitum for 8 weeks. Body weight and feed consumption were determined at weekly intervals. Egg weight, albumen height and shell thickness were determined on three eggs per hen at two week intervals. The Haugh unit was calculated using the egg weight and albumen height according to the method of Haugh (1937) as a measure for interior quality of eggs. Egg production was recorded daily. For the determination of fertility and hatchability, hens were inseminated every 4-5 days with 0.06
630
CHI, MIROCHA, KURTZ, WEAVER, BATES AND SHIMODA
ml. of pooled semen from S.C.W.L. males. Eggs were collected four times daily with individual identification and stored at a temperature of 12.8-15.6° C. Eggs were set within 5 days after production in a forced draft incubator 5 with a separate hatching compartment. All eggs were candled on the 7th day and eggs appearing infertile were broken to verify infertility. Eggs were transferred to the hatching compartment on the 18th day. At the termination of incubation, dead embryos were recorded and examined for abnormality. Hatchability was calculated on the fertile eggs. The progeny of the initial and the last set of eggs was tested for 3 weeks following hatching.
the histopathological examination of tissues, two birds from each level of T-2 toxin were randomly selected. The dissected tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 microns and stained with hematoxylin and eosin in order to evaluate the morphological cellular changes. Tissues examined microscopically were myocardium, liver, kidney, brain, ovary, oviduct, bursa of Fabricius, crop, gizzard and intestines. The weight of the liver, heart, gizzard and spleen was determined and expressed per 100 grams of body weight. Peripheral blood and bone marrow smears were stained with modified WrightGiemsa stain.
Birds were closely observed daily, examined grossly at each weighing for any clinical signs including incidence of oral lesions, and recorded. At the end of the experiment, blood samples were collected by cardiac puncture. Two ml. of blood was delivered to a test tube in which a drop of dipotassium ethylenediamine tetraacetate was placed and evaporated. Whole blood erythrocyte counts, leukocyte counts, hemoglobin, hematocrit and differential leukocyte counts were estimated on this sample by standard methods (Schalm, 1965). The remaining blood was centrifuged, without addition of anticoagulant, to separate serum which was kept frozen until analysis. The blood serum was analyzed for total protein, cholesterol, uric acid, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactic dehydrogenase (LDH) and alkaline phosphatase according to the methods described by Henry (1964).
Statistical analysis of data was performed using an analysis of variance technique and comparisons among treatment means were made using the honestly significant difference (hsd) procedure (Steel and Torrie, 1960).
After sampling blood, hens were necropsied and bone marrow smears were made from tibia. Hens were grossly examined for the presence of any pathological changes. For
5. Robbins hatch-matic®, .Robbins Incubator Co., Denver, Colorado.
RESULTS Hens fed a diet containing 8 p.p.m. T-2 toxin consumed significantly (P < 0.05) less feed than hens fed the control diet while hens fed a diet containing 1 p.p.m. consumed significantly (P < 0.05) greater amount of feed than the control group (Table 2). Body weight change of hens fed T-2 toxin was not different from that of control hens. Egg production of hens fed a diet containing 8 p.p.m. T-2 toxin was significantly (P < 0.05) less than that of hens fed the control diet or diets containing 0.5 and 1 p.p.m. (Table 2). The most marked effect of T-2 toxin on egg production was observed during the last period of the experiment in hens fed 8 p.p.m. resulting in 11.3% lower egg production as compared with the control group (Figure 1). Feed conversion of hens fed a diet containing 2 p.p.m. T-2 toxin was inferior (P < 0.05) to that of hens fed the control diet or a diet containing 0.5 p.p.m., but hens fed 4 or 8 p.p.m. T-2 toxin had a feed conversion ratio
631
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similar to the control group. Egg weight and the Haugh unit were not significantly influenced by T-2 toxin. Hens fed 8 p.p.m. T-2 toxin produced eggs with thinner (P < 0.05) shells than hens fed other treatment diets. Fertility was not different between treatments (Table 2); hatchability, however, of hens fed 8 p.p.m. T-2 toxin was lower (P < 0.05) than that of control groups or hens fed 0.5 p.p.m. The hatchability of hens fed 2
and 4 p.p.m. T-2 toxin was also lower than that of control group, but the difference between the control group and 4 p.p.m. group was not significant. The relatively lower hatchability of hens fed 2 to 8 p.p.m. T-2 toxin was observed from the first 2 week period (Figure 2) and hens fed 8 p.p.m. had the lowest hatchability at the last 2 week period. Death of the embryo mostly occurred in the early stage of incubation. No evidence of malformation in dead embryos or hatched
632
CHI, MIROCHA, KURTZ, WEAVER, BATES AND SHIMODA
TABLE 2.—Reproductive performance parameters of laying hens fed diets containing varying amounts of T-2 toxin'-2 Dietary level of T-2 toxin (p.p. m.) 0 (Control) Body weight gain, g . / h e n / 2 weeks Feed consumption, g./hen/day Egg production, % Feed conversion; kg. feed/dozen eggs g. feed/g. egg Egg weight, g. Haugh unit Shell thickness, mm. Fertility, % Hatchability, 3 %
12.8 100.0b 89.25b 1.34a 2.17ab 51.61 90.32 0.3187b 95.25 96.85c
0.5 12.3 99.5b 90.33b 1.33a 2.12a 52.23 92.85 0.3255b 95.85 95.84bc
1.0 19.5 104.0c 89.56b 1.40ab 2.20ab 52.88 93.13 0.3261b 95.49 94.80abc
2.0 22.3 101.2bc 85.75ab 1.42b 2.25b 52.47 93.24 0.3216b 93.62 91.15ab
4.0 11.0
8.0 2.6
99.9b 87.05ab
94.4a 84.20a
1.38ab 2.20ab 52.19 92.10 0.3209b 96.27 92.78abc
1.35ab 2.14ab 52.66 93.36 0.3060a 95.54 89.38a
1 Mean value of 6 replicated groups with 4 hens each at each level of T-2 toxin. 2 Means in the same line with different letters differ significantly (P < 0.05).. 3
Hatchability of fertile eggs.
chicks was found in T-2 treated groups. The growth rate and feed /gain ratio of progeny from hens fed T-2 toxin were similar to that of progeny from the control group (Table 3). Chicks hatched from eggs of hens fed 8 p.p.m. T-2 toxin had smaller (P < 0.05) body weights at 21 days than those hatched from eggs of hens fed 0.5, 1 or 4 p.p.m. The weights of the liver, heart, gizzard and spleen were not influenced by the dietary T-2 toxin (Table 4). Hens fed diets containing 0.5 and 1 p.p.m. T-2 toxin had a lower (P < 0.05) leukocyte count than control hens (Table 5). Hens fed 8 p.p.m. T-2 toxin also had a lower leukocyte level than the control group, but the difference was not statistically significant. The number of erythrocytes of T-2 treated hens was not significantly different from that of control hens, but hens fed 1 p.p.m. T-2 toxin had a lower erythrocyte level than hens fed 2 or 4 p.p.m. Hematocrit, hemoglobin and differential leukocyte counts were not affected by dietary T-2 toxin. Serum alkaline phosphatase and SGPT were not affected by T-2 toxin at concentra-
tions lower than 4 p.p.m. of the diet; hens fed 8 p.p.m. had higher (P < 0.05) serum alkaline phosphatase and lower (P < 0.05) SGPT than the control group (Table 6). The serum LDH level was higher (P < 0.05) in hens fed 2, 4 and 8 p.p.m. T-2 toxin as compared with control hens or hens fed 0.5 and 1 p.p.m. Serum cholesterol was not significantly changed in T-2 treated hens as compared with control hens. However, hens fed 0.5 p.p.m. had lower serum cholesterol than hens fed 2 and 4 p.p.m. T-2 toxin. Serum uricacidof hens 8 p.p.m. T-2 toxin was higher (P < 0.05) than that of the control group. Serum GOT and total protein were not significantly changed in T-2 treated hens. Oral lesions were observed from the 2nd week in hens fed 4 and 8 p.p.m. T-2 toxin. After the 3rd week, even hens fed 0.5 p.p.m. had small oral lesions (Table 7). At the level of 8 p.p.m., nearly all of the hens in the group had oral lesions. The lesions were circumscribed, proliferative, caseous plaques and occurred at the margin of the beak, the mucosa of the hard palate, angle of the mouth and on or under the tongue. Several of these
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lesions coalesced on the oral mucosa. The severity and incidence of the lesions were proportional to the amount of the toxin in the diet. Small mucosal ulcers in the anterior portion of the gizzard were observed in many
of the hens fed 4 and 8 p.p.m. T-2 toxin. Other than these observations, no pathological or clinical signs were noted in necropsy examinations. Microscopic studies of the various organs revealed no significant patho-
CHI, MIROCHA, KURTZ, WEAVER, BATES AND SHIMODA
634
TABLE 3.—The growth rate and feed/gain ratio of progeny from hens fed varying amounts of T-2 toxin' Dietary level of T-2 toxin (p.p.m.)
21-day body weight (g.): First setting of egg Last setting of egg Avg. 3 Feed/gain ratio First setting of egg Last setting of egg Avg.
0 (Control)
0.5
1.0
2.0
4.0
8.0
Avg. 2
226.0 225.0 225.5ab
221.0 237.8 229.4a
229.4 235.3 232.4a
220.7 236.0 228.4ab
222.8 237.0 229.9a
218.2 219.3 218.7b
223.0 231.7**
1.80 1.85 1.83
1.78 1.93 1.86
1.80 1.92 1.86
1.81 1.94 1.88
1.77 1.91 1.84
1.81 1.85 1.83
1.80 1.90**
'Mean values of 6 replicated groups of 10 chicks each. The averages of first and last setting of eggs were compared by t-test; **, significantly different at 3P < 0.01. The averages at dietary levels of T-2 toxin were compared using the hsd procedure; means bearing different letters differ significantly (P < 0.05). 2
TABLE 4.- -Organ weights of laying hens fed diets containing varying amounts of T-2 toxin
Dietary level of T-2 toxin (p.p.m.) 0 (Control) 0.5 2.42 2.37 2.47 2.30 Liver weight, g. 2.01 0.49 0.48 Heart weight, g. 0.43 0.48 0.42 1.74 Gizzard weight, g. 1.76 1.77 1.71 1.66 0.097 Spleen weight, g. 0.097 0.095 0.120 0.120 1 Mean values of 6 replicated groups with 4 hens each at each level of T-2 toxin. 2 Organ weights expressed per 100 grams of body weight.
logical changes in various organs except necrotic lesions in the crop and gizzard of groups fed high concentrations of T-2 toxin. DISCUSSION The toxic metabolites of fungi have been implicated in poor reproductive performance of laying hens. Hamilton and Garlich (1972) reported a delayed and decreased egg production in commercial laying hens fed anatoxin; hatchability was also reduced (Kratzer et al., 1969). The feeding of ochratoxin to laying hens resulted in a delay in sexual maturity, decrease in egg production and hatchability and depression in subsequent performance of the progeny for their first 2-week period (Choudhury, 1971). Zearalenone appeared to have very little detrimental effects on reproductive performance of laying hens at relatively high concentrations (Speers
2.56 0.48 1.67 0.105
et al., 1971) even though it had caused profound reproductive disorders in swine (Mirocha et al., 1971). It is evident from the data of this study that T-2 toxin had detrimental effects on egg production, shell quality and hatchability at the level of 8 p.p.m. of the diet. The egg production and hatchability were depressed almost immediately but more severely after 6 weeks. It is not known at this time whether the decrease in hatchability was due to transmission of T-2 toxin or its toxic metabolites into the egg at a concentration sufficient to kill the embryo. The possibility exists that T-2 toxin might alter the nutritional status of the egg within the hen while on a T-2 toxin diet resulting in reduced hatchability. Hens fed 0.5 p.p.m. T-2 toxin slightly improved their egg production without any deleterious effects on other reproductive per-
T-2
TOXIN
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TABLE 5.—Hematological findings for laying hens fed diets containing varying amounts of T-2 toxin' Dietary level of T-2 (p.p.m.) 0 0.5 1.0 2.0 4.0 8.0 'Mean < 0.05).
R.B.C. W.B.C. Based on 300 cell count (%) Hemoglobin (mil(thouHematocrit lions/ sands/ Total (8./ Mono mm.3) mm.3) Lympho neutro Baso 100 ml.) (%) 26.4 1.1 8.79 2.42ab 24.9 5.0 28.4b 69.0 29.3 9.29 2.43ab 17.9a 62.9 32.1 3.9 1.1 26.8 8.93 2.09a 14.9a 62.1 30.9 5.8 1.2 28.1 9.57 2.71b 28.8b 68.5 25.4 5.4 0.7 27.6 9.41 2.68b 28.1b 70.1 23.3 5.5 1.1 26.3 8.83 2.52ab 21.5ab 72.2 19.7 6.5 1.6 values of 12 hens. Means in the same column with different letters differ significantly (P
TABLE 6.—Blood chemistry findings for laying hens fed diets containing varying amounts of T-2 toxin' Total Dietary Alkaline SGOT SGPT level phosphatase (R.F. (R.F. LDH protein Cholesterol Uric acid of T-2 (B. units/ units/ units/ (I.U./ (mg./ (mg./ (g./ (p.p.m.) 100 ml.) ml.) ml.) ml.) 100 ml.) 100 ml.) 100 ml.) 0 50.8a 257.5 7.02 19.17b 855a 98.2ab 5.42a 0.5 75.0ab 215.0 16.67ab 585a 6.27 90.3a 6.53ab 1.0 45.0a 208.0 17.33b 797a 6.55 95.lab 5.63ab 2.0 52.5a 217.5 20.08b 1959b 6.69 112.4b 4.81a 4.0 62.5a 230.0 20.17b 1680b 6.59 116.5b 6.18ab 8.0 129.2b 201.7 11.25a 1435b 7.44 110.8ab 7.68b 'Mean values of 12 hens. Means in the same column with different letters differ significantly (P < 0.05).
formance criteria. A similar effect was seen on growth of rainbow trout (Marasas et aL, 1969). The possibility of an antibiotic effect of the toxin on the microflora in the feed or intestinal tract exists. Oral lesions, a most prominent sign, were similar to those observed in broiler chicks fed T-2 toxin (Wyatt et al., 1972; Chi et al., 1977). Wyatt et al. (1972) examined the oral lesions histopathologically and found an intense inflammation of the tissue and local necrosis. The outer layers of the lesion consisted of fibrinous material which sloughed off easily while the under-lying tissue was heavily infiltrated with granulated leukocytes. Large numbers of bacteria were also found in the eroded area. T-2 toxin apparently is a contact corrosive as evidenced by the formation of oral lesions and necrosis of the gizzard and crop seen in many of the hens fed high levels of T-2 toxin. There was no
death during the experimental period. Histopathological examination of organs from treated birds agreed with the results of gross examination; no significant lesions were found except in the crop and gizzard. There was an indication of lymphocytic infiltration in several tissues, probably due to a low grade viral infection in many of the hens fed T-2 toxin; however, no lesions attributable to the toxin alone were found. The presence of this low grade viral infection should not have interfered with the formation of any lesions that might have been caused by the T-2 toxin. The leukocyte count in chicks fed a low concentration of T-2 toxin (0.2 p.p.m.) increased over that of control chicks with no significant changes at concentrations of 0.4 to4 p.p.m. (Chi et al., 1977). When T-2 toxin was fed to hens at 0.5 and 1.0 p.p.m., the leukocyte count decreased below that of the
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control group while at 2 to 8 p.p.m., no difference was observed. Since there was no evidence of pathological changes in bone marrow and organ tissues, a decrease in the leukocyte level at the low concentrations of the toxin may not be the result of T-2 toxicosis. Blood enzymes, used as a criterion of hepatic and myocardial tissue damages, did not change sufficiently to suggest that pathological changes occurred in the organs after exposure to T-2 toxin; this agreed with the results of gross and histopathological examinations of liver, heart and kidney in which clinical signs were not evident. The major sources of serum alkaline phosphatase are bone, intestine and liver (Fishman and Ghosh, 1967). Serum alkaline phosphatase increases in calcium-deficient hens and an increase in alkaline phosphatase levels is associated with decalcification of bone (Hurwitz and Griminger, 1961). The changes in intestinal alkaline phosphatase is associated with calcium and phosphorus absorption (Moog and Glazier, 1972). In this study, the increase of serum alkaline phosphatase in T-2 toxin treated hens was observed at a concentration (8 p.p.m.) which also resulted in thin egg shells; this suggests that T-2 toxin interferes with calcium metabolism, probably by reduced absorption in the intestine. Even though T-2 toxin did not cause any significant pathological signs in organs, the toxin appeared inflammatory and did affect reproductive performance of laying hens in terms of decreased egg production, hatchability and thin egg shell formation. In addition, there are possibilities that cereal grains and animal feed contaminated with species of Fusarium often contain other toxic metabolites in addition to T-2 toxin. The contamination of animal feeds with T-2 and other mycotoxins associated with it should be avoided in order to safeguard animal and public health.
T-2 TOXIN
REFERENCES Bamburg, J. R., N. V. Riggs and F. M. Strong, 1968. The structures of toxins from two strains of Fusarium tricinctum. Tetrahedron, 23: 3329-3336. Brook, P. J., and E. P. White, 1966. Fungus toxins affecting mammals. Ann. Rev. Phytopathol. 4: 171194. Burmeister, H. R., 1971. T-2 toxin production by Fusarium tricinctum on solid substrate. Appl. Microbiol. 21: 739-742. Chi, M. S., C. J. Mirocha, H. J. Kurtz, G. Weaver, F. Bates and W. Shimoda, 1977. Subacute toxicity of T-2 toxin in broiler chicks. Poultry Sci. 56: 306-313. Choudhury, H., C. W. Carlson and G. Semeniuk, 1971. A study of ochratoxin toxicity in hens. Poultry Sci. 50: 1855-1858. Christensen, C. M., R. A. Meronuck, G. H. Nelson and J. C. Behrens, 1972. Effects on turkey poults of rations containing corn invaded by Fusarium tricinctum (Cda.) Sny. & Hans. Appl. Microbiol. 23: 177-179. Fishman, W. H., and N. K. Ghosh, 1967. Isoenzymes in human alkaline phosphatase. Advances Clin. Chem. 10: 255-370. Forgacs, J., 1964. Stachybotryotoxicosis and moldy corn toxicosis. In: Mycotoxins in Foodstuffs, Ed. G. N. Wogan, M.I.T. Press, Cambridge, Mass. p. 87-104. Forgacs, J., H. Koch, W. T. Carle and R. H. WhiteStevens, 1962. Mycotoxicoses. I. Relationship of toxic fungi to moldy-feed toxicosis in poultry. Avian Dis. 6: 363-380. Gilgan, M. W., E. B. Smalley and F. M. Strong, 1966. Isolation and partial characterization of a toxin from Fusarium tricinctum on moldy corn. Arch. Biochem. Biophys. 114: 1-3. Hamilton, P. B., and J. D. Garlich, 1972. Failure of vitamin supplementation to alter the fatty liver syndrome caused by aflatoxin. Poultry Sci. 51: 688-692. Haugh, R. R., 1937. The Haugh unit for measuring egg quality. U.S. Egg Poultry Mag. 43: 552-555 and 572-573. Henry, R. J., 1964. Clinical Chemistry, Principles and Techniques. Harper and Row, New York, N.Y. Hsu, I. C , E. B. Smalley, F. M. Strong and W. E. Rebelin, 1972. Identification of T-2 toxin in moldy corn associated with a lethal toxicosis in dairy cattle. Appl. Microbiol. 24: 684-690. Hurwitz, S., and P. Griminger, 1961. The response of plasma alkaline phosphatase, parathyroids and
637
blood and bone minerals to calcium intake in the fowl. J. Nutrition, 73: 177-185. Joffe, A. Z., 1964. Toxin production in cereal fungi causing toxic alimentary aleukia in man. In: Mycotoxins in Foodstuffs, Ed. G. N. Wogan, M.I.T. Press, Cambridge, Mass. p. 77-86. Kratzer, F. H., D. Bandy, M. Wiley and A. N. Booth, 1969. Aflatoxin effects in poultry. Proc. Soc. Exp. Biol. Med. 131: 1281-1284. Marasas, W. F. O., J. R. Bamburg, E. B. Smalley, F. M. Strong, W. L. Ragland and P. E. Degurse, 1969. Low dose, long term effects on trout and rats of T-2 toxin produced by the fungus Fusarium tricinctum. Toxicol. Appl. Pharmacol. 15: 471-482. Mirocha, C. J., C. M. Christensen and G. H. Nelson, 1971. F-2 (Zearalenone) estrogenic mycotoxin from Fusarium. In: Microbial Toxins, Vol. 7, Algal and Fungal Toxins, Ed. S. Kadis, A. Ciegler and S. J. Ajl, Academic Press, New York, N.Y. p. 107-138. Moog, F. and H. S. Glazier, 1972. Phosphate absorption and alkaline phosphatase activity in the small intestine of the adult mouse and of the chick embryo and hatched chick. Comp. Biochem. Physiol. 42: 321-336. Schalm, O. W., 1965. Veterinary Hematology. 2nd Ed. Lea and Febiger, Philadelphia, Pennsylvania. Smalley, E. B., W. F. P. Marasas, F. M. Strong, J. R. Bamburg, R. E. Nichols and N. R. Kosuri, 1970. Mycotoxins associated with moldy corn. Proc. First U.S.—Japan Conf. Toxic Microorganisms, Honolulu, Hawaii. Unnumbered publication. U.S. Dept. of Interior and U.J.N.R. Panels on Toxic Microorganisms, Washington, D.C. p. 163-173. Speers, G. M., R. S. Meronuck, D. M. Barnes and C. J. Mirocha, 1971. Effect of feeding Fusarium roseum, F. sp. graminearum contaminated corn and the mycotoxin F-2 on the growing chick and laying hen. Poultry Sci. 50: 627-633. Stanford, G. K., R. D. Hood and A. W. Hayes, 1975. Effect of prenatal administration of T-2 toxin to mice. Res. Commum. Chem. Path. Pharmacol. 10: 743-746. Steel, R. G. D. and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, N.Y. Wyatt, R. D., W. M. Colwell, P. B. Hamilton and H. R. Burmeister, 1973. Neural disturbances in chickens caused by dietary T-2 toxin. Appl. Microbiol. 26: 757-761. Wyatt, R. D., B. A. Weeks, P. B. Hamilton and H. R. Burmeister, 1972. Severe oral lesions in chickens caused by ingestion of dietary fusariatoxin T-2. Appl. Microbiol. 24: 251-257.
SEPTEMBER 17-21, 1978. XVI WORLD'S POULTRY CONGRESS, RIO DE JANEIRO, BRAZIL
S. C H I , C. J. MIROCHA, H . J. K U R T Z , G.
W E A V E R , F . BATES AND W .
SHIMODA 3
Departments of Animal Science, Plant Pathology, Veterinary Biology and Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul, Minnesota 55108 (Received for publication August 19, 1976)
ABSTRACT Purified T-2 toxin was fed to S.C.W.L. hens at levels of 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 p.p.m. of an otherwise balanced diet. Feed consumption, egg production and shell thickness were significantly (P < 0.05) decreased in hens fed 8 p.p.m. as compared with control hens. The fertility and progeny performance were not depressed by feeding T-2 toxin, but the hatchability of fertile eggs of hens fed 2 and 8 p.p.m. was significantly (P < 0.05) lower than that of hens fed the control diet. The weights of liver, heart, gizzard and spleen were not influenced by T-2 toxin. Serum levels of alkaline phosphatase, LDH and uric acid of hens fed high concentrations of T-2 toxin were greater than those of control hens. SGPT in hens fed 8.0 p.p.m. was lower when compared with ccntrol hens. No outward changes in hematocrit, hemoglobin, erythrocyte, leukocyte and differential leukocyte counts were noted with feeding T-2 toxin. Most hens fed T-2 toxin developed oral lesions: circumscribed proliferative yellow caseous plaques at the margin of the beak, mucosa of the hard palate and angle of the mouth, and tongue. The incidence and severity of lesions were proportional to the dietary level of T-2 toxin. The only other lesion observed in necropsy examination at the end of the experiment was the small mucosal ulcer in the anterior portion of the gizzard in hens fed high levels of T-2 toxin. Microscopic examination of bone marrow and various organs (liver, heart, kidney, lung, brain, gastrointestinal tract, etc.) revealed no significant pathological change except the necrotic lesions in the gizzard and crop. POULTRY SCIENCE 56: 628-637,
INTRODUCTION
1977
correlated with moldy corn toxicosis in farm animals (Gilgan et ai, 1966; Smalley et ai, 1970; H s u et al, 1972).
T
H E contamination of corn and other cereal grains with certain fungal species and the s u b s e q u e n t development of their toxic metabolites have been recently recognized to cause disease (mycotoxicosis) in farm animals (Forgacs et a/., 1962; F o r g a c s , 1964; Gilgan et ai, 1966; Brook and White, 1966) and h u m a n s (Joffe, 1964). T h e fungal flora and resulting toxic metabolites naturally occuring in cereal grains were closely related to the prevailing environmental conditions. Fusarium tricinctum was reportedly most prominent on moldy corn in low-temperature storage and its detection in moldy corn was
A m o n g the several mycotoxins elaborated by strains of F. tricinctum a major metabolite was identified to be 3-hydroxy-4, 15-diacetoxy-8-(3-methylbutyry loxy)-12,13-epoxy-A 9 trichothecene which was designated as T-2 toxin (Bamburg et ai, 1968). An application of T-2 toxin to the shaved skin of laboratory animals resulted in an inflammatory skin response and the severity of the response was proportional to the a m o u n t of the toxin applied suggesting usefulness as a semiquantitative biological assay (Bamburg et al, 1968). Hsu et al. (1972) chemically identified T-2 toxin at a concentration of 2 p . p . m . in moldy corn being fed to a herd of dairy cattle, 20% of which died of u n k n o w n causes over a 5-month period. In poultry T-2 toxin caused a decrease of weight gain, oral lesions (Christensen et ai, 1972; Wyatt et al., 1972; Chi
1. Published as paper No. 9651, Scientific Series of the Minnesota Agricultural Experiment Station. 2. Supported by Research Grant RFP-641-4-191 from Food and Drug Administration. 3. Food and Drug Administration, Beltsville, Maryland. 628
629
T-2 TOXIN
et al., 1977) and neural disturbances (Wyatt era/., 1973). Limited information is available regarding the toxic effects of dietary T-2 toxin on reporduction of farm animals and poultry. Intraperitoneal injections of T-2 toxin at a level of 1 mg./kg. body weight in mice resulted in an increased maternal mortality and decreased prenatal survival (Stanford et al., 1975). Several types of gross malformations were noted in that study; limb and bone malformations and retarded jaw development. Choudhury et al. (1971) noticed that when ochratoxin, a toxic metabolite of Aspergillus ochraceus, was fed to laying hens, it reduced egg production and hatchability of fertile eggs, and depressed subsequent performance of the progeny for their first two weeks of life. The objective of this investigation was to study the subacute effects of T-2 toxin on reproductive performance and health of laying hens. MATERIALS AND METHODS T-2 toxin was produced by growing Fusarium tricinctum NRRL 3299 on rice or corn grit substrate, extracted and purified as described by Chi et al. (1977) and Burmeister (1971). Single Comb White Leghorn hens 4 were housed in individual cages at 22 weeks of age and fed a basal diet (Table 1) until 27 weeks of age. Egg production record during this period was kept individually. At 27 weeks of age, hens were selected by body weight and the rate of egg production and divided into groups of four hens each so that each group had similar body weight and an egg production rate. The dietary treatments consisted of 6 concentrations of T-2 toxin: 0 (control), 0.5, 1, 2, 4, and 8 p.p.m. Each
4. Shaver Starcross 288, Silver Lake Hatchery and Breeding Farm, Silver Lake, Minnesota.
TABLE 1.—Composition of the basal diet Ingredient
%
Corn, No. 1 food grade, ground Soybean meal, solv. extracted, 44% protein Meat and bone meal, 50% protein Alfalfa meal, dehydrated, 17% protein Animal fat, stabilized Dicalcium phosphate Limestone Salt, iodized Trace mineral mix 1 Vitamin mix (VMH-69) 2 Di_-methionine
59.20 22.50 4.00 2.50 4.00 1.00 6.00 0.40 0.10 0.25 0.05
1
Trace mineral mix provided in mg. per kilogram of the diet: zinc, 60.0; manganese, 60.0; iron, 20.0; copper, 2.0; iodine, 1.2; cobalt, 0.2. 2 Vitamin mix VMH-69 provided per kg. of the diet: vitamin A, 5,000 I.U.; vitamin D 3 , 1,500 I.U.; vitamin K, 1.0 mg.; riboflavin, 4.0 mg.; calcium pantothenate, 8.0 mg.; niacin, 20.0 mg.; choline chloride, 250 mg.; vitamin B 1 2 , 5.0 n-g.
treatment was randomly assigned to 6 groups of hens. T-2 toxin was prepared as a premix by dissolving it in 50% ethanol, spraying onto finely ground corn and evaporating the ethanol. This premix was then substituted into the basal diet for an equivalent amount of ground corn to provide each concentration of T-2 toxin in the final diet. The basal diet (Table 1) was based on corn and soybean meal without supplements of antibiotics or any other drugs to prevent interactions with T-2 toxin. Experimental diets and water were provided ad libitum for 8 weeks. Body weight and feed consumption were determined at weekly intervals. Egg weight, albumen height and shell thickness were determined on three eggs per hen at two week intervals. The Haugh unit was calculated using the egg weight and albumen height according to the method of Haugh (1937) as a measure for interior quality of eggs. Egg production was recorded daily. For the determination of fertility and hatchability, hens were inseminated every 4-5 days with 0.06
630
CHI, MIROCHA, KURTZ, WEAVER, BATES AND SHIMODA
ml. of pooled semen from S.C.W.L. males. Eggs were collected four times daily with individual identification and stored at a temperature of 12.8-15.6° C. Eggs were set within 5 days after production in a forced draft incubator 5 with a separate hatching compartment. All eggs were candled on the 7th day and eggs appearing infertile were broken to verify infertility. Eggs were transferred to the hatching compartment on the 18th day. At the termination of incubation, dead embryos were recorded and examined for abnormality. Hatchability was calculated on the fertile eggs. The progeny of the initial and the last set of eggs was tested for 3 weeks following hatching.
the histopathological examination of tissues, two birds from each level of T-2 toxin were randomly selected. The dissected tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 microns and stained with hematoxylin and eosin in order to evaluate the morphological cellular changes. Tissues examined microscopically were myocardium, liver, kidney, brain, ovary, oviduct, bursa of Fabricius, crop, gizzard and intestines. The weight of the liver, heart, gizzard and spleen was determined and expressed per 100 grams of body weight. Peripheral blood and bone marrow smears were stained with modified WrightGiemsa stain.
Birds were closely observed daily, examined grossly at each weighing for any clinical signs including incidence of oral lesions, and recorded. At the end of the experiment, blood samples were collected by cardiac puncture. Two ml. of blood was delivered to a test tube in which a drop of dipotassium ethylenediamine tetraacetate was placed and evaporated. Whole blood erythrocyte counts, leukocyte counts, hemoglobin, hematocrit and differential leukocyte counts were estimated on this sample by standard methods (Schalm, 1965). The remaining blood was centrifuged, without addition of anticoagulant, to separate serum which was kept frozen until analysis. The blood serum was analyzed for total protein, cholesterol, uric acid, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactic dehydrogenase (LDH) and alkaline phosphatase according to the methods described by Henry (1964).
Statistical analysis of data was performed using an analysis of variance technique and comparisons among treatment means were made using the honestly significant difference (hsd) procedure (Steel and Torrie, 1960).
After sampling blood, hens were necropsied and bone marrow smears were made from tibia. Hens were grossly examined for the presence of any pathological changes. For
5. Robbins hatch-matic®, .Robbins Incubator Co., Denver, Colorado.
RESULTS Hens fed a diet containing 8 p.p.m. T-2 toxin consumed significantly (P < 0.05) less feed than hens fed the control diet while hens fed a diet containing 1 p.p.m. consumed significantly (P < 0.05) greater amount of feed than the control group (Table 2). Body weight change of hens fed T-2 toxin was not different from that of control hens. Egg production of hens fed a diet containing 8 p.p.m. T-2 toxin was significantly (P < 0.05) less than that of hens fed the control diet or diets containing 0.5 and 1 p.p.m. (Table 2). The most marked effect of T-2 toxin on egg production was observed during the last period of the experiment in hens fed 8 p.p.m. resulting in 11.3% lower egg production as compared with the control group (Figure 1). Feed conversion of hens fed a diet containing 2 p.p.m. T-2 toxin was inferior (P < 0.05) to that of hens fed the control diet or a diet containing 0.5 p.p.m., but hens fed 4 or 8 p.p.m. T-2 toxin had a feed conversion ratio
631
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similar to the control group. Egg weight and the Haugh unit were not significantly influenced by T-2 toxin. Hens fed 8 p.p.m. T-2 toxin produced eggs with thinner (P < 0.05) shells than hens fed other treatment diets. Fertility was not different between treatments (Table 2); hatchability, however, of hens fed 8 p.p.m. T-2 toxin was lower (P < 0.05) than that of control groups or hens fed 0.5 p.p.m. The hatchability of hens fed 2
and 4 p.p.m. T-2 toxin was also lower than that of control group, but the difference between the control group and 4 p.p.m. group was not significant. The relatively lower hatchability of hens fed 2 to 8 p.p.m. T-2 toxin was observed from the first 2 week period (Figure 2) and hens fed 8 p.p.m. had the lowest hatchability at the last 2 week period. Death of the embryo mostly occurred in the early stage of incubation. No evidence of malformation in dead embryos or hatched
632
CHI, MIROCHA, KURTZ, WEAVER, BATES AND SHIMODA
TABLE 2.—Reproductive performance parameters of laying hens fed diets containing varying amounts of T-2 toxin'-2 Dietary level of T-2 toxin (p.p. m.) 0 (Control) Body weight gain, g . / h e n / 2 weeks Feed consumption, g./hen/day Egg production, % Feed conversion; kg. feed/dozen eggs g. feed/g. egg Egg weight, g. Haugh unit Shell thickness, mm. Fertility, % Hatchability, 3 %
12.8 100.0b 89.25b 1.34a 2.17ab 51.61 90.32 0.3187b 95.25 96.85c
0.5 12.3 99.5b 90.33b 1.33a 2.12a 52.23 92.85 0.3255b 95.85 95.84bc
1.0 19.5 104.0c 89.56b 1.40ab 2.20ab 52.88 93.13 0.3261b 95.49 94.80abc
2.0 22.3 101.2bc 85.75ab 1.42b 2.25b 52.47 93.24 0.3216b 93.62 91.15ab
4.0 11.0
8.0 2.6
99.9b 87.05ab
94.4a 84.20a
1.38ab 2.20ab 52.19 92.10 0.3209b 96.27 92.78abc
1.35ab 2.14ab 52.66 93.36 0.3060a 95.54 89.38a
1 Mean value of 6 replicated groups with 4 hens each at each level of T-2 toxin. 2 Means in the same line with different letters differ significantly (P < 0.05).. 3
Hatchability of fertile eggs.
chicks was found in T-2 treated groups. The growth rate and feed /gain ratio of progeny from hens fed T-2 toxin were similar to that of progeny from the control group (Table 3). Chicks hatched from eggs of hens fed 8 p.p.m. T-2 toxin had smaller (P < 0.05) body weights at 21 days than those hatched from eggs of hens fed 0.5, 1 or 4 p.p.m. The weights of the liver, heart, gizzard and spleen were not influenced by the dietary T-2 toxin (Table 4). Hens fed diets containing 0.5 and 1 p.p.m. T-2 toxin had a lower (P < 0.05) leukocyte count than control hens (Table 5). Hens fed 8 p.p.m. T-2 toxin also had a lower leukocyte level than the control group, but the difference was not statistically significant. The number of erythrocytes of T-2 treated hens was not significantly different from that of control hens, but hens fed 1 p.p.m. T-2 toxin had a lower erythrocyte level than hens fed 2 or 4 p.p.m. Hematocrit, hemoglobin and differential leukocyte counts were not affected by dietary T-2 toxin. Serum alkaline phosphatase and SGPT were not affected by T-2 toxin at concentra-
tions lower than 4 p.p.m. of the diet; hens fed 8 p.p.m. had higher (P < 0.05) serum alkaline phosphatase and lower (P < 0.05) SGPT than the control group (Table 6). The serum LDH level was higher (P < 0.05) in hens fed 2, 4 and 8 p.p.m. T-2 toxin as compared with control hens or hens fed 0.5 and 1 p.p.m. Serum cholesterol was not significantly changed in T-2 treated hens as compared with control hens. However, hens fed 0.5 p.p.m. had lower serum cholesterol than hens fed 2 and 4 p.p.m. T-2 toxin. Serum uricacidof hens 8 p.p.m. T-2 toxin was higher (P < 0.05) than that of the control group. Serum GOT and total protein were not significantly changed in T-2 treated hens. Oral lesions were observed from the 2nd week in hens fed 4 and 8 p.p.m. T-2 toxin. After the 3rd week, even hens fed 0.5 p.p.m. had small oral lesions (Table 7). At the level of 8 p.p.m., nearly all of the hens in the group had oral lesions. The lesions were circumscribed, proliferative, caseous plaques and occurred at the margin of the beak, the mucosa of the hard palate, angle of the mouth and on or under the tongue. Several of these
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lesions coalesced on the oral mucosa. The severity and incidence of the lesions were proportional to the amount of the toxin in the diet. Small mucosal ulcers in the anterior portion of the gizzard were observed in many
of the hens fed 4 and 8 p.p.m. T-2 toxin. Other than these observations, no pathological or clinical signs were noted in necropsy examinations. Microscopic studies of the various organs revealed no significant patho-
CHI, MIROCHA, KURTZ, WEAVER, BATES AND SHIMODA
634
TABLE 3.—The growth rate and feed/gain ratio of progeny from hens fed varying amounts of T-2 toxin' Dietary level of T-2 toxin (p.p.m.)
21-day body weight (g.): First setting of egg Last setting of egg Avg. 3 Feed/gain ratio First setting of egg Last setting of egg Avg.
0 (Control)
0.5
1.0
2.0
4.0
8.0
Avg. 2
226.0 225.0 225.5ab
221.0 237.8 229.4a
229.4 235.3 232.4a
220.7 236.0 228.4ab
222.8 237.0 229.9a
218.2 219.3 218.7b
223.0 231.7**
1.80 1.85 1.83
1.78 1.93 1.86
1.80 1.92 1.86
1.81 1.94 1.88
1.77 1.91 1.84
1.81 1.85 1.83
1.80 1.90**
'Mean values of 6 replicated groups of 10 chicks each. The averages of first and last setting of eggs were compared by t-test; **, significantly different at 3P < 0.01. The averages at dietary levels of T-2 toxin were compared using the hsd procedure; means bearing different letters differ significantly (P < 0.05). 2
TABLE 4.- -Organ weights of laying hens fed diets containing varying amounts of T-2 toxin
Dietary level of T-2 toxin (p.p.m.) 0 (Control) 0.5 2.42 2.37 2.47 2.30 Liver weight, g. 2.01 0.49 0.48 Heart weight, g. 0.43 0.48 0.42 1.74 Gizzard weight, g. 1.76 1.77 1.71 1.66 0.097 Spleen weight, g. 0.097 0.095 0.120 0.120 1 Mean values of 6 replicated groups with 4 hens each at each level of T-2 toxin. 2 Organ weights expressed per 100 grams of body weight.
logical changes in various organs except necrotic lesions in the crop and gizzard of groups fed high concentrations of T-2 toxin. DISCUSSION The toxic metabolites of fungi have been implicated in poor reproductive performance of laying hens. Hamilton and Garlich (1972) reported a delayed and decreased egg production in commercial laying hens fed anatoxin; hatchability was also reduced (Kratzer et al., 1969). The feeding of ochratoxin to laying hens resulted in a delay in sexual maturity, decrease in egg production and hatchability and depression in subsequent performance of the progeny for their first 2-week period (Choudhury, 1971). Zearalenone appeared to have very little detrimental effects on reproductive performance of laying hens at relatively high concentrations (Speers
2.56 0.48 1.67 0.105
et al., 1971) even though it had caused profound reproductive disorders in swine (Mirocha et al., 1971). It is evident from the data of this study that T-2 toxin had detrimental effects on egg production, shell quality and hatchability at the level of 8 p.p.m. of the diet. The egg production and hatchability were depressed almost immediately but more severely after 6 weeks. It is not known at this time whether the decrease in hatchability was due to transmission of T-2 toxin or its toxic metabolites into the egg at a concentration sufficient to kill the embryo. The possibility exists that T-2 toxin might alter the nutritional status of the egg within the hen while on a T-2 toxin diet resulting in reduced hatchability. Hens fed 0.5 p.p.m. T-2 toxin slightly improved their egg production without any deleterious effects on other reproductive per-
T-2
TOXIN
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TABLE 5.—Hematological findings for laying hens fed diets containing varying amounts of T-2 toxin' Dietary level of T-2 (p.p.m.) 0 0.5 1.0 2.0 4.0 8.0 'Mean < 0.05).
R.B.C. W.B.C. Based on 300 cell count (%) Hemoglobin (mil(thouHematocrit lions/ sands/ Total (8./ Mono mm.3) mm.3) Lympho neutro Baso 100 ml.) (%) 26.4 1.1 8.79 2.42ab 24.9 5.0 28.4b 69.0 29.3 9.29 2.43ab 17.9a 62.9 32.1 3.9 1.1 26.8 8.93 2.09a 14.9a 62.1 30.9 5.8 1.2 28.1 9.57 2.71b 28.8b 68.5 25.4 5.4 0.7 27.6 9.41 2.68b 28.1b 70.1 23.3 5.5 1.1 26.3 8.83 2.52ab 21.5ab 72.2 19.7 6.5 1.6 values of 12 hens. Means in the same column with different letters differ significantly (P
TABLE 6.—Blood chemistry findings for laying hens fed diets containing varying amounts of T-2 toxin' Total Dietary Alkaline SGOT SGPT level phosphatase (R.F. (R.F. LDH protein Cholesterol Uric acid of T-2 (B. units/ units/ units/ (I.U./ (mg./ (mg./ (g./ (p.p.m.) 100 ml.) ml.) ml.) ml.) 100 ml.) 100 ml.) 100 ml.) 0 50.8a 257.5 7.02 19.17b 855a 98.2ab 5.42a 0.5 75.0ab 215.0 16.67ab 585a 6.27 90.3a 6.53ab 1.0 45.0a 208.0 17.33b 797a 6.55 95.lab 5.63ab 2.0 52.5a 217.5 20.08b 1959b 6.69 112.4b 4.81a 4.0 62.5a 230.0 20.17b 1680b 6.59 116.5b 6.18ab 8.0 129.2b 201.7 11.25a 1435b 7.44 110.8ab 7.68b 'Mean values of 12 hens. Means in the same column with different letters differ significantly (P < 0.05).
formance criteria. A similar effect was seen on growth of rainbow trout (Marasas et aL, 1969). The possibility of an antibiotic effect of the toxin on the microflora in the feed or intestinal tract exists. Oral lesions, a most prominent sign, were similar to those observed in broiler chicks fed T-2 toxin (Wyatt et al., 1972; Chi et al., 1977). Wyatt et al. (1972) examined the oral lesions histopathologically and found an intense inflammation of the tissue and local necrosis. The outer layers of the lesion consisted of fibrinous material which sloughed off easily while the under-lying tissue was heavily infiltrated with granulated leukocytes. Large numbers of bacteria were also found in the eroded area. T-2 toxin apparently is a contact corrosive as evidenced by the formation of oral lesions and necrosis of the gizzard and crop seen in many of the hens fed high levels of T-2 toxin. There was no
death during the experimental period. Histopathological examination of organs from treated birds agreed with the results of gross examination; no significant lesions were found except in the crop and gizzard. There was an indication of lymphocytic infiltration in several tissues, probably due to a low grade viral infection in many of the hens fed T-2 toxin; however, no lesions attributable to the toxin alone were found. The presence of this low grade viral infection should not have interfered with the formation of any lesions that might have been caused by the T-2 toxin. The leukocyte count in chicks fed a low concentration of T-2 toxin (0.2 p.p.m.) increased over that of control chicks with no significant changes at concentrations of 0.4 to4 p.p.m. (Chi et al., 1977). When T-2 toxin was fed to hens at 0.5 and 1.0 p.p.m., the leukocyte count decreased below that of the
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control group while at 2 to 8 p.p.m., no difference was observed. Since there was no evidence of pathological changes in bone marrow and organ tissues, a decrease in the leukocyte level at the low concentrations of the toxin may not be the result of T-2 toxicosis. Blood enzymes, used as a criterion of hepatic and myocardial tissue damages, did not change sufficiently to suggest that pathological changes occurred in the organs after exposure to T-2 toxin; this agreed with the results of gross and histopathological examinations of liver, heart and kidney in which clinical signs were not evident. The major sources of serum alkaline phosphatase are bone, intestine and liver (Fishman and Ghosh, 1967). Serum alkaline phosphatase increases in calcium-deficient hens and an increase in alkaline phosphatase levels is associated with decalcification of bone (Hurwitz and Griminger, 1961). The changes in intestinal alkaline phosphatase is associated with calcium and phosphorus absorption (Moog and Glazier, 1972). In this study, the increase of serum alkaline phosphatase in T-2 toxin treated hens was observed at a concentration (8 p.p.m.) which also resulted in thin egg shells; this suggests that T-2 toxin interferes with calcium metabolism, probably by reduced absorption in the intestine. Even though T-2 toxin did not cause any significant pathological signs in organs, the toxin appeared inflammatory and did affect reproductive performance of laying hens in terms of decreased egg production, hatchability and thin egg shell formation. In addition, there are possibilities that cereal grains and animal feed contaminated with species of Fusarium often contain other toxic metabolites in addition to T-2 toxin. The contamination of animal feeds with T-2 and other mycotoxins associated with it should be avoided in order to safeguard animal and public health.
T-2 TOXIN
REFERENCES Bamburg, J. R., N. V. Riggs and F. M. Strong, 1968. The structures of toxins from two strains of Fusarium tricinctum. Tetrahedron, 23: 3329-3336. Brook, P. J., and E. P. White, 1966. Fungus toxins affecting mammals. Ann. Rev. Phytopathol. 4: 171194. Burmeister, H. R., 1971. T-2 toxin production by Fusarium tricinctum on solid substrate. Appl. Microbiol. 21: 739-742. Chi, M. S., C. J. Mirocha, H. J. Kurtz, G. Weaver, F. Bates and W. Shimoda, 1977. Subacute toxicity of T-2 toxin in broiler chicks. Poultry Sci. 56: 306-313. Choudhury, H., C. W. Carlson and G. Semeniuk, 1971. A study of ochratoxin toxicity in hens. Poultry Sci. 50: 1855-1858. Christensen, C. M., R. A. Meronuck, G. H. Nelson and J. C. Behrens, 1972. Effects on turkey poults of rations containing corn invaded by Fusarium tricinctum (Cda.) Sny. & Hans. Appl. Microbiol. 23: 177-179. Fishman, W. H., and N. K. Ghosh, 1967. Isoenzymes in human alkaline phosphatase. Advances Clin. Chem. 10: 255-370. Forgacs, J., 1964. Stachybotryotoxicosis and moldy corn toxicosis. In: Mycotoxins in Foodstuffs, Ed. G. N. Wogan, M.I.T. Press, Cambridge, Mass. p. 87-104. Forgacs, J., H. Koch, W. T. Carle and R. H. WhiteStevens, 1962. Mycotoxicoses. I. Relationship of toxic fungi to moldy-feed toxicosis in poultry. Avian Dis. 6: 363-380. Gilgan, M. W., E. B. Smalley and F. M. Strong, 1966. Isolation and partial characterization of a toxin from Fusarium tricinctum on moldy corn. Arch. Biochem. Biophys. 114: 1-3. Hamilton, P. B., and J. D. Garlich, 1972. Failure of vitamin supplementation to alter the fatty liver syndrome caused by aflatoxin. Poultry Sci. 51: 688-692. Haugh, R. R., 1937. The Haugh unit for measuring egg quality. U.S. Egg Poultry Mag. 43: 552-555 and 572-573. Henry, R. J., 1964. Clinical Chemistry, Principles and Techniques. Harper and Row, New York, N.Y. Hsu, I. C , E. B. Smalley, F. M. Strong and W. E. Rebelin, 1972. Identification of T-2 toxin in moldy corn associated with a lethal toxicosis in dairy cattle. Appl. Microbiol. 24: 684-690. Hurwitz, S., and P. Griminger, 1961. The response of plasma alkaline phosphatase, parathyroids and
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blood and bone minerals to calcium intake in the fowl. J. Nutrition, 73: 177-185. Joffe, A. Z., 1964. Toxin production in cereal fungi causing toxic alimentary aleukia in man. In: Mycotoxins in Foodstuffs, Ed. G. N. Wogan, M.I.T. Press, Cambridge, Mass. p. 77-86. Kratzer, F. H., D. Bandy, M. Wiley and A. N. Booth, 1969. Aflatoxin effects in poultry. Proc. Soc. Exp. Biol. Med. 131: 1281-1284. Marasas, W. F. O., J. R. Bamburg, E. B. Smalley, F. M. Strong, W. L. Ragland and P. E. Degurse, 1969. Low dose, long term effects on trout and rats of T-2 toxin produced by the fungus Fusarium tricinctum. Toxicol. Appl. Pharmacol. 15: 471-482. Mirocha, C. J., C. M. Christensen and G. H. Nelson, 1971. F-2 (Zearalenone) estrogenic mycotoxin from Fusarium. In: Microbial Toxins, Vol. 7, Algal and Fungal Toxins, Ed. S. Kadis, A. Ciegler and S. J. Ajl, Academic Press, New York, N.Y. p. 107-138. Moog, F. and H. S. Glazier, 1972. Phosphate absorption and alkaline phosphatase activity in the small intestine of the adult mouse and of the chick embryo and hatched chick. Comp. Biochem. Physiol. 42: 321-336. Schalm, O. W., 1965. Veterinary Hematology. 2nd Ed. Lea and Febiger, Philadelphia, Pennsylvania. Smalley, E. B., W. F. P. Marasas, F. M. Strong, J. R. Bamburg, R. E. Nichols and N. R. Kosuri, 1970. Mycotoxins associated with moldy corn. Proc. First U.S.—Japan Conf. Toxic Microorganisms, Honolulu, Hawaii. Unnumbered publication. U.S. Dept. of Interior and U.J.N.R. Panels on Toxic Microorganisms, Washington, D.C. p. 163-173. Speers, G. M., R. S. Meronuck, D. M. Barnes and C. J. Mirocha, 1971. Effect of feeding Fusarium roseum, F. sp. graminearum contaminated corn and the mycotoxin F-2 on the growing chick and laying hen. Poultry Sci. 50: 627-633. Stanford, G. K., R. D. Hood and A. W. Hayes, 1975. Effect of prenatal administration of T-2 toxin to mice. Res. Commum. Chem. Path. Pharmacol. 10: 743-746. Steel, R. G. D. and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, N.Y. Wyatt, R. D., W. M. Colwell, P. B. Hamilton and H. R. Burmeister, 1973. Neural disturbances in chickens caused by dietary T-2 toxin. Appl. Microbiol. 26: 757-761. Wyatt, R. D., B. A. Weeks, P. B. Hamilton and H. R. Burmeister, 1972. Severe oral lesions in chickens caused by ingestion of dietary fusariatoxin T-2. Appl. Microbiol. 24: 251-257.
SEPTEMBER 17-21, 1978. XVI WORLD'S POULTRY CONGRESS, RIO DE JANEIRO, BRAZIL