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Influence of Ochratoxin A and T-2 Toxin Singly and in Combination on Broiler Chickens1
ENVIRONMENT AND HEALTH
Influence of Ochratoxin A and T-2 Toxin Singly and in Combination on Broiler Chickens 1 L. F. KUBENA, R. B. HARVEY, W. E. HUFF, and D. E. CORRIER U.S. Department of Agriculture, Agricultural Research Service, Veterinary Toxicology & Entomology Research Laboratory, College Station, Texas 7784] T. D. PHILLIPS Department of Veterinary Public Health, Texas A & M University, College Station, Texas 77843
Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211 (Received for publication May 9, 1988) ABSTRACT Effects of feeding diets containing ochratoxin A (OA) at 2.0 mg/kg and T-2 toxin at 4.0 mg/kg singly and in combination were characterized in male broiler chicks from 1 day to 3 wk of age. Body weights were depressed by feeding OA singly, T-2 singly, and the OA/T-2 combination. The efficiency of feed utilization was reduced in the OA and OA/T-2 combination-fed groups. The T-2 toxin caused oral lesions, anemia, and changes in some serum biochemical values. Feeding OA resulted in increases in relative liver, kidney, gizzard, and pancreas weights, a microcytic hypochromic anemia, and changed serum chemistries. The OA/T-2 combination caused increases in relative liver, kidney, proventriculus, and gizzard weights and variable serum biochemistries. The effects of OA and T-2 were additive for reduced body weight gains, mean corpuscular volume, and for reduced serum levels of total protein, and for reduced lactate dehydrogenase activity. A significant interaction occurred between OA and T-2 causing elevated serum triglyceride levels and decreased gamma glutamyl transferase activity and calcium levels. Degenerative renal tubular changes were observed in some OA and OA/T-2 combination-fed chicks. These data indicate that OA and T-2 in combination may be more toxic for some parameters than the individual mycotoxins, and may pose a greater problem for the poultry industry than either of the mycotoxins individually. (Key words: ochratoxin A, T-2 toxin, mycotoxins, chickens, interactions) 1989 Poultry Science 68:867-872 INTRODUCTION _ , . . ,„
^ n t i o n of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion ofother products that may be suitable.
mental feeding of OA has been shown (Kubena et al., 1988) to have detrimental effects Qn m c h i c k s i n d i c a t i n g O A is a , m h a z a l d tQ producers. ^ . Zmyaxnm produced by T_2 ^ n , , _, „ . ,D * species of the genus Fusanwn (Barn^ , e ' ?> 1 9 7 0 > m a t « « fo™d m ^ cereals feeds and vegetables Research has shown m a t T 2 toxm causes " reductions in feed consumption and weight gam (Wyatt et al., 1973b; Chi et al., 1977; Hoerr et al., 1982; Huff et al., 1988), severe oral lesions in broilers (Wyatt et al., 1972, 1973b; Hoerr et al., 1982), abnormal behavior (Wyatt et al., 1973a), altered feathering (Wyatt et al, 1975), md a c o a g u i a p a t h y (Doerr et al., 1981). T o the j . , ;, , j ,->» J T <-> . • t. authors knowledge, OA and T-2 toxin have not been reported to occur simultaneously in a single feedstuff; however, it is not unusual to
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use multiple grain sources in poultry and livestock diets. Thus the possibility exists for feeding diets contaminated with both OA and T-2 toxin. The effects of OA and T-2 toxin in combination have not been investigated in poultry. The purpose of this research was to investigate the toxicity and describe the major effects of feeding male broiler chicks diets containing OA and T-2 toxin singly and in combination from 1 day to 3 wk of age.
Day-old male broiler Hubbard x Hubbard chicks (x weight = 43.5 g) were obtained from a commercial hatchery and individually weighed and wingbanded. Chicks were placed into electrically heated batteries under fluorescent lighting with feed and water available ad libitum. A completely randomized 2 x 2 factorial experimental design was used, consisting of four dietary treatments: 1) control, with 0 mg OA, 0 mg T-2 toxin/kg of diet; 2) 0 mg OA, 4 mg T-2 toxin/kg of diet; 3) 2 mg OA, 0 mg T-2 toxin/kg of diet; and 4) 2 mg OA plus 4 mg T-2 toxin/kg of diet. There were 6 replicates of 10 broilers/dietary treatment, for a total of 240 chicks. Broilers were maintained on these treatments to 3 wk of age. Chicks were fed a commercial, unmedicated cornsoybean meal-based diet that contained or exceeded levels of critical nutrients recommended by the National Research Council (1984). The OA was produced, extracted, and purified by methods previously described by Huff et al. (1974). The T-2 toxin was kindly provided by G. E. Rottinghaus, Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO. Crystalline OA was dissolved in 95% ethanol, and the T-2 toxin was dissolved in 95% ethanol. Appropriate quantities for each diet were mixed with 1 kg of the diet, which was dried and mixed with the rest of the diet to produce the three treatments containing added toxins. Chicks were individually weighed weekly; feed consumption was recorded for each replicate. Deaths were recorded as they occurred during the study. After 3 wk, the feeding trial was terminated and 20 broilers (2 replicates of 10 chicks each) from each treatment were bled by cardiac puncture for hematological and serum biochemical ana-
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on September 27, 2016
MATERIALS AND METHODS
lyses. Thirty broilers (five chicks from each replicate) were killed by cervical dislocation. The liver, kidney, spleen, pancreas, proventriculus, gizzard, and bursa of Fabricius were removed and weighed. Samples of the kidney were immediately taken from four randomly selected chicks per treatment and placed in 10% neutral buffered formalin. Paraffin sections were cut at 5 um and stained with hematoxylin and eosin for histopathological examination. At the termination of the study, all chicks were visually scored for oral lesions (using a 4-point scoring system ranging from 1 to 4) by the same individual without knowledge as to treatment groups. A lesion scored as 1 had no visible lesions; a lesion scored as 2 had one or two mouth lesions clearly visible on either the lower or upper mandible; a lesion score of 4 had large lesions occurring at several sites within the mouth, principally on the upper and lower mandibles, the corners of the mouth, and the back of the tongue; lesions scored as 3 were intermediate in appearance to lesions scored 2 or 4. Hemoglobin was measured as cyanmethhemoglobin (Coulter Electronics, Hileah, FL). Erythrocyte count, mean corpuscular volume, and hematocrit were determined with a Coulter Model ZBI Counter equipped with a mean corpuscular volume/hematocrit computer and a channelyzer (Coulter Electronics, Hileah, FL). The mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated. Automated equipment previously described by Kubena et al. (1985) was used to measure serum levels of uric acid, creatinine, urea nitrogen, glucose, inorganic phosphorus, total protein, albumin, cholesterol, triglycerides, alkaline phosphatase, cholinesterase, lactate dehydrogenase, aspartate aminotransferase, gamma glutamyl transferase, and creatine kinase. Data for all response variables were subjected to analysis of variance (Snedecor and Cochran, 1967) using the general linear model procedure in the SAS software (SAS, 1982). Main effects were levels of OA and T-2 toxin and their interactions. Means for treatments showing significant differences in the analysis of variance were compared; differences were indicated using Duncan's new multiple range procedure (Duncan, 1955). All statements of significance are based on the .05 level of probability.
OCHRATOXIN A AND T-2 TOXIN IN COMBINATION
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The effects of OA and T-2 toxin singly and in combination are shown in Table 1. Except in one instance (T-2 at 15 to 21 days), both OA and T-2 toxin significantly reduced body weight gains when fed alone. Body weight gains were further reduced by me OA/T-2 toxin combination, indicating an additive effect. The efficiency of feed utilization was reduced by OA singly and to a similar degree by the OA/T-2 toxin combination treatment. Mortality ranged from 3% to 10% and could not be attributed to treatment. Seventy percent of the chicks receiving either T-2 toxin singly or in combination with OA had oral lesions. When compared with controls, relative weights of the liver, kidney, gizzard, and pancreas were increased by feeding OA singly, whereas no changes were observed in relative organ weights for the chicks receiving T-2 toxin singly (Table 2). Feeding the OA/T-2 toxin combination resulted in increases in relative liver, kidney, gizzard, and proventriculus weights. The relative weights of the spleen and bursa of Fabricius were not significantly different from those of the control in any of the treatments (data not shown). Microscopic lesions consisting of swollen epithelial cells and condensed dark nuclei, indicative of early degeneration, were occasionally present in the proximal convoluted renal tubules of chicks receiving OA or the OA/T-2 toxin combination. Similar lesions were not observed in the control group nor in the T-2 toxin-treated group. The mild degenerative tubular changes were compatible with ochratoxicosis. There were no histopathological changes suggestive of an additive or synergistic relationship between OA and T-2 toxin. The individual and combined effects of OA and T-2 toxin on hematological and serum mineral values are shown in Table 3. The OA caused a significant decrease in hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin concentration. The T-2 toxin caused a decrease in hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration. With the exception of erythrocyte count (data not shown), all hematological parameters were significantly decreased in the OA/T-2 toxin combination group when compared with controls. Feeding OA at 2 mg/kg of diet resulted in decreased serum levels of calcium and
870
KUBENA ET AL. TABLE 2. Effects of dietary ochratoxin A (OA), T-2 toxin, and OAIT-2 toxin combination on relative organ weights (21 days)1
Treatment OA 0 2 0 2
T-2 (mg/kg) 0 0 4 4
Liver 3.44 4.36 3.33 4.74
Kidney ± ± ± ±
.10b .17* .09b .23*
.51 .79 .52 .78
± ± ± ±
.01 b .03* .01 b .03*
Proventriculus
Gizzard 2.63 2.94 2.76 3.30
± ± ± ±
.04° .lO6 .06*° .11*
.66 .72 .65 .73
± ± ± ±
SO*0 .03*" .02 c .02*
Pancreas .38 .46 .37 .41
± ± ± ±
.01 b .02* .01 b .01*b
phosphorus, whereas feeding T-2 toxin at 4 mg/kg of diet resulted in decreased serum levels of calcium and magnesium. Feeding the OA/T-2 toxin combination resulted in a decrease only in serum phosphorus. A significant interaction, characterized as antagonistic, between OA and T-2 toxin occurred to increase serum calcium levels to control levels. There were no changes in serum levels of potassium and sodium for any of the treatments (data not shown). The effects of dietary OA and T-2 toxin and the OA/T-2 toxin combination on serum biochemical values are shown in Table 4. When compared with controls, feeding OA resulted in a significant increase in serum uric acid, creatinine, gamma glutamyl transferase, and alkaline phosphatase and decreases in urea nitrogen, cholesterol, total protein, albumin, and lactic dehydrogenase. The T-2 toxin caused an increase in gamma glutamyl transferase and decreases in total protein, albumin, creatine kinase, and lactate dehydrogenase. The OA/T-2 toxin combination caused increases, in uric acid, creatinine, and triglycerides and decreases in urea nitrogen, cholesterol, total protein, albumin, creatine kinase, aspartate aminotransferase, and lactate dehydrogenase. The OA and T-2 toxins were additive in the effects on serum total protein and lactate dehydrogenase. A significant interaction between OA and T-2 toxin was observed for the serum triglyceride and gamma glutamyl transferase measurements.
feedstuffs that are used in poultry and livestock feeds. The individual effects of OA have been well documented (Huff et al., 1974, 1975; Prior et al., 1980; Huff and Doerr, 1981; Campbell et al., 1983; Kubena et al., 1983, 1985, 1986, 1988). The effects of T-2 toxin singly, although not as pronounced for some parameters as those of OA, still represent significant toxicity. Reduced performance due to T-2 toxin observed in the present study supports the previous work of Wyatt et al. (1973b), Chi et al. (1977), Hoerr et al. (1982), and Huff et al. (1988). The OA/T-2 toxin combination treatment effects in young broiler chicks can be characterized by reduced growth rates; decreased efficiency of feed utilization; decreased hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration; decreased total protein, albumin, creatine kinase, aspartate aminotransferase, and lactate dehydrogenase; and possible association with the inhibition of protein biosynthesis by these mycotoxins. The increased relative liver weights as well as the increase in serum triglyceride levels might be associated with alterations in lipid metabolism, primarily a result of impaired lipid transport. The reduction in serum cholesterol is most likely due to the inhibition of cholesterol biosynthesis, with liver involvement and perhaps a shift of concentration from the blood to the liver. The increased relative kidney weights, increased serum uric acid, and creatinine levels indicate impaired renal function due to the ingestion of DISCUSSION OA, as measurements of these parameters did Ochratoxin A is an important mycotoxin not differ significantly from those of the chicks receiving OA alone. Relative gizzard and due to its toxicity and its occurrence in
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a-c Values within columns with no common superscripts are significantly different (P<.05) according to Duncan's multiple range test. Values represent the x ± SEM of six groups of five broilers/treatment
0 2 0 2
OA
0 2 0 2
7.76 6.60 7.31 6.20
(g/dL) —• ± .09' ± .22c ± .12b ± .14°
Hemoglobin
25.45 23.35 25.25 22.25
± ± ± ±
.46' .70b .26' .63 b
(%)
Hematocrit -
(Hm3) 140.65 ± .91' 133.25 ± .88b 135.00 ± .69b 129.35 ± 1.19°
Volume 36.61 32.23 35.80 31.20
(pg) ± .73' ± .55 b ± 1.51' ± .61 b
Hemoglobin
Mean corpuscular
30.63 28.27 28.92 27.99
± ± ± ±
.50' .42b .30 b .51 b
(%)
Hemoglobin concentration 10. 9. 9. 10.
Cal
Values represent the x ± SEM of 2 groups of 10 broilers/treatment.
Values within columns with no common superscripts are significantly different (P<.05) according to Duncan's multi
(mg/kg) 0 0 4 4
T-2
Treatment
11.52 16.66 10.59 15.13
± ± ± ±
Uric acid
.56 b 1.67' .45 b 1.42a
2.35 1.62 2.22 1.09
± ± ± ±
Urea N
.23? .34 ± .14b .41 ± .31 ± • 27 hb .07 .42 ±
.02 b .02' .01 b .02'
Creatinine 214 204 173 305
± ± ± ±
28 b 25 b 12 b 48'*
Triglycerides 151 108 141 113
± ± ± ±
4' 6b 6' 7b
Cholesterol 3.01 2.09 2.76 1.86
± ± ± ±
Total protein
.05' 1.32 ± .02' .10f .84 ± .04' .06° 1.18 ± .03 b .09 d .82 ± .06°
Albumin
9.42 11.82 10.07 10.15
± ± ± ±
•Significant OA x T-2 interaction (P<.05).
Values represent the x ± SEM of 2 groups of 10 broilers/treatment
.42 b .72' .48° J0b*
Gamma glutamyl transferase
Values within columns with no common superscripts are significantly different (P<.05) according to Duncan's multiple range test.
- (mg/kg) 0 0 4 4
T-2
Treatment
4 5 3 3
A p
TABLE 4. Effects of dietary ochratoxin A {OA), T-2 toxin, and OAIT-2 toxin combination on serum bioche
•Significant OA x T-2 interaction (P<.05).
OA
Hematologic parameter
TABLE 3. Effects of dietary ochratoxin A (OA), T-2 toxin, and OAIT-2 toxin combination on hematol
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Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. 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. Hoerr, F. J., W. W. Carlton, B. Yagen, and A. Z. Joffe, 1982. Mycotoxicosis caused by either T-2 toxin or diacetoxyscirpenol in the diet of broiler chickens. Fundam. Appl. Toxicol. 2:121-124. Huff, W. E., and J. A. Doerr, 1981. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poultry Sci. 60:550-555. Huff, W. E., R. B. Harvey, L. F. Kubena, and G. E. Rottinghaus, 1988. Toxic synergism between aflatoxin and T-2 toxin in broiler chickens. Poultry Sci. 67: 1418-1423. Huff, W. E., R. D. Wyatt, and P. B. Hamilton, 1975. Nephrotoxicity of dietary ochratoxin A in broiler chickens. Appl. Microbiol. 30:48-51. Huff, W. E., R. D. Wyatt, T. L. Tucker, and P. B. Hamilton, 1974. Ochratoxicosis in the broiler chicken. Poultry Sci. 53:1585-1591. Krogh, P., F. Elling, B. Hald, B. Tylling, V. E. Peterson, E. Sbadhauge, and C. K. Svendsen, 1976. Experimental avian nephropathy. Acta Pathol. Microbiol. Scand. Sect. A. 84:215-221. Kubena, L. F., R. B. Harvey, O. J. Fletcher, T. D. Phillips, H. H. Mollenhauer, D. A. Witzel, and N. D. Heidelbaugh, 1985. Toxicity of ochratoxin A and vanadium to growing chicks. Poultry Sci. 64:620-628. Kubena, L. F., R. B. Harvey, T. D. Phillips, and O. J. Fletcher, 1986. Influence of ochratoxin A and vanadium on various parameters in growing chicks. Poultry Sci. 65:1671-1678. Kubena, L. F., W. E. Huff, R. B. Harvey, D. E. Corrier, T. D. Phillips, and C. R. Creger, 1988. Influence of ochratoxin A and deoxynivalenol on growing broiler chicks. Poultry Sci. 67:253-260. Kubena, L. F., T. D. Phillips, C. R. Creger, D. A. Witzel, and N. D. Heidelbaugh, 1983. Toxicity of ochratoxin A and tannic acid to growing chicks. Poultry Sci. 62: 1786-1792. National Research Council, 1984. Nutrient Requirements of Poultry. 1. Nutrient Requirements of Domestic AniACKNOWLEDGMENTS mals. 8th ed. Natl. Acad. Sci., Washington, DC. Prior, M. G., 1976. Mycotoxin determination on animal The authors gratefully acknowledge the feedstuffs and tissues in Western Canada. Can. J. Comp. Med. 40:73-79. excellent technical assistance of Maurice Connell, Florence Fair, Earl Munson, Albert Prior, M. G., J. B. O'Neill, and C. S. Sisodia, 1980. Effects of ochratoxin A on growth response and residues in Blanks, and Ellen Moore. broilers. Poultry Sci. 59:1254-1257. SAS, 1982. SAS User's guide: Statistics. SAS Inst Inc., Cary, NC. REFERENCES Snedecor, G. W., and W. E. Cochran, 1967. Statistical Methods. 6th ed. The Iowa State University Press, Bamburg, J. R., F. M. Strong, and E. B. Smalley, 1970. Ames, IA. Toxins from moldy feed cereals. J. Agric. Food Chem. Wyatt, R. D., W. M. Colwell, P. B. Hamilton, and H. R. 17:443-450. Burmeister, 1973a. Neural disturbances in chickens Campbell, M. L., J. D. May, W. E. Huff, and J. A Doerr, caused by dietary T-2 toxin. Appl. Microbiol. 26: 1983. Evaluation of immunity of young broiler chick757-761. ens during simultaneous aflatoxicosis and ochratoxi- Wyatt, R. D., P. B. Hamilton, and H. R. Burmeister, 1973b. cosis. Poultry Sci. 62:2138-2144. The effects of T-2 toxin in broiler chickens. Poultry Chi, M. S., C. J. Mirocha, H. J. Kurtz, G. Weaver, F. Bates, Sci. 52:1853-1859. and W. Shimoda, 1977. Subacute toxicity of T-2 toxin Wyatt, R. D., P. B. Hamilton, and H. R. Burmeister, 1975. in broiler chicks. Poultry Sci. 56:306-313. Altered feathering of chicks caused by T-2 toxin. Chu, F. S., 1974. Studies on ochratoxicosis. Grit. Rev. Poultry Sci. 54:1042-1045. Toxicol. 3:499-524. Wyatt, R. D., B. A. Weeks, P. B. Hamilton, and H. R. Doerr, J. A., P. B. Hamilton, and H. R. Burmeister, 1981. T-2 Burmeister, 1972. Severe oral lesions in chickens caused by ingestion of dietary fusariotoxin T-2. Appl. toxicosis and blood coagulation in young chickens. Microbiol. 24:251-257. Toxicol. Appl. Pharmacol. 60:157-162.
proventriculus weights were significantly greater in the OA/T-2 toxin combination group than in the other treatments, possibly due to overall irritative properties of the mycotoxins. The occurrence and severity of the oral lesions appear to reflect the effect of T-2 toxin, as lesion scores of those in the combination group did not differ significantly from those of the chicks receiving T-2 toxin alone. Additive OA/ T-2 toxin effects were observed for decreases in body weight gains, mean corpuscular volume, and serum levels of total protein and lactate dehydrogenase activity. A significant interaction occurred between OA and T-2 toxin, causing significantly elevated serum triglyceride levels and decreased gamma glutamyl transferase and calcium levels. The interaction can be characterized as synergistic for triglyceride levels and as antagonistic for gamma glutamyl transferase and calcium levels. These data indicate that under the experimental conditions employed, OA and T-2 toxin in combination may be more toxic than the individual mycotoxins for some parameters. Poultry and livestock may be more susceptible to OA and T-2 toxin as well as other mycotoxins if nutritional, environmental, health, or other stress factors are involved. Differences in susceptibility due to breed or strains might also occur, making it difficult to dismiss the potential effects of these two mycotoxins when present in combination.
Influence of Ochratoxin A and T-2 Toxin Singly and in Combination on Broiler Chickens 1 L. F. KUBENA, R. B. HARVEY, W. E. HUFF, and D. E. CORRIER U.S. Department of Agriculture, Agricultural Research Service, Veterinary Toxicology & Entomology Research Laboratory, College Station, Texas 7784] T. D. PHILLIPS Department of Veterinary Public Health, Texas A & M University, College Station, Texas 77843
Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211 (Received for publication May 9, 1988) ABSTRACT Effects of feeding diets containing ochratoxin A (OA) at 2.0 mg/kg and T-2 toxin at 4.0 mg/kg singly and in combination were characterized in male broiler chicks from 1 day to 3 wk of age. Body weights were depressed by feeding OA singly, T-2 singly, and the OA/T-2 combination. The efficiency of feed utilization was reduced in the OA and OA/T-2 combination-fed groups. The T-2 toxin caused oral lesions, anemia, and changes in some serum biochemical values. Feeding OA resulted in increases in relative liver, kidney, gizzard, and pancreas weights, a microcytic hypochromic anemia, and changed serum chemistries. The OA/T-2 combination caused increases in relative liver, kidney, proventriculus, and gizzard weights and variable serum biochemistries. The effects of OA and T-2 were additive for reduced body weight gains, mean corpuscular volume, and for reduced serum levels of total protein, and for reduced lactate dehydrogenase activity. A significant interaction occurred between OA and T-2 causing elevated serum triglyceride levels and decreased gamma glutamyl transferase activity and calcium levels. Degenerative renal tubular changes were observed in some OA and OA/T-2 combination-fed chicks. These data indicate that OA and T-2 in combination may be more toxic for some parameters than the individual mycotoxins, and may pose a greater problem for the poultry industry than either of the mycotoxins individually. (Key words: ochratoxin A, T-2 toxin, mycotoxins, chickens, interactions) 1989 Poultry Science 68:867-872 INTRODUCTION _ , . . ,„
^ n t i o n of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion ofother products that may be suitable.
mental feeding of OA has been shown (Kubena et al., 1988) to have detrimental effects Qn m c h i c k s i n d i c a t i n g O A is a , m h a z a l d tQ producers. ^ . Zmyaxnm produced by T_2 ^ n , , _, „ . ,D * species of the genus Fusanwn (Barn^ , e ' ?> 1 9 7 0 > m a t « « fo™d m ^ cereals feeds and vegetables Research has shown m a t T 2 toxm causes " reductions in feed consumption and weight gam (Wyatt et al., 1973b; Chi et al., 1977; Hoerr et al., 1982; Huff et al., 1988), severe oral lesions in broilers (Wyatt et al., 1972, 1973b; Hoerr et al., 1982), abnormal behavior (Wyatt et al., 1973a), altered feathering (Wyatt et al, 1975), md a c o a g u i a p a t h y (Doerr et al., 1981). T o the j . , ;, , j ,->» J T <-> . • t. authors knowledge, OA and T-2 toxin have not been reported to occur simultaneously in a single feedstuff; however, it is not unusual to
867
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on September 27, 2016
G. E. ROTTINGHAUS
868
KUBENA ET AL.
use multiple grain sources in poultry and livestock diets. Thus the possibility exists for feeding diets contaminated with both OA and T-2 toxin. The effects of OA and T-2 toxin in combination have not been investigated in poultry. The purpose of this research was to investigate the toxicity and describe the major effects of feeding male broiler chicks diets containing OA and T-2 toxin singly and in combination from 1 day to 3 wk of age.
Day-old male broiler Hubbard x Hubbard chicks (x weight = 43.5 g) were obtained from a commercial hatchery and individually weighed and wingbanded. Chicks were placed into electrically heated batteries under fluorescent lighting with feed and water available ad libitum. A completely randomized 2 x 2 factorial experimental design was used, consisting of four dietary treatments: 1) control, with 0 mg OA, 0 mg T-2 toxin/kg of diet; 2) 0 mg OA, 4 mg T-2 toxin/kg of diet; 3) 2 mg OA, 0 mg T-2 toxin/kg of diet; and 4) 2 mg OA plus 4 mg T-2 toxin/kg of diet. There were 6 replicates of 10 broilers/dietary treatment, for a total of 240 chicks. Broilers were maintained on these treatments to 3 wk of age. Chicks were fed a commercial, unmedicated cornsoybean meal-based diet that contained or exceeded levels of critical nutrients recommended by the National Research Council (1984). The OA was produced, extracted, and purified by methods previously described by Huff et al. (1974). The T-2 toxin was kindly provided by G. E. Rottinghaus, Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO. Crystalline OA was dissolved in 95% ethanol, and the T-2 toxin was dissolved in 95% ethanol. Appropriate quantities for each diet were mixed with 1 kg of the diet, which was dried and mixed with the rest of the diet to produce the three treatments containing added toxins. Chicks were individually weighed weekly; feed consumption was recorded for each replicate. Deaths were recorded as they occurred during the study. After 3 wk, the feeding trial was terminated and 20 broilers (2 replicates of 10 chicks each) from each treatment were bled by cardiac puncture for hematological and serum biochemical ana-
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on September 27, 2016
MATERIALS AND METHODS
lyses. Thirty broilers (five chicks from each replicate) were killed by cervical dislocation. The liver, kidney, spleen, pancreas, proventriculus, gizzard, and bursa of Fabricius were removed and weighed. Samples of the kidney were immediately taken from four randomly selected chicks per treatment and placed in 10% neutral buffered formalin. Paraffin sections were cut at 5 um and stained with hematoxylin and eosin for histopathological examination. At the termination of the study, all chicks were visually scored for oral lesions (using a 4-point scoring system ranging from 1 to 4) by the same individual without knowledge as to treatment groups. A lesion scored as 1 had no visible lesions; a lesion scored as 2 had one or two mouth lesions clearly visible on either the lower or upper mandible; a lesion score of 4 had large lesions occurring at several sites within the mouth, principally on the upper and lower mandibles, the corners of the mouth, and the back of the tongue; lesions scored as 3 were intermediate in appearance to lesions scored 2 or 4. Hemoglobin was measured as cyanmethhemoglobin (Coulter Electronics, Hileah, FL). Erythrocyte count, mean corpuscular volume, and hematocrit were determined with a Coulter Model ZBI Counter equipped with a mean corpuscular volume/hematocrit computer and a channelyzer (Coulter Electronics, Hileah, FL). The mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated. Automated equipment previously described by Kubena et al. (1985) was used to measure serum levels of uric acid, creatinine, urea nitrogen, glucose, inorganic phosphorus, total protein, albumin, cholesterol, triglycerides, alkaline phosphatase, cholinesterase, lactate dehydrogenase, aspartate aminotransferase, gamma glutamyl transferase, and creatine kinase. Data for all response variables were subjected to analysis of variance (Snedecor and Cochran, 1967) using the general linear model procedure in the SAS software (SAS, 1982). Main effects were levels of OA and T-2 toxin and their interactions. Means for treatments showing significant differences in the analysis of variance were compared; differences were indicated using Duncan's new multiple range procedure (Duncan, 1955). All statements of significance are based on the .05 level of probability.
OCHRATOXIN A AND T-2 TOXIN IN COMBINATION
869
RESULTS
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The effects of OA and T-2 toxin singly and in combination are shown in Table 1. Except in one instance (T-2 at 15 to 21 days), both OA and T-2 toxin significantly reduced body weight gains when fed alone. Body weight gains were further reduced by me OA/T-2 toxin combination, indicating an additive effect. The efficiency of feed utilization was reduced by OA singly and to a similar degree by the OA/T-2 toxin combination treatment. Mortality ranged from 3% to 10% and could not be attributed to treatment. Seventy percent of the chicks receiving either T-2 toxin singly or in combination with OA had oral lesions. When compared with controls, relative weights of the liver, kidney, gizzard, and pancreas were increased by feeding OA singly, whereas no changes were observed in relative organ weights for the chicks receiving T-2 toxin singly (Table 2). Feeding the OA/T-2 toxin combination resulted in increases in relative liver, kidney, gizzard, and proventriculus weights. The relative weights of the spleen and bursa of Fabricius were not significantly different from those of the control in any of the treatments (data not shown). Microscopic lesions consisting of swollen epithelial cells and condensed dark nuclei, indicative of early degeneration, were occasionally present in the proximal convoluted renal tubules of chicks receiving OA or the OA/T-2 toxin combination. Similar lesions were not observed in the control group nor in the T-2 toxin-treated group. The mild degenerative tubular changes were compatible with ochratoxicosis. There were no histopathological changes suggestive of an additive or synergistic relationship between OA and T-2 toxin. The individual and combined effects of OA and T-2 toxin on hematological and serum mineral values are shown in Table 3. The OA caused a significant decrease in hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin concentration. The T-2 toxin caused a decrease in hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration. With the exception of erythrocyte count (data not shown), all hematological parameters were significantly decreased in the OA/T-2 toxin combination group when compared with controls. Feeding OA at 2 mg/kg of diet resulted in decreased serum levels of calcium and
870
KUBENA ET AL. TABLE 2. Effects of dietary ochratoxin A (OA), T-2 toxin, and OAIT-2 toxin combination on relative organ weights (21 days)1
Treatment OA 0 2 0 2
T-2 (mg/kg) 0 0 4 4
Liver 3.44 4.36 3.33 4.74
Kidney ± ± ± ±
.10b .17* .09b .23*
.51 .79 .52 .78
± ± ± ±
.01 b .03* .01 b .03*
Proventriculus
Gizzard 2.63 2.94 2.76 3.30
± ± ± ±
.04° .lO6 .06*° .11*
.66 .72 .65 .73
± ± ± ±
SO*0 .03*" .02 c .02*
Pancreas .38 .46 .37 .41
± ± ± ±
.01 b .02* .01 b .01*b
phosphorus, whereas feeding T-2 toxin at 4 mg/kg of diet resulted in decreased serum levels of calcium and magnesium. Feeding the OA/T-2 toxin combination resulted in a decrease only in serum phosphorus. A significant interaction, characterized as antagonistic, between OA and T-2 toxin occurred to increase serum calcium levels to control levels. There were no changes in serum levels of potassium and sodium for any of the treatments (data not shown). The effects of dietary OA and T-2 toxin and the OA/T-2 toxin combination on serum biochemical values are shown in Table 4. When compared with controls, feeding OA resulted in a significant increase in serum uric acid, creatinine, gamma glutamyl transferase, and alkaline phosphatase and decreases in urea nitrogen, cholesterol, total protein, albumin, and lactic dehydrogenase. The T-2 toxin caused an increase in gamma glutamyl transferase and decreases in total protein, albumin, creatine kinase, and lactate dehydrogenase. The OA/T-2 toxin combination caused increases, in uric acid, creatinine, and triglycerides and decreases in urea nitrogen, cholesterol, total protein, albumin, creatine kinase, aspartate aminotransferase, and lactate dehydrogenase. The OA and T-2 toxins were additive in the effects on serum total protein and lactate dehydrogenase. A significant interaction between OA and T-2 toxin was observed for the serum triglyceride and gamma glutamyl transferase measurements.
feedstuffs that are used in poultry and livestock feeds. The individual effects of OA have been well documented (Huff et al., 1974, 1975; Prior et al., 1980; Huff and Doerr, 1981; Campbell et al., 1983; Kubena et al., 1983, 1985, 1986, 1988). The effects of T-2 toxin singly, although not as pronounced for some parameters as those of OA, still represent significant toxicity. Reduced performance due to T-2 toxin observed in the present study supports the previous work of Wyatt et al. (1973b), Chi et al. (1977), Hoerr et al. (1982), and Huff et al. (1988). The OA/T-2 toxin combination treatment effects in young broiler chicks can be characterized by reduced growth rates; decreased efficiency of feed utilization; decreased hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration; decreased total protein, albumin, creatine kinase, aspartate aminotransferase, and lactate dehydrogenase; and possible association with the inhibition of protein biosynthesis by these mycotoxins. The increased relative liver weights as well as the increase in serum triglyceride levels might be associated with alterations in lipid metabolism, primarily a result of impaired lipid transport. The reduction in serum cholesterol is most likely due to the inhibition of cholesterol biosynthesis, with liver involvement and perhaps a shift of concentration from the blood to the liver. The increased relative kidney weights, increased serum uric acid, and creatinine levels indicate impaired renal function due to the ingestion of DISCUSSION OA, as measurements of these parameters did Ochratoxin A is an important mycotoxin not differ significantly from those of the chicks receiving OA alone. Relative gizzard and due to its toxicity and its occurrence in
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a-c Values within columns with no common superscripts are significantly different (P<.05) according to Duncan's multiple range test. Values represent the x ± SEM of six groups of five broilers/treatment
0 2 0 2
OA
0 2 0 2
7.76 6.60 7.31 6.20
(g/dL) —• ± .09' ± .22c ± .12b ± .14°
Hemoglobin
25.45 23.35 25.25 22.25
± ± ± ±
.46' .70b .26' .63 b
(%)
Hematocrit -
(Hm3) 140.65 ± .91' 133.25 ± .88b 135.00 ± .69b 129.35 ± 1.19°
Volume 36.61 32.23 35.80 31.20
(pg) ± .73' ± .55 b ± 1.51' ± .61 b
Hemoglobin
Mean corpuscular
30.63 28.27 28.92 27.99
± ± ± ±
.50' .42b .30 b .51 b
(%)
Hemoglobin concentration 10. 9. 9. 10.
Cal
Values represent the x ± SEM of 2 groups of 10 broilers/treatment.
Values within columns with no common superscripts are significantly different (P<.05) according to Duncan's multi
(mg/kg) 0 0 4 4
T-2
Treatment
11.52 16.66 10.59 15.13
± ± ± ±
Uric acid
.56 b 1.67' .45 b 1.42a
2.35 1.62 2.22 1.09
± ± ± ±
Urea N
.23? .34 ± .14b .41 ± .31 ± • 27 hb .07 .42 ±
.02 b .02' .01 b .02'
Creatinine 214 204 173 305
± ± ± ±
28 b 25 b 12 b 48'*
Triglycerides 151 108 141 113
± ± ± ±
4' 6b 6' 7b
Cholesterol 3.01 2.09 2.76 1.86
± ± ± ±
Total protein
.05' 1.32 ± .02' .10f .84 ± .04' .06° 1.18 ± .03 b .09 d .82 ± .06°
Albumin
9.42 11.82 10.07 10.15
± ± ± ±
•Significant OA x T-2 interaction (P<.05).
Values represent the x ± SEM of 2 groups of 10 broilers/treatment
.42 b .72' .48° J0b*
Gamma glutamyl transferase
Values within columns with no common superscripts are significantly different (P<.05) according to Duncan's multiple range test.
- (mg/kg) 0 0 4 4
T-2
Treatment
4 5 3 3
A p
TABLE 4. Effects of dietary ochratoxin A {OA), T-2 toxin, and OAIT-2 toxin combination on serum bioche
•Significant OA x T-2 interaction (P<.05).
OA
Hematologic parameter
TABLE 3. Effects of dietary ochratoxin A (OA), T-2 toxin, and OAIT-2 toxin combination on hematol
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KUBENA EI AL.
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Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. 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. Hoerr, F. J., W. W. Carlton, B. Yagen, and A. Z. Joffe, 1982. Mycotoxicosis caused by either T-2 toxin or diacetoxyscirpenol in the diet of broiler chickens. Fundam. Appl. Toxicol. 2:121-124. Huff, W. E., and J. A. Doerr, 1981. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poultry Sci. 60:550-555. Huff, W. E., R. B. Harvey, L. F. Kubena, and G. E. Rottinghaus, 1988. Toxic synergism between aflatoxin and T-2 toxin in broiler chickens. Poultry Sci. 67: 1418-1423. Huff, W. E., R. D. Wyatt, and P. B. Hamilton, 1975. Nephrotoxicity of dietary ochratoxin A in broiler chickens. Appl. Microbiol. 30:48-51. Huff, W. E., R. D. Wyatt, T. L. Tucker, and P. B. Hamilton, 1974. Ochratoxicosis in the broiler chicken. Poultry Sci. 53:1585-1591. Krogh, P., F. Elling, B. Hald, B. Tylling, V. E. Peterson, E. Sbadhauge, and C. K. Svendsen, 1976. Experimental avian nephropathy. Acta Pathol. Microbiol. Scand. Sect. A. 84:215-221. Kubena, L. F., R. B. Harvey, O. J. Fletcher, T. D. Phillips, H. H. Mollenhauer, D. A. Witzel, and N. D. Heidelbaugh, 1985. Toxicity of ochratoxin A and vanadium to growing chicks. Poultry Sci. 64:620-628. Kubena, L. F., R. B. Harvey, T. D. Phillips, and O. J. Fletcher, 1986. Influence of ochratoxin A and vanadium on various parameters in growing chicks. Poultry Sci. 65:1671-1678. Kubena, L. F., W. E. Huff, R. B. Harvey, D. E. Corrier, T. D. Phillips, and C. R. Creger, 1988. Influence of ochratoxin A and deoxynivalenol on growing broiler chicks. Poultry Sci. 67:253-260. Kubena, L. F., T. D. Phillips, C. R. Creger, D. A. Witzel, and N. D. Heidelbaugh, 1983. Toxicity of ochratoxin A and tannic acid to growing chicks. Poultry Sci. 62: 1786-1792. National Research Council, 1984. Nutrient Requirements of Poultry. 1. Nutrient Requirements of Domestic AniACKNOWLEDGMENTS mals. 8th ed. Natl. Acad. Sci., Washington, DC. Prior, M. G., 1976. Mycotoxin determination on animal The authors gratefully acknowledge the feedstuffs and tissues in Western Canada. Can. J. Comp. Med. 40:73-79. excellent technical assistance of Maurice Connell, Florence Fair, Earl Munson, Albert Prior, M. G., J. B. O'Neill, and C. S. Sisodia, 1980. Effects of ochratoxin A on growth response and residues in Blanks, and Ellen Moore. broilers. Poultry Sci. 59:1254-1257. SAS, 1982. SAS User's guide: Statistics. SAS Inst Inc., Cary, NC. REFERENCES Snedecor, G. W., and W. E. Cochran, 1967. Statistical Methods. 6th ed. The Iowa State University Press, Bamburg, J. R., F. M. Strong, and E. B. Smalley, 1970. Ames, IA. Toxins from moldy feed cereals. J. Agric. Food Chem. Wyatt, R. D., W. M. Colwell, P. B. Hamilton, and H. R. 17:443-450. Burmeister, 1973a. Neural disturbances in chickens Campbell, M. L., J. D. May, W. E. Huff, and J. A Doerr, caused by dietary T-2 toxin. Appl. Microbiol. 26: 1983. Evaluation of immunity of young broiler chick757-761. ens during simultaneous aflatoxicosis and ochratoxi- Wyatt, R. D., P. B. Hamilton, and H. R. Burmeister, 1973b. cosis. Poultry Sci. 62:2138-2144. The effects of T-2 toxin in broiler chickens. Poultry Chi, M. S., C. J. Mirocha, H. J. Kurtz, G. Weaver, F. Bates, Sci. 52:1853-1859. and W. Shimoda, 1977. Subacute toxicity of T-2 toxin Wyatt, R. D., P. B. Hamilton, and H. R. Burmeister, 1975. in broiler chicks. Poultry Sci. 56:306-313. Altered feathering of chicks caused by T-2 toxin. Chu, F. S., 1974. Studies on ochratoxicosis. Grit. Rev. Poultry Sci. 54:1042-1045. Toxicol. 3:499-524. Wyatt, R. D., B. A. Weeks, P. B. Hamilton, and H. R. Doerr, J. A., P. B. Hamilton, and H. R. Burmeister, 1981. T-2 Burmeister, 1972. Severe oral lesions in chickens caused by ingestion of dietary fusariotoxin T-2. Appl. toxicosis and blood coagulation in young chickens. Microbiol. 24:251-257. Toxicol. Appl. Pharmacol. 60:157-162.
proventriculus weights were significantly greater in the OA/T-2 toxin combination group than in the other treatments, possibly due to overall irritative properties of the mycotoxins. The occurrence and severity of the oral lesions appear to reflect the effect of T-2 toxin, as lesion scores of those in the combination group did not differ significantly from those of the chicks receiving T-2 toxin alone. Additive OA/ T-2 toxin effects were observed for decreases in body weight gains, mean corpuscular volume, and serum levels of total protein and lactate dehydrogenase activity. A significant interaction occurred between OA and T-2 toxin, causing significantly elevated serum triglyceride levels and decreased gamma glutamyl transferase and calcium levels. The interaction can be characterized as synergistic for triglyceride levels and as antagonistic for gamma glutamyl transferase and calcium levels. These data indicate that under the experimental conditions employed, OA and T-2 toxin in combination may be more toxic than the individual mycotoxins for some parameters. Poultry and livestock may be more susceptible to OA and T-2 toxin as well as other mycotoxins if nutritional, environmental, health, or other stress factors are involved. Differences in susceptibility due to breed or strains might also occur, making it difficult to dismiss the potential effects of these two mycotoxins when present in combination.