Evaluation of the Efficacy of a Feed Additive to Ameliorate the Toxic Effects of 4,15-Diacetoxiscirpenol in Growing Chicks G. J. Diaz1 Laboratorio de Toxicologı´a, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogota´, D.C., Colombia DAS was added to the diet, supplementation of Mycofix protected against the adverse effects of DAS on feed intake and BW at both levels of inclusion (0.75 and 1.5 g/kg); however, no protection against oral lesions was obtained by Mycofix supplementation. This finding suggests that the adverse effect of DAS on performance is not due to the oral lesions per se but it is likely the result of the systemic absorption of the mycotoxin. When 2 ppm dietary DAS was present in the diet, only partial protection on BW and feed intake was obtained by Mycofix supplementation. More studies are required to determine if a higher dose of Mycofix could be capable of counteracting the adverse effects of 2 ppm dietary DAS on chicken performance.
(Key words: 4,15-diacetoxiscirpenol, mycotoxin control, feed additive, broiler, Eubacterium) 2002 Poultry Science 81:1492–1495
INTRODUCTION Mycotoxins are common food and feed contaminants produced by certain fungi. Mycotoxin exposure can affect the health and performance of farmed animals, causing reduced weight gain, decreased productivity, reduced immune response, and even death (Leeson et al., 1995). The trichothecene mycotoxins are a group of fungal metabolites with the same basic structure. The chemistry, natural occurrence, and toxicology of trichothecenes were reviewed by Leeson et al. (1995). Although the number of known trichothecenes is currently over 100, information on their natural occurrence in agricultural products indicates that the most prevalent are deoxynivalenol, T2 toxin, HT-2 toxin, 4,15-diacetoxyscirpenol (DAS), and 15-monoacetoxyscirpenol (Bauer et al., 1989). In terms of LD50, DAS is the most toxic trichothecene for poultry species. The LD50 values for DAS, T-2 toxin, HT-2 toxin, and neosolaniol in day-old chicks are 2.0, 5.0, 7.2, and 24.9 mg/kg, respectively (Leeson et al., 1995). Surveys conducted to determine the natural occurrence of DAS
2002 Poultry Science Association, Inc. Received for publication September 19, 2001. Accepted for publication June 3, 2002. 1 To whom correspondence should be addressed: dgjdiaz@ veterinaria.unal.edu.co.
indicate that levels in mixed feeds and grain range from 0.1 to 2.3 ppm. Bata et al. (1983) reported DAS levels from 0.2 to 2.1 ppm, while Stratton et al. (1993) found DAS contamination ranging from 0.1 to 2.3 ppm. In this later survey, DAS was present in less than 10% of the 150 grain samples analyzed. The major findings in chickens receiving diets containing DAS are oral lesions, abnormal feathering, and decreased feed intake and weight gain (Chi and Mirocha, 1978; Hoerr et al., 1981; Ademoyero and Hamilton, 1991a,b; Parkhurst et al., 1992). Dietary DAS may also affect layer performance. Diaz et al. (1994) reported a significant decrease in egg production, and feed intake in commercial layers fed a diet containing 2 ppm DAS for 24 d. A common treatment for trichothecene toxicosis in poultry has been removal of the source of toxin coupled with symptomatic and supportive therapy (Leeson et al., 1995). In the case of chronically exposed birds, the usual approach is the administration of a highly nutritious diet free from mycotoxins, avoidance of stresses, and control of secondary infectious diseases; acutely exposed birds can be treated with oral adsorbents in order to minimize absorption from the gastrointestinal tract (Leeson et al., 1995). A more specific dietary treatment has been recently
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Abbreviation Key: DAS = diacetoxiscirpenol.
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ABSTRACT The possible protective effect of a feed additive (Mycofix) against the toxic effects of 4,15-diacetoxiscirpenol (DAS) in growing broiler chickens was investigated in a 21-d fully randomized trial consisting of seven dietary treatments (control with no DAS or Mycofix added, 1 ppm DAS alone, 1 ppm DAS supplemented with 0.75 g/kg Mycofix, 1 ppm DAS supplemented with 1.5 g/ kg Mycofix, 2 ppm DAS alone, 2 ppm DAS supplemented with 0.75 g/kg Mycofix, and 2 ppm DAS supplemented with 1.5 g/kg Mycofix). When no feed additive was included, both levels of dietary DAS significantly decreased BW and feed intake and caused oral lesions, with the effect of 2 ppm DAS being more severe. When 1 ppm
EFFICACY OF A FEED ADDITIVE AGAINST DAS
MATERIALS AND METHODS A total of 140 male day-old broiler chicks (Ross) obtained from a commercial hatchery was used. The chicks were individually weighed and wing-banded and randomly allotted into 28 pens, with five chicks per replicate pen. The chicks were housed in electrically heated batteries under continuous fluorescent light with feed and water provided ad libitum. A completely randomized arrangement of treatments was used with seven treatment groups and four replicate pens per treatment. The seven treatment groups were each given a different experimental diet that consisted of a commercial starter ration mixed with the following inclusion rates of DAS or Mycofix: Group one (control), no DAS or Mycofix added; group two: 1 ppm DAS + no Mycofix; group three: 1 ppm DAS + 0.75 g/kg Mycofix; group four: 1 ppm DAS + 1.5 g/kg Mycofix; group five: 2 ppm DAS + no Mycofix; group six: 2 ppm DAS + 0.75 g/kg Mycofix; and group seven: 2 ppm DAS + 1.5 g/kg Mycofix. Purified DAS was prepared from broth cultures of Fusarium sambucinum NRRL 13495, according to the method described by Richardson and Hamilton (1987). Toxin purity greater than 95% was confirmed by thin-layer chromatography. The basal commercial diet was analyzed for aflatoxin, ochratoxin A, and four type-A trichothecene mycotoxins (T-2 toxin, DAS, HT-2 toxin, and neosolaniol), and no detectable levels of these mycotoxins were found. The experimental diets were fed for 3 wk (Day 1 to 21 of age). The chicks were weighed on an individual basis, and feed consumption for each replicate was recorded weekly. The oral cavity was checked daily for oral lesions. At the end of the experiment (Day 21), five chicks from each treatment were taken at random and sacrificed by cervical dislocation. The liver, heart, spleen, proventriculus, and gizzard were removed and weighed to determine the relative weight of these organs.
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Biomin GTI Gesunde, Herzogenburg, Austria.
Statistical Analysis Data (pen means) for all response variables were subjected to ANOVA for a complete randomized design, using the general linear models procedure of SAS software (SAS Institute, 1987). The main effect was treatment within each week. Error was estimated from variation among pens. Variable means from treatments showing significant differences in the ANOVA were compared using the Fisher’s protected least significant difference procedure. Statements of significance are based on P ≤ 0.05.
RESULTS AND DISCUSSION The effect of dietary DAS with and without Mycofix supplementation on BW is shown in Table 1. Compared with controls, chicks receiving 1 ppm dietary DAS without Mycofix showed a significantly lower BW after 21 d of dietary exposure; however, no difference in BW was seen between chicks receiving 1 ppm DAS supplemented with Mycofix (at both inclusion levels of 0.75 and 1.5 g/ kg) and controls. Chicks receiving 2 ppm DAS without Mycofix in the diet had a significantly lower BW than controls at Day 7, 14 and 21. Chicks receiving 2 ppm DAS supplemented with 1.5 g/kg Mycofix had a significantly reduced BW at 14 and 21 d of age, and chicks receiving 2 ppm DAS supplemented with 0.75 g/kg Mycofix had a significantly lower BW after 21 d of exposure. Table 2 summarizes the performance of broiler chicks fed diets contaminated with DAS with and without supplementation of Mycofix. The 21-d weight gain was significantly lower in chicks receiving 1 ppm DAS without Mycofix supplementation and in all groups receiving 2 ppm DAS, regardless of Mycofix supplementation. Feed intake was also significantly lower in these four treatment groups, and the lowest feed intake was observed in chicks fed 2 ppm DAS without Mycofix supplementation (90.6% of controls). Feed:gain ratios were not significantly different among the seven experimental treatments. Oral lesions typical of type-A trichothecene exposure (Ademoyero and Hamilton, 1991a,b) were observed in all groups receiving DAS, regardless of Mycofix supplementation. The incidence and severity of the oral lesions were greater in those chicks receiving 2 ppm DAS than in those fed 1 ppm DAS. Interestingly, chicks receiving DAS had necrotic lesions at the tip of the tongue resembling the so-called “black tongues” reported by field veterinarians in Mexico, Colombia, and other countries (Gonzalez-Escobar, 2001). These lesions were visible as dark areas of variable size located at the tip of the tongue (Figure 1). Both levels of DAS produced “black tongue” lesions, but it took more days of exposure for the lower dose to cause these lesions (10 to 12 d) than for the higher dose (6 to 8 d). No attempt was made to obtain quantitative data from these lesions. No significant differences in the relative weights of heart, liver, spleen, proventriculus, and gizzard were ob-
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developed using microorganisms capable of inactivating the trichothecenes through enzymatic modification of the basic trichothecene structure. The inhibitory activity of trichothecenes requires the presence of an intact 9,10double bond and the C-12,13 epoxide, and opening of the 12,13-epoxide ring results in loss of any apparent toxicity (Ehrlich and Daigle, 1987). Binder et al. (2000) isolated a new species of bacterium of the genus Eubacterium capable of biotransforming the epoxide group of trichothecenes into a diene. A fermentation and stabilization process was established to use this bacterium as a feed additive. The encapsulated bacterium has been included in a commercial formulation designed for poultry and swine diets (Mycofix2). The aim of the present study was to determine the possible effectiveness of the dietary supplementation of Mycofix against the adverse effects of dietary DAS in growing broiler chickens.
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DIAZ TABLE 1. Effect of dietary DAS with and without the addition of Mycofix on BW in broiler chickens1 Dietary DAS or Mycofix Treatment
DAS (mg/kg)
1 2 3 4 5 6 7
0 1 1 1 2 2 2
Days of exposure to the experimental diets
Mycofix (g/kg)
0
0 0 0.75 1.5 0 0.75 1.5
± ± ± ± ± ± ±
43.0 43.8 45.0 43.3 43.8 43.8 43.3
7 a
0.9 0.8a 0.6a 0.9a 0.6a 0.5a 0.8a
157.5 148.8 157.3 157.3 146.3 154.0 152.5
± ± ± ± ± ± ±
14 a
2.7 3.5ab 2.5a 1.9a 2.7b 1.4ab 0.8ab
365.3 342.5 353.5 368.3 332.8 340.0 336.8
± ± ± ± ± ± ±
21 a
5.3 4.7ab 8.1ab 8.8a 7.7b 5.5ab 3.7b
718.0 640.0 698.5 723.0 611.0 601.8 638.5
± ± ± ± ± ± ±
6.1a 7.9b 11.9a 17.2a 9.6b 32.2b 27.8b
Means within a column lacking a common superscript differ (P < 0.05). Values are means ± SEM of four replicate pens per treatment. DAS = 4,15-diacetoxiscirpenol.
a,b 1
served after 21 d of dietary exposure to 1 ppm DAS; however, only 7 d were required to produce a significant reduction in BW (7%) when 2 ppm DAS was given. The feed additive tested in this study (Mycofix) protected against the adverse effects of 1 ppm dietary DAS on feed intake and BW at both levels of inclusion tested; however, only partial protection was obtained when 2 ppm dietary DAS was included in the diet. Further, no protection against the adverse effect of 2 ppm DAS on the 21-d BW gain was obtained by using any of the two inclusion levels of Mycofix. More studies are required to determine if a higher dose of Mycofix could be capable of counteracting the adverse effects of 2 ppm dietary DAS on chicken performance. Also, additional studies are required to determine if the product is capable of protecting the birds for longer term exposures to the toxin. Oral lesions typical of dietary trichothecene exposure were seen in all chicks receiving DAS, regardless of Mycofix supplementation. However, chicks receiving 1 ppm DAS along with Mycofix did not show a significant decrease in performance compared with controls. Studies conducted in several animal models indicate that there is a cellular mechanism capable of quickly transporting the trichothecenes through the cell membrane, which lead to a rapid absorption of the toxin from the exposed areas (Leeson et al., 1995). The presence of oral lesions in chickens showing no detrimental effects on performance suggests that the oral lesions per se are probably not the cause of the decreased performance reported in broilers
TABLE 2. Performance of broiler chickens fed diets contaminated with DAS with and without the addition of Mycofix1 Dietary DAS or Mycofix Treatment 1 2 3 4 5 6 7
DAS (mg/kg)
Mycofix (g/kg)
0 1 1 1 2 2 2
0 0 0.75 1.5 0 0.75 1.5
Gain (g) 1 to 21 d Means ± SEM
% of control
± ± ± ± ± ± ±
100.0 88.3 96.8 100.7 84.1 82.3 88.1
675.0 596.3 653.5 679.8 567.5 558.0 594.8
5.8a 8.6b 11.8a 17.8a 9.7b 32.2b 28.0b
Feed intake (g) Means ± SEM
% of control
Feed:gain ratio (g:g)
± ± ± ± ± ± ±
100.0 94.4 99.8 101.3 90.6 95.4 94.9
1.37a 1.48a 1.42a 1.38a 1.47a 1.49a 1.49a
926.0 874.3 924.0 938.3 839.3 883.3 878.5
10.3a 9.9c 19.6a 8.7a 11.9c 17.2b 9.4b
Means within a column lacking a common superscript differ (P < 0.05). Values are means ± SEM of four replicate pens per treatment. DAS = 4,15-diacetoxiscirpenol.
a–c 1
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served among the seven experimental groups (data not shown). Several studies have shown the adverse effects of DAS on chicken performance. Chi and Mirocha (1978) reported a 25% decrease in growth in chicks fed 5 ppm dietary DAS for 3 wk, while Ademoyero and Hamilton (1991a) found a 20% decrease in growth in chicks fed 4 ppm DAS for 3 wk. In another study by Ademoyero and Hamilton (1991b), chicks fed 1, 2 or 4 ppm dietary DAS for 3 wk had a 3, 9, and 27% decrease in BW, respectively. In this later study, the effect of 1 ppm dietary DAS on BW was not significant. The results of the present study confirm the dose-dependent effect of dietary DAS on chicken performance. However, in the present study, 1 ppm dietary DAS (without the addition of Mycofix) caused a significant decrease in BW (11%) compared with a nonsignificant 3% decrease in the 3-wk BW reported by Ademoyero and Hamilton (1991b). Further, in the present trial, 2 ppm DAS (without the addition of Mycofix) caused a 15% decrease in the 3-wk BW compared with a 9% decrease reported by Ademoyero and Hamilton (1991b). A possible explanation for these differences may be the difference in feed consumption between the 1991 strains of broiler chickens and current strains. Current broiler chicken strains grow faster, eat more feed, and therefore consume more toxin during the same time-period than older strains. The present study also shows that the adverse effect of DAS is not only dose-dependent but also timedependent. A significant reduction in BW (11%) was ob-
EFFICACY OF A FEED ADDITIVE AGAINST DAS
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supplementation of 0.75 or 1.5 g/kg Mycofix. However, when 2 ppm DAS is present in the diet, only partial protection is afforded by up to 1.5 g/kg Mycofix supplementation. More studies are needed to determine if a higher dose of Mycofix can completely overcome the adverse effects of 2 ppm DAS.
ACKNOWLEDGMENTS Thanks are due to Hernan Morales for his help with the production of purified DAS. The expert technical assistance of Norma S. Perilla is also gratefully acknowledged.
REFERENCES
exposed to dietary DAS. More likely, the systemically absorbed mycotoxin is responsible for the reduced performance. In chicks receiving 1 ppm DAS along with Mycofix, the trichothecene was presumably inactivated in the gastrointestinal tract thanks to the enzymatic action of the feed additive (Binder et al., 2000). This postulated lack of systemic absorption of the intact toxin from the gastrointestinal tract may have been the reason for the lack of detrimental effects on performance seen in chicks receiving 1 ppm DAS supplemented with Mycofix. The partial protection afforded by Mycofix when 2 ppm dietary DAS was used suggests that a higher level than 1.5 g/kg of inclusion is needed to counteract the effect of 2 ppm DAS. However, further studies are required to test this hypothesis. A condition known as “black tongues” in poultry is a problem recently reported by field veterinarians working in Mexico and other Latin American countries (GonzalezEscobar, 2001). The etiology of the problem is still unknown, but it has been postulated that type-A trichothecene mycotoxins such as T-2 toxin or DAS may be involved (Gonzalez-Escobar, 2001). The observation of “black tongue” lesions in chicks receiving dietary DAS in the present study indicates that these lesions can be produced by dietary exposure to DAS. Type-A trichothecenes need to be considered among the differential diagnosis of “black tongues,” and it might be useful for field veterinarians to submit feed samples from flocks exhibiting “black tongue” lesions for type-A trichothecene analysis. In summary, the results of the present study confirm previous studies showing that dietary DAS can adversely affect chicken performance. The present trial also shows that levels as low as 1 ppm can significantly decrease feed intake and BW in broilers, an effect that had not been reported. The adverse effects of 1 ppm dietary DAS on feed intake and BW can be counteracted by dietary
Ademoyero, A. A., and P. B. Hamilton. 1991a. High dietary fat increases the toxicity of diacetoxyscirpenol in chickens. Poult. Sci. 70:2271–2274. Ademoyero, A. A., and P. B. Hamilton. 1991b. Mouth lesions in broiler chickens caused by scirpenol mycotoxins. Poult. Sci. 70:2082–2089. Bata, A., S. Vanyi, and R. Lasztity. 1983. Simultaneous detection of some fusariotoxins by gas-liquid chromatography. J. Assoc. Off. Anal. Chem. 66:577–581. Bauer, J., M. Gareis, and B. Gedek. 1989. Metabolism of the trichothecenes T-2 toxin, diacetoxyscirpenol, and deoxynivalenol by farm animals. Pages 139–165 in Fusarium: Mycotoxins, Taxonomy, and Pathogenicity. J. Chelkowski, ed. Elsevier, Amsterdam. Binder, E. M., D. Heidler, G. Schatzmayr, N. Thimm, E. Fuchs, R. Krska, and J. Binder. 2000. Microbial detoxification of mycotoxins in animal feed in Proceedings of the 10th International IUPAC Symposium on Mycotoxins and Phycotoxins, W. J. de Koe, The Netherlands. Chi, M. S., and C. J. Mirocha. 1978. Necrotic oral lesions in chickens fed diacetoxiscirpenol, T-2 toxin, and crotocin. Poult. Sci. 57:807–808. Diaz, G. J., E. J. Squires, R. J. Julian, and H. J. Boermans. 1994. Individual and combined effects of T-2 toxin and DAS in laying hens. Br. Poult. Sci. 35:393–405. Ehrlich, K. C., and K. W. Daigle. 1987. Protein synthesis inhibition by 8-oxo-12,13-epoxytrichothecenes. Biochim. Biophys. Acta 923:206–213. Gonzalez-Escobar, E. 2001. Lenguas negras: clasificacio´n para hablar el mismo idioma. Tecnologı´a Avipecuaria en Latinoame´rica 13:54–56. Hoerr, F. J., W. W. Carlton, and B. Yagen. 1981. Mycotoxicosis caused by a single dose of T-2 toxin or diacetoxyscirpenol in broiler chickens. Vet. Pathol. 18:652–664. Leeson, S., G. J. Diaz, and J. D. Summers. 1995. Trichothecenes. Pages 190–226 in Poultry Metabolic Disorders and Mycotoxins. University Books, Guelph. Parkhurst, C. R., P. B. Hamilton, and A. A. Ademoyero. 1992. Abnormal feathering of chicks caused by scirpenol mycotoxins differing in degree of acetylation. Poult. Sci. 71:833–837. Richardson, K. E., and P. B. Hamilton. 1987. Preparation of 4,15diacetoxyscirpenol from cultures of Fusarium sambucinum NRRL 13495. Appl. Environ. Microbiol. 53:460–462. SAS Institute. 1987. SAS/STAT Guide for Personal Computers. Version 6.02 Edition. SAS Institute Inc., Cary, NC. Stratton, G. W., A. R. Robinson, H. C. Smith, L. Kittilsen, and M. Barbour. 1993. Levels of five mycotoxins in grains harvested in Atlantic Canada as measured by high-performance liquid chromatography. Arch. Environ. Contam. Toxicol. 24:399–409.
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FIGURE 1. Chick fed 2 ppm dietary 4,15-diacetoxiscirpenol showing a dark discoloration located at the tip of the tongue (“black tongue”) and a typical type-A trichothecene lesion at the base of the tongue.