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Efficacy of sodium bentonite as a detoxifier of broiler feed contaminated with aflatoxin and fumonisin
ENVIRONMENT, WELL-BEING, AND BEHAVIOR Efficacy of Sodium Bentonite as a Detoxifier of Broiler Feed Contaminated with Aflatoxin and Fumonisin R. Miazzo,* M. F. Peralta,* C. Magnoli,† M. Salvano,‡ S. Ferrero,§ S. M. Chiacchiera,|| E. C. Q. Carvalho,# C. A. R. Rosa,** and A. Dalcero†,1 *Departamento de Produccio´n Animal, Facultad de Agronomı´a y Veterinaria, †Departamento de Microbiologı´a e Inmunologı´a, ‡Departamento de Biologı´a Molecular, §Departamento de Matema´ticas, and 储Departamento de Quı´mica, Facultad de Ciencias Exactas Fı´sico-Quı´micas y Naturales, Universidad Nacional de Rı´o Cuarto, (5800) Rı´o Cuarto, Co´rdoba, Argentina; #Departamento de Patologı´a, Facultade de Veterinaria, Universidade Federal Fluminense, Brazil; and **Departamento de Microbiologı`a e Imunologı´a Instituto de Veterinaria Universidade Federal Rural do Rı´o de Janeiro, Brazil the relative weights of liver, kidney, and spleen. Addition of SB to diets containing AFB1 and FB1 only decreased liver weights. In relation to the control, lower serum levels of total protein, albumin, and globulins were observed for all AFB1 containing diets without SB addition, whereas all other treatments were not altered. Livers of birds fed diets containing AFB1 and a combination of AFB1 and FB1 were enlarged, yellowish, friable, and had rounded borders. The histopathology of them, stained with hematoxylin and eosin, showed multifocal and varied cytoplasmatic vacuolization with perilobular location. Incorporation of SB reduced the incidence and severity of the hepatic histopathology changes associated with aflatoxicosis.
(Key words: aflatoxin, fumonisin, broiler, bentonite, detoxification) 2005 Poultry Science 84:1–8
mal chronic diseases. One of the myriad effects of mycotoxins is the ability to impair the immune system in fowl. Aflatoxin B1 (AFB1) is the best-known mycotoxin for its ability to affect the primary immune response (Thaxton et al., 1974). Aflatoxins are secondary metabolites produced by species of Aspergillus and are natural contaminants of animal feeds (Edds and Bortell, 1983; Dalcero et al., 1997, 1998; Magnoli et al., 1998). They are potent liver carcinogens in laboratory animals (Wogan, 1973, 1992; Wogan et al., 1974). AFB1 has been implicated as one of the etiological factors, apart from hepatitis B virus, in the development of human liver cancer (van Rensburg et al., 1985; Peers et al., 1987). Fumonisins are produced by Fusarium spp. (Gelderblom et al., 1988) that contaminate corn worldwide (Rheeder et al., 1992; Chulze et al., 1996). Long-term studies have indicated that fumonisin B1 (FB1) is hepatocarcinogenic in rats, whereas a recent study reported its nephro-
INTRODUCTION The adverse effects of naturally occurring mutagens and carcinogens and their possible effects on human and animal health are observed by investigating the biological effects of purified compounds in experimental animals. In contrast very little is known about the interaction of complex mixtures in biological systems. A single compound may affect different reactions within one biological system, whereas it may also exhibit additive, antagonistic, or synergistic interactions with other compounds (Bata et al., 1996). The occurrence of mycotoxins together with various endogenous and exogenous risk factors, such as nutrition or virus infections, may modulate metabolism. Mycotoxins act synergistically to influence the risk of several ani-
2005 Poultry Science Association, Inc. Received for publication April 15, 2004. Accepted for publication September 9, 2004. 1 To whom correspondence should be addressed: adalcero@exa. unrc.edu.ar.
Abbreviation Key: AFB1=aflatoxin B1; ALB = albumin; FB1 = fumonisin B1; GLOB = globulin; SB = sodium bentonite.
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ABSTRACT Sodium bentonite (SB) was evaluated for its ability to reduce the deleterious effects of fumonisin B1 (FB1) and aflatoxin B1 (AFB1) in broiler diets. It was incorporated into the diets (0.3%) containing 2.5 mg/kg AFB1, 200 mg/kg FB1, or a combination of 2.5 mg/kg AFB1 and 200 mg/kg FB1. Aflatoxin B1 significantly diminished body weight gain, whereas FB1 or the combination of FB1 and SB had no effect. Addition of SB in the diets significantly diminished the inhibitory effects of dietary AFB1. Feeding AFB1 alone caused significant increases in the relative weights of most observed organs. Feeding FB1 alone did not alter relative weights of any organs. In the combined diet (AFB1 plus FB1) relative weights of the liver, kidney, gizzard, and spleen were increased. Addition of SB to the diet containing AFB1 diminished
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MIAZZO ET AL. TABLE 1. Composition (g/kg diet) and proximate analyses of the experimental diets Diet Grower
Finisher
Yellow corn Soybean oil meal Soybeans, heat treated Meat and bone meal Vitamin and mineral mix1 NaCl Oystershell Sunflower oil DL-Methionine L-Lysine Monensin Total Proximate composition (g/kg diet) of the test diets Crude protein Crude fat Crude fiber Calcium Total phosphorus Lysine Methionine Tryptophan ME, kcal.kg
629.0 226.9 55.0 69.0 1.50 2.00 3.50 10.0 1.6 1.0 0.5 1,000.0
672.0 190.0 50.0 70.0 1.50 2.00 3.00 10.0 1.0 — 0.5 1,000.0
203.3 54.7 33.4 9.7 5.9 11.4 5.0 2.4 3,047.0
189.0 55.3 30.8 9.5 5.7 9.3 4.2 2.2 3,062.0
1 Vitamin and mineral mix provided (mg/kg of feed): vitamin A, 8,325 IU; vitamin D3, 2,075 IU; vitamin K3, 1.25 mg; vitamin B1, 1 mg; vitamin B2, 2.5 mg; vitamin B6, 2.5 mg; vitamin B12, 0.0125 mg; folic acid, 0.25 mg; nicotinic acid, 25 mg; calcium pantothenate, 10 mg; biotin, 0.01 mg; choline chloride, 240 mg; manganese, 87.5 mg; iron, 60 mg; copper, 7.5 mg; zinc, 68.75 mg; I, 1.0 mg; Se, 0.2 mg; and butylated hydroxytoluene, 0.312 mg.
carcinogenicity and cancer-promoting activity in rats and in the livers of mice (Gelderblom et al., 1988, 1991, 2001). Several studies have reported the co-occurrence of AFB1, FB1, and other mycotoxins in feedstuffs (Huff et al., 1992; Dalcero et al., 1997, 1998; Ueno et al., 1997; Magnoli et al., 1999). At present, little is known about the interaction between AFB1 and FB1 with regard to their toxic properties. Numerous strategies for detoxification or inactivation of mycotoxin in contaminated feedstuffs have been used. Many techniques are impractical or ineffective (Piva et al., 1995; Ramos and Herna´ndez, 1997). One approach to the detoxification of mycotoxin is use of nonnutritive absorptive materials in the diet to reduce the absorption of mycotoxins from the gastrointestinal tract. In vitro and in vivo studies have indicated that natural sodium bentonite (SB) from southern Argentina has a strong ability to absorb AFB1 from aqueous solution (Miazzo et al., 2000). However, previous in vitro studies carried out with aluminosilicate, a synthetic NaA zeolite, showed that FB1 can significantly reduce the efficacy of the sorbent to sequester AFB1 (Kikot et al., 2002). Therefore, the need of
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Sigma Chemical Co., St. Louis, MO. Labconco Corp., Kansas City, MO. 4 Castiglioni yacimientos, Pes y Cı´a S. A. F. I. A. M. I, Bs, As, Argentina. 5 From the FusariumResearch Center, Pennsylvania State University, University Park, PA. 3
MATERIALS AND METHODS Chemicals The purities of purchased AFB1 and FB1 standards2 were assayed by HPLC. Their purities were confirmed as being greater than 99%. Demineralized water (HPLC grade) was obtained with Labconco equipment (model 90901-01).3 Commercial SB del Lago4 (SB) was obtained from a mine in Cinco Saltos, Province of Rı´o Negro, Argentina. The chemical composition of the SB was 54.91% SiO2, 21.41% Al2O3, traces of Fe2O3, 0.01% MnO, 0.1% TiO2, traces of CaO, 2.81% MgO, 1.70% Na2O, 0.16% K2O, traces of SO3, 0.05% P2O5, and 5.59% H2O. The mean particle size was 53 µm (99.5%). A 6% SB aqueous solution had pH of 8.5, and the swelling capacity after 24 h was 16 cm3/g. The batch of SB was previously assayed in vitro and in vivo for its ability to adsorb AFB1 (Rosa et al., 2001).
AFB1 and FB1 Production Aflatoxins were produced via fermentation of milled corn by Aspergillus parasiticus NRRL 3000. The sterile substrate, placed in Erlenmeyer flasks, was inoculated with 2 mL of the mold aqueous suspension containing 106 spores/mL. Cultures were allowed to grow for 7 d at 25°C in darkness. On the d 7 Erlenmeyer flasks were autoclaved, and culture material was dried at 40°C in a forced-air oven for 48 h. The AFB1 content in the milled corn was 40.9, 4.99, 2.99, and 0.98 mg/kg of AFB1, AFG1, AFB2, and AFG2, respectively. The milled corn was incorporated into the basal diet to provide 2.5 mg of AFB1/ kg of diet. Fumonisins were produced via fermentation of milled corn by Fusarium verticillioides M 70755 following the procedure described by Ledoux et al. (1995). Ground culture material containing 4,700 mg of FB1, 930 mg of FB2, and 430 mg of FB3/kg of corn was incorporated to the basal diet to provide the desired level of FB1 (200 mg/kg of diet).
Mycotoxin Analyses Aflatoxin and fumonisin analyses were performed by HPLC, following the methodology proposed by Trucksess et al. (1994) and Shephard et al. (1990), respectively. Zearalenone and deoxynivalenol were analyzed by thinlayer chromatography according to AOAC (1995).
Experimental Design and Chicks Ross male broiler chicks obtained from a commercial hatchery were kept in a poultry house during the experi-
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Ingredient
carrying out an interaction study was observed. In the present study, the in vivo ability of SB to prevent gastrointestinal absorption of AFB1, FB1, or a combination of AFB1 and FB1 was assayed.
AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE
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ment. Birds were maintained with food and water ad libitum for 22 d under standard management conditions. Treatments started on d 23. A grower diet was given from d 1 to 32, and a finisher diet was provided from d 33 until the end of the experiment. Diets were formulated to meet the levels of critical nutrients recommended by the NRC (1994). The composition and proximate analyses of both experimental diets are presented in Table 1. The starting mean BW at d 23 was 748 ± 28 g. During the experimental period, the control diet was screened for aflatoxins, zearalenone, deoxynivalenol, and fumonisins. Assayed levels of naturally occurring AFB1 and FB1 ranged from 14 to 25 ppb and 50 to 134 ppb, respectively. The rest of the toxins were below the detection limits of previously described techniques (Dalcero et al., 1997; Magnoli et al., 2002). Downloaded from http://ps.oxfordjournals.org/ at Carleton University on November 28, 2014
Housing The poultry house was divided into 40 pens (1 × 1 m; height: 100°cm) built side by side along a corridor where the pens were accessible. The pens were separated by plywood walls (height: 190°cm). The walls were made of wire mesh and reinforced with plywood up to 0.6 m, which allow auditory contact but no visual contact among the groups. Each pen was littered with pine wood shavings and equipped with a suspended bell-drinker and a feeder. The birds were maintained on a 12L:12D schedule with free access to water and diets. Average daily temperature ranged from 15 to 20°C. A total of 160 chicks was randomly divided into 8 treatments with 4 replicate groups per treatment and 5 chicks per replicate group. From d 23 to 50 of age, birds were kept under ideal conditions and fed with the following experimental diets: 1) control feedstuffs without addition of SB, AFB1, or FB1; 2) 0.3% SB; 3) 2.5 mg of AFB1/ kg of feed; 4) 2.5 mg of AFB1/kg of feed and 0.3% SB; 5) 200 mg of FB1/kg of feed; 6) 200 mg of FB1/kg of feed and 0.3% SB; 7) 2.5 mg of AFB1/kg and 200 mg of FB1/ kg of feed; 8) 2.5 mg of AFB1/kg, 200 mg of FB1/kg of feed and 0.3% SB. Chickens were monitored daily for signs of morbidity and mortality. The effect of SB on mycotoxicoses was determined by measuring BW gain; feed:gain ratio; total protein, albumin (ALB), and globulin (GLOB) concentrations in serum of broilers in each group. The aforementioned serum parameter concentrations were determined with a clinical chemistry analyzer6 according to the manufacturer’s recommended procedure. The serum biochemical values of individual birds within a replicate group were pooled and expressed as mean ± standard errors. The birds were killed by cervical dislocation and were necropsied and examined for gross lesions in liver, heart, kidney, gizzard, and spleen.
6 Metrolab 2100, Express Healthcare Management (expresshealthcare mgmt.com).
FIGURE 1. Effects of sodium bentonite (SB) on biochemical indicators of broiler chick feed diets containing 2.5 mg of aflatoxin B1 (AFB1) and 200 mg of fumonisin B1 (FB1) per kilogram of feed, singly or in combination. (A) albumin (ALB), (B) globulin (GLOB), and (C) total protein. Treatments: ◆ aControl, 〫 0.3% SB,a ▲ 2.5 mg of AFB1/kg of feed;b 䊉 2.5 mg of AFB1/kg of feed plus 0.3% SB;a 䊊 200 mg of FB1/ kg of feed;a 䉭 200 mg of FB1/kg of feed plus 0.3% SB;a 䊏 2.5 mg of AFB1/kg plus 200 mg of FB1/kg of feed;b 䊐 2.5 mg of AFB1/kg plus 200 mg of FB1/kg of feed plus 0.3% SB. a,bValues without a common superscript differ (P < 0.05) according to the Tukey test.
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MIAZZO ET AL. TABLE 2. Effects of aflatoxin B1 (AFB1), fumonisin B1 (FB1), sodium bentonite (SB), and their combination on BW gains and feed conversions of broiler chicks fed diets BW gain1
Treatment SB (%)
AFB1 (mg/kg)
FB1 (mg/kg)
0 0.3 0 0.3 0 0.3 0 0.3
0 0 2.5 2.5 0 0 2.5 2.5
0 0 0 0 200 200 200 200
23 to 50 d old (g) ± ± ± ± ± ± ± ±
1,865 1,895 1,463 1,805 1,812 1,855 1,338 1,505
Feed:gain Ratio2 (kg:kg)
Change from control (%)
29a 26a 23b 21a 23a 29a 29b 21b
0 +1.6 −21.6 −3.2 −2.8 −0.5 −28.3 −19.3
2.30 2.33 2.31 2.25 2.32 2.27 2.94 2.87
± ± ± ± ± ± ± ±
0.05a 0.08a 0.04a 0.06a 0.05a 0.05a 0.03b 0.01b
a,b Means values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. 1 Results are reported as mean ± SD for 20 birds. 2 Results are reported as mean ± SD for 4 lots.
At the termination of the study, livers were excised, weighed, and fixed in 10% neutral buffered formalin. Fixed tissues were trimmed, embedded in paraffin, and stained with hematoxylin and eosin stain for histopathological examination. Liver sections of all birds were microscopically examined.
Statistical Analysis A completely randomized experimental design was used, and the GLM procedure of SAS software (SAS Institute, 1995) was used to evaluate the data. Tukey’s significant difference test was applied in the event of significant global effects. Although birds were reared in groups, the average BW gain of each treatment was computed on individual basis with the aim of a better statistical evaluation of the parameter.
RESULTS The effects of AFB1 and FB1 singly and in combination on BW gains and feed conversions are shown in Table 2. Mortality did not occur. When compared with the control, the results showed that AFB1 intake diminished to about 22% BW. Addition of 200 mg/kg FB1 singly or FB1 plus
SB did not significantly alter BW. Addition of 0.3% SB to diets significantly diminished the inhibitory effects of feeding 2.5 mg of AFB1/kg of diet. The feed conversion (kg of feed/kg of gain) increased when AFB1 was combined with FB1 with or without SB. Data presented in Table 3 show the effects of dietary treatment on relative organ weights. Feeding AFB1 alone caused significant increases in the relative weights of most of observed organs (liver, kidney, gizzard, and spleen). None of the treatments induced changes in heart weights. Feeding FB1 alone did not alter relative weights of any organs. In the combined diet (AFB1 plus FB1) the relative weights of liver, kidney, gizzard, and spleen were increased. Addition of 0.3% SB to the diet containing AFB1 alone diminished the toxic effect of AFB1 on relative weights of some organs (liver, kidney, and spleen). However, SB in diets with AFB1 plus FB1 only decreased the weights of livers. The changes in the biochemical parameters for different treatments are shown in Figure 1. Compared with the control, alterations in the serum levels of the total protein, ALB, and GLOB were observed for all diets containing AFB1 without SB addition. The effect became more remarkable at d 50 and coincided with longer exposure to AFB1. Other treatments were not altered. Figure 2 shows the macroscopic appearances of livers from chicks that had been fed with the control diet, SB,
TABLE 3. Effect of a sodium bentonite (SB) on relative organ weights of broiler chicks fed diets containing aflatoxin B1 (AFB1) and fumonisin B1 (FB1) alone or in combination1 SB (%)
AFB1 (mg/kg)
FB1 (mg/kg)
0 0.3 0 0.3 0 0.3 0 0.3
0 0 2.5 2.5 0 0 2.5 2.5
0 0 0 0 200 200 200 200
Liver (g/100 g of BW) 2.89 2.90 3.43 2.89 2.86 2.85 3.84 2.90
± ± ± ± ± ± ± ±
0.05a 0.03a 0.04b 0.05a 0.02a 0.03a 0.02b 0.04a
Kidney (g/100 g of BW) 0.41 0.43 0.68 0.40 0.42 0.41 0.72 0.71
± ± ± ± ± ± ± ±
0.03a 0.03a 0.02b 0.01a 0.03a 0.02a 0.03b 0.02b
Heart (g/100 g of BW) 0.63 0.65 0.64 0.61 0.62 0.62 0.64 0.67
± ± ± ± ± ± ± ±
0.02a 0.02a 0.04a 0.03a 0.02a 0.03a 0.02a 0.01a
Gizzard (g/100 g of BW) 2.33 2.30 2.73 2.72 2.31 2.30 2.64 2.96
± ± ± ± ± ± ± ±
Spleen (g/100 g of BW)
0.05a 0.03a 0.04b 0.05b 0.02a 0.03a 0.02b 0.04b
Means values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. Values represent means of 8 groups of 5 chicks each per treatment.
a,b 1
0.06 0.07 0.13 0.08 0.06 0.05 0.15 0.13
± ± ± ± ± ± ± ±
0.03a 0.02a 0.03b 0.02a 0.01a 0.03a 0.02b 0.03b
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Histopathology
AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE
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AFB1, and a combination of AFB1 and FB1 diets. Livers from the last 2 treatments were enlarged, yellowish, friable, and with rounded borders. Livers from other treatments were not included because they had normal appearances. When compared with the control (Figure 3A), histopathologies of livers from birds fed with AFB1 alone or AFB1 plus FB1 showed multifocal and varied cytoplasmatic vacuolization with perilobular location when stained with hematoxylin and eosin (Figure 3B and C, respectively). Liver samples of broilers consuming the FB1 diet or other SB-supplemented diets (SB, AFB1 + SB, FB1 + SB, or AFB1 + FB1 + SB) all revealed discrete and diffuse cellular swelling as depicted in Figure 3D.
DISCUSSION Aflatoxin and fumonisin produce severe economic losses and health problems in the poultry industry because of their toxicity and frequency of occurrence in feedstuffs (Kubena et al., 1993). The current study demonstrated the toxicity of AFB1 combined with FB1 in growing broiler chicks and a reduction in the ability of SB to decrease the toxicity of AFB1 in the presence of FB1. In vitro studies have shown that SB is strong binder of AFB1
(Veldman et al., 1992; Rosa et al., 2001). An inert, stable, and insoluble complex between SB and AFB1 was assumed to be the responsible for preventing toxin absorption in the intestine. Body weight reduction, caused by intake of feed contaminated with AFB1, has already been reported in previous studies (Rosa et al., 2001). Sodium bentonite was inert and nontoxic. Olver (1997) and Parlat et al. (1999) reported similar results for clinoptilolite, a natural zeolite. The present results agreed with our earlier findings about the ability of SB to ameliorate the adverse effects of AFB1 in growing broilers (Rosa et al., 2001). However, addition of SB did not have the same effect on birds fed a combined diet (AFB1 plus FB1). Previous in vitro studies involving NaA zeolite as an adsorbent have shown that the presence of FB1 could significantly decrease the ability of sorbent to capture AFB1 (Kikot et al., 2002). At pH 2, a competitive adsorption between both toxins was observed, but FB1 was released from the surface when the pH was increased to 6, value at which AFB1 is not able to be absorbed. Kubena et al. (1990a,b) reported additive toxicity in the majority of the mycotoxins when acting in combination. These authors reported that a decrease in serum total protein and ALB in broilers, as a result of aflatoxicosis, was not alleviated by hydrated sodium calcium aluminosilicate.
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FIGURE 2. Representative livers from broiler chickens fed with diets: A) control, B) 0.3% sodium bentonite (SB), C) 2.5 mg of aflatoxin B1 (AFB1) and 200 mg of fumonisin (FB1)/kg of feed, and D) 2.5 mg of AFB1/kg of feed.
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MIAZZO ET AL.
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FIGURE 3. Photomicrographs of hematoxylin and eosin stained liver sections of chicks fed with the following diets: A) control, 0.3% sodium bentonite (SB), B) 2.5 mg of aflatoxin B1 (AFB1)/kg feed, C) 2.5 mg of AFB1 and 200 mg of fumonisin B1 (FB1)/kg of feed, and D) 0.3% SB. Bars equal 10 µm.
AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE
ACKNOWLEDGMENTS The authors are grateful to the SECyT (Secretarı´a de Ciencia y Te´cnica, Universidad Nacional de Rı´o Cuarto) Res. No. 129/01, 077/03, and CONICET (Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas) PIP Res. No. 962/01, which supported this study through grants. Carina Magnoli thanks CONICET for fellowship support. Ana Marı´a Dalcero and Stella Maris Chiacchiera held positions at CONICET. We thank Castiglioni, Pes y Cı´a SAFIAMI for providing the sodium bentonite.
REFERENCES AOAC. 1995. Sections 975.35, 976.22 in Official Methods of Analysis. Association of Official Analytical Chemists, Gaithersburg, MD. Bata, A., R. Glavits, A. Vanyi, and G. Salyi. 1996. More important mycotoxicoses of Poult. clı´nico-pathological. Review article. Magy. Allatorv. Lapja 51:395–408. Chulze, S., M. L. Ramirez, M. C. Farnochi, M. Pascale, A. Visconti, and G. March. 1996. Fusarium and fumonisins occurrence in Argentinian corn at different ear maturity stages. J. Agric. Food Chem. 44:2797–2801. Dalcero, A., C. Magnoli, S. M. Chiacchiera, G. Palacio, and M. M. Reynoso. 1997. Mycoflora and incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina. Mycopathologia 137:179–184. Dalcero, A., C. Magnoli, M. Luna, G. Ancasi, M. M. Reynoso, S. Chiacchiera, R Miazzo, and G. Palacio. 1998. Mycoflora and naturally ocurring mycotoxins in poultry feeds in Argentina. Mycopathologia 141:37–43. Edds, G. T., and R. A. Bortell. 1983. Biological effects of aflatoxinas: Poultry. Pages 56–61 in Aflatoxin and Aspergillus flavus in corn. U. L. Diener, R. L. Asquity, and J. W. Dickens, ed. Southern Cooperative Series Bulletin 279. Auburn University, Auburn, Alabama. Gelderblom, W. C., S. Abel, C. M. Smuts, J. Marnewick, W. F. Marasas, E. R. Lemmer, and D. Ramljak. 2001. Fumonisininduced hepatocarcinogenesis: Mechanisms related to cancer initiation and promotion. Environ. Health Perspect. 109:291–300. Gelderblom, W. C. A., K. Jaskiewicz, W. F. O. Marasas, P. G. Thiel, M. J. Horak, R. Vieggaar, and N. P. J. Kriek. 1988. Fumonisins—Novel mycotoxins with cancer promoting activity produced by Fusarium moniliforme. Appl. Environ. Microbiol. 54:1806–1811. Gelderblom, W. C. A., N. P. J. Kriek, W. F. O. .Marasas, and P. G. Thiel. 1991. Toxicity and carcinogenicity of the Fusarium moniliforme metabolite fumonisin B1 in rats. Carcinogenesis 12:1247–1251. Huff, W. E., L. F. Kubena, R. B. Harvey, and T. D. Phillips. 1992. Efficacy of hidrated sodium calcium aluminosilicate to reduce the individual and combined toxicity of aflatoxin and ochratoxin A. Poult. Sci. 71:64–69. Kececi, T., H. Oguz, V. Kurtoglu, and O. Demet. 1998. Effects of polyvynylpoly-pyrrolidone, synthetic zeolite and bentonite on serum biochemical and haematological characters of broiler chickens during aflatoxicosis. Br. Poult. Sci. 39:452– 458. Kikot, A., C. Magnoli, S. Chiacchiera, A. Dalcero, R. Miazzo, and E. I. Basaldella. 2002. Effect of fumonisin B1 on NaA zeolite effectiveness to adsorb aflatoxin B1. Metal Ions Biol. Med. 7:242–246. Kubena, L. F., R. B. Harvey, R. H. Baile, S. A. Buckley, and R. G. Rottinghaus. 1998. Effects of hydrateted sodium calcium aluminosilicate [T-Bind ] on mycotoxicosis in young broiler chickens. Poult. Sci. 77:1502–1509.
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Protective effects of the nonnutritive additive (SB) on gross hepatic changes produced by AFB1 or by a combination of AFB1 and FB1 were observed. A relative increase in liver weight was caused by AFB1 or by a combination of AFB1 and FB1. Addition of SB ameliorated the observed effects. Chronic mycotoxicoses may be diagnosed by determining serum biochemical alterations even before major clinical symptoms appear (Oguz et al., 2000). Reductions in concentrations of serum total protein and ALB, as indicators of protein synthesis, and GLOB were observed in chicks consuming diets containing AFB1 alone and combined with FB1 (Tung et al., 1975; Huff et al, 1992; Kubena et al., 1994). As expected, all of these values in the present results were sensibly affected by AFB1. The stronger effect at d 50 might have coincided with longer exposure to AFB1. Changes in concentrations of serum total protein, ALB, and GLOB were reversed by SB in chicks fed diets containing AFB1 alone or in combination with FB1. Increases in serum total protein, ALB, and GLOB observed at d 43 might have been due to dietary changes at d 32. Fumonisin B1 did not affect any of these parameters. Taking into account that the biochemical parameters for mycotoxicoses seemed to depend on bird age and nutritional status at the time of exposure to contaminated feed, we suggest that further in vivo experiments with this adsorbent should be conducted. Our results agreed with those reported by Kubena et al. (1998), who found significant decreases in these biochemical parameters at exposure levels of AFB1 ranging from 2.5 to 5 ppm. Kececi et al. (1998) and Oguz et al. (2000) showed that some serum biochemical changes could be ameliorated by 0.5% SB administration to the diet of broiler chickens fed with 2.5 mg of AFB1/kg of diet. However, Oguz et al. (2002) reported that the use of 50 and 100 ppb of AFB1 did not cause any significant change in the total protein and ALB, probably due to the low mycotoxin dose in the assay. In our previous studies the biochemical parameters for broilers fed a diet containing 0.3% SB and 5 mg of AFB1/ kg of diet did not completely return to normal values, showing an inhibition of protein synthesis (Rosa et al., 2001). In the present report, a complete return to normal biochemical parameters was observed, which could have been due to the lower AFB1 exposure dose compared with the former. It should be stressed that this research showed that SB was effective to counteract only some of the AFB1promoted effects. In fact, addition of SB to a diet containing a combination of AFB1 and FB1 was not able to return BW gain to equal that of the control. This result could be attributed to a competition between FB1 and AFB1 for the active surface sites of SB, rendering a greater bioavailability of AFB1 in the presence of high dose of FB1. The current data indicate toxicity for chicks of AFB1 alone or in combination with FB1. Neither additive nor synergistic toxic interaction between AFB1 and FB1 seems to be operating as shown with most of the parameters.
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MIAZZO ET AL. Peers, F., X. Bosch, J. Kaldor, A. Linsell, and M. Pluijmen. 1987. Aflatoxin exposure, hepatitis B virus infection and liver cancer in Swaziland. Int. J. Cancer 39:545-553. Piva, G., F. P. F. Galvano, A. Pietri, and A. Piva. 1995. Detoxification methods of aflatoxins. A Review. Nutr. Res.15:767–776. Ramos, A. J., and E. Herna´ndez. 1997. Prevention of aflatoxicosis in farm animals by means hydrated sodium calcium aluminosilication addition to feedstuffs. A review. Anim. Feed Sci. Technol. 65:197–206. Rheeder, J. P., W. F. O. Marasas, P. G. Thiel, E. W. Sydenham, G. S. Shephard, and D. J. van Schalwyk. 1992. Fusarium moniliforme and fumonisins in corn in relation to human esophageal cancer in Tanskei. Phytopathology 82:353–357. Rosa, C. A. R., R. Miazzo, C. Magnoli, M. Salvano, S. M. Chiacchiera, S. Ferrero, M. Saenz, C. Q. Carvalho, and A. Dalcero. 2001. Evaluation of the efficacy of bentonite from the south of Argentina to ameliorate the toxic effects of aflatoxin in broilers. Poult. Sci. 80:139–144. SAS Institute. 1995. SAS User’s Guide: Statistics. Edition. SAS Institute Inc., Cary, NC. Shephard, G. S., E. W. Sydenham, P. G. Thiel, W. A. C. Gelderblom. 1990. Quantitative determination of fumonisins B1 and B2 by high-performance liquid chromatography with fluorescence detection. J. Liquid Chromatogr. 3:2077–2080. Thaxton, J. P., H. T. Tung, and P. B. Hamilton. 1974. Immunosuppression in chickens by aflatoxin. Poult. Sci. 53:721–725. Trucksess, M. W., M. E. Stack, S. Nesheim, R. Albert, and T. Romer. 1994. Multifunctional column coupled with liquid chromatography for determination of aflatoxins B1, B2, G1 and G2 in corn, almonds, Brazil nuts, peanuts, and pistachio nuts: Collaborative study. J. AOAC Int. 77:1512–1521. Tung, H. T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton. 1975. Concentrations of serum proteins during aflatoxicosis. Toxicol. Appl. Pharmacol. 34:320–326. Ueno, Y., K. Lijima, S. D Wang, Y. Sugiura, M. Sekijima, T. Tanaka, C. Chen, and S. Z. Yu. 1997. Fumonisins as a possible contributory risk factor for primary liver cancer: A 3-year study of corn harvested in Haimen, China by HPLC and ELISA. Food Chem. Toxicol. 35:1143–1150. van Rensburg, S. J., P. Cook-Mozaffari , D. J. van Schalkwyk, J. J. van der Watt, T. J. Vincent, and I. F. Purchase. 1985. Hepatocellular carcinoma and dietary aflatoxin in Mozambique and Transkei. Br. J. Cancer 51:713–726. Veldman, A., G. J. Borggreve, E. J. Mulders, D. van der Lagemaat. 1992. Occurrence of the mycotoxins ochratoxin A, zearalenone and deoxynivalenol in feed components. Food Addit. Contam. 6:647–655. Wogan, G. N. 1973. Aflatoxin carcino´genesis. Pages 309–344 in Methods in Cancer Research. Academic Press, New York. Wogan, G. N, S. Paglialunga, and P. M. Newberne. 1974. Carcinogenic effects of low dietary levels of aflatoxin B1 in rats. Food Cosmet. Toxicol. 12:681–685. Wogan, G. N. 1992. Aflatoxins as risk factors for hepatocellular carcinoma in humans. Cancer Res. 52:2114s–2118s.
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Kubena, L. F., R. B. Harvey, W. Huff, D. E. Corrier, T. D. Phillips, and R. G. Rottinghaus. 1990a. Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and T-2 toxin. Poult. Sci. 69:1078–1086. Kubena, L. F., R. B. Harvey, W. Huff, M. H. Elissalde, A. G. Yersin, T. D. Phillips, and G. E. Rottinghaus. 1993. Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and diacetoxyscirpenol. Poult. Sci. 72:51–59. Kubena, L. F., R. B. Harvey, T. D. Phillips, D. E. Corrier, and W. Huff. 1990b. Diminution of aflatoxicosis in growing chickens by the dietary addition of a hydrated, sodium calcium aluminosilicate. Poult. Sci. 69:727–735. Kubena, L. F., E. E. Smith, A. Gentles, R. B. Harvey., T. S. Edrington, T. D. Phillips, and G. E. Rottinghaus. 1994. Individual and combined toxicity of T-2 toxin and cyclo-piazonic acid in broiler chicks. Poult. Sci. 73:1390–1397. Ledoux, D. R., A. J. Bermudez, G. E. Rottinghaus, J. Broomhead, and G. A. Bennett. 1995. Effects of feeding Fusarium fujikuroi culture material, containing known levels of moniliformin, in young broiler chicks. Poult. Sci. 74:297–305. Magnoli, C., S. M. Chiacchiera, R. Miazzo, G. Palacio, A. Angeletti, C. Hallak, and A. Dalcero. 2002. The mycoflora and toxicity of feedstuffs from a production plant in Co´rdoba, Argentina. Mycotoxin Res. 18:8–22. Magnoli, C., A. M Dalcero., S. M. Chiacchiera, R Miazzo, and M. A. Saenz. 1998. Enumeration and identification of Aspergillus group and Penicillium species in poultry feeds from Argentina. Mycopathologia 142: 27–32. Magnoli, C. E., M. A. Saenz, S. Ferrero, S. M. Chiacchiera, .and A. M. Dalcero. 1999. Natural occurrence of Fusarium species and fumonisins production by toxigenic strains isolated from poultry feeds in Argentina. Mycopathologia 145:35–41. Miazzo, R. D., C. A. R. Rosa, E. C. De Queiroz Carvalho, C. Magnoli, S. M. Chiacchiera, G. Palacio, M. Saenz, A. Kikot, E. Basaldela, and A. Dalcero. 2000. Efficacy of synthetic zeolite to reduce the toxicity of aflatoxin in broiler chicks. Poult. Sci. 79:1–6. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Oguz, Y., L. Doganci, F. Bulucu, C. Can, C. Oktenli, M. Yenicesu, and A. Vural. 2002. Acute pyelonephritis causing acute renal allograft dysfunction. Int. Urol. Nephrol. 34:299–301. Oguz, H., T. Kececi, Y. O. Birdane, F. Onder, and V. Kurtoglu. 2000. Effect of clinoptilolite on serum biochemical and haematological characters of broiler chickens during experimental aflatoxicosis. Res.Vet. Sci. 69:89–93. Olver, M. D. 1997. Effect of feeding clinoptilolite (zeolite) on the performance of three strains of laying hens. Br. Poult. Sci. 38:220–222. Parlat, S. S., A. O. H. Yildiz, and H. Oguz. 1999. Effect of clinoptilolite on fattening performance of Japanese quail (Coturinix coturnix japonica) during experimental aflatoxicosis. Br. Poult. Sci. 40:495–500.
(Key words: aflatoxin, fumonisin, broiler, bentonite, detoxification) 2005 Poultry Science 84:1–8
mal chronic diseases. One of the myriad effects of mycotoxins is the ability to impair the immune system in fowl. Aflatoxin B1 (AFB1) is the best-known mycotoxin for its ability to affect the primary immune response (Thaxton et al., 1974). Aflatoxins are secondary metabolites produced by species of Aspergillus and are natural contaminants of animal feeds (Edds and Bortell, 1983; Dalcero et al., 1997, 1998; Magnoli et al., 1998). They are potent liver carcinogens in laboratory animals (Wogan, 1973, 1992; Wogan et al., 1974). AFB1 has been implicated as one of the etiological factors, apart from hepatitis B virus, in the development of human liver cancer (van Rensburg et al., 1985; Peers et al., 1987). Fumonisins are produced by Fusarium spp. (Gelderblom et al., 1988) that contaminate corn worldwide (Rheeder et al., 1992; Chulze et al., 1996). Long-term studies have indicated that fumonisin B1 (FB1) is hepatocarcinogenic in rats, whereas a recent study reported its nephro-
INTRODUCTION The adverse effects of naturally occurring mutagens and carcinogens and their possible effects on human and animal health are observed by investigating the biological effects of purified compounds in experimental animals. In contrast very little is known about the interaction of complex mixtures in biological systems. A single compound may affect different reactions within one biological system, whereas it may also exhibit additive, antagonistic, or synergistic interactions with other compounds (Bata et al., 1996). The occurrence of mycotoxins together with various endogenous and exogenous risk factors, such as nutrition or virus infections, may modulate metabolism. Mycotoxins act synergistically to influence the risk of several ani-
2005 Poultry Science Association, Inc. Received for publication April 15, 2004. Accepted for publication September 9, 2004. 1 To whom correspondence should be addressed: adalcero@exa. unrc.edu.ar.
Abbreviation Key: AFB1=aflatoxin B1; ALB = albumin; FB1 = fumonisin B1; GLOB = globulin; SB = sodium bentonite.
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ABSTRACT Sodium bentonite (SB) was evaluated for its ability to reduce the deleterious effects of fumonisin B1 (FB1) and aflatoxin B1 (AFB1) in broiler diets. It was incorporated into the diets (0.3%) containing 2.5 mg/kg AFB1, 200 mg/kg FB1, or a combination of 2.5 mg/kg AFB1 and 200 mg/kg FB1. Aflatoxin B1 significantly diminished body weight gain, whereas FB1 or the combination of FB1 and SB had no effect. Addition of SB in the diets significantly diminished the inhibitory effects of dietary AFB1. Feeding AFB1 alone caused significant increases in the relative weights of most observed organs. Feeding FB1 alone did not alter relative weights of any organs. In the combined diet (AFB1 plus FB1) relative weights of the liver, kidney, gizzard, and spleen were increased. Addition of SB to the diet containing AFB1 diminished
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MIAZZO ET AL. TABLE 1. Composition (g/kg diet) and proximate analyses of the experimental diets Diet Grower
Finisher
Yellow corn Soybean oil meal Soybeans, heat treated Meat and bone meal Vitamin and mineral mix1 NaCl Oystershell Sunflower oil DL-Methionine L-Lysine Monensin Total Proximate composition (g/kg diet) of the test diets Crude protein Crude fat Crude fiber Calcium Total phosphorus Lysine Methionine Tryptophan ME, kcal.kg
629.0 226.9 55.0 69.0 1.50 2.00 3.50 10.0 1.6 1.0 0.5 1,000.0
672.0 190.0 50.0 70.0 1.50 2.00 3.00 10.0 1.0 — 0.5 1,000.0
203.3 54.7 33.4 9.7 5.9 11.4 5.0 2.4 3,047.0
189.0 55.3 30.8 9.5 5.7 9.3 4.2 2.2 3,062.0
1 Vitamin and mineral mix provided (mg/kg of feed): vitamin A, 8,325 IU; vitamin D3, 2,075 IU; vitamin K3, 1.25 mg; vitamin B1, 1 mg; vitamin B2, 2.5 mg; vitamin B6, 2.5 mg; vitamin B12, 0.0125 mg; folic acid, 0.25 mg; nicotinic acid, 25 mg; calcium pantothenate, 10 mg; biotin, 0.01 mg; choline chloride, 240 mg; manganese, 87.5 mg; iron, 60 mg; copper, 7.5 mg; zinc, 68.75 mg; I, 1.0 mg; Se, 0.2 mg; and butylated hydroxytoluene, 0.312 mg.
carcinogenicity and cancer-promoting activity in rats and in the livers of mice (Gelderblom et al., 1988, 1991, 2001). Several studies have reported the co-occurrence of AFB1, FB1, and other mycotoxins in feedstuffs (Huff et al., 1992; Dalcero et al., 1997, 1998; Ueno et al., 1997; Magnoli et al., 1999). At present, little is known about the interaction between AFB1 and FB1 with regard to their toxic properties. Numerous strategies for detoxification or inactivation of mycotoxin in contaminated feedstuffs have been used. Many techniques are impractical or ineffective (Piva et al., 1995; Ramos and Herna´ndez, 1997). One approach to the detoxification of mycotoxin is use of nonnutritive absorptive materials in the diet to reduce the absorption of mycotoxins from the gastrointestinal tract. In vitro and in vivo studies have indicated that natural sodium bentonite (SB) from southern Argentina has a strong ability to absorb AFB1 from aqueous solution (Miazzo et al., 2000). However, previous in vitro studies carried out with aluminosilicate, a synthetic NaA zeolite, showed that FB1 can significantly reduce the efficacy of the sorbent to sequester AFB1 (Kikot et al., 2002). Therefore, the need of
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Sigma Chemical Co., St. Louis, MO. Labconco Corp., Kansas City, MO. 4 Castiglioni yacimientos, Pes y Cı´a S. A. F. I. A. M. I, Bs, As, Argentina. 5 From the FusariumResearch Center, Pennsylvania State University, University Park, PA. 3
MATERIALS AND METHODS Chemicals The purities of purchased AFB1 and FB1 standards2 were assayed by HPLC. Their purities were confirmed as being greater than 99%. Demineralized water (HPLC grade) was obtained with Labconco equipment (model 90901-01).3 Commercial SB del Lago4 (SB) was obtained from a mine in Cinco Saltos, Province of Rı´o Negro, Argentina. The chemical composition of the SB was 54.91% SiO2, 21.41% Al2O3, traces of Fe2O3, 0.01% MnO, 0.1% TiO2, traces of CaO, 2.81% MgO, 1.70% Na2O, 0.16% K2O, traces of SO3, 0.05% P2O5, and 5.59% H2O. The mean particle size was 53 µm (99.5%). A 6% SB aqueous solution had pH of 8.5, and the swelling capacity after 24 h was 16 cm3/g. The batch of SB was previously assayed in vitro and in vivo for its ability to adsorb AFB1 (Rosa et al., 2001).
AFB1 and FB1 Production Aflatoxins were produced via fermentation of milled corn by Aspergillus parasiticus NRRL 3000. The sterile substrate, placed in Erlenmeyer flasks, was inoculated with 2 mL of the mold aqueous suspension containing 106 spores/mL. Cultures were allowed to grow for 7 d at 25°C in darkness. On the d 7 Erlenmeyer flasks were autoclaved, and culture material was dried at 40°C in a forced-air oven for 48 h. The AFB1 content in the milled corn was 40.9, 4.99, 2.99, and 0.98 mg/kg of AFB1, AFG1, AFB2, and AFG2, respectively. The milled corn was incorporated into the basal diet to provide 2.5 mg of AFB1/ kg of diet. Fumonisins were produced via fermentation of milled corn by Fusarium verticillioides M 70755 following the procedure described by Ledoux et al. (1995). Ground culture material containing 4,700 mg of FB1, 930 mg of FB2, and 430 mg of FB3/kg of corn was incorporated to the basal diet to provide the desired level of FB1 (200 mg/kg of diet).
Mycotoxin Analyses Aflatoxin and fumonisin analyses were performed by HPLC, following the methodology proposed by Trucksess et al. (1994) and Shephard et al. (1990), respectively. Zearalenone and deoxynivalenol were analyzed by thinlayer chromatography according to AOAC (1995).
Experimental Design and Chicks Ross male broiler chicks obtained from a commercial hatchery were kept in a poultry house during the experi-
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Ingredient
carrying out an interaction study was observed. In the present study, the in vivo ability of SB to prevent gastrointestinal absorption of AFB1, FB1, or a combination of AFB1 and FB1 was assayed.
AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE
3
ment. Birds were maintained with food and water ad libitum for 22 d under standard management conditions. Treatments started on d 23. A grower diet was given from d 1 to 32, and a finisher diet was provided from d 33 until the end of the experiment. Diets were formulated to meet the levels of critical nutrients recommended by the NRC (1994). The composition and proximate analyses of both experimental diets are presented in Table 1. The starting mean BW at d 23 was 748 ± 28 g. During the experimental period, the control diet was screened for aflatoxins, zearalenone, deoxynivalenol, and fumonisins. Assayed levels of naturally occurring AFB1 and FB1 ranged from 14 to 25 ppb and 50 to 134 ppb, respectively. The rest of the toxins were below the detection limits of previously described techniques (Dalcero et al., 1997; Magnoli et al., 2002). Downloaded from http://ps.oxfordjournals.org/ at Carleton University on November 28, 2014
Housing The poultry house was divided into 40 pens (1 × 1 m; height: 100°cm) built side by side along a corridor where the pens were accessible. The pens were separated by plywood walls (height: 190°cm). The walls were made of wire mesh and reinforced with plywood up to 0.6 m, which allow auditory contact but no visual contact among the groups. Each pen was littered with pine wood shavings and equipped with a suspended bell-drinker and a feeder. The birds were maintained on a 12L:12D schedule with free access to water and diets. Average daily temperature ranged from 15 to 20°C. A total of 160 chicks was randomly divided into 8 treatments with 4 replicate groups per treatment and 5 chicks per replicate group. From d 23 to 50 of age, birds were kept under ideal conditions and fed with the following experimental diets: 1) control feedstuffs without addition of SB, AFB1, or FB1; 2) 0.3% SB; 3) 2.5 mg of AFB1/ kg of feed; 4) 2.5 mg of AFB1/kg of feed and 0.3% SB; 5) 200 mg of FB1/kg of feed; 6) 200 mg of FB1/kg of feed and 0.3% SB; 7) 2.5 mg of AFB1/kg and 200 mg of FB1/ kg of feed; 8) 2.5 mg of AFB1/kg, 200 mg of FB1/kg of feed and 0.3% SB. Chickens were monitored daily for signs of morbidity and mortality. The effect of SB on mycotoxicoses was determined by measuring BW gain; feed:gain ratio; total protein, albumin (ALB), and globulin (GLOB) concentrations in serum of broilers in each group. The aforementioned serum parameter concentrations were determined with a clinical chemistry analyzer6 according to the manufacturer’s recommended procedure. The serum biochemical values of individual birds within a replicate group were pooled and expressed as mean ± standard errors. The birds were killed by cervical dislocation and were necropsied and examined for gross lesions in liver, heart, kidney, gizzard, and spleen.
6 Metrolab 2100, Express Healthcare Management (expresshealthcare mgmt.com).
FIGURE 1. Effects of sodium bentonite (SB) on biochemical indicators of broiler chick feed diets containing 2.5 mg of aflatoxin B1 (AFB1) and 200 mg of fumonisin B1 (FB1) per kilogram of feed, singly or in combination. (A) albumin (ALB), (B) globulin (GLOB), and (C) total protein. Treatments: ◆ aControl, 〫 0.3% SB,a ▲ 2.5 mg of AFB1/kg of feed;b 䊉 2.5 mg of AFB1/kg of feed plus 0.3% SB;a 䊊 200 mg of FB1/ kg of feed;a 䉭 200 mg of FB1/kg of feed plus 0.3% SB;a 䊏 2.5 mg of AFB1/kg plus 200 mg of FB1/kg of feed;b 䊐 2.5 mg of AFB1/kg plus 200 mg of FB1/kg of feed plus 0.3% SB. a,bValues without a common superscript differ (P < 0.05) according to the Tukey test.
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MIAZZO ET AL. TABLE 2. Effects of aflatoxin B1 (AFB1), fumonisin B1 (FB1), sodium bentonite (SB), and their combination on BW gains and feed conversions of broiler chicks fed diets BW gain1
Treatment SB (%)
AFB1 (mg/kg)
FB1 (mg/kg)
0 0.3 0 0.3 0 0.3 0 0.3
0 0 2.5 2.5 0 0 2.5 2.5
0 0 0 0 200 200 200 200
23 to 50 d old (g) ± ± ± ± ± ± ± ±
1,865 1,895 1,463 1,805 1,812 1,855 1,338 1,505
Feed:gain Ratio2 (kg:kg)
Change from control (%)
29a 26a 23b 21a 23a 29a 29b 21b
0 +1.6 −21.6 −3.2 −2.8 −0.5 −28.3 −19.3
2.30 2.33 2.31 2.25 2.32 2.27 2.94 2.87
± ± ± ± ± ± ± ±
0.05a 0.08a 0.04a 0.06a 0.05a 0.05a 0.03b 0.01b
a,b Means values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. 1 Results are reported as mean ± SD for 20 birds. 2 Results are reported as mean ± SD for 4 lots.
At the termination of the study, livers were excised, weighed, and fixed in 10% neutral buffered formalin. Fixed tissues were trimmed, embedded in paraffin, and stained with hematoxylin and eosin stain for histopathological examination. Liver sections of all birds were microscopically examined.
Statistical Analysis A completely randomized experimental design was used, and the GLM procedure of SAS software (SAS Institute, 1995) was used to evaluate the data. Tukey’s significant difference test was applied in the event of significant global effects. Although birds were reared in groups, the average BW gain of each treatment was computed on individual basis with the aim of a better statistical evaluation of the parameter.
RESULTS The effects of AFB1 and FB1 singly and in combination on BW gains and feed conversions are shown in Table 2. Mortality did not occur. When compared with the control, the results showed that AFB1 intake diminished to about 22% BW. Addition of 200 mg/kg FB1 singly or FB1 plus
SB did not significantly alter BW. Addition of 0.3% SB to diets significantly diminished the inhibitory effects of feeding 2.5 mg of AFB1/kg of diet. The feed conversion (kg of feed/kg of gain) increased when AFB1 was combined with FB1 with or without SB. Data presented in Table 3 show the effects of dietary treatment on relative organ weights. Feeding AFB1 alone caused significant increases in the relative weights of most of observed organs (liver, kidney, gizzard, and spleen). None of the treatments induced changes in heart weights. Feeding FB1 alone did not alter relative weights of any organs. In the combined diet (AFB1 plus FB1) the relative weights of liver, kidney, gizzard, and spleen were increased. Addition of 0.3% SB to the diet containing AFB1 alone diminished the toxic effect of AFB1 on relative weights of some organs (liver, kidney, and spleen). However, SB in diets with AFB1 plus FB1 only decreased the weights of livers. The changes in the biochemical parameters for different treatments are shown in Figure 1. Compared with the control, alterations in the serum levels of the total protein, ALB, and GLOB were observed for all diets containing AFB1 without SB addition. The effect became more remarkable at d 50 and coincided with longer exposure to AFB1. Other treatments were not altered. Figure 2 shows the macroscopic appearances of livers from chicks that had been fed with the control diet, SB,
TABLE 3. Effect of a sodium bentonite (SB) on relative organ weights of broiler chicks fed diets containing aflatoxin B1 (AFB1) and fumonisin B1 (FB1) alone or in combination1 SB (%)
AFB1 (mg/kg)
FB1 (mg/kg)
0 0.3 0 0.3 0 0.3 0 0.3
0 0 2.5 2.5 0 0 2.5 2.5
0 0 0 0 200 200 200 200
Liver (g/100 g of BW) 2.89 2.90 3.43 2.89 2.86 2.85 3.84 2.90
± ± ± ± ± ± ± ±
0.05a 0.03a 0.04b 0.05a 0.02a 0.03a 0.02b 0.04a
Kidney (g/100 g of BW) 0.41 0.43 0.68 0.40 0.42 0.41 0.72 0.71
± ± ± ± ± ± ± ±
0.03a 0.03a 0.02b 0.01a 0.03a 0.02a 0.03b 0.02b
Heart (g/100 g of BW) 0.63 0.65 0.64 0.61 0.62 0.62 0.64 0.67
± ± ± ± ± ± ± ±
0.02a 0.02a 0.04a 0.03a 0.02a 0.03a 0.02a 0.01a
Gizzard (g/100 g of BW) 2.33 2.30 2.73 2.72 2.31 2.30 2.64 2.96
± ± ± ± ± ± ± ±
Spleen (g/100 g of BW)
0.05a 0.03a 0.04b 0.05b 0.02a 0.03a 0.02b 0.04b
Means values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. Values represent means of 8 groups of 5 chicks each per treatment.
a,b 1
0.06 0.07 0.13 0.08 0.06 0.05 0.15 0.13
± ± ± ± ± ± ± ±
0.03a 0.02a 0.03b 0.02a 0.01a 0.03a 0.02b 0.03b
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Histopathology
AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE
5
AFB1, and a combination of AFB1 and FB1 diets. Livers from the last 2 treatments were enlarged, yellowish, friable, and with rounded borders. Livers from other treatments were not included because they had normal appearances. When compared with the control (Figure 3A), histopathologies of livers from birds fed with AFB1 alone or AFB1 plus FB1 showed multifocal and varied cytoplasmatic vacuolization with perilobular location when stained with hematoxylin and eosin (Figure 3B and C, respectively). Liver samples of broilers consuming the FB1 diet or other SB-supplemented diets (SB, AFB1 + SB, FB1 + SB, or AFB1 + FB1 + SB) all revealed discrete and diffuse cellular swelling as depicted in Figure 3D.
DISCUSSION Aflatoxin and fumonisin produce severe economic losses and health problems in the poultry industry because of their toxicity and frequency of occurrence in feedstuffs (Kubena et al., 1993). The current study demonstrated the toxicity of AFB1 combined with FB1 in growing broiler chicks and a reduction in the ability of SB to decrease the toxicity of AFB1 in the presence of FB1. In vitro studies have shown that SB is strong binder of AFB1
(Veldman et al., 1992; Rosa et al., 2001). An inert, stable, and insoluble complex between SB and AFB1 was assumed to be the responsible for preventing toxin absorption in the intestine. Body weight reduction, caused by intake of feed contaminated with AFB1, has already been reported in previous studies (Rosa et al., 2001). Sodium bentonite was inert and nontoxic. Olver (1997) and Parlat et al. (1999) reported similar results for clinoptilolite, a natural zeolite. The present results agreed with our earlier findings about the ability of SB to ameliorate the adverse effects of AFB1 in growing broilers (Rosa et al., 2001). However, addition of SB did not have the same effect on birds fed a combined diet (AFB1 plus FB1). Previous in vitro studies involving NaA zeolite as an adsorbent have shown that the presence of FB1 could significantly decrease the ability of sorbent to capture AFB1 (Kikot et al., 2002). At pH 2, a competitive adsorption between both toxins was observed, but FB1 was released from the surface when the pH was increased to 6, value at which AFB1 is not able to be absorbed. Kubena et al. (1990a,b) reported additive toxicity in the majority of the mycotoxins when acting in combination. These authors reported that a decrease in serum total protein and ALB in broilers, as a result of aflatoxicosis, was not alleviated by hydrated sodium calcium aluminosilicate.
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FIGURE 2. Representative livers from broiler chickens fed with diets: A) control, B) 0.3% sodium bentonite (SB), C) 2.5 mg of aflatoxin B1 (AFB1) and 200 mg of fumonisin (FB1)/kg of feed, and D) 2.5 mg of AFB1/kg of feed.
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MIAZZO ET AL.
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FIGURE 3. Photomicrographs of hematoxylin and eosin stained liver sections of chicks fed with the following diets: A) control, 0.3% sodium bentonite (SB), B) 2.5 mg of aflatoxin B1 (AFB1)/kg feed, C) 2.5 mg of AFB1 and 200 mg of fumonisin B1 (FB1)/kg of feed, and D) 0.3% SB. Bars equal 10 µm.
AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE
ACKNOWLEDGMENTS The authors are grateful to the SECyT (Secretarı´a de Ciencia y Te´cnica, Universidad Nacional de Rı´o Cuarto) Res. No. 129/01, 077/03, and CONICET (Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas) PIP Res. No. 962/01, which supported this study through grants. Carina Magnoli thanks CONICET for fellowship support. Ana Marı´a Dalcero and Stella Maris Chiacchiera held positions at CONICET. We thank Castiglioni, Pes y Cı´a SAFIAMI for providing the sodium bentonite.
REFERENCES AOAC. 1995. Sections 975.35, 976.22 in Official Methods of Analysis. Association of Official Analytical Chemists, Gaithersburg, MD. Bata, A., R. Glavits, A. Vanyi, and G. Salyi. 1996. More important mycotoxicoses of Poult. clı´nico-pathological. Review article. Magy. Allatorv. Lapja 51:395–408. Chulze, S., M. L. Ramirez, M. C. Farnochi, M. Pascale, A. Visconti, and G. March. 1996. Fusarium and fumonisins occurrence in Argentinian corn at different ear maturity stages. J. Agric. Food Chem. 44:2797–2801. Dalcero, A., C. Magnoli, S. M. Chiacchiera, G. Palacio, and M. M. Reynoso. 1997. Mycoflora and incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina. Mycopathologia 137:179–184. Dalcero, A., C. Magnoli, M. Luna, G. Ancasi, M. M. Reynoso, S. Chiacchiera, R Miazzo, and G. Palacio. 1998. Mycoflora and naturally ocurring mycotoxins in poultry feeds in Argentina. Mycopathologia 141:37–43. Edds, G. T., and R. A. Bortell. 1983. Biological effects of aflatoxinas: Poultry. Pages 56–61 in Aflatoxin and Aspergillus flavus in corn. U. L. Diener, R. L. Asquity, and J. W. Dickens, ed. Southern Cooperative Series Bulletin 279. Auburn University, Auburn, Alabama. Gelderblom, W. C., S. Abel, C. M. Smuts, J. Marnewick, W. F. Marasas, E. R. Lemmer, and D. Ramljak. 2001. Fumonisininduced hepatocarcinogenesis: Mechanisms related to cancer initiation and promotion. Environ. Health Perspect. 109:291–300. Gelderblom, W. C. A., K. Jaskiewicz, W. F. O. Marasas, P. G. Thiel, M. J. Horak, R. Vieggaar, and N. P. J. Kriek. 1988. Fumonisins—Novel mycotoxins with cancer promoting activity produced by Fusarium moniliforme. Appl. Environ. Microbiol. 54:1806–1811. Gelderblom, W. C. A., N. P. J. Kriek, W. F. O. .Marasas, and P. G. Thiel. 1991. Toxicity and carcinogenicity of the Fusarium moniliforme metabolite fumonisin B1 in rats. Carcinogenesis 12:1247–1251. Huff, W. E., L. F. Kubena, R. B. Harvey, and T. D. Phillips. 1992. Efficacy of hidrated sodium calcium aluminosilicate to reduce the individual and combined toxicity of aflatoxin and ochratoxin A. Poult. Sci. 71:64–69. Kececi, T., H. Oguz, V. Kurtoglu, and O. Demet. 1998. Effects of polyvynylpoly-pyrrolidone, synthetic zeolite and bentonite on serum biochemical and haematological characters of broiler chickens during aflatoxicosis. Br. Poult. Sci. 39:452– 458. Kikot, A., C. Magnoli, S. Chiacchiera, A. Dalcero, R. Miazzo, and E. I. Basaldella. 2002. Effect of fumonisin B1 on NaA zeolite effectiveness to adsorb aflatoxin B1. Metal Ions Biol. Med. 7:242–246. Kubena, L. F., R. B. Harvey, R. H. Baile, S. A. Buckley, and R. G. Rottinghaus. 1998. Effects of hydrateted sodium calcium aluminosilicate [T-Bind ] on mycotoxicosis in young broiler chickens. Poult. Sci. 77:1502–1509.
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Protective effects of the nonnutritive additive (SB) on gross hepatic changes produced by AFB1 or by a combination of AFB1 and FB1 were observed. A relative increase in liver weight was caused by AFB1 or by a combination of AFB1 and FB1. Addition of SB ameliorated the observed effects. Chronic mycotoxicoses may be diagnosed by determining serum biochemical alterations even before major clinical symptoms appear (Oguz et al., 2000). Reductions in concentrations of serum total protein and ALB, as indicators of protein synthesis, and GLOB were observed in chicks consuming diets containing AFB1 alone and combined with FB1 (Tung et al., 1975; Huff et al, 1992; Kubena et al., 1994). As expected, all of these values in the present results were sensibly affected by AFB1. The stronger effect at d 50 might have coincided with longer exposure to AFB1. Changes in concentrations of serum total protein, ALB, and GLOB were reversed by SB in chicks fed diets containing AFB1 alone or in combination with FB1. Increases in serum total protein, ALB, and GLOB observed at d 43 might have been due to dietary changes at d 32. Fumonisin B1 did not affect any of these parameters. Taking into account that the biochemical parameters for mycotoxicoses seemed to depend on bird age and nutritional status at the time of exposure to contaminated feed, we suggest that further in vivo experiments with this adsorbent should be conducted. Our results agreed with those reported by Kubena et al. (1998), who found significant decreases in these biochemical parameters at exposure levels of AFB1 ranging from 2.5 to 5 ppm. Kececi et al. (1998) and Oguz et al. (2000) showed that some serum biochemical changes could be ameliorated by 0.5% SB administration to the diet of broiler chickens fed with 2.5 mg of AFB1/kg of diet. However, Oguz et al. (2002) reported that the use of 50 and 100 ppb of AFB1 did not cause any significant change in the total protein and ALB, probably due to the low mycotoxin dose in the assay. In our previous studies the biochemical parameters for broilers fed a diet containing 0.3% SB and 5 mg of AFB1/ kg of diet did not completely return to normal values, showing an inhibition of protein synthesis (Rosa et al., 2001). In the present report, a complete return to normal biochemical parameters was observed, which could have been due to the lower AFB1 exposure dose compared with the former. It should be stressed that this research showed that SB was effective to counteract only some of the AFB1promoted effects. In fact, addition of SB to a diet containing a combination of AFB1 and FB1 was not able to return BW gain to equal that of the control. This result could be attributed to a competition between FB1 and AFB1 for the active surface sites of SB, rendering a greater bioavailability of AFB1 in the presence of high dose of FB1. The current data indicate toxicity for chicks of AFB1 alone or in combination with FB1. Neither additive nor synergistic toxic interaction between AFB1 and FB1 seems to be operating as shown with most of the parameters.
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