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In vitro and in vivo efficacy of a hydrated sodium calcium aluminosilicate to bind and reduce aflatoxin residues in tissues of broiler chicks fed aflatoxin B1
Research Notes In vitro and in vivo efficacy of a hydrated sodium calcium aluminosilicate to bind and reduce aflatoxin residues in tissues of broiler chicks fed aflatoxin B1 D. V. Neeff,* D. R. Ledoux,† G. E. Rottinghaus,‡ A. J. Bermudez,‡ A. Dakovic,§ R. A. Murarolli,† and C. A. F. Oliveira*1 *Department of Food Engineering, School of Animal Science and Food Engineering, University of São Paulo, Pirassununga, SP, 13630-000, Brazil; †Animal Sciences Department, and ‡Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia 65211; and §Institute for Technology of Nuclear and Other Mineral Raw Materials, PO Box 390, 11000, Belgrade, Serbia each treatment were anesthetized with carbon dioxide, killed by cervical dislocation, and samples of liver and kidney were collected for analysis of AFB1 residues. The percentage of AFB1 bound for each concentration of adsorbent (100, 10, 1, 0.5, 0.25, and 0.05 mg/10 mL) was 100, 91.1, 81.8, 75.4, 40.1, and 8.8%, respectively. Concentrations of aflatoxin residues (AFB1, aflatoxicol, aflatoxins B2 and G1) were lower (P < 0.05) in livers and kidneys of birds fed AFB1 plus HSCAS (diet D), when compared with birds fed AFB1 alone (diet C). However, histopathology data from the in vivo study indicated that HSCAS did not prevent lesions associated with aflatoxicosis. The decrease in the bioavailability of AFB1 caused by the HSCAS reduced aflatoxin residues in liver and kidney, but not enough to completely prevent the toxic effects of AFB1 in broilers.
Key words: aflatoxin B1, residue, adsorbent, liver, broiler 2013 Poultry Science 92:131–137 http://dx.doi.org/10.3382/ps.2012-02510
INTRODUCTION
the bioavailability of aflatoxin in the gastrointestinal tract (Phillips, 1999). The liver is the target organ of AFB1 in broilers and is characterized by a severe hepatic enlargement and fatty infiltration (Huff et al., 1986). The liver and kidney are the main organs involved in the detoxification of AFB1, and also where most residues accumulate (Hussain et al., 2010). The AFB1 is primarily biotransformated in the liver by cytochrome P-450 associated enzymes, which generate hydroxylated metabolites such as aflatoxins M1 (AFM1) and B2a (AFB2a; Biehl and Buck, 1987). Aflatoxicol (AFL) can also be formed by the reduction of AFB1 by an NADPH-dependent cytoplasmic enzyme present in the soluble fraction of liver homogenates (Biehl and Buck, 1987). However, these compounds are also a potential human health risk because they can be carried over into animal-derived food products and some of them can still induce toxic effects in experimental models (Fernandez et al., 1994).
Aflatoxins are fungal metabolites found as contaminants in a wide range of food and agricultural products. Aflatoxin B1 (AFB1), the most commonly occurring aflatoxin, is a potent mutagen and hepatocarcinogen to a wide range of animal species (Biehl and Buck, 1987). Chronic exposure to aflatoxins may not only significantly alter productivity and animal farming trends, but may also impose a risk to the consumer from direct exposure to aflatoxin-contaminated food commodities (Ramos and Hernández, 1996). The addition of hydrated sodium calcium aluminosilicate (HSCAS) to AFB1 contaminated diets has been shown to greatly reduce ©2013 Poultry Science Association Inc. Received June 1, 2012. Accepted October 5, 2012. 1 Corresponding author: [email protected]
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ABSTRACT The aim of this study was to determine the binding capacity of a hydrated sodium calcium aluminosilicate (HSCAS) for aflatoxin B1 (AFB1), and the efficacy of the HSCAS to reduce the concentrations of residual AFB1 and its metabolites in the liver and kidney of broilers fed AFB1. One hundred 1-d-old male broilers (Ross 708) were maintained in chick batteries and allowed ad libitum access to feed and water. A completely randomized design was used with 5 replicate pens of 5 chicks assigned to each of 4 dietary treatments from hatch to 21 d. Dietary treatments included the following: A) basal diet (BD), with no HSCAS or AFB1, B) BD supplemented with 0.5% HSCAS only, C) BD supplemented with 2.5 mg of AFB1/kg of feed, and D) BD supplemented with 2.5 mg of AFB1/ kg of feed and 0.5% HSCAS. On d 21, 5 chicks from
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MATERIALS AND METHODS Experimental Design and Birds The experiment was conducted in the Animal Science Research Center, Department of Animal Science, University of Missouri, Columbia. A total of one hundred 1-d-old male broilers (Ross 708) were maintained in chick batteries and allowed ad libitum access to feed and water. A completely randomized design was used with 5 replicate pens of 5 chicks assigned to each of 4 dietary treatments from hatch to d 21. Treatments included the following: A) basal diet (BD), with no HSCAS or AFB1, B) BD supplemented with 0.5% HSCAS, C) BD supplemented with 2.5 mg of AFB1/kg of feed, and D) BD supplemented with 2.5 mg of AFB1/ kg of feed and 0.5% HSCAS. Chicks were weighed at the beginning and the end of the experiment. Feed consumption was determined at the end of the experiment, and feed conversion (FC) was calculated. Mortality was recorded as it occurred, and dead birds were necropsied to determine cause of death. In addition, chicks were inspected daily and any health-related problems were recorded.
AFB1 Aflatoxin B1 was produced in the Veterinary Medical Diagnostic Laboratory, through rice fermentation by Aspergillus parasiticus NRRL 2999 by methods described by Shotwell et al. (1966). Aflatoxin B1 was quantified using an HPLC system (Hitachi High Technologies America, Schaumburg, IL) equipped with a model L-7100 pump, model L-7485 fluorescence detector (ex 365 nm; em 430 nm), model L-7200 autosampler with D-7000 data acquisition interface and ConcertChrom
software on a microcomputer. The column was a Phenomenex (Torrance, CA) 100 × 4.6 mm Hyperclone C18 (3 µm) with a mobile phase of water:acetonitrile (70:30) at a flow rate of 1.0 mL/min.
Adsorbent The experimental adsorbent used was HSCAS, obtained from a proprietary source. The name HSCAS is a general classification that includes different adsorbents rather than a specific substance. It can be defined as any clay material containing aluminum and silica, exchangeable sodium and calcium cations, and waters of hydration.
Diet Preparation The BD was a commercial corn soybean meal-type diet formulated to meet or exceed the nutritional requirements of growing chicks as recommended by the NRC (1994). The diets were prepared in a horizontal/ helicoidal mixer, for 15 min at the concentrations proposed for the treatments. After preparation of the experimental diets, a 1-kg sample was collected from each treatment and analyzed for confirmation of aflatoxin B1 level. The AFB1 was extracted from samples according to the method of Rottinghaus et al. (1982). Additionally, all diets were screened (Rottinghaus et al., 1982, 1992) and found to be free of the following mycotoxins: ochratoxin A, vomitoxin, zearalenone, and fumonisin B1. The assay detection limits were 50 µg/kg, 500 µg/g, 500 µg/kg, and 1.0 µg/g, respectively, for ochratoxin A, vomitoxin, zearalenone, and fumonisin B1.
In Vitro Analysis The in vitro analysis was conducted using procedures described by Ledoux and Rottinghaus (1999). Different concentrations of the HSCAS (100, 10, 1, 0.5, 0.25, and 0.05 mg/10 mL) were added to tubes containing 0.1 M phosphate buffer adjusted to pH 3.0, and containing 2 mg/L of AFB1. Samples were then put on a rotator shaker for 30 min at room temperature, centrifuged at 1,000 × g for 5 min at 25°C, and 1.0 mL of the supernatant analyzed by HPLC, using the same chromatographic conditions as previously described for quantification of AFB1 in culture material. To measure loss due to nonspecific binding and to eliminate exogenous peaks, a control was prepared (10 mL of 0.1 M phosphate buffer at pH 3 and the HSCAS). An aliquot of the original pH 3 buffered AFB1 test solution was used as the standard.
In Vivo Analysis Performance. Feed intake (FI), BW gain (BWG), FC, mortality percent, relative kidney weight, and rela-
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In addition, residual AFB1 may be also present in edible products intended for human consumption, including liver and eggs (Park and Pohland, 1986). Among the detoxification methods reported experimentally, the addition of sorbent compounds to feeds is one of the most effective and economical procedures because AFB1 binds chemically to the sorbent and therefore reduces its bioavailability (Phillips, 1999). In vitro analysis of mycotoxin sequestration can be very useful in identifying potential dietary sequestering agents, and in helping to determine mechanisms and conditions favorable for sequestration to occur (Ledoux and Rottinghaus, 1999). However, in vivo studies are needed to confirm the efficacy of adsorbents for mycotoxins. The objectives of this study were 1) to determine the binding capacity of a HSCAS for AFB1, 2) to determine the efficacy of the HSCAS to ameliorate the toxic effects of AFB1 on broiler performance and serum chemistries, and 3) to determine the efficacy of the HSCAS to reduce residual concentrations of AFB1 metabolites in the liver and kidney of broilers fed AFB1.
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RESEARCH NOTE
• 1 = liver section unremarkable; • 2 = lesions in liver section are compatible with mild aflatoxicosis, affecting less than 20% of the hepatic parenchyma; • 3 = lesions in liver section are compatible with moderate aflatoxicosis, affecting 20 to 60% of the hepatic parenchyma; and • 4 = lesions in liver section are compatible with severe aflatoxicosis, affecting more than 60% of the hepatic parenchyma. Birds were examined for signs of gross pathology due to aflatoxin or resulting from nutritional deficiencies caused by addition of test products. Broiler serum analyses were performed using an automatic analyzer (Eastman Kodak Co., Rochester, NY), and the following chemistries were determined: proteins (globulin [GLOB], total protein [TP], albumin [ALB]), enzymes (aspartate aminotransferase [AST], gamma-glutamyltransferase [GGT]), glucose (GLU), uric acid (UA), and calcium (Ca). Aflatoxin Residue Analysis in Liver and Kidney. Five samples of liver or kidney from each treatment (one sample of each organ per replicate) were collected for analysis of AFB1 residues (AFB1, AFB2, AFM1, AFG1, AFG2, and AFL), according to the method by Chiavaro et al. (2005), with some adaptations proposed by the manufacturer of the immunoaffinity columns (AFLAPREP M, Biopharm Rhône Ltd., Glasgow, UK). Five grams of frozen liver or kidney samples were ground with 25 mL of methanol-water (80:20) in a mixer for 2 to 3 min, and the resulting slurry centrifuged (Sorvall RC 3B Plus) at 3,600 × g. An aliquot (4 mL) of the supernatant was diluted with 25 mL of PBS and passed through an AFLAPREP M (R-Biopharm Rhône Ltd.) immunoaffinity cleanup column. The column was
washed 2 times with 10 mL of PBS followed by 2 mL of distilled water. After the washing steps, the aflatoxins were eluted with 3 mL of acetonitrile, dried, and 200 µL of acetonitrile and 800 µL of water were added to the sample vial. Final extracts were injected into the HPLC with fluorescence detection at 365 nm excitation and 440 nm emission. The detection limit was set at 1.0 µg/kg of aflatoxin. Calibration curves of aflatoxins were prepared using standard solutions of AFB1, AFB2, AFG1, AFG2, AFM1 (Trilogy, Washington, DC) and AFL (Sigma-Aldrich, St. Louis, MO) previously evaluated according to Scott (1990). The approximate retention times for AFB1, AFB2, AFM1, AFG1, AFG2, and AFL were 15, 11, 6.30, 10, 7.5, and 24.35 min, respectively.
Statistical Analysis Data were analyzed as a 2 × 2 factorial by ANOVA using the GLM procedure of SAS software (SAS Institute Inc., 1992). The means for treatments showing significant differences in the ANOVA were compared using Tukey’s test (Snedecor and Cochran, 1967). Statistical significance was accepted at P < 0.05.
RESULTS AND DISCUSSION In Vitro Assays The binding percentages of different amounts of HSCAS in 2 mg/L of AFB1 solution at pH 3 are presented in Table 1. The percentage of AFB1 bound for each concentration of the adsorbent (100, 10, 1, 0.5, 0.25, and 0.05 mg/10 mL) was, 100, 91.1, 81.8, 75.4, 40.1, and 8.8%, respectively. The higher the concentration of the HSCAS, the greater was its ability to bind AFB1. This result agrees with Phillips et al. (1995), who found that addition of HSCAS to the diet of animal species could protect the animals from the toxic effects of aflatoxin. According to Ramos and Hernández (1996), the adsorbent should have a high affinity for the specific mycotoxin, resulting in the formation of a strong complex, thus reducing the possibility of any rupture of the complex, which would result in the subsequent absorption of these fungal metabolites. The adsorbent should also have a high binding capacity to prevent saturation of the adsorbent. Protection from aflatoxin results from the binding of aflatoxins to HSCAS in the gastrointestinal tract, thereby reducing the bioavailability of the aflatoxins (Phillips et al., 1995).
Growth Performance and Organ Weights The effects of dietary treatments on growth performance and liver and kidney weights are shown in Table 2. Feed intake, BWG, FC, and relative weights of liver and kidneys of birds fed HSCAS alone were not different (P > 0.05) from those of control birds. These
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tive liver weight were calculated. Body weight gain was measured weekly, FI was determined at the end of the experiment, and FC was calculated. Serological and Histopathological Analysis. On d 21, all chicks were anesthetized with carbon dioxide. Ten chicks from each treatment (2 chicks from each replicate) were bled by cardiac puncture, and were the blood samples submitted for serum chemistries and enzyme levels. The remaining chicks (15 per treatment, 3 chicks per replicate) were killed by cervical dislocation, and the liver and kidney were removed and weighed. Liver samples were fixed in formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin stain for histopathological analysis (Luna, 1968). Liver sections from all treatment groups were examined microscopically. Individual sample numerical scores were reported using the following scoring system, based on the severity of the main aflatoxin-associated lesions (biliary hyperplasia, periportal swelling, vacuolar degeneration of hepatocytes, and periportal heterophil infiltration):
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Neeff et al. Table 1. Binding efficacy of different amounts of hydrated sodium calcium aluminosilicate (HSCAS) in 2 mg/L of aflatoxin B1 (AFB1) solution at pH 3 HSCAS amount (mg/10 mL)
Starting AFB1 concentration (µg/mL)
AFB1 in solution (HPLC) (µg/L)
AFB1 bound (%)
AFB1 adsorbed (mg/g)
2.0 2.0 2.0 2.0 2.0 2.0
0.010 0.178 0.365 0.492 1.198 1.825
100 91.1 81.8 75.4 40.1 8.80
0.20 1.82 16.35 30.16 32.07 35.05
100 10 1 0.5 0.25 0.05
Compared with the current study, the adsorbents used in studies by Ledoux et al. (1999) and Shi et al. (2006) were effective in ameliorating the toxic effects of aflatoxin on relative liver and kidney weights. Similar to the current study, Kubena et al. (1998) also did not observe any benefits to relative organ weights using 0.25% of an adsorbent. The liver is considered the target organ during aflatoxicosis in poultry because the relative liver weight is significantly increased by lower levels of aflatoxin compared with any other organ (Huff et al., 1986) and becomes pale with rounded margins (Ledoux et al., 1999).
Serum Biochemistry The results of serum biochemistry are summarized in Table 3. Significant differences were observed between the 4 treatments for GLU, ALB, TP, GLOB, and Ca, and the birds fed 2.5 mg/kg of AFB1 and 0.5% HSCAS plus 2.5 mg/kg of AFB1 had the worst results. The most sensitive parameter to aflatoxin-induced change in a study by Huff et al. (1986) was serum ALB levels. In the control group, ALB levels increased between d 3 and 6, then remained constant through d 21 (approximately 1.30 g/100 mL). Decreases in levels of serum TP and ALB are indicators of aflatoxicosis, and increased
Table 2. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to ameliorate the toxic effects of aflatoxin B1 (AFB1) on performance of broiler chicks from hatch to d 211 Treatment AFB1 (mg/kg) HSCAS (%) 0 0 0 0.5 2.5 0 2.5 0.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 0 2.5 HSCAS 0 0.5
Feed intake1 (g)
BW gain1 (g)
Feed conversion1 (g:g)
993a 970a 747b 794b 19
800a 772a 581b 651b 22
1.243 1.272 1.396 1.246 0.064
<0.0001 0.5440 0.0804 981.63a 770.54b 870.23a 881.94a
<0.0001 0.3355 0.0381
0.3351 0.3535 0.1821
785.85a 616.09b 690.26a 711.68a
1.26a 1.32a 1.32a 1.26a
Mortality1 (%) 0 4 12 8 0.10 0.1362 1.0000 0.4442 2.0a 10.0a 6.0a 6.0a
Relative liver weight2 Relative kidney weight2 (g/100 g of BW) (g/100 g of BW) 2.668b 2.735b 4.012a 3.886a 0.089
0.804b 0.771b 1.277a 1.354a 0.043
<0.0001 0.7454 0.2971
<0.0001 0.6221 0.2241
2.70b 3.95a 3.34a 3.31a
0.79b 1.32a 1.04a 1.06a
a,bValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 5 chicks each. 2Data are means of 5 replicate pens of 3 chicks each.
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results indicate that the HSCAS at 0.5% of the diet did not negatively affect the nutritional integrity of the diet, indicating that this dietary concentration of the HSCAS was safe to feed. Compared with controls, birds fed 2.5 mg of AFB1/kg of diet alone had reduced (P < 0.05) FI and BWG, poor FC, and increased (P < 0.05) relative liver and kidney weights. The addition of 0.5% HSCAS to the AFB1 diet was not effective (P > 0.05) statistically in reducing or preventing the negative effects of AFB1 on growth performance or relative organ weights. However, it should be noted that birds fed AFB1 alone had a 27% reduction in BWG, whereas the birds fed both AFB1 and HSCAS only had an 18% reduction in BWG, suggesting that the HSCAS was partially effective in alleviating the decrease in BWG caused by AFB1. The most significant economic effect of aflatoxicosis in poultry is the reduced growth rate (Huff et al., 1986). The current results are in contrast to a previous study by Ledoux et al. (1999) in which 1% of an HSCAS was completely effective in preventing the toxic effects of aflatoxin. The higher concentration of HSCAS (1%) used by Ledoux et al. (1999) may well account for the lack of efficacy of the HSCAS used in the current study. However, it has been shown that not all HSCAS are equally effective and some are more effective at higher concentrations.
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Table 3. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to ameliorate the toxic effects of aflatoxin B1 (AFB1) on blood chemistries of broiler chicks1 Treatment AFB1 (mg/kg)
GLU (mg/dL)
ALB (g/dL)
TP (g/dL)
GLOB (g/dL)
CA (mg/dL)
0 0.5 0 0.5
351ab 419a 226c 264bc 26
0.90a 0.94a 0.42b 0.56b 0.04
2.04a 2.32a 1.14b 1.40b 0.08
1.14b 1.40a 0.74c 0.88c 0.05
10.52ab 10.76a 9.28c 9.62bc 0.28
284 283 216 218 40
11.4 12.6 12.2 12.8 1.0
8.26 9.00 7.84 8.78 0.94
<0.0001 0.0612 0.5970 385.00a 245.00b 288.50a 341.50a
<0.0001 0.0416 0.2361 0.92a 0.49b 0.66b 0.75a
<0.0001 0.0029 0.8980 2.18a 1.27b 1.59b 1.86a
<0.0001 0.0013 0.2609 1.27a 0.81b 0.94b 1.14a
0.0006 0.3114 0.8592 10.64a 9.45b 9.90a 10.19a
0.1179 0.9844 0.9688 283.60a 217.00a 249.90a 250.70a
0.6195 0.3756 0.7652 12.00a 12.50a 11.80a 12.70a
0.7372 0.3833 0.9163 8.63a 8.31a 8.05a 8.89a
0 2.5 0 0.5
AST (U/L)
GGT (U/L)
UA (mg/dL)
a–cValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 2 chicks per pen. GLU = glucose; ALB = albumin; TP = total protein; GLOB = globulin; CA = calcium; AST = aspartate aminotransferase; GGT = gamma glutamyl transferase; UA = uric acid.
activities of serum GGT and AST have been reported to be sensitive serological indicators of liver and kidney toxicity. It should be pointed out that the clinical signs for aflatoxicosis depend on bird age and nutritional or health status at the time of exposure to contaminated feed (Shi et al., 2006). The toxic effects of AFB1 on serum chemistries were, in general, according to previous studies (Huff et al., 1986; Ledoux et al., 1999; Shi et al., 2006). Kubena et al. (1990) reported that the alterations in some serum biochemical and enzymatic parameters as a result of aflatoxicosis were not alleviated by HSCAS; our results are in agreement with theirs.
Histopathology No significant microscopic lesions were evident in liver sections of controls or birds fed HSCAS alone. Mild, moderate, or severe lesions indicative of aflatoxicosis were seen in liver sections examined in birds fed AFB1 alone or birds fed HSCAS plus AFB1. These hepatic lesions included moderate biliary hyperplasia, mild to severe periportal swelling, and vacuolar degeneration of hepatocytes, mild to moderate regeneration of hepatocytes, and mild to severe periportal heterophil infiltration. The mean liver lesion score of birds from control and HSCAS groups was 1.0. The addition of the HSCAS to the AFB1 diet was not effective in reducing the severity of the liver lesions caused by AFB1, as the mean liver score of groups treated with AFB1 alone or in combination with HSCAS was 3.0. These results are in contrast to a previous study by Ledoux et al. (1999), who reported that 1% HSCAS was completely effective in preventing liver lesions caused by 4 mg of AFB1/kg of diet.
Aflatoxin Residues The values for AFB1 residues in liver and kidney of broilers fed dietary treatments are presented in Tables 4 and 5, respectively. Concentrations of AFB1, AFL, AFG1, and AFB2 were lower (P < 0.05) in livers of birds fed AFB1 plus HSCAS compared with birds fed AFB1 alone. Concentrations of AFM1 were all lower in birds fed AFB1 plus HSCAS compared with birds fed AFB1 alone. No AFB1 residues were found in the livers of birds fed the BD (diet A) or the birds fed the BD plus HSCAS. Concentrations of AFB1 were also lower (P < 0.05) in kidneys of birds fed AFB1 plus HSCAS compared with those fed AFB1 only. Concentrations of AFM1, AFG1, AFG2, and AFB2 were numerically, but not statistically, lower in birds fed AFB1 plus HSCAS compared with birds fed AFB1 alone. The AFL was the only residue in the kidney that was not significantly different (P > 0.05) among treatments. No AFB1 residues were found in the kidney of birds fed BD or birds fed BD plus HSCAS. Fernández et al. (1994) fed broiler chickens a diet containing 2.5 mg/kg of AFB1 for 32 d, and the mean aflatoxin residues found in liver were 0.23 µg/kg of AFB1, 0.06 µg/kg of AFM1, 0.23 µg/kg of AFL, and 0.16 µg/kg of AFG1. In kidney, they found 0.25 µg/ kg of AFB1 and 0.12 µg/kg of AFM1. These values are much lower compared with our results for the diet containing 2.5 mg/kg of aflatoxin. Other studies have also reported lower concentrations of AFB1 residues after ingestion of aflatoxin containing feeds. Bintvihok and Kositcharoenkul (2006), fed broilers diets containing AFB1, and AFB1 plus calcium propionate for 3 d, and reported AFB1 residues of 0.05 ±
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0 0 2.5 2.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 HSCAS
HSCAS (%)
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Table 4. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to reduce aflatoxin residues in liver of broiler chicks fed aflatoxin B1 (AFB1) from hatch to d 211 Treatment AFB1 (mg/kg)
HSCAS (%)
AFB1 (µg/kg)
AFB2 (µg/kg)
AFL (µg/kg)
AFG1 (µg/kg)
AFG2 (µg/kg)
0b 0b 0.21a 0.12a 0.02
0b 0b 8.32a 1.49b 1.08 <0.0001 0.0741 0.0741 0.000b 0.163a 0.104a 0.059a
0b 0b 0.48a 0.07b 0.09 <0.0001 0.0008 0.0008 0.000b 2.243a 1.974a 0.269b
0b 0b 2.34a 0b 0.28 0.0003 0.0062 0.0062 0.000b 4.906a 4.162a 0.744b
0b 0b 3.95a 0.54b 0.41 0.1220 0.1220 0.1220 0.000a 0.014a 0.014a 0.000a
0a 0a 0.03a 0a 0.01 0.0007 0.0007 0.0007 0.000b 1.172a 1.172a 0.000b
1.0 1.0 1.0 428.80a 428.80a 428.80a 428.80a
a,bValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 3 chicks per pen. AFM = aflatoxin M ; AFL = aflatoxicol; AFG = aflatoxin G ; AFG = aflatoxin G ; 1 1 2 2 1 1 AFB2 = aflatoxin B2.
0.03 µg/kg and 0.13 ± 0.05 µg/kg in livers of birds fed 50 µg/kg of AFB1 and 100 µg/kg of AFB1, respectively. For AFM1 residues in liver, the values were 0.10 ± 0.04 µg/kg, 0.32 ± 0.13 µg/kg, 0.05 ± 0.03 µg/kg, and 0.05 ± 0.04 µg/kg in birds fed 50 µg/kg of AFB1, 100 µg/kg of AFB1, 100 µg/kg of AFB1 + 0.25% calcium propionate, and 100 µg/kg of AFB1 + 0.5% calcium propionate, respectively. Hussain et al. (2010) fed birds for 7, 14, and 28 d of age with 0, 1.6, 3.2, and 6.4 mg/ kg of AFB1 for 7 d and detected AFB1 residues in liver from all treatments, with the highest value (3.74 µg/ kg) observed in birds at 7 d of age. They found that older birds had lower tissue residues of AFB1 compared with younger birds, suggesting that older birds developed a more efficient mechanism of metabolizing AFB1.
Another possibility is that tissue dilution occurred due to the larger bird size. According to Phillips (1999), the protective effect of HSCAS is the result of rapid binding of aflatoxin by HSCAS in the gastrointestinal tract of chickens, thus preventing its absorption and normal distribution to the liver. Our results showed that HSCAS was effective in reducing aflatoxin residues, especially in liver. The biotransformation of AFB1 into AFM1 and AFL in the liver also decreased in broilers receiving HSCAS, therefore lowering the toxicity of AFB1. In conclusion, the results demonstrate that HSCAS was effective in binding AFB1 in vitro. The addition of HSCAS to the feed would potentially prevent aflatoxicosis by inducing a HSCAS-AFB1 complex, which
Table 5. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to reduce aflatoxin residues in kidney of broiler chicks fed aflatoxin B1 (AFB1) from hatch to d 211 Treatment AFB1 (mg/kg)
HSCAS (%)
0 0 0 0.5 2.5 0 2.5 0.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 0 2.5 HSCAS 0 0.5
AFM1 (µg/kg)
AFB1 (µg/kg)
AFB2 (µg/kg)
AFL (µg/kg)
AFG1 (µg/kg)
AFG2 (µg/kg)
0b 0b 0.68a 0.42a 0.09 <0.0001 0.1441 0.1441 0.000b 0.550a 0.341a 0.209a
0c 0c 16.16a 7.52b 1.59 <0.0001 0.0152 0.0152 0.000b 11.84a 8.080a 3.762b
0b 0b 0.91a 0.66ab 0.19 0.0008 0.5189 0.5189 0.000b 0.786a 0.456a 0.330a
0a 0a 0.48a 0.15a 0.14 0.0446 0.2632 0.2632 0.000b 0.312a 0.239a 0.073a
0b 0b 6.94a 4.19a 0.73 <0.0001 0.0796 0.0796 0.000b 5.566a 3.470a 2.096a
0b 0b 0.16a 0.12ab 0.03 0.0004 0.5384 0.5384 0.000b 0.142a 0.081a 0.061a
a–cValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 3 chicks per pen. AFM = aflatoxin M ; AFL = aflatoxicol; AFG = aflatoxin G ; AFG = aflatoxin G ; 1 1 2 2 1 1 AFB2 = aflatoxin B2.
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0 0 0 0.5 2.5 0 2.5 0.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 0 2.5 HSCAS 0 0.5
AFM1 (µg/kg)
RESEARCH NOTE
would be able to restrict gastrointestinal absorption of AFB1. However, histopathology data from the in vivo study indicated that HSCAS did not completely protect broilers against aflatoxicosis, but was effective in reducing aflatoxin residues in liver and kidney of chicks fed 2.5 mg of AFB1/kg of diet from hatch to d 21. The decrease in the bioavailability of AFB1 caused by the HSCAS led to a reduction in the aflatoxin residues in liver and kidney, but was not enough to prevent the toxic effects of AFB1 in broilers.
ACKNOWLEDGMENTS
REFERENCES Biehl, M. L., and W. B. Buck. 1987. Chemical contaminants: Their metabolism and their residues. J. Food Prot. 50:1058–1073. Bintvihok, A., and S. Kositcharoenkul. 2006. Effect of dietary calcium propionate on performance, hepatic enzyme activities and aflatoxin residues in broilers fed a diet containing low levels of aflatoxin B1. Toxicon 47:41–46. Chiavaro, E., C. Cacchioli, E. Berni, and E. Spotti. 2005. Immunoaffinity clean-up and direct fluorescence measurement of aflatoxin B1 and M1 in pig liver: comparison with high-performance liquid chromatography determination. Food Addit. Contam. 22:1154– 1161. Fernández, A., M. T. Verde, M. Gascón, J. J. Ramos, and J. Gomez. 1994. Aflatoxin and its metabolites in tissues from laying hens and broiler chickens fed a contaminated diet. J. Sci. Food Agric. 65:407–414. Huff, W. E., L. F. Kubena, R. B. Harvey, D. E. Corrier, and H. H. Mollenhauer. 1986. Progression of aflatoxicosis in broiler chickens. Poult. Sci. 65:1891–1899. Hussain, Z., M. Z. Khan, A. Khan, I. Javed, M. K. Saleemi, S. Mahmood, and M. R. Asi. 2010. Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels. Food Chem. Toxicol. 48:3304–3307. Kubena, L. F., R. B. Harvey, R. H. Bailey, S. A. Buckley, and G. E. Rottinghaus. 1998. Effects of a hydrated sodium calcium aluminosilicate (T-Bind) on mycotoxicosis in young broiler chickens. Poult. Sci. 77:1502–1509. Kubena, L. F., R. B. Harvey, T. D. Phillips, D. E. Corrier, and W. E. Huff. 1990. Diminution of aflatoxicosis in growing chikens by
the dietary addition of a hydrated sodium calcium aluminosilicate. Poult. Sci. 69:727–735. Ledoux, D. R., and G. E. Rottinghaus. 1999. In vitro and in vivo testing of adsorbents for detoxifying mycotoxins in contaminated feedstuffs. Pages 369–379 in Biotechnology in the Feed Industry. Proceedings of the 15th Annual Symposium. T. P. Lyons and K. A. Jacques, ed. Nottingham University Press, Nottingham, UK. Ledoux, D. R., G. E. Rottinghaus, A. J. Bermudez, and M. AlonsoDebolt. 1999. Efficacy of hydrated sodium calcium aluminosilicate to ameliorate the toxic effects of aflatoxin in broiler chicks. Poult. Sci. 78:204–210. Luna, G. L. 1968. Manual of Histologic Staining Methods of the Armed Forces. 3rd ed. McGraw-Hill, New York, NY. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Park, D. L., and A. E. Pohland. 1986. A rationale for the control of aflatoxin in animal feeds. Pages 473–482 in Mycotoxins and Phycotoxins. P. S. Steyn and R. Vleggaar, ed. Elsevier Applied Science, Amsterdam, the Netherlands. Phillips, T. D. 1999. Dietary clay in the chemoprevention of aflatoxin-induced disease. Toxicol. Sci. 52:118–126. Phillips, T. D., A. B. Sarr, and P. G. Grant. 1995. Selective chemisorption and detoxification of aflatoxins by phillosilicate clay. Nat. Toxins 3:204–213. Ramos, A. J., and E. Hernández. 1996. In vitro aflatoxin adsorption by means of montmorillonite silicate. A study of adsorption isotherms. Anim. Feed Sci. Technol. 62:263–269. Rottinghaus, G. E., C. E. Coatney, and H. C. Minor. 1992. A rapid sensitive thin layer chromatography procedure for the detection of fumonisin B1 and B2. J. Vet. Diagn. Invest. 4:326–329. Rottinghaus, G. E., B. Olsen, and G. D. Osweiler. 1982. Rapid screening method for aflatoxin B1, zearalenone, ochratoxin A, T-2 toxin, diacetoxyscirpenol and vomitoxin. Pages 477–484 in Proceedings of the 25th Animal American Association of Veterinary Laboratory Diagnosticians, Nashville, TN. SAS Institute Inc. 1992. SAS® User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. Scott, P. M. 1990. Natural poisons. Pages 1184–1213 in Official Methods of Analysis of the Association of Official Analytical Chemists. 15th ed. K. Helrich, ed. Assoc. Off. Anal. Chem., Arlington, VA. Shi, Y. H., Z. R. Xu, J. L. Feng, and C. Z. Wang. 2006. Efficacy of modified montmorillonite nanocomposite to reduce the toxicity of aflatoxin in broiler chicks. Anim. Feed Sci. Technol. 129:138–148. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson. 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425–428. Snedecor, G. W., and W. G. Cochran. 1967. Statistical Methods. 6th ed. Iowa State Univ. Press, Ames.
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The authors thank Conselho Nacional de Desenvolvimento Científico (CNPq), Brazil, grant no. 134068/2010-6, for financial support.
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Key words: aflatoxin B1, residue, adsorbent, liver, broiler 2013 Poultry Science 92:131–137 http://dx.doi.org/10.3382/ps.2012-02510
INTRODUCTION
the bioavailability of aflatoxin in the gastrointestinal tract (Phillips, 1999). The liver is the target organ of AFB1 in broilers and is characterized by a severe hepatic enlargement and fatty infiltration (Huff et al., 1986). The liver and kidney are the main organs involved in the detoxification of AFB1, and also where most residues accumulate (Hussain et al., 2010). The AFB1 is primarily biotransformated in the liver by cytochrome P-450 associated enzymes, which generate hydroxylated metabolites such as aflatoxins M1 (AFM1) and B2a (AFB2a; Biehl and Buck, 1987). Aflatoxicol (AFL) can also be formed by the reduction of AFB1 by an NADPH-dependent cytoplasmic enzyme present in the soluble fraction of liver homogenates (Biehl and Buck, 1987). However, these compounds are also a potential human health risk because they can be carried over into animal-derived food products and some of them can still induce toxic effects in experimental models (Fernandez et al., 1994).
Aflatoxins are fungal metabolites found as contaminants in a wide range of food and agricultural products. Aflatoxin B1 (AFB1), the most commonly occurring aflatoxin, is a potent mutagen and hepatocarcinogen to a wide range of animal species (Biehl and Buck, 1987). Chronic exposure to aflatoxins may not only significantly alter productivity and animal farming trends, but may also impose a risk to the consumer from direct exposure to aflatoxin-contaminated food commodities (Ramos and Hernández, 1996). The addition of hydrated sodium calcium aluminosilicate (HSCAS) to AFB1 contaminated diets has been shown to greatly reduce ©2013 Poultry Science Association Inc. Received June 1, 2012. Accepted October 5, 2012. 1 Corresponding author: [email protected]
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ABSTRACT The aim of this study was to determine the binding capacity of a hydrated sodium calcium aluminosilicate (HSCAS) for aflatoxin B1 (AFB1), and the efficacy of the HSCAS to reduce the concentrations of residual AFB1 and its metabolites in the liver and kidney of broilers fed AFB1. One hundred 1-d-old male broilers (Ross 708) were maintained in chick batteries and allowed ad libitum access to feed and water. A completely randomized design was used with 5 replicate pens of 5 chicks assigned to each of 4 dietary treatments from hatch to 21 d. Dietary treatments included the following: A) basal diet (BD), with no HSCAS or AFB1, B) BD supplemented with 0.5% HSCAS only, C) BD supplemented with 2.5 mg of AFB1/kg of feed, and D) BD supplemented with 2.5 mg of AFB1/ kg of feed and 0.5% HSCAS. On d 21, 5 chicks from
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MATERIALS AND METHODS Experimental Design and Birds The experiment was conducted in the Animal Science Research Center, Department of Animal Science, University of Missouri, Columbia. A total of one hundred 1-d-old male broilers (Ross 708) were maintained in chick batteries and allowed ad libitum access to feed and water. A completely randomized design was used with 5 replicate pens of 5 chicks assigned to each of 4 dietary treatments from hatch to d 21. Treatments included the following: A) basal diet (BD), with no HSCAS or AFB1, B) BD supplemented with 0.5% HSCAS, C) BD supplemented with 2.5 mg of AFB1/kg of feed, and D) BD supplemented with 2.5 mg of AFB1/ kg of feed and 0.5% HSCAS. Chicks were weighed at the beginning and the end of the experiment. Feed consumption was determined at the end of the experiment, and feed conversion (FC) was calculated. Mortality was recorded as it occurred, and dead birds were necropsied to determine cause of death. In addition, chicks were inspected daily and any health-related problems were recorded.
AFB1 Aflatoxin B1 was produced in the Veterinary Medical Diagnostic Laboratory, through rice fermentation by Aspergillus parasiticus NRRL 2999 by methods described by Shotwell et al. (1966). Aflatoxin B1 was quantified using an HPLC system (Hitachi High Technologies America, Schaumburg, IL) equipped with a model L-7100 pump, model L-7485 fluorescence detector (ex 365 nm; em 430 nm), model L-7200 autosampler with D-7000 data acquisition interface and ConcertChrom
software on a microcomputer. The column was a Phenomenex (Torrance, CA) 100 × 4.6 mm Hyperclone C18 (3 µm) with a mobile phase of water:acetonitrile (70:30) at a flow rate of 1.0 mL/min.
Adsorbent The experimental adsorbent used was HSCAS, obtained from a proprietary source. The name HSCAS is a general classification that includes different adsorbents rather than a specific substance. It can be defined as any clay material containing aluminum and silica, exchangeable sodium and calcium cations, and waters of hydration.
Diet Preparation The BD was a commercial corn soybean meal-type diet formulated to meet or exceed the nutritional requirements of growing chicks as recommended by the NRC (1994). The diets were prepared in a horizontal/ helicoidal mixer, for 15 min at the concentrations proposed for the treatments. After preparation of the experimental diets, a 1-kg sample was collected from each treatment and analyzed for confirmation of aflatoxin B1 level. The AFB1 was extracted from samples according to the method of Rottinghaus et al. (1982). Additionally, all diets were screened (Rottinghaus et al., 1982, 1992) and found to be free of the following mycotoxins: ochratoxin A, vomitoxin, zearalenone, and fumonisin B1. The assay detection limits were 50 µg/kg, 500 µg/g, 500 µg/kg, and 1.0 µg/g, respectively, for ochratoxin A, vomitoxin, zearalenone, and fumonisin B1.
In Vitro Analysis The in vitro analysis was conducted using procedures described by Ledoux and Rottinghaus (1999). Different concentrations of the HSCAS (100, 10, 1, 0.5, 0.25, and 0.05 mg/10 mL) were added to tubes containing 0.1 M phosphate buffer adjusted to pH 3.0, and containing 2 mg/L of AFB1. Samples were then put on a rotator shaker for 30 min at room temperature, centrifuged at 1,000 × g for 5 min at 25°C, and 1.0 mL of the supernatant analyzed by HPLC, using the same chromatographic conditions as previously described for quantification of AFB1 in culture material. To measure loss due to nonspecific binding and to eliminate exogenous peaks, a control was prepared (10 mL of 0.1 M phosphate buffer at pH 3 and the HSCAS). An aliquot of the original pH 3 buffered AFB1 test solution was used as the standard.
In Vivo Analysis Performance. Feed intake (FI), BW gain (BWG), FC, mortality percent, relative kidney weight, and rela-
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In addition, residual AFB1 may be also present in edible products intended for human consumption, including liver and eggs (Park and Pohland, 1986). Among the detoxification methods reported experimentally, the addition of sorbent compounds to feeds is one of the most effective and economical procedures because AFB1 binds chemically to the sorbent and therefore reduces its bioavailability (Phillips, 1999). In vitro analysis of mycotoxin sequestration can be very useful in identifying potential dietary sequestering agents, and in helping to determine mechanisms and conditions favorable for sequestration to occur (Ledoux and Rottinghaus, 1999). However, in vivo studies are needed to confirm the efficacy of adsorbents for mycotoxins. The objectives of this study were 1) to determine the binding capacity of a HSCAS for AFB1, 2) to determine the efficacy of the HSCAS to ameliorate the toxic effects of AFB1 on broiler performance and serum chemistries, and 3) to determine the efficacy of the HSCAS to reduce residual concentrations of AFB1 metabolites in the liver and kidney of broilers fed AFB1.
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RESEARCH NOTE
• 1 = liver section unremarkable; • 2 = lesions in liver section are compatible with mild aflatoxicosis, affecting less than 20% of the hepatic parenchyma; • 3 = lesions in liver section are compatible with moderate aflatoxicosis, affecting 20 to 60% of the hepatic parenchyma; and • 4 = lesions in liver section are compatible with severe aflatoxicosis, affecting more than 60% of the hepatic parenchyma. Birds were examined for signs of gross pathology due to aflatoxin or resulting from nutritional deficiencies caused by addition of test products. Broiler serum analyses were performed using an automatic analyzer (Eastman Kodak Co., Rochester, NY), and the following chemistries were determined: proteins (globulin [GLOB], total protein [TP], albumin [ALB]), enzymes (aspartate aminotransferase [AST], gamma-glutamyltransferase [GGT]), glucose (GLU), uric acid (UA), and calcium (Ca). Aflatoxin Residue Analysis in Liver and Kidney. Five samples of liver or kidney from each treatment (one sample of each organ per replicate) were collected for analysis of AFB1 residues (AFB1, AFB2, AFM1, AFG1, AFG2, and AFL), according to the method by Chiavaro et al. (2005), with some adaptations proposed by the manufacturer of the immunoaffinity columns (AFLAPREP M, Biopharm Rhône Ltd., Glasgow, UK). Five grams of frozen liver or kidney samples were ground with 25 mL of methanol-water (80:20) in a mixer for 2 to 3 min, and the resulting slurry centrifuged (Sorvall RC 3B Plus) at 3,600 × g. An aliquot (4 mL) of the supernatant was diluted with 25 mL of PBS and passed through an AFLAPREP M (R-Biopharm Rhône Ltd.) immunoaffinity cleanup column. The column was
washed 2 times with 10 mL of PBS followed by 2 mL of distilled water. After the washing steps, the aflatoxins were eluted with 3 mL of acetonitrile, dried, and 200 µL of acetonitrile and 800 µL of water were added to the sample vial. Final extracts were injected into the HPLC with fluorescence detection at 365 nm excitation and 440 nm emission. The detection limit was set at 1.0 µg/kg of aflatoxin. Calibration curves of aflatoxins were prepared using standard solutions of AFB1, AFB2, AFG1, AFG2, AFM1 (Trilogy, Washington, DC) and AFL (Sigma-Aldrich, St. Louis, MO) previously evaluated according to Scott (1990). The approximate retention times for AFB1, AFB2, AFM1, AFG1, AFG2, and AFL were 15, 11, 6.30, 10, 7.5, and 24.35 min, respectively.
Statistical Analysis Data were analyzed as a 2 × 2 factorial by ANOVA using the GLM procedure of SAS software (SAS Institute Inc., 1992). The means for treatments showing significant differences in the ANOVA were compared using Tukey’s test (Snedecor and Cochran, 1967). Statistical significance was accepted at P < 0.05.
RESULTS AND DISCUSSION In Vitro Assays The binding percentages of different amounts of HSCAS in 2 mg/L of AFB1 solution at pH 3 are presented in Table 1. The percentage of AFB1 bound for each concentration of the adsorbent (100, 10, 1, 0.5, 0.25, and 0.05 mg/10 mL) was, 100, 91.1, 81.8, 75.4, 40.1, and 8.8%, respectively. The higher the concentration of the HSCAS, the greater was its ability to bind AFB1. This result agrees with Phillips et al. (1995), who found that addition of HSCAS to the diet of animal species could protect the animals from the toxic effects of aflatoxin. According to Ramos and Hernández (1996), the adsorbent should have a high affinity for the specific mycotoxin, resulting in the formation of a strong complex, thus reducing the possibility of any rupture of the complex, which would result in the subsequent absorption of these fungal metabolites. The adsorbent should also have a high binding capacity to prevent saturation of the adsorbent. Protection from aflatoxin results from the binding of aflatoxins to HSCAS in the gastrointestinal tract, thereby reducing the bioavailability of the aflatoxins (Phillips et al., 1995).
Growth Performance and Organ Weights The effects of dietary treatments on growth performance and liver and kidney weights are shown in Table 2. Feed intake, BWG, FC, and relative weights of liver and kidneys of birds fed HSCAS alone were not different (P > 0.05) from those of control birds. These
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tive liver weight were calculated. Body weight gain was measured weekly, FI was determined at the end of the experiment, and FC was calculated. Serological and Histopathological Analysis. On d 21, all chicks were anesthetized with carbon dioxide. Ten chicks from each treatment (2 chicks from each replicate) were bled by cardiac puncture, and were the blood samples submitted for serum chemistries and enzyme levels. The remaining chicks (15 per treatment, 3 chicks per replicate) were killed by cervical dislocation, and the liver and kidney were removed and weighed. Liver samples were fixed in formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin stain for histopathological analysis (Luna, 1968). Liver sections from all treatment groups were examined microscopically. Individual sample numerical scores were reported using the following scoring system, based on the severity of the main aflatoxin-associated lesions (biliary hyperplasia, periportal swelling, vacuolar degeneration of hepatocytes, and periportal heterophil infiltration):
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Neeff et al. Table 1. Binding efficacy of different amounts of hydrated sodium calcium aluminosilicate (HSCAS) in 2 mg/L of aflatoxin B1 (AFB1) solution at pH 3 HSCAS amount (mg/10 mL)
Starting AFB1 concentration (µg/mL)
AFB1 in solution (HPLC) (µg/L)
AFB1 bound (%)
AFB1 adsorbed (mg/g)
2.0 2.0 2.0 2.0 2.0 2.0
0.010 0.178 0.365 0.492 1.198 1.825
100 91.1 81.8 75.4 40.1 8.80
0.20 1.82 16.35 30.16 32.07 35.05
100 10 1 0.5 0.25 0.05
Compared with the current study, the adsorbents used in studies by Ledoux et al. (1999) and Shi et al. (2006) were effective in ameliorating the toxic effects of aflatoxin on relative liver and kidney weights. Similar to the current study, Kubena et al. (1998) also did not observe any benefits to relative organ weights using 0.25% of an adsorbent. The liver is considered the target organ during aflatoxicosis in poultry because the relative liver weight is significantly increased by lower levels of aflatoxin compared with any other organ (Huff et al., 1986) and becomes pale with rounded margins (Ledoux et al., 1999).
Serum Biochemistry The results of serum biochemistry are summarized in Table 3. Significant differences were observed between the 4 treatments for GLU, ALB, TP, GLOB, and Ca, and the birds fed 2.5 mg/kg of AFB1 and 0.5% HSCAS plus 2.5 mg/kg of AFB1 had the worst results. The most sensitive parameter to aflatoxin-induced change in a study by Huff et al. (1986) was serum ALB levels. In the control group, ALB levels increased between d 3 and 6, then remained constant through d 21 (approximately 1.30 g/100 mL). Decreases in levels of serum TP and ALB are indicators of aflatoxicosis, and increased
Table 2. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to ameliorate the toxic effects of aflatoxin B1 (AFB1) on performance of broiler chicks from hatch to d 211 Treatment AFB1 (mg/kg) HSCAS (%) 0 0 0 0.5 2.5 0 2.5 0.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 0 2.5 HSCAS 0 0.5
Feed intake1 (g)
BW gain1 (g)
Feed conversion1 (g:g)
993a 970a 747b 794b 19
800a 772a 581b 651b 22
1.243 1.272 1.396 1.246 0.064
<0.0001 0.5440 0.0804 981.63a 770.54b 870.23a 881.94a
<0.0001 0.3355 0.0381
0.3351 0.3535 0.1821
785.85a 616.09b 690.26a 711.68a
1.26a 1.32a 1.32a 1.26a
Mortality1 (%) 0 4 12 8 0.10 0.1362 1.0000 0.4442 2.0a 10.0a 6.0a 6.0a
Relative liver weight2 Relative kidney weight2 (g/100 g of BW) (g/100 g of BW) 2.668b 2.735b 4.012a 3.886a 0.089
0.804b 0.771b 1.277a 1.354a 0.043
<0.0001 0.7454 0.2971
<0.0001 0.6221 0.2241
2.70b 3.95a 3.34a 3.31a
0.79b 1.32a 1.04a 1.06a
a,bValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 5 chicks each. 2Data are means of 5 replicate pens of 3 chicks each.
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results indicate that the HSCAS at 0.5% of the diet did not negatively affect the nutritional integrity of the diet, indicating that this dietary concentration of the HSCAS was safe to feed. Compared with controls, birds fed 2.5 mg of AFB1/kg of diet alone had reduced (P < 0.05) FI and BWG, poor FC, and increased (P < 0.05) relative liver and kidney weights. The addition of 0.5% HSCAS to the AFB1 diet was not effective (P > 0.05) statistically in reducing or preventing the negative effects of AFB1 on growth performance or relative organ weights. However, it should be noted that birds fed AFB1 alone had a 27% reduction in BWG, whereas the birds fed both AFB1 and HSCAS only had an 18% reduction in BWG, suggesting that the HSCAS was partially effective in alleviating the decrease in BWG caused by AFB1. The most significant economic effect of aflatoxicosis in poultry is the reduced growth rate (Huff et al., 1986). The current results are in contrast to a previous study by Ledoux et al. (1999) in which 1% of an HSCAS was completely effective in preventing the toxic effects of aflatoxin. The higher concentration of HSCAS (1%) used by Ledoux et al. (1999) may well account for the lack of efficacy of the HSCAS used in the current study. However, it has been shown that not all HSCAS are equally effective and some are more effective at higher concentrations.
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Table 3. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to ameliorate the toxic effects of aflatoxin B1 (AFB1) on blood chemistries of broiler chicks1 Treatment AFB1 (mg/kg)
GLU (mg/dL)
ALB (g/dL)
TP (g/dL)
GLOB (g/dL)
CA (mg/dL)
0 0.5 0 0.5
351ab 419a 226c 264bc 26
0.90a 0.94a 0.42b 0.56b 0.04
2.04a 2.32a 1.14b 1.40b 0.08
1.14b 1.40a 0.74c 0.88c 0.05
10.52ab 10.76a 9.28c 9.62bc 0.28
284 283 216 218 40
11.4 12.6 12.2 12.8 1.0
8.26 9.00 7.84 8.78 0.94
<0.0001 0.0612 0.5970 385.00a 245.00b 288.50a 341.50a
<0.0001 0.0416 0.2361 0.92a 0.49b 0.66b 0.75a
<0.0001 0.0029 0.8980 2.18a 1.27b 1.59b 1.86a
<0.0001 0.0013 0.2609 1.27a 0.81b 0.94b 1.14a
0.0006 0.3114 0.8592 10.64a 9.45b 9.90a 10.19a
0.1179 0.9844 0.9688 283.60a 217.00a 249.90a 250.70a
0.6195 0.3756 0.7652 12.00a 12.50a 11.80a 12.70a
0.7372 0.3833 0.9163 8.63a 8.31a 8.05a 8.89a
0 2.5 0 0.5
AST (U/L)
GGT (U/L)
UA (mg/dL)
a–cValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 2 chicks per pen. GLU = glucose; ALB = albumin; TP = total protein; GLOB = globulin; CA = calcium; AST = aspartate aminotransferase; GGT = gamma glutamyl transferase; UA = uric acid.
activities of serum GGT and AST have been reported to be sensitive serological indicators of liver and kidney toxicity. It should be pointed out that the clinical signs for aflatoxicosis depend on bird age and nutritional or health status at the time of exposure to contaminated feed (Shi et al., 2006). The toxic effects of AFB1 on serum chemistries were, in general, according to previous studies (Huff et al., 1986; Ledoux et al., 1999; Shi et al., 2006). Kubena et al. (1990) reported that the alterations in some serum biochemical and enzymatic parameters as a result of aflatoxicosis were not alleviated by HSCAS; our results are in agreement with theirs.
Histopathology No significant microscopic lesions were evident in liver sections of controls or birds fed HSCAS alone. Mild, moderate, or severe lesions indicative of aflatoxicosis were seen in liver sections examined in birds fed AFB1 alone or birds fed HSCAS plus AFB1. These hepatic lesions included moderate biliary hyperplasia, mild to severe periportal swelling, and vacuolar degeneration of hepatocytes, mild to moderate regeneration of hepatocytes, and mild to severe periportal heterophil infiltration. The mean liver lesion score of birds from control and HSCAS groups was 1.0. The addition of the HSCAS to the AFB1 diet was not effective in reducing the severity of the liver lesions caused by AFB1, as the mean liver score of groups treated with AFB1 alone or in combination with HSCAS was 3.0. These results are in contrast to a previous study by Ledoux et al. (1999), who reported that 1% HSCAS was completely effective in preventing liver lesions caused by 4 mg of AFB1/kg of diet.
Aflatoxin Residues The values for AFB1 residues in liver and kidney of broilers fed dietary treatments are presented in Tables 4 and 5, respectively. Concentrations of AFB1, AFL, AFG1, and AFB2 were lower (P < 0.05) in livers of birds fed AFB1 plus HSCAS compared with birds fed AFB1 alone. Concentrations of AFM1 were all lower in birds fed AFB1 plus HSCAS compared with birds fed AFB1 alone. No AFB1 residues were found in the livers of birds fed the BD (diet A) or the birds fed the BD plus HSCAS. Concentrations of AFB1 were also lower (P < 0.05) in kidneys of birds fed AFB1 plus HSCAS compared with those fed AFB1 only. Concentrations of AFM1, AFG1, AFG2, and AFB2 were numerically, but not statistically, lower in birds fed AFB1 plus HSCAS compared with birds fed AFB1 alone. The AFL was the only residue in the kidney that was not significantly different (P > 0.05) among treatments. No AFB1 residues were found in the kidney of birds fed BD or birds fed BD plus HSCAS. Fernández et al. (1994) fed broiler chickens a diet containing 2.5 mg/kg of AFB1 for 32 d, and the mean aflatoxin residues found in liver were 0.23 µg/kg of AFB1, 0.06 µg/kg of AFM1, 0.23 µg/kg of AFL, and 0.16 µg/kg of AFG1. In kidney, they found 0.25 µg/ kg of AFB1 and 0.12 µg/kg of AFM1. These values are much lower compared with our results for the diet containing 2.5 mg/kg of aflatoxin. Other studies have also reported lower concentrations of AFB1 residues after ingestion of aflatoxin containing feeds. Bintvihok and Kositcharoenkul (2006), fed broilers diets containing AFB1, and AFB1 plus calcium propionate for 3 d, and reported AFB1 residues of 0.05 ±
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0 0 2.5 2.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 HSCAS
HSCAS (%)
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Table 4. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to reduce aflatoxin residues in liver of broiler chicks fed aflatoxin B1 (AFB1) from hatch to d 211 Treatment AFB1 (mg/kg)
HSCAS (%)
AFB1 (µg/kg)
AFB2 (µg/kg)
AFL (µg/kg)
AFG1 (µg/kg)
AFG2 (µg/kg)
0b 0b 0.21a 0.12a 0.02
0b 0b 8.32a 1.49b 1.08 <0.0001 0.0741 0.0741 0.000b 0.163a 0.104a 0.059a
0b 0b 0.48a 0.07b 0.09 <0.0001 0.0008 0.0008 0.000b 2.243a 1.974a 0.269b
0b 0b 2.34a 0b 0.28 0.0003 0.0062 0.0062 0.000b 4.906a 4.162a 0.744b
0b 0b 3.95a 0.54b 0.41 0.1220 0.1220 0.1220 0.000a 0.014a 0.014a 0.000a
0a 0a 0.03a 0a 0.01 0.0007 0.0007 0.0007 0.000b 1.172a 1.172a 0.000b
1.0 1.0 1.0 428.80a 428.80a 428.80a 428.80a
a,bValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 3 chicks per pen. AFM = aflatoxin M ; AFL = aflatoxicol; AFG = aflatoxin G ; AFG = aflatoxin G ; 1 1 2 2 1 1 AFB2 = aflatoxin B2.
0.03 µg/kg and 0.13 ± 0.05 µg/kg in livers of birds fed 50 µg/kg of AFB1 and 100 µg/kg of AFB1, respectively. For AFM1 residues in liver, the values were 0.10 ± 0.04 µg/kg, 0.32 ± 0.13 µg/kg, 0.05 ± 0.03 µg/kg, and 0.05 ± 0.04 µg/kg in birds fed 50 µg/kg of AFB1, 100 µg/kg of AFB1, 100 µg/kg of AFB1 + 0.25% calcium propionate, and 100 µg/kg of AFB1 + 0.5% calcium propionate, respectively. Hussain et al. (2010) fed birds for 7, 14, and 28 d of age with 0, 1.6, 3.2, and 6.4 mg/ kg of AFB1 for 7 d and detected AFB1 residues in liver from all treatments, with the highest value (3.74 µg/ kg) observed in birds at 7 d of age. They found that older birds had lower tissue residues of AFB1 compared with younger birds, suggesting that older birds developed a more efficient mechanism of metabolizing AFB1.
Another possibility is that tissue dilution occurred due to the larger bird size. According to Phillips (1999), the protective effect of HSCAS is the result of rapid binding of aflatoxin by HSCAS in the gastrointestinal tract of chickens, thus preventing its absorption and normal distribution to the liver. Our results showed that HSCAS was effective in reducing aflatoxin residues, especially in liver. The biotransformation of AFB1 into AFM1 and AFL in the liver also decreased in broilers receiving HSCAS, therefore lowering the toxicity of AFB1. In conclusion, the results demonstrate that HSCAS was effective in binding AFB1 in vitro. The addition of HSCAS to the feed would potentially prevent aflatoxicosis by inducing a HSCAS-AFB1 complex, which
Table 5. Efficacy of hydrated sodium calcium aluminosilicate (HSCAS) to reduce aflatoxin residues in kidney of broiler chicks fed aflatoxin B1 (AFB1) from hatch to d 211 Treatment AFB1 (mg/kg)
HSCAS (%)
0 0 0 0.5 2.5 0 2.5 0.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 0 2.5 HSCAS 0 0.5
AFM1 (µg/kg)
AFB1 (µg/kg)
AFB2 (µg/kg)
AFL (µg/kg)
AFG1 (µg/kg)
AFG2 (µg/kg)
0b 0b 0.68a 0.42a 0.09 <0.0001 0.1441 0.1441 0.000b 0.550a 0.341a 0.209a
0c 0c 16.16a 7.52b 1.59 <0.0001 0.0152 0.0152 0.000b 11.84a 8.080a 3.762b
0b 0b 0.91a 0.66ab 0.19 0.0008 0.5189 0.5189 0.000b 0.786a 0.456a 0.330a
0a 0a 0.48a 0.15a 0.14 0.0446 0.2632 0.2632 0.000b 0.312a 0.239a 0.073a
0b 0b 6.94a 4.19a 0.73 <0.0001 0.0796 0.0796 0.000b 5.566a 3.470a 2.096a
0b 0b 0.16a 0.12ab 0.03 0.0004 0.5384 0.5384 0.000b 0.142a 0.081a 0.061a
a–cValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.05). 1Data are means of 5 replicate pens of 3 chicks per pen. AFM = aflatoxin M ; AFL = aflatoxicol; AFG = aflatoxin G ; AFG = aflatoxin G ; 1 1 2 2 1 1 AFB2 = aflatoxin B2.
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0 0 0 0.5 2.5 0 2.5 0.5 SEM Source of variation (P-value) AFB1 HSCAS AFB1 × HSCAS Main effect mean AFB1 0 2.5 HSCAS 0 0.5
AFM1 (µg/kg)
RESEARCH NOTE
would be able to restrict gastrointestinal absorption of AFB1. However, histopathology data from the in vivo study indicated that HSCAS did not completely protect broilers against aflatoxicosis, but was effective in reducing aflatoxin residues in liver and kidney of chicks fed 2.5 mg of AFB1/kg of diet from hatch to d 21. The decrease in the bioavailability of AFB1 caused by the HSCAS led to a reduction in the aflatoxin residues in liver and kidney, but was not enough to prevent the toxic effects of AFB1 in broilers.
ACKNOWLEDGMENTS
REFERENCES Biehl, M. L., and W. B. Buck. 1987. Chemical contaminants: Their metabolism and their residues. J. Food Prot. 50:1058–1073. Bintvihok, A., and S. Kositcharoenkul. 2006. Effect of dietary calcium propionate on performance, hepatic enzyme activities and aflatoxin residues in broilers fed a diet containing low levels of aflatoxin B1. Toxicon 47:41–46. Chiavaro, E., C. Cacchioli, E. Berni, and E. Spotti. 2005. Immunoaffinity clean-up and direct fluorescence measurement of aflatoxin B1 and M1 in pig liver: comparison with high-performance liquid chromatography determination. Food Addit. Contam. 22:1154– 1161. Fernández, A., M. T. Verde, M. Gascón, J. J. Ramos, and J. Gomez. 1994. Aflatoxin and its metabolites in tissues from laying hens and broiler chickens fed a contaminated diet. J. Sci. Food Agric. 65:407–414. Huff, W. E., L. F. Kubena, R. B. Harvey, D. E. Corrier, and H. H. Mollenhauer. 1986. Progression of aflatoxicosis in broiler chickens. Poult. Sci. 65:1891–1899. Hussain, Z., M. Z. Khan, A. Khan, I. Javed, M. K. Saleemi, S. Mahmood, and M. R. Asi. 2010. Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels. Food Chem. Toxicol. 48:3304–3307. Kubena, L. F., R. B. Harvey, R. H. Bailey, S. A. Buckley, and G. E. Rottinghaus. 1998. Effects of a hydrated sodium calcium aluminosilicate (T-Bind) on mycotoxicosis in young broiler chickens. Poult. Sci. 77:1502–1509. Kubena, L. F., R. B. Harvey, T. D. Phillips, D. E. Corrier, and W. E. Huff. 1990. Diminution of aflatoxicosis in growing chikens by
the dietary addition of a hydrated sodium calcium aluminosilicate. Poult. Sci. 69:727–735. Ledoux, D. R., and G. E. Rottinghaus. 1999. In vitro and in vivo testing of adsorbents for detoxifying mycotoxins in contaminated feedstuffs. Pages 369–379 in Biotechnology in the Feed Industry. Proceedings of the 15th Annual Symposium. T. P. Lyons and K. A. Jacques, ed. Nottingham University Press, Nottingham, UK. Ledoux, D. R., G. E. Rottinghaus, A. J. Bermudez, and M. AlonsoDebolt. 1999. Efficacy of hydrated sodium calcium aluminosilicate to ameliorate the toxic effects of aflatoxin in broiler chicks. Poult. Sci. 78:204–210. Luna, G. L. 1968. Manual of Histologic Staining Methods of the Armed Forces. 3rd ed. McGraw-Hill, New York, NY. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Park, D. L., and A. E. Pohland. 1986. A rationale for the control of aflatoxin in animal feeds. Pages 473–482 in Mycotoxins and Phycotoxins. P. S. Steyn and R. Vleggaar, ed. Elsevier Applied Science, Amsterdam, the Netherlands. Phillips, T. D. 1999. Dietary clay in the chemoprevention of aflatoxin-induced disease. Toxicol. Sci. 52:118–126. Phillips, T. D., A. B. Sarr, and P. G. Grant. 1995. Selective chemisorption and detoxification of aflatoxins by phillosilicate clay. Nat. Toxins 3:204–213. Ramos, A. J., and E. Hernández. 1996. In vitro aflatoxin adsorption by means of montmorillonite silicate. A study of adsorption isotherms. Anim. Feed Sci. Technol. 62:263–269. Rottinghaus, G. E., C. E. Coatney, and H. C. Minor. 1992. A rapid sensitive thin layer chromatography procedure for the detection of fumonisin B1 and B2. J. Vet. Diagn. Invest. 4:326–329. Rottinghaus, G. E., B. Olsen, and G. D. Osweiler. 1982. Rapid screening method for aflatoxin B1, zearalenone, ochratoxin A, T-2 toxin, diacetoxyscirpenol and vomitoxin. Pages 477–484 in Proceedings of the 25th Animal American Association of Veterinary Laboratory Diagnosticians, Nashville, TN. SAS Institute Inc. 1992. SAS® User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. Scott, P. M. 1990. Natural poisons. Pages 1184–1213 in Official Methods of Analysis of the Association of Official Analytical Chemists. 15th ed. K. Helrich, ed. Assoc. Off. Anal. Chem., Arlington, VA. Shi, Y. H., Z. R. Xu, J. L. Feng, and C. Z. Wang. 2006. Efficacy of modified montmorillonite nanocomposite to reduce the toxicity of aflatoxin in broiler chicks. Anim. Feed Sci. Technol. 129:138–148. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson. 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425–428. Snedecor, G. W., and W. G. Cochran. 1967. Statistical Methods. 6th ed. Iowa State Univ. Press, Ames.
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
The authors thank Conselho Nacional de Desenvolvimento Científico (CNPq), Brazil, grant no. 134068/2010-6, for financial support.
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