Efficacy of a Hydrated Sodium Calcium Aluminosilicate to Ameliorate the Toxic Effects of Aflatoxin in Broiler Chicks 1

Transcription

1 Efficacy of a Hydrated Sodium Calcium Aluminosilicate to Ameliorate the Toxic Effects of Aflatoxin in Broiler Chicks 1 D. R. LEDOUX,*,2,3 G. E. ROTTINGHAUS,*,4 A. J. BERMUDEZ,*,4 and M. ALONSO-DEBOLT *Fusarium\Poultry Research Laboratory, University of Missouri, Columbia, Missouri 65211, and Milwhite, Inc., Houston, Texas ABSTRACT In vitro and in vivo studies were conducted to evaluate the efficacy of a hydrated sodium calcium aluminosilicate (Improved Milbond-TX, IMTX) to alleviate the toxic effects of aflatoxin (AF) B 1 in chicks. In vitro results indicated that IMTX was able to bind 100% of AFB 1 at ph 3 to 9. In the in vivo study, five pen replicates of six chicks were assigned to each of four dietary treatments, which included: 1) basal diet containing neither IMTX nor AFB 1 (control); 2) basal diet supplemented with 1% IMTX; 3) basal diet supplemented with 4 mg AFB 1 /kg diet; and 4) basal diet supplemented with 1% IMTX and 4 mg AFB 1 /kg diet. The addition of IMTX to chick diets at a level of 1% did not negatively affect chick performance, organ weights, or serum chemistry, or cause pathological changes. Improved Milbond-TX completely prevented the reduced performance, changes in organ weights, serum chemistry changes, and gross pathology observed in chicks fed AFB 1. The IMTX dramatically reduced the incidence and severity of the hepatic histopathology changes associated with aflatoxicosis and completely prevented the renal lesions of aflatoxicosis. These results indicated that IMTX was effective in preventing the toxic effects of AF that may be present in poultry rations at levels up to 4 mg/kg feed. (Key words: adsorbent, aflatoxin, chick, aluminosilicate) 1998 Poultry Science 77: INTRODUCTION Aflatoxins (AF), a class of mycotoxins produced by fungal species of the genus Aspergillus (A. flavus and A. parasiticus), are contaminants in feed ingredients routinely used for poultry rations. Major forms of AF include B 1,B 2,G 1, and G 2, with AFB 1 being the most common and biologically active component (Busby and Wogan, 1981). Aflatoxins cause a variety of effects in poultry, including poor performance, liver pathology, immunosuppression, and changes in relative organ weights (Edds and Bortell, 1983; Kubena et al., 1990, 1993). Livers characteristically are pale and enlarged as a result of aflatoxicosis, with microscopic changes including fatty change, hepatic necrosis, and biliary hyperplasia (Hoerr, 1997). Measures used by the livestock industry to protect animals from the toxic effects of AF include grain testing, use of mold inhibitors, fermentation, microbial Received for publication July 6, Accepted for publication October 6, Contribution Number of the Missouri Agricultural Experiment Station. 2Department of Animal Sciences. 3 To whom correspondence should be addressed: LedouxD@missouri.edu 4Veterinary Medical Diagnostic Laboratory. Abbreviation Key: AF = aflatoxin; IMTX = Improved Milbond-TX. 204 inactivation, physical separation, thermal inactivation, irradiation, ammoniation (CAST, 1989), ozone degradation (Mckenzie et al., 1997), and the use of adsorbents (Masimanco et al., 1973; Phillips et al., 1990a,b). Unfortunately, most of these measures are costly, timeconsuming, and only partially effective. At the present time, one of the more promising and practical approaches is the use of adsorbents. Selected adsorbents added to AF-contaminated feeds can sequester AF during the digestive process, allowing the mycotoxin to pass harmlessly through the animal (Davidson et al., 1987; Phillips et al., 1990a,b). The major advantages of these adsorbents include expense, safety, and easy administration through addition to animal feeds. However, not all adsorbents are equally effective in protecting poultry against the toxic effects of AF and several adsorbents have been shown to impair nutrient utilization (Chung et al., 1990; Kubena et al., 1993; Scheideler, 1993). Recently, Dale (1998) noted that many of the adsorbents on the market today have not been adequately tested for in vivo efficacy, but are used based only on in vitro data. However, as Scheideler (1993) suggested, in vitro tests may not always be a reliable indicator of an adsorbent s ability to bind AF. Therefore,

2 it is important that adsorbent supplementation be subjected to in vivo evaluation both with respect to efficacy and to determine whether impaired nutrient utilization from diets can occur. The objectives of this research were to determine the efficacy of Improved Milbond-TX (IMTX) to ameliorate the toxic effects of AFB 1 present in poultry rations and to demonstrate that the addition of IMTX to poultry diets would not negatively affect poultry performance. MATERIALS AND METHODS In Vitro Studies Aflatoxin B 1 was purchased from Sigma Chemical Co.5 A primary stock solution of AFB 1 (1,000 mg/l) was prepared in methanol. Aflatoxin test solutions for adsorption tests were prepared by adding methanol stock solutions to 0.1 M phosphate buffer adjusted to the desired ph. The binding ability of IMTX was tested using the following general procedure. Improved Milbond-TX (0.5-g aliquots, run in duplicate) was placed in screw cap polypropylene bottles and 50, 100, or 250 ml of 0.1 M phosphate buffer (ph 3) containing 5 mg/ml AFB 1 was added. The bottles were placed on a rotator shaker for 30 min at room temperature. A 5-mL aliquot of the AFB 1 test solution was then centrifuged at 3,000 rpm for 10 min and 2 ml of the aqueous supernatant was removed for AFB 1 analysis. An aliquot of the original buffered AFB 1 test solution was used as the HPLC standard. Improved Milbond-TX was tested for its ability to bind AFB 1 at ph 3, 5, 7, and 9 using the following general procedure. Duplicate aliquots of 0.1 M phosphate buffer (adjusted to ph 3, 5, 7, and 9) containing 2 mg/ml AFB 1 in solution (50 ml) was added to 100 ml screw cap polypropylene bottles to which had been added 0.5 g IMTX. The bottles were placed on a rotator shaker for 30 min at room temperature. A 5-mL aliquot of AFB 1 test solution was then centrifuged at 3,000 rpm for 10 min and 2 ml of the aqueous supernatant was removed for AFB 1 analysis. An aliquot of the original buffered AFB 1 test solution was used as the HPLC standard at each ph. Analysis of AFB 1 by HPLC analysis was performed on a Perkin-Elmer Model 250 liquid chromatograph pump equipped with an ISS200 Perkin-Elmer autosampler, a Perkin-Elmer 8.3 cm C 18 cartridge (3 mm particle size), and fluorescence detection with a Perkin-Elmer LS-4 fluorescence spectrophotometer.6 Flow rate of mobile phase was 1 ml/min. The mobile phase was a 40:17:2 mixture of water:methanol:isopropanol and detection wavelengths were 365 nm (excitation) and 430 nm (emission). 5Sigma Chemical Co., St. Louis, MO Perkin-Elmer, Norwalk, CT Kodak Ektachem Analyzer, Eastman Kodak Co., Rochester, NY ALUMINOSILICATE AND AFLATOXICOSIS 205 In Vivo Study One hundred and twenty day-old male broiler chicks were purchased from a commercial hatchery, weighed, wing-banded, and assigned to pens in stainless steel chick batteries. Birds were maintained on a 24-h continuouslight schedule and allowed ad libitum access to feed and water. The animal care and use protocol was reviewed and approved by the University of Missouri-Columbia Animal Care and Use Committee. A completely randomized design was used with five pen replicates of six chicks assigned to each of four dietary treatments. Dietary treatments evaluated included: 1) basal diet containing neither IMTX nor AFB 1 (control); 2) basal diet supplemented with 1% IMTX; 3) basal diet supplemented with 4 mg AFB 1 /kg diet; and 4) basal diet supplemented with 1% IMTX and 4 mg AFB 1 /kg diet. The basal diet was a commercial corn-soybean meal diet formulated to meet or exceed the nutritional requirements of growing chicks as recommended by the NRC (1994). Dietary AFB 1 concentrations were confirmed by analysis and all diets were screened by the method of Rottinghaus et al. (1982) for the presence of AF, citrinin, T-2 toxin, vomitoxin, sterigmatocystin, zearalenone, the fumonisins, diacetoxyscirpenol, and ochratoxin A prior to the start of the experiment. Birds were individually weighed on a weekly basis with feed consumption recorded weekly and mortality recorded as it occurred. In addition, birds were inspected daily and any health-related problems recorded. On Day 21, 10 chicks (five replicates of 2 chicks each) from each treatment were bled by cardiac puncture and samples used for serum biochemistry analyses including albumin, total protein, uric acid, cholesterol, triglycerides, creatinine, glucose, calcium, phosphorus, urea nitrogen, aspartate transaminase, and gamma glutamyltransferase.7 Fifteen chicks (five replicates of three chicks each) from each treatment were killed by cervical dislocation and the liver, kidney, spleen, pancreas, bursa of Fabricius, proventriculus, and gizzard were removed and weighed. Samples of the liver, kidney, spleen, brain, pancreas, bursa of Fabricius, proximal tibia, esophagus, duodenum, jejunum, ileum, cecum, skeletal muscle, and thymus were taken from five randomly selected birds per treatment for histopathological examination. Tissue samples were fixed in 10% neutral buffered formalin. Fixed tissues were trimmed, embedded in paraffin, sectioned at 4 mm, and stained with hematoxylin and eosin. All birds were examined for signs of gross pathology due either to AF or resulting from nutritional deficiencies that may have been caused by addition of IMTX. Brains were examined for the lesions of cerebellar swelling, edema, and hemorrhage as seen with vitamin E deficiency (Austic and Scott, 1997). The mucosal surface of the crop and esophagus was examined for the multiple white foci, indicative of squamous metaplasia of the subepithelial mucous glands, observed with vitamin A deficiency (Austic and Scott, 1997). The proximal tibial growth plate

3 206 was examined for widening of the zones of proliferation and hypertrophy, as seen in cases of vitamin D, calcium, or phosphorus deficiency (Austic and Scott, 1997). The tibia was also broken in order to evaluate bone strength. The skeletal muscles and abdominal cavity was examined for the hemorrhages observed in cases of vitamin K deficiency (Austic and Scott, 1997). The legs were also examined for bowing or perosis, which can occur with deficiencies in nicotinic acid, choline, or manganese (Austic and Scott, 1997). The skin and feathers of the chicks were examined for indications of pantothenic acid and biotin deficiency (Austic and Scott, 1997). Data were analyzed as a 2 2 factorial by analysis of variance using the General Linear Models procedure of SAS (SAS Institute, 1985). The means for treatments showing significant differences in the analysis of variance were compared using the Fisher s protected least significant difference procedure (Snedecor and Cochran, 1967). All statements of significance are based on the 0.05 level of probability. In Vitro Studies RESULTS Improved Milbond-TX was able to bind 100% of AFB 1 at ph 3, and the binding ability of IMTX for AFB 1 did not change as the ph increased from 3 to 9. In order to determine the binding ability of IMTX, the clay was exposed to three levels of AFB 1 (250, 500, and 1,250 mg) at ph 3. Aflatoxin B 1 was effectively removed (100%) at all three levels, suggesting that the clay would be a good candidate for further in vivo testing. LEDOUX ET AL. In Vivo Study Feed ingredients used to prepare diets were negative for AF, citrinin, T-2 toxin, vomitoxin, sterigmatocystin, zearalenone, the fumonisins, diacetoxyscirpenol, and ochratoxin A. The effects of dietary treatments on chick performance from Day 1 to 21 are presented in Table 1. Chicks fed IMTX consumed similar amounts of feed (979 vs 964 g), grew as well (788 vs 779 g), and were as efficient (1.24 vs 1.24 g:g) as chicks fed the control diet. In contrast, chicks fed AFB 1 alone consumed 22% less feed (P < 0.05; 746 vs 964 g) and gained 25% less weight (P < 0.05; 583 vs 779 g) than control chicks. Chicks fed the combination AFB 1 /IMTX diet consumed similar amounts of feed (1000 vs 964 g), grew as well (796 vs 779 g), and were as efficient (1.26 vs 1.24 g:g) as control chicks. Chicks fed the IMTX diet had organ weights similar to those of control chicks (Table 2). Compared with controls, chicks fed AFB 1 alone had heavier (P < 0.05) relative liver, heart, kidney, proventriculus, and pancreas weights, whereas chicks fed the combination AFB 1 /IMTX diet had similar organ weights to those of control chicks (Table 2). Relative weights of spleen, bursa of Fabricius, and gizzard were not affected (P > 0.05) by dietary treatments (data not shown). Effects of dietary treatments on serum chemistry are presented in Table 3. Serum chemistry values of chicks fed IMTX alone were similar to those of control chicks. Compared to controls, serum concentrations of calcium, phosphorus, cholesterol, glucose, albumen, total protein, and globulin were decreased (P < 0.05) in chicks fed AFB 1 alone, whereas serum concentrations of sodium and chloride were increased (P < 0.05). In contrast, with the exception of glucose and cholesterol, serum chemistry TABLE 1. Effects of Improved Milbond-TX (IMTX) on performance of broilers fed aflatoxin B 1 (AFB 1 ) 1 Treatment Feed Body weight AFB 1 IMTX intake gain Feed:gain (mg/kg) (%) (g) (g:g) A 779 A A 788 A B 583 B ,000 A 796 A 1.26 SEM Probabilities Source of variation AFB IMTX AFB 1 IMTX Main effect means AFB A 784 A B 689 B 1.24 IMTX B 681 B A 792 A 1.25 A,BValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < 0.001). 1Data are means of five replicate pens of six chicks each.

4 ALUMINOSILICATE AND AFLATOXICOSIS 207 TABLE 2. Effects of Improved Milbond-TX (IMTX) on relative liver, heart, kidney, proventriculus, and pancreas weights of broilers fed aflatoxin B 1 (AFB 1 ) 1 Treatment AFB 1 IMTX Liver Heart Kidney Proventriculus Pancreas (mg/kg) (%) (g/100 g BW) B 0.66 B 0.92 B 0.52 B 0.28 B B 0.68 B 0.93 B 0.51 B 0.28 B A 0.85 A 1.53 A 0.65 A 0.33 A B 0.69 B 0.94 B 0.50 B 0.26 B SEM Probabilities Source of variation AFB IMTX AFB 1 IMTX Main effect means AFB B 0.67 B 0.93 B 0.51 B A 0.77 A 1.24 A 0.58 A 0.30 IMTX A 0.75 A 1.23 A 0.58 A 0.30 A B 0.69 B 0.93 B 0.51 B 0.27 B A,BValues within each column for interactive means or between each pair of main effect means with no common superscript differ significantly (P < ). 1Data are means of five replicate pens of three chicks each. values of chicks fed the combination AFB 1 /IMTX were similar to those of control chicks. No signs of nutritional deficiencies were found in any of the organs examined in any of the treatments. A total of five chicks (two controls; two AFB 1 ; and one AFB 1 /IMTX) died during the course of the study. These deaths did not appear to be related to treatment. No treatment-related gross lesions were observed in control chicks, chicks fed IMTX, or chicks fed the AFB 1 /IMTX combination diet. Chicks fed AFB 1 alone had enlarged pale livers with rounded margins. Microscopic sections of brain, bursa of Fabricius, thymus, spleen, duodenum, pancreas, esophagus, jejunum, cecum, ileum, skeletal muscle, and proximal tibia were unremarkable in all treatment groups. Sections of liver and kidney were also unremarkable in the chicks fed the control diet and the 1% IMTX diet. Moderate lesions of aflatoxicosis were evident in all chicks fed AFB 1 alone and mild microscopic lesions of aflatoxicosis were noted in two of five of the chicks fed the AFB 1 /IMTX diet. Chicks fed AFB 1 alone had moderate to severe periportal cytoplasmic vacuolation of hepatocytes (fatty change) TABLE 3. Effects of Improved Milbond-TX (IMTX) on serum chemistry of broilers fed aflatoxin B 1 (AFB 1 ) 1 Treatment AFB 1 IMTX Ca P CHOL GLC Na Cl ALB TPROT GLOB (mg/kg) (%) (mg/dl) (mmol/l) (g/dl) ab 8.17 a 102 b 357 a 139 b 110 b 1.26 a 2.58 a 1.33 a b 7.35 ab 106 ab 288 ab 140 b 112 ab 1.27 a 2.63 a 1.37 a c 6.67 b 62 c 230 b 144 a 115 a 0.91 b 1.67 b 0.76 b a 7.57 ab 117 a 250 b 139 b 112 ab 1.28 a 2.63 a 1.33 a SEM Probabilities Source of variation AFB IMTX AFB 1 IMTX Main effect means AFB a 104 a 322 a 139 b 111 b 1.27 a 2.61 a 1.35 a b 89 b 240 b 142 a 114 a 1.10 b 2.15 b 1.05 b IMTX b b a b 2.12 b 1.05 b a a b a 2.63 a 1.35 a 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 five replicate pens of three chicks each. CHOL = cholesterol; GLC = glucose; ALB = albumin; TPROT = total protein; GLOB = globulin.

5 208 LEDOUX ET AL. FIGURE 1. Photomicrographs (hematoxylin and eosin) of liver sections from chicks fed control ration (A), ration containing aflatoxin (AF)B 1 (B), and ration containing AFB 1 /Improved Milbond-TX (IMTX) (C). Liver sections from chicks fed AFB 1 had moderate to severe periportal cytoplasmic vacuolation of hepatocytes (indicated by arrows). Liver sections in the other two treatments were either normal (as demonstrated in photomicrographs A and C) or in two of five sections of the AFB 1 /IMTX treatment, had mild biliary hyperplasia. Bar equals 20 mm. with moderate periportal heterophil infiltration (Figure 1). Two of five chicks fed the AFB 1 /IMTX diet had mild biliary hyperplasia. Chicks fed AFB 1 alone also had significant renal pathology with thickening of the glomerular capillary basement membrane (Figure 2) and mild heterophil infiltration of the glomeruli. Sections of kidney were normal in chicks fed the AFB 1 /IMTX diet. DISCUSSION In vitro studies showed that AFB 1 was completely bound by IMTX at the concentrations (250 to 1,250 mg) tested and over a ph range of 3 to 9. These results suggested that IMTX would be a good candidate for further in vivo testing. FIGURE 2. Photomicrographs (hematoxylin and eosin) of kidney sections from chicks fed control ration (A), ration containing aflatoxin (AF) B 1 (B), and ration containing AFB 1 and Improved Milbond-Tex (IMTX) (C). Kidney sections from chicks fed AFB 1 had a marked thickening of the glomerular capillary basement membrane (indicated by arrows), whereas kidney sections in the other two treatments were normal. Bar equals 15 mm.

6 ALUMINOSILICATE AND AFLATOXICOSIS 209 Chick performance was not negatively affected by the addition of 1% IMTX to the basal diet, and the absence of any signs of nutritional deficiencies in any of the organs examined indicated that IMTX did not negatively affect dietary nutrients (such as minerals and vitamins) at this dietary inclusion level. Lower feed intakes (22%) and body weight gains (25%) observed in chicks fed AFB 1 alone were consistent with previous reports on the performance depressing effects of AF (Edds and Bortel, 1983; Kubena et al., 1990). The addition of IMTX to the AFB 1 diet completely reversed the growth-depressing effects of AFB 1. Amelioration of the growth-depressing effect of AFB 1 has been observed in chicks fed adsorbents such as the hydrated sodium calcium aluminosilicates. These adsorbents have been shown to bind AF in the digestive tract, allowing the mycotoxin to pass harmlessly through the animal (Davidson et al., 1987; Phillips et al., 1990a,b). Increases in the relative weights of liver, heart, kidney, proventriculus, and pancreas of broilers fed AFB 1 alone in the present study have been reported previously (Kubena et al., 1993). In the present study, addition of IMTX to the AFB 1 diet prevented the increased organ weights observed in chicks fed AFB 1 alone. In general, adsorbents have been effective in preventing or ameliorating changes in organ weights observed in chicks fed AF (Kubena et al., 1990, 1993). Decreases in serum albumin, globulin, and total protein are indicators of impaired protein synthesis (Tung et al., 1975), and have been observed in chicks suffering from aflatoxicosis (Kubena et al., 1993). The addition of IMTX to the AFB 1 diet prevented the decrease in these AF-sensitive serum proteins in the present study. Improved Milbond-TX was also effective in preventing changes in other serum chemistries observed in chicks fed AFB 1 alone. Gross and histopathology lesions observed with AFB 1 in this study are similar to those reported in the literature (Hoerr, 1997; Randall and Reece, 1996). The liver is considered to be the primary target organ of AF; however, kidney lesions similar to those observed in this study have been observed previously (Randall and Reece, 1996). The addition of IMTX to the AFB 1 diet largely prevented the AFB 1 -associated pathology in the liver and completely prevented renal pathology. The mild biliary hyperplasia noted in two of five chicks in the AFB 1 /IMTX diet did not have an adverse effect on production parameters in this treatment group. The addition of IMTX to chick diets at a level of 1% did not negatively affect chick performance, organ weights, or serum chemistry, or cause any pathological changes. This result indicates that IMTX did not negatively affect dietary nutrients at this dietary inclusion level. 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