Effects of inorganic adsorbents and cyclopiazonic acid in broiler chickens

ENVIRONMENT AND HEALTH Effects of Inorganic Adsorbents and Cyclopiazonic Acid in Broiler Chickens1,2 M. R. DWYER,* L. F. KUBENA,† R. B. HARVEY,† K. MAYURA,* A. B. SARR,* S. BUCKLEY,† R. H. BAILEY,* and T. D. PHILLIPS*,3 *Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, †USDA, Agricultural Research Service, Food Animal Protection Research Laboratory, 2881 F&B Road, College Station, Texas 77845 proventriculus, and gizzard. Also, there were some alterations in hematology, serum biochemical values, and enzyme activities. Treatment with inorganic adsorbents did not effectively diminish the growth-inhibitory effects of CPA or the increased weights of organs, although there was some protection from hematological, serum biochemical, and enzymatic changes produced by CPA. The results of this study suggest that in vitro binding of CPA to clay does not accurately forecast its efficacy in vivo; the reasons for this discrepancy are not clear, but they may be related to differences in clay binding capacity and ligand selectivity for CPA in vitro vs in vivo. Predictions about the ability of inorganic adsorbents to protect chickens from the adverse effects of mycotoxins should be approached with caution and should be confirmed in vivo, paying particular attention to the potential for nutrient interactions.

(Key words: mycotoxin, cyclopiazonic acid, toxicity, inorganic adsorbents, chicken) 1997 Poultry Science 76:1141–1149

INTRODUCTION Cyclopiazonic acid (CPA), chemically classified as an indole tetramic acid, is a mycotoxin produced by several species of Aspergillus and Penicillium fungi (Cole, 1984; Dorner et al., 1984; Trucksess et al., 1987; Frisvad, 1989). The importance of CPA is implied by its natural occurrence in corn (Gallagher et al., 1978; Widiastuti et al.. 1988; Lee and Hagler, 1991; Urano et al., 1992), cheese (LeBars, 1979), and peanuts (Lansden and Davidson, 1983; Urano et al., 1992) and also its production by many strains of Aspergillus flavus alone or in combination with the aflatoxins (Gallagher et al., 1978). Aspergillus flavus is

Received for publication August 19, 1996. Accepted for publication March 12, 1997. 1Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by USDA or Texas A&M University and does not imply its approval to the exclusion of other products that may be suitable. 2Abstract published in The Toxicologist 1996; Fund. Appl. Toxicol. 30:213 (Abstr.). 3To whom correspondence should be addressed.

a major constituent of the mycoflora of corn in the southern U.S.; hence there is potential for contamination of CPA in poultry feed. Gallagher et al. (1978) tested 54 strains of A. flavus and found that 14 produced CPA alone, whereas 14 produced both CPA and aflatoxin, and 4 produced only aflatoxin. Widiastuti et al. (1988) reported levels up to 9 ppm of CPA in approximately 81% of samples analyzed at a poultry feed mill in Indonesia. Ross et al. (1991) detected natural occurrence of CPA (approximately 10 ppm) in sunflower seeds in North Dakota, after seeds were fed to sows that experienced conception problems and refusal of feed. Urano and coworkers (1992) reported co-occurrence of CPA and aflatoxins in corn and peanuts in the U.S. They found, that of 45 corn samples analyzed, 51% contained CPA, and of 50 peanut samples analyzed, 90% contained CPA. Cyclopiazonic acid has been shown to be moderately toxic to several species of animals (Purchase, 1971; Dorner et al., 1983; Lomax et al., 1984; Morrissey et al., 1985; Cullen et al., 1988; Pier et al., 1989; Smith et al., 1992). The toxic effects of CPA in broiler chicks have

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ABSTRACT Previous studies with cyclopiazonic acid (CPA) have indicated that this mycotoxin strongly adsorbs onto the surface of a naturally acidic phyllosilicate clay (AC). The objective of this study was to determine whether AC (and similar adsorbents) could protect against the toxicity of CPA in vivo. Acidic phyllosilicate clay, neutral phyllosilicate clay (NC, or hydrated sodium calcium aluminosilicate), and a common zeolite (CZ, or clinoptilolite) were evaluated. Oneday-old broiler chicks consumed diets containing 0 or 45 mg/kg CPA alone or in combination with 1% AC, NC, or CZ ad libitum from Day 1 to 21. Body weight, feed consumption, feed:gain, hematology, serum biochemical values, and enzyme activities were evaluated. Compared to controls, CPA alone reduced body weight at Day 21 by a total of 26% and resulted in a significantly higher feed:gain ratio. Toxicity of CPA was also expressed through increased relative weights of kidney,

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4Kindly provided by S. W. Peterson, USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 N. University St., Peoria, IL 61604. 5Kindly provided by Engelhard Corp., Cleveland, OH 44122. 6Kindly provided by Zeotech Corp., Albuquerque, NM 87107. 7Burdick and Jackson, Muskegon, MI 49442. 8Hewlett Packard, Palo Alto, CA 94304. 9Supelco, Inc., Supelco Chromatography Products, Supelco Park, Bellefonte, PA 16823. 10Waters Corp., Milford, MA 01757.

effectiveness of selected clay minerals to adsorb ligands such as CPA, which possess functional groups similar to the aflatoxins (Dwyer et al., 1994, 1995). These studies have indicated that an acidic phyllosilicate (AC) strongly adsorbs CPA and can prevent its toxicity in the hydra bioassay (Dwyer et al., 1994). We proposed that the low pH of AC could catalyze the conversion of the keto-enol group in CPA to a diketone similar to the b-keto lactone group in aflatoxins. Based on these findings, the objective of this research was to evaluate the ability of AC and other adsorbents to protect against CPA toxicity in vivo (i.e., broiler chicks).

MATERIALS AND METHODS

Chemicals A pure isolate of Penicillium griseofulvum (NRRL 3523)4 was obtained and CPA was biosynthesized in our laboratory. Acidic phyllosilicate (AC) and NC were obtained from Engelhard Corporation.5 Clinoptilolite (CZ) was obtained from Zeotech Corporation.6 Solvents were spectral grade from Burdick and Jackson.7 Reagents were of the highest purity that was commercially available.

Biosynthesis of CPA The synthetic medium used in the biosynthesis of CPA was as described by Neethling and McGrath (1977), and the procedure for the production and purification was a modification of the method described by Dorner et al. (1983). Chemical confirmation and purity of CPA were determined using gas chromatography/mass spectroscopy, HPLC, scanning UV-visible spectrophotometry, and melting point. Low resolution mass spectra were obtained with a Hewlett Packard gas chromatography HP 5890A,8 with a capillary on the column injector, mass selective spectrometer detector HP 5970A.8 A fused silica capillary SPB-5 (15 m × 0.32 i.d. × 1 mm) column was purchased from Supelco Inc.9 The ionization voltage was 70 eV and the ion source temperature was 280 C. The ramp program consisted of an initial temperature of 40 C and a final temperature of 280 C at a rate of 15 C/min. There was a solvent delay of 2 min and the total run time was 40 min. The HPLC procedure for the analysis of CPA was a modification of the method of Urano et al. (1992). Cyclopiazonic acid was analyzed using Waters equipment,10 including model 510 pumps, an automatic sampler (WISP model 710), a variable UV wavelength absorbance detector (model 486, wavelength 284 nm), a photodiode array detector (model 996), and a digital computer equipped with Millennium software. Separation was obtained using a reverse phase ODS (C18, 10 m) column operated at a flow rate of 2 mL/min. The mobile phase consisted of 85% methanol and an aqueous solution of 4 mM zinc sulfate. The UV spectrum of CPA in methanol was determined with a Beckman DU 65

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been reported by Dorner et al. (1983), Cullen et al. (1988), Wilson et al. (1990), Smith et al. (1992), Kubena et al. (1994), and Balachandran and Parthasarathy (1996). Cyclopiazonic acid has been suspected in the induction of mycotoxicoses in quail in Indonesia (Stoltz et al., 1988). The neurotoxic effects of CPA (opisthotonos, catalepsy, hypothermia, and sedation) closely resemble some of the symptoms observed by Blount (1961) for Turkey “X” disease. As a result, Cole (1986) suggested that it is highly possible that CPA may have been involved in Turkey “X” disease as well. Cyclopiazonic acid has been reported to markedly distribute to the skeletal tissue in animals (Norred et al., 1985, 1988; Norred, 1990). Following either oral or parenteral administration of [14C] CPA to rats, as much as 50% of the radioactive dose could be detected in muscle, with significant quantities persisting for as long as 72 h (Norred et al., 1985). To assess the extent to which this distribution of CPA could occur in poultry, Norred and coworkers (1988) orally dosed chicks with CPA and found that CPA accumulated in edible tissues. Also recently, Dorner and coworkers (1994) reported that CPA was found in milk and eggs after oral administration of CPA to lactating ewes and laying hens. These findings indicate a potential source of human exposure via ingestion of contaminated animal meat and byproducts. In light of these facts, it is desirable to develop strategies to prevent production of mycotoxins and to develop viable methods to detoxify contaminated food and feed. At present there are no practical detoxification methods available for CPA. Clays have been utilized as chemisorbents in the diet to immobilize and detoxify other mycotoxins. Dietary additions of zeolite (Smith, 1980), bentonite (Carson, 1982), and spent bleaching clay from canola refining (Smith, 1984), have been shown to reduce some of the toxic effects of zearalenone and T-2 toxin in rats and immature swine. More recently, hydrated sodium calcium aluminosilicate (NC), a neutral phyllosilicate clay, was reported to selectively adsorb the aflatoxins in aqueous solutions in vitro, including milk (Phillips et al., 1988; Ellis et al., 1990), and also protect against aflatoxin in animals, i.e., rats, cattle, chickens, goats, lamb, turkey, and swine (Phillips et al., 1988, 1991, 1994; Colvin et al., 1989; Harvey et al., 1989, 1991a,b, 1994; Kubena et al., 1990, 1991, 1993; Lindmann et al., 1993; Smith et al., 1994, Sarr et al., 1995). Recent in vitro studies in our laboratory have evaluated the

INORGANIC ADSORBENTS AND CYCLOPIAZONIC ACID

spectrophotometer11 (Cole and Cox, 1981). Determination of purity was based on its extinction coefficient at 284 nm. Melting point was determined using a standard electrothermal melting point apparatus.

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were added. The diet contained or exceeded the levels of critical nutrients recommended by the National Research Council (1994).

Experimental Design Chemisorption Determination

One-day-old chicks were individually weighed, wingbanded, and randomly distributed to the different treatment groups (four replicates of five chicks per dietary treatment). Chicks were grouped based on the following dietary treatments: 1) basal feed free of toxin (Control); 2) basal feed containing 1% AC; 3) basal feed containing 1% NC; 4) basal feed containing 1% CZ; 5) basal feed containing 45 mg CPA/kg; 6) basal feed containing 1% AC + 45 mg CPA/kg; 7) basal feed containing 1% NC + 45 mg CPA/kg; 8) basal feed containing 1% CZ + 45 mg CPA/ kg. Broilers were provided with these feeds to 3 wk of age. Water and feed were consumed ad libitum for the duration of the experiment.

Measurements

Male broiler chicks (1-d-old Peterson × Hubbard) were obtained from a commercial hatchery. Chicks were maintained in wire-floored brooding cages, with continuous fluorescent illumination and forced ventilation.

Body weight and feed consumption were measured weekly and mortality was recorded as it occurred. Liver, kidney, spleen, pancreas, proventriculus, gizzard, bursa of Fabricius, and heart were evaluated for weight changes. Serum concentrations of total protein, albumin, glucose, cholesterol, triglycerides, uric acid, blood nitrogen, inorganic phosphorus, and activities of lactate dehydrogenase, alkaline phosphatase, g-glutamyl- transferase, aspartate aminotransferase, alanine aminotransferase, and creatine kinase were determined on a clinical chemistry analyzer.12 Hemoglobin was measured as cyanmethemoglobin.13 Erythrocyte count, mean corpuscular volume, and hematocrit were determined with a Coulter Model ZBI counter13 equipped with a mean corpuscular volume and hematocrit computer and channelizer. The mean corpuscular hemoglobin and mean corpuscular hemoglobin concentrations were calculated.

Preparation of Toxin

Statistical Analysis

Cyclopiazonic acid was incorporated into the diet by dissolving the toxin in 1N sodium bicarbonate and then mixing the appropriate quantities with 1 kg of the diet. After drying, the 1 kg of the diet containing the toxin was mixed with the basal feed to produce the selected treatment of CPA for the experiment. The basal diet consisted of a corn and soybean meal that was free of detectable mycotoxin contamination, and no antibiotics, coccidiostats, growth promoters, or inorganic sorbents

Data were analyzed for all variables using PCSAS Version 6.02.14 Data were subjected to ANOVA (Snedecor and Cochran, 1967) using the General Linear Models procedure to establish differences between means. Means showing significant differences in ANOVA were compared using Fisher’s protected least significant difference procedure (Snedecor and Cochran, 1967). All statements of differences were based on significance of P < 0.05.

Experimental Animals

RESULTS 11Beckman Instruments, Inc., Scientific Instruments Division, Fullerton, CA 92634. 12Gilford Impact, 400E, Ciba Corning Diagnostics Corp., Gilford Systems, Oberlin, OH 44774. 13Coulter Electronics, Hialeah, FL 33012. 14SAS Institute Inc., Cary, NC 27512.

The metabolite that was isolated and purified from cultures of Penicillium griseofulvum was identified and confirmed to be CPA. The purity of CPA was determined to be 98%. In vitro adsorption studies demonstrated that binding of CPA to AC, NC, and CZ was

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The strength of adsorption of CPA to test samples was determined using methods previously described in our laboratory (Phillips et al., 1988). Cyclopiazonic acid was added to disposable glass tubes containing 1% clay (wt/ vol) in water and then mixed at 600 rpm for 1 h on a shaker (three replicates per test sample). After centrifugation at 1,500 rpm for 10 min, the supernatant was transferred to another test tube and extracted four times with chloroform. The chloroform extract was evaporated to dryness under a slow stream of nitrogen, redissolved in methanol, and analyzed by HPLC. The HPLC procedure for the analysis of CPA was a modification of method of Urano et al. (1992). The stability of the CPA-clay complex was determined by washing the clay pellet with an eluotropic series of solvents (polar to nonpolar). The amount of CPA desorbed from the pellet was determined by sequentially adding 2 mL of methanol, followed by methylene chloride. Following centrifugation, the supernatant was transferred to another test tube, evaporated, redissolved in methanol, and injected onto the HPLC. The strength of adsorption of CPA at equilibrium was expressed as a chemisorption index (Ca), which was defined as the ratio of the difference between the amount bound (Cb) and the amount desorbed (Cd) to the initial amount added (Ci), i.e., (Cb – Cd )/Ci.

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DWYER ET AL. TABLE 1. Binding of cyclopiazonic acid (CPA) by selected clays:chemisorption index1

Test clays1

Percentage initial binding2

Percentage desorbed3

Chemisorption index4

Acidic phyllosilicate (AC) Neutral phyllosilicate (NC) Clinoptilolite (CZ)

95.41 ± 0.95 43.03 ± 3.89 13.78 ± 1.35

2.03 ± 0.12 8.11 ± 0.46 3.83 ± 0.27

0.93 0.35 0.10

represent the x of three replicates for each test clay ± SE. (1%) were prepared in test tubes containing CPA (40 mg) in 5 mL of water. Samples were shaken for 1 h, and the supernatant was removed and extracted with chloroform to determine initial adsorption. 3Desorption was determined by washing pellets with methanol and methylene chloride sequentially. 4The strength of binding was defined as the ratio of the difference between the amount bound and the amount desorbed to the initial concentration of ligand added. 1Values 2Clays

TABLE 2. Effects of selected clays and cyclopiazonic acid (CPA) on body weights, feed:gain, and mortality in broiler chicks1

Treatment

Body weight

CPA

CZ2

NC3

(mg/kg) 0 0 0 0 45 45 45 45 LSD5

0 1 0 0 0 1 0 0

0 0 1 0 0 0 1 0

a–cMeans

AC4

Day old

Week 1

0 0 0 1 0 0 0 1

46 46 46 46 46 46 46 47 0.6

159a 158a 151a 149ab 135bc 134c 128c 132c 15

(%)

Week 2

Week 3

404a 389a 387a 398a 322b 326b 325b 314b 41

767a 715a 705a 725a 567b 593b 565b 580b 72

(g)

within column with no common superscript differ significantly (P < 0.05). represent the x of four groups of five broiler chicks each per treatment less mortality. 2CZ = clinoptilolite, zeolite. 3NC = neutral phyllosilicate, hydrated sodium calcium aluminosilicate. 4AC = acidic phyllosilicate. 5LSD = least significant difference as determined by Fisher’s protected LSD procedure. 1Values

Week 3 BW change from control (%) 0 –7 –8 –5 –26 –23 –26 –24

Feed:gain

Mortality

(kg:kg) 1.51c 1.63bc 1.61bc 1.55c 1.88a 1.70abc 1.83ab 1.65abc 0.25

(%) 5 10 0 0 0 0 0 5

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inhibitory effects produced by CPA (Table 2). Overall there were higher feed:gain ratios for CPA-treated birds (i.e., with and without clay), with the birds given CPA alone having the highest feed:gain ratio. Mortality was observed in the controls, CZ alone, and the AC plus CPA treatments. The effects of clays and CPA on relative organ weights are shown in Table 3. When compared with controls, there were increased relative weights of kidney, proventriculus, and gizzard for the chicks treated with CPA alone. The significant increases in relative organ weights were not prevented by the clays tested. There was no significant difference between the chicks that were fed clay plus CPA and the chicks fed diets containing CPA alone. Generally, the chicks that received clays alone were not significantly different from controls for relative organ weights, except for the NC-treated chicks. The relative weights of spleen and heart in this treatment were increased significantly compared to controls. None of the treatments altered relative weights of liver and bursa of Fabricius (data not shown).

markedly different. Based on chemisorption indices, CPA possessed the highest propensity and tightest binding for AC (Ca = 0.93), followed by NC (Ca = 0.35), with very little adsorption to CZ (Ca = 0.10) (Table 1). Based on these in vitro findings, we predicted that AC might be more effective than either NC or CZ in adsorbing and inactivating the toxin in the gastrointestinal tract of chicks. In vivo results from this study indicated that CPA can significantly affect broiler health and production. The effects of clay and CPA on body weights, feed:gain, and mortality are shown in Table 2. Body weights were significantly reduced after the 1st wk for all chicks fed diets containing 45 mg/kg of CPA. The percentage reduction in body weight produced by CPA alone at Week 3 was 26%. Body weights of birds fed diets containing sorbents alone (i.e., AC, NC, and CZ) were not significantly different from controls. Body weights of the chicks in the clay plus CPA treatment groups were significantly different from controls and were similar to the body weights of birds given CPA alone. The clays that were evaluated did not prevent the growth

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INORGANIC ADSORBENTS AND CYCLOPIAZONIC ACID TABLE 3. Effects of selected clays and cyclopiazonic acid (CPA) on relative organ weights of broiler chicks1 Treatment CPA

CZ2

NC3

(mg/kg) 0 0 0 0 45 45 45 45 LSD5

0 1 0 0 0 1 0 0

0 0 1 0 0 0 1 0

AC4

Kidney

Spleen

Pancreas

0 0 0 1 0 0 0 1

0.463c 0.483bc 0.518abc 0.466bc 0.521ab 0.521ab 0.519abc 0.542a 0.057

0.101bc 0.097bc 0.131a 0.091c 0.099bc 0.119ab 0.101bc 0.114abc 0.03

(g/100 g BW) 0.35c 0.65b 0.35c 0.65b 0.35bc 0.66b 0.36bc 0.66b 0.40abc 1.14a 0.37abc 1.09a 0.42ab 0.95a 0.42a 1.00a 0.06 0.20

(%)

Proventriculus Gizzard 2.61c 2.73bc 2.64bc 2.62bc 2.99a 2.85ab 2.85ab 3.02a 0.24

Heart 0.73b 0.79ab 0.86a 0.77ab 0.80ab 0.81ab 0.81ab 0.81ab 0.10

a–cMeans

within column with no common superscript differ significantly (P < 0.05). represent the x of four groups of three broiler chicks each per treatment. 2CZ = clinoptilolite, zeolite. 3NC = neutral phyllosilicate, hydrated sodium calcium aluminosilicate. 4AC = acidic phyllosilicate. 5LSD = least significant difference as determined by Fisher’s protected LSD procedure. 1Values

cant difference between chicks receiving CZ plus CPA or chicks receiving AC plus CPA chicks and controls. The chicks treated with CZ plus CPA and AC plus CPA were also not significantly different from chicks treated with CPA alone. The chicks treated with NC plus CPA were significantly different from controls. The toxicity of CPA was also expressed through alterations in hematology values. There was a significant difference between controls and chicks treated with CPA alone for mean red blood cell and hematocrit values (Table 5). When compared to controls, mean red blood cell counts and hematocrit values were increased in the chicks treated with CPA alone. For mean red blood cell count, all the sorbents plus CPA treated groups indicated no significant difference when compared to controls and also were not significantly different from

TABLE 4. Effects of selected clays and cyclopiazonic acid (CPA) on serum biochemical values of broiler chicks1 Treatment CPA

CZ2

NC3

(mg/kg) 0 0 0 0 45 45 45 45 LSD5

0 1 0 0 0 1 0 0

0 0 1 0 0 0 1 0

a–cMeans

AC4

Cholesterol

Glucose

0 0 0 1 0 0 0 1

161c 177abc 175abc 168bc 185abc 182abc 187ab 197a 26

280ab 284ab 279ab 277ab 310a 274ab 274ab 270b 37

(%)

Phosphorus (mg/dL) 6.15b 6.14b 6.35ab 6.19ab 6.93a 5.80bc 5.08c 5.90b 0.78

within column with no common superscript differ significantly (P < 0.05). represent the x of four groups of three broiler chicks each per treatment. 2CZ = clinoptilolite, zeolite. 3NC = neutral phyllosilicate, hydrated sodium calcium aluminosilicate. 4AC = acidic phyllosilicate. 5LSD = least significant difference as determined by Fisher’s protected LSD procedure. 1Values

Blood urea nitrogen

Albumin

1.98b 1.85b 2.00b 1.64b 2.81a 1.91b 1.93b 2.02b 0.70

(g/dL) 1.19ab 1.25a 1.19ab 1.23a 1.12b 1.09b 1.15ab 1.18ab 0.11

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The effect of clays and CPA on serum biochemical values are presented in Table 4. Significant serum biochemical alterations produced by CPA alone were increased phosphorus and blood urea nitrogen. The increase in blood urea nitrogen was shown to be inhibited by all the clays evaluated; that is, there was no significant difference when compared to controls. For serum phosphorus, when compared to controls, there was no significant difference for AC plus CPA and CZ plus CPA treatments. Toxicity of CPA was also expressed through significant changes in serum enzymatic activities and hematology values (Table 5). When compared to controls, alanine aminotransferase activity was significantly increased in chicks fed diets containing CPA alone. For alanine aminotransferase activity, there was no signifi-

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TABLE 5. Effects of selected clays and cyclopiazonic acid (CPA) on serum enzyme activities and hematology values of broiler chicks1 Treatment CPA (mg/kg) 0 0 0 0 45 45 45 45 LSD5

CZ2

NC3

AC4

g-Glutamyl transferase

(%) 0 1 0 0 0 1 0 0

0 0 1 0 0 0 1 0

0 0 0 1 0 0 0 1

11.11a 10.62a 11.14a 8.99a 9.78a 9.08a 8.18ab 5.54b 3.08

Alkaline phosphatase (IU/L) 9,606b 12,719ab 12,070b 14,182ab 14,242ab 19,360a 10,381b 10,159b 6,005

Alanine aminotransferase

Mean red blood cell 106

20.31bc 19.66c 29.37a 26.28ab 26.95a 25.37abc 26.87a 23.72abc 6.00

(× 1.80b 1.90ab 1.92ab 1.87ab 2.00a 1.88ab 1.90ab 1.86ab 0.17

mm3)

Hematocrit (%) 27.63b 28.75ab 29.38ab 28.00b 31.25a 30.13ab 28.13b 27.38b 2.79

a–cMeans

within column with no common superscript differ significantly (P < 0.05). represent the x of four groups of three broiler chicks each per treatment. 2CZ = clinoptilolite, zeolite. 3NC = neutral phyllosilicate, hydrated sodium calcium aluminosilicate. 4AC = acidic phyllosilicate. 5LSD = least significant difference as determined by Fisher’s protected LSD procedure. 1Values

DISCUSSION In this study, the toxic effects of CPA were expressed as reduced body weights (–26% at Week 3), higher feed: gain ratio, increased relative organ weights (i.e., kidney, proventriculus and gizzard), increased serum phosphorus and blood urea nitrogen, increased activity of alanine aminotransferase, increased mean red blood cell counts and hematocrit values. The toxic effects produced by CPA were in general agreement with previous reports (Dorner et al., 1983; Cullen et al., 1988; Smith et al., 1992; Kubena et al., 1994). The changes in body weights that were produced by CPA are consistent with earlier data reported by Wilson et al. (1986), Smith et al. (1992) and Kubena et al. (1994). These studies reported a dose-dependent reduction in weight gain for CPAtreated birds. In contrast, Dorner et al. (1983) did not observe any significant differences in weight gain when CPA was fed at 50 mg/kg to broiler chickens. The relative weights of proventriculus and gizzard were significantly increased in our study. Mycotoxins have been known to irritate the proventriculus and gizzard of the gastrointestinal tract, thus causing an increase in the relative weights of these organs (Huff and Doerr, 1981). Increased alanine aminotransferase activity and increased blood urea nitrogen have been

reported to be sensitive serological indicators of liver and kidney toxicity, respectively. These parameters were significantly increased in this study, suggesting that CPA may be producing critical injury to these organs. There was a significant increase in relative weights of kidney, but not a significant increase in relative liver weights. Cullen and coworkers (1988) reported that after orally dosing broiler chicks with CPA, the most common lesions consisted of necrosis and hemorrhage of the mucosa of the proventriculus and hepatocellular vacuolation. Dorner et al. (1983) also reported that after feeding chicks 50 and 100 ppm of CPA, there was hyperplasia of the proventriculus and mucosal necrosis in the gizzard. After orally dosing pigs with CPA (10 mg/kg), the target organs were the gastrointestinal tract, liver and kidney (Lomax et al., 1984). The changes in relative organ weights in this study were consistent with reports on the histopathological changes in organs of broilers dosed with CPA. Overall, the test clays were not toxic to the broilers at the level of 1% in the total diet. There were no significant differences between controls and broilers fed clays alone for most parameters evaluated. Also test clays did not exhibit a distinction in their ability to protect chicks against CPA, suggesting a lack of predictability between in vitro chemisorption data and the in vivo results. The inability of (AC) to protect chicks from the effects of CPA may have been influenced by a number of factors, including: 1) lack of selectivity of the acidic clay and competition with other ligands in the gastrointestinal tract, and 2) binding capacity of the clay. It has been shown that a variety of functional properties of clay and zeolitic minerals (i.e., cation exchange capacities, plateau surface densities, surface pH, porosities, predominant exchangeable cation, ligand specificities, ligand capacity, and heat of adsorption) are critical for the immobilization of diverse ligands. These properties have been shown to be important in predicting the efficacy of NC in vivo.

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CPA alone treated. For hematocrit values, NC plus CPA and AC plus CPA were not significantly different from controls. The CZ treated group was not significantly different from controls, but was also not significantly different from CPA alone treated chicks. When compared with controls, there were no significant differences in serum values for uric acid, total protein, triglycerides, creatine kinase, aspartate aminotransferase, lactate dehydrogenase, mean corpuscular volume, and mean corpuscular hemoglobin concentration (data not shown).

INORGANIC ADSORBENTS AND CYCLOPIAZONIC ACID

ACKNOWLEDGMENTS This study was supported by TAES H-6215, ATP 9999002-004, and NIH P42-ES04917. The authors gratefully acknowledge the excellent technical assistance of Maurice Connell, Laura Rippley, and Scott Schroeder.

REFERENCES Balachandran, C., and K. R. Parthasarathy, 1996. Influence of dietary rice culture material containing cyclopiazonic acid on certain biochemical parameters of broiler chickens. Mycopathologia 132:161–166. Blount, W. P., 1961. Turkey “X” disease. Turkeys 9:52–77. Carson, M. S., 1982. The effect of dietary fiber and nonnutritive mineral additives on T-2 toxicoses in rats. M.Sc. Thesis. Department of Nutrition, University of Guelph, ON, Canada. Cole, R. J., 1984. Cyclopiazonic acid and related toxins. Pages 404–414 in: Mycotoxins, Production, Isolation, Separation and Purification. V. Betina, ed. Elsevier Science Publishers, Amsterdam, The Netherlands. Cole, R. J., 1986. Etiology of Turkey “X” disease in retrospect: A case for the involvement of cyclopiazonic acid. Mycotox. Res. 2:3–7. Cole, R. J., and R. H. Cox, 1981. Tremorgen group. Pages 497–499 in: Handbook of Toxic Fungal Metabolites. R. J. Cole and R. H. Cox, ed. Academic Press, New York, NY. Colvin, B.M.L.T. Sangster, K. D. Hayden, R. W. Bequer, and D. M. Wilson, 1989. Effect of a high affinity aluminosilicate sorbent on prevention of aflatoxicosis in growing pigs. Vet. Hum. Toxicol. 31:46–48. Cullen, J. M., M. Wilson, W. M. Halger, Jr., J. F. Ort, and R. J. Cole, 1988. Histologic lesions in broiler chicks given cyclopiazonic acid orally. Am. J. Vet. Res. 49:728–731. Decker, W. J., 1980. Activated charcoal adsorbs aflatoxin B1. Vet. Human Toxicol. 22:388–389. Dorner, J. W., R. J. Cole, and U. L. Diener, 1984. The relationship of Aspergillus flavus and Aspergillus parasiticus with reference to production of aflatoxins and cyclopiazonic acid. Mycopathologia 87:13–15. Dorner, J. W., R. J. Cole, D. J. Erlington, S. Susksupath, G. H. McDowell, and W. L. Bryden, 1994. Cyclopiazonic acid residues in milk and eggs. J. Agric. Food Chem. 42: 1516–1518. Dorner, J. W., R. J. Cole, L. G. Lomax, H. S. Grosser, and U. L. Diener, 1983. Cyclopiazonic acid production by Aspergillus flavus and its effects on broiler chickens. Appl. Environ. Microbiol. 46:698–703. Dwyer, M. R., A. B. Sarr, K. Mayura, K. S. Washburn, and T. D. Phillips, 1994. Evaluation of the binding ability of different sorbents for cyclopiazonic acid. Toxicologist 14: 212. (Abstr.) Dwyer, M. R., A. B. Sarr, K. Mayura, K. S. Washburn, and T. D. Phillips, 1995. A clay-based method for the adsorption and inactivation of cyclopiazonic acid. Toxicologist 15:74. (Abstr.) Ellis, J. A., R. B. Harvey, L. F. Kubena, R. H. Bailey, B. A. Clement, and T. D. Phillips, 1990. Reduction of aflatoxin M1 residues in milk utilizing hydrated sodium calcium aluminosilicate. Toxicologist 10:163. (Abstr.) Frisvad, J. C., 1989. The connection between Penicillia and Aspergillia mycotoxins with special emphasis on misidentified isolates. Arch. Environ. Contam. Toxicol. 18:452–467. Gallagher, R. T., J. L. Richard, H. M. Stahr, and R. J. Cole, 1978. Cyclopiazonic acid production by aflatoxigenic and nonaflatoxigenic strains of Aspergillus flavus. Mycopathologia 66:31–36. Harvey, R. B., L. F. Kubena, M. H. Elissalde, D. E. Corrier, and T. D. Phillips, 1994. Comparison of two hydrated sodium calcium aluminosilicate compounds to experimentally protect growing barrows from aflatoxicosis. J. Vet. Diagn. Invest. 6:88–92.

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For example, Kubena and coworkers (1990) compared the protective effects of NC and charcoal in aflatoxintreated broiler chicks. They found that NC diminished aflatoxicosis, but charcoal, which effectively binds aflatoxin in vitro (Decker, 1980), did not show any protective effects in chicks. Apparently, for an inorganic adsorbent to reduce the bioavailability and toxicity of a poison such as CPA, may require ligand specificity and covalent interaction (i.e., tight binding). A high affinity of CPA for binding sites may also be necessary for efficacy in vivo, due to chemicals in the diet and gastrointestinal tract that may antagonize the binding of CPA. The binding capacity of the clay is also important, since it may be possible to exceed the binding maximum. In this study, a high level of toxin was used to elicit toxicity (i.e., 45 mg CPA/kg). This may suggest that higher levels of clay would be required to produce more effective protection from CPA toxicity in vivo. It is also possible that the level of CPA used in this study may be higher than that found to routinely occur in naturally contaminated poultry feed; however, in corn the concentration of CPA has not been reported to exceed 9 ppm (Widiastuti et al., 1988; Lee and Hagler, 1991; Urano et al., 1992). In comparison, aflatoxin B1 has a much higher binding capacity and affinity for NC vs CPA for AC (i.e., 420 nmol aflatoxin B1 bound/mg of NC vs 130 nmol of CPA bound/mg of AC) (Phillips et al., 1995). This difference could be attributed to the inability of CPA to effectively access active binding sites within the interlayer of the clay due to size or stearic factors that could hinder docking. If CPA is unable to access the interlayer of the clay, and binding is exclusively on the surface of the clay, then a low binding capacity would not be unexpected. Studies are in progress to determine the binding sites for CPA on particles of AC. The results from this study indicate that CPA can significantly reduce body weight and affect overall broiler health and performance. In general, test clays were not effective in diminishing the growth inhibitory effects of CPA and the increased relative weights of organs at the levels used in these studies. There was, however, apparent protection noted for some of the hematological, serum biochemical and enzymatic changes associated with CPA toxicity. The clays that were effective in binding CPA in vitro did not significantly prevent CPA toxicity in the broilers. Our results suggest that in vitro binding of CPA to clay does not accurately forecast its efficacy in vivo. Predictions about the ability of inorganic adsorbents to prevent the adverse effects of mycotoxins in vivo, should be approached with caution, and should be confirmed in vivo, paying particular attention to the potential for nutrient interactions.

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