ANIMAL FEED SCIENCE AND TECHNOlOCY

ELSEVIER Animal Feed Science Technology 65 (1997) 197-206

Prevention of aflatoxicosis in farm animals by means of hydrated sodium calcium aluminosilicate

addition to feedstuffs: a review

A.J. Ramos a3*, E. Hemhdez b a Department of Food Technology, University of Lleida, Food Technology Area, UdL-IRTA centre R + D,

CeRTA. Ac. Alcalde Rouira Roure 177, 2.5198 Lleidu, Spain b Department of Biotechnology, Polytechnic Uniuersity of Valencia, Camino de Vera, 14, 46022 Valencia,

Spain

Accepted 1 August 1996

Abstract

Mycotoxins are a wide group of fungal toxins that have been associated with severe toxic effects in man and animals. Aflatoxins are the most dangerous of these fungal secondary metabolites. Because there is no definitive way in which complete detoxification of food and feed contaminated with mycotoxins can be achieved, new methods to eliminate mycotoxicosis are sought.

Hydrated sodium calcium aluminosilicate (HSCAS), a sorbent compound obtained from natural zeolite, has demonstrated an ability to sorb mycotoxins with a high affinity. Addition of this compound to feedstuffs contaminated with aflatoxins has shown a protective effect against the development of aflatoxicosis in farm animals.

Several authors have postulated that the main mechanism implicated in this process could be chemisorption of the toxins through the formation of a stable complex comprising HSCAS and the mycotoxins. This complex is not able to cross the luminal membrane of the gastrointestinal tract and therefore the bioavailability of aflatoxins is reduced in a dose-dependent manner.

This review comments on the in vitro and in vivo application of HSCAS to sorb aflatoxins and other mycotoxins. The effect on animal performance of dietary addition of HSCAS to feedstuffs contaminated with mycotoxins is discussed in a variety of farm animals. 0 1997 Elsevier Science B.V.

Keywords: Aflatoxins; Mycotoxins; Hydrated sodium calcium aluminosilicate; HSCAS; Adsorption

* Corresponding author. Tel.: 34.73-702OCNl ext. 5011; fax: 34-73-238264; e-mail: ajramos@tecal.udl.es

0377-8401/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO377-8401(96)01084-X

198 A.J. Ramos, E. Hetindez/Animal Feed Science Technolog?; 65 (1997) 197-206

1. Introduction

Mycotoxins comprise a structurally diverse family of naturally occurring, fungal- elaborated toxins, many of which have been strongly implicated as chemical progenitors of toxicity in man and animals. Undoubtedly, the most thoroughly studied and best understood of the mycotoxins are the aflatoxins.

The aflatoxins are a group of structurally similar polysubstituted coumarins. These extremely toxic metabolites are produced by the common molds Aspergillus,flavus Link and Aspergillus parasiticus Speare. As A. jlavus is considered to be one of the most common storage molds, the occurrence of aflatoxins in feedstuffs is common throughout the world.

There are four major naturally occurring aflatoxins, the most hepatotoxic being aflatoxin B,; the other three, structurally similar, compounds are the aflatoxins B,, G, and G?. All four have been detected as contaminants of crops before harvest, between harvesting and drying, during storage, and after processing and manufacturing (Council for Agricultural Science and Technology, 1989). The carry-over of aflatoxin B, and its metabolic transformation product, the aflatoxin M,, into tissues suitable for human consumption, eggs and milk of food-producing animals, has been described for different animal species (Rodricks and Stoloff, 1977; Patterson, 1978; Stoloff, 1979; Shreeve et al., 1979; Patterson et al., 1980; Munksgaard et al., 1987; Van Egmond, 1989; Veldman et al., 1992).

The main biological effects of aflatoxins are carcinogenicity, immunosuppression, mutagenicity and teratogenicity (Betina, 1989). Chronic exposure to aflatoxins may significantly alter productivity and animal farming trends. In addition, direct exposure to aflatoxin B, contaminated agricultural food commodities, together with an undesirable mycotoxin contamination of foods of animal origin, may impose a great risk to the consumer. Consequently, to reduce and/or prevent human exposure via the secondary route, practical and effective methods to detoxify aflatoxin-containing feedstuffs are in great demand.

A variety of physical, chemical and biological approaches to counteract the aflatoxin problem have been reported in the literature on mycotoxins (Doyle et al., 1982; Samarajeewa et al., 1990; Ramos and Hem&ndez, 19911, but large-scale, practical and cost-effective methods for detoxifying aflatoxin-containing feedstuffs are currently not available.

One of the most recent approaches to the problem has been the use of nutritionally inert sorbents in the diet that sequester aflatoxins and reduce the absorption of these mycotoxins from the gastrointestinal tract, avoiding toxic effects for livestock and the carry-over of the toxins to animal products.

This review comments on the in vitro efficacy of the most thoroughly studied adsorbent, a hydrated sodium calcium aluminosilicate (HSCAS), that has demonstrated a high affinity for aflatoxins, and its in vivo application in a varied number of farm animals will be discussed.

A.J. Ramos. E. Hemcindez / Animal Feed Science Technology 65 (1997) 197-206 199

2. Hydrated sodium calcium aluminosilicate (HSCAS)

Among all aluminosilicates tested with regard to aflatoxin adsorption, a hydrated sodium calcium aluminosilicate (HSCAS) from natural zeolite (NovaSil’“, Engelhard Corp., Cleveland, OH) has been the most extensively studied because of its promising aflatoxin-binding capacity. This is a phyllosilicate whose positive charge deficiencies create the potential for sorbing positively charged or cationic compounds (Theng, 1974). Phyllosilicates are layered structures formed by the condensation of sheets of linked silicate tetrahedra with linked aluminate octahedra; zeolite is an aluminosilicate whose structure contains channels (known as molecular sieves in synthetic zeolites) filled with water and alkali and alkaline earth exchangeable cations (Mumpton and Fishman, 1977). When these channels are free of water the adsorptive properties of the aluminosilicate are augmented.

First reports of the use of HSCAS to adsorb aflatoxins were made in 1987 (Davidson et al., 1987; Phillips et al., 1987) and since then more than 25 papers have been published on this subject, an indication of the increasing interest this compound has generated.

Phillips et al. (1987) briefly reported the discovery of the adsorbent properties of HSCAS in vitro and in vivo and postulated its use as a sequestrant in the gastrointestinal tract. At the same time, Davidson et al. (1987) reported that the use of 0.1 and 0.5% HSCAS in the diets of chicks fed an aflatoxin-contaminated feedstuff (20 and 80 pg aflatoxins kg- ’ feed) was able to reduce the bioavailability of aflatoxin in a dose-depen- dent manner.

The in vitro experiments carried out with radiolabeled aflatoxin B, (Phillips et al., 1988) demonstrated that of 38 different adsorbents tested (a variety of aluminas, zeolites, silicas, phyllosilicates and chemically modified phyllosilicates), HSCAS was the best aflatoxin B,-adsorbing compound as it was able to form the most stable complex with this mycotoxin (with a sorption of more than 80% of aflatoxin B, present in the medium). The complex was also demonstrated to be stable in water at pH 2, 7 and IO and at temperatures of 25 and 37°C. The stability of the HSCAS-aflatoxin B, sorption complex was evaluated by extraction with an elutropic series of solvents. As less than 10% of aflatoxin B, was extracted, they suggested that the mechanism implicated in this process could be chemisorption with the formation of strong bonds between the molecules, but later on, a more specific mechanism was proposed (Phillips et al., 1990b; Sarr et al., 1990). This mechanism was the formation of a complex by the B-carbonyl system of the aflatoxin with uncoordinated edge site aluminum ions in HSCAS. Phillips et al. (1990a) reported that the reaction between the aflatoxin B, and HSCAS reached equilibrium after 30 min. Calculations based on the equilibrium binding experiments indicated that approximately 200-232 nmol of aflatoxin could be maxi- mally bound per milligram of HSCAS.

Scheideler (1993) found different results in an in vitro experiment with HSCAS. HSCAS (100 mg HSCAS ml-’ test solution) was able to adsorb only 55% of radiolabeled aflatoxin B , (200 ng aflatoxin B 1 ml - ’ test solution) when the test solution was 80% methanol, whereas the percentage of adsorption diminished to 36.74% when the test solution was water containing 3 g of intestinal contents isolated from the intestine of 2-week-old broilers.

200 A.J. Ramos, E. Hemrindez/Animal Feed Science Technology 65 (1997) 197-206

HSCAS, fed mainly at a concentration of 0.5% of the diet, has been assayed in a great variety of farm animals, including chicken, turkey poults, pigs, minks, cows and lambs.

2.1. Chickens

Davidson et al. (1987) demonstrated that 0.5% HSCAS was able to reduce the bioavailability of aflatoxins (20 and 80 pg aflatoxins kg-’ feed tested) in the liver and blood of Arbor Acres X Peterson broiler chicks in a 14day experiment.

Phillips et al. (1988) showed that 0.5% HSCAS fed to broiler and Leghorn chicks significantly decreased the growth inhibitory effect of feeding 7.5 mg aflatoxin B, kg- ’ feed. The same results were found by Kubena et al. (1988) who demonstrated that the addition of 0.5% HSCAS diminished the growth inhibitory effect of aflatoxin B, (7.5 mg aflatoxin B, kg-’ feed) or total aflatoxins (5.0 mg total aflatoxins kg-’ feed, produced by Aspergillus parasiticus on rice powder) in broiler and Leghorn chicks by 55-100%. With the same experiment design, Kubena et al. (1990b) confirmed that 0.5% HSCAS diminished the toxic impact on weight gains produced by aflatoxin B r. Except for the bursa fabricia, relative weights of the liver, kidney, proventriculus and gizzard were not affected by 5 mg aflatoxins in the diet when 0.5% HSCAS was added to the feedstuff. Substantial protection was provided by HSCAS on the concentrations of serum total protein and albumin, and serum GGT was not affected when HSCAS was given in conjunction with aflatoxins.

Doerr (1989) demonstrated that as little as 0.125% of HSCAS was able to protect young broilers against 2 mg aflatoxins kg- ’ feed (50% improvement), although complete protection was obtained only with levels up to 0.5%. Similarly, feed conver- sions improved in a dose-dependent fashion when HSCAS was added to diets containing aflatoxins.

Scheideler (1989) showed that HSCAS alleviated depression of growth in the presence of 2.5 mg aflatoxin B, kg- ’ feed, and suppressed the increase of liver lipids due to treatment with the mycotoxin, while an increment of serum phosphorus was found with this treatment. Later, the same author (Scheideler, 1993) found, in an in vivo study on broiler chicks, that the addition of 1% HSCAS to diets contaminated with 2.5 mg aflatoxin B , kg-’ feed alleviated the depression in growth and suppressed the increase in liver lipids caused by the mycotoxin. However, the addition of the HSCAS alone caused bone ash to increase and serum Cl to decrease.

Araba and Wyatt (1991) showed that when HSCAS was added to broiler chicken diets contaminated with 5 mg aflatoxins kg -’ feed, toxicity was reduced. Thus, 0.5% HSCAS reduced the effects of aflatoxins on feed intake, body weight gain, liver weight and liver lipids by 40%, 38%, 37% and 4%, respectively; corresponding values for 1% HSCAS were lOO%, 84%, 80% and 49%.

Recently, Kubena et al. (1993b) assayed NovaSil” HSCAS-1 and two newly constructed and modified HSCAS compounds (referred to as HSCAS-2 and HSCAS-3) that exhibited enhanced capacities for binding aflatoxin B, in vitro. They incorporated these three HSCAS compounds into chick diets (0.5%) containing 0, 2.5 or 5.0 mg aflatoxins kg- ’ feed during a 3 week study. The relative weights of the liver, kidney and proventriculus were protected by the three HSCAS compounds. Compounds

A.J. Ramos, E. Herndndez/Animl Feed Science Technolog)a 65 (1997) 197-206 201

HSCAS-2 and HSCAS-3 provided significant protection against the changes in concen- tration of total protein, albumin and cholesterol and the activity of aspartate amino transferase, whereas HSCAS- 1 provided protection only against the change in activity of the latter. These results emphasize the fact that silicate-type sorbents differ in their ability to protect against aflatoxicosis. Thus, HSCAS-2 and HSCAS-3, created with additional binding sites, seemed to be more protective than HSCAS- 1.

2.2. cows

Harvey et al. (1991b) demonstrated that HSCAS was capable of reducing the secretion of aflatoxin M, into milk. Lactating dairy cows were fed a diet containing 200 mg aflatoxins kg ’ + 0.5% HSCAS for 7 days. Aflatoxin M, excretion was reduced in 24%, and the carry-over of the mycotoxin was reduced in 44% when the aflatoxin contamination was 100 mg kg-’ feed and the amount of HSCAS used was 1.0%.

2.3. Goats

Smith et al. (1994) demonstrated in two different experiments that HSCAS added to feedstuffs of lactating dairy goats was able to reduce aflatoxin M, residue in milk. Thus, g-day addition of 4% HSCAS to a feed contaminated with 200 p,g total aflatoxin (79% aflatoxin B ,, 16% aflatoxin G,, 4% aflatoxin B,, 1% aflatoxin G,) per kilogram of feed was responsible for an 86.9% aflatoxin reduction, whereas l-2% HSCAS added during a 12&y treatment to a diet contaminated with 100 pg aflatoxins kg-’ feed resulted in 5 1.9-82.2% reduction, respectively. The HSCAS treatments did not affect the nutri- tional quality of goat’s milk. Feed intake and milk production were also not altered.

2.4. Lambs

Harvey et al. (1991a) added 2.0% HSCAS to the diets of growing wethers contami- nated with 2.6 mg aflatoxins kg ’ feed in a 6 week study. HSCAS was able to protect against changes in body weight and weight gain of lambs caused by aflatoxins as well as against variations in serum AST and GGT activities.

2.5. Minks

Bonna et al. (1991) investigated the influence of HSCAS in the toxicity of aflatoxin to mink during an 11 week study. They reported that 0.5% HSCAS prevented the mortality of mink fed with 34 or 102 pg aflatoxins (aflatoxin B, + B,) kg- ’ feed. HSCAS ameliorated the adverse effects of aflatoxins in feed consumption and mink body weight, but these parameters did not reach the values observed in the control group. Addition of HSCAS to the diets containing 102 p,g aflatoxins kg-’ feed reduced or eliminated the histopathologic lesions observed in the livers of the aflatoxin-alone group. Changes in the animals that received the high-dose of aflatoxin plus HSCAS were similar to those seen in the low-dose aflatoxin group.

2.6. Pigs

Harvey et al. (1989a,b) showed that addition of 0.5 or 2.0% HSCAS to the diets of pigs contaminated with 3 mg aflatoxins kg-’ feed in a 4 week study effectively

202 A.J. Ramos, E. Hemrinde:/Animal Feed Science Technology 65 (1997) 197-206

protected the pigs against the adverse effects of aflatoxins. There were no differences in weight gain, feed conversion, hematology, serum biochemistry or the results of macro- scopic and microscopic pathologic examination between the control and HSCAS-treated groups, whereas these parameters were drastically altered in the aflatoxin-alone group. They postulated that HSCAS can modulate the toxicity of aflatoxin in growing barrows via sequestration and reduction of bioavailability in vivo, and may offer a novel approach to the preventive management of aflatoxins in animals.

Colvin et al. (1989) demonstrated that addition of 0.5% HSCAS to the diet of growing pigs (contaminated with 525 p,g aflatoxins kg- ’ feed) in a 35-day experiment resulted in a reduction of the detrimental effects of the mycotoxin on liver weight gains and feed intake. Thus, pigs receiving HSCAS had weight gains equal to 94% of those measured in control animals (aflatoxin alone in the diet caused a 33% reduction in liver weight gains), and consumed 90% of the amount of feed consumed by control animals (reduced by 28% in aflatoxin-alone group). Postmortem gross pathological examination of pig livers showed that livers from control and aflatoxin + HSCAS groups were indistinguishable, whereas in the aflatoxin-alone group some histopathological changes, such as karyomegaly, were found.

Lindemann et al. (1989) showed that inclusion of 0.5% HSCAS in a diet of crossbred pigs contaminated with 840 p,g aflatoxin B, kg-’ feed during 49 days resulted in an improvement of daily gains compared with the group of animals consuming aflatoxin B , alone. HSCAS was more effective in the amelioration of aflatoxicosis than the addition of folic acid or selenium, and provided an improvement in blood clinical chemistry values and in cell-mediated immune function, in comparison with the aflatoxin B, control group. In another report (Lindemann et al., 1993), the same authors demon- strated that, in a 42-day trial, HSCAS was able to improve all clinical chemistry indicators that had been negatively affected by a contaminated diet (800 kg aflatoxin B, kg-‘).

Beaver et al. (1990) demonstrated in a 35-day study that addition of 0.5% HSCAS to a contaminated diet (524 pg aflatoxin B, + B, kg-’ feed) of growing pigs significantly reduced the amount of aflatoxin M, found in liver, kidney and muscle tissue, and decreased the amount of aflatoxin B2 in the muscle and kidney and of aflatoxin B, only in the muscle.

Schell et al. (1993) reported that 0.5% HSCAS addition to a weanling pigs diet containing 500-800 kg aflatoxins kg-’ feed resulted in a reduction in the changes in the serum measurements normally affected by aflatoxin, including albumin, total protein, GGT and ALP, and in an improvement of the average daily gain and gain:feed ratios of the animals. They demonstrated that the addition of 0.5% Ca bentonite results in a better performance than that obtained by 0.5% HSCAS or 0.5% sepiolite, but that these two clays (both natural phyllosilicates) give a better result than palygorskite, another natural clay.

2.7. Turkey poults

Kubena et al. (1991) added 0.5% HSCAS to diets containing 1 or 0.5 mg aflatoxins kg-’ feed in growing male turkey poults during 3 weeks. The addition of the adsorbent material resulted in a 68% decrease in mortality for the 3 week experimental period. The

A.J. Rnmos, E. Hem&k/Animal Feed Science Technology 65 (19971 197-206 203

HSCAS only treatment did not alter any of the common parameters studied. The HSCAS diminished the adverse effects of aflatoxin on body weight gains, most relative organ weights, hematological values, serum biochemical values and enzyme activities associated with 0.5 but not 1 mg aflatoxins kg-’ feed. They postulated that the fact that 100% protection was not obtained for same parameters when 0.5% HSCAS was added to diets containing 0.5 mg aflatoxins kg-’ feed indicates that not all of the aflatoxin was being bound to the adsorbent. The small quantity of unbound aflatoxin could cause the increased toxicity observed at 1 mg aflatoxins kg -’ feed. These findings suggest that HSCAS is only able to reduce the adverse effects of aflatoxins in an aflatoxin-sensitive species such as the turkey.

2.8. HSCAS and other mycotoxins

As a result of the promising results obtained with the aflatoxins, the addition of HSCAS to foodstuffs has been evaluated using a wide variety of mycotoxins. Thus, Bursian et al. (1992) demonstrated that 0.5% HSCAS added to the diets of minks contaminated with the estrogenic mycotoxin zearalenone (lo-20 mg kg-’ feed, during 24 days) can alleviate some of the undesirable reproductive effects of zearalenone which are not related to its hyperestrogenic action. HSCAS protected against the significant increase in gestation length, the decrease in litter size and the increase in kit mortality observed in the zearalenone control group (20 mg zearalenone kg ’ feed).

Huff et al. (1992) demonstrated in a 3 week experiment that addition of 0.5% HSCAS to a diet of broilers contaminated with ochratoxin A (2.0 g kg- ’ feed) and with or without aflatoxins (3.5 g kg-’ feed) had little effect on the toxicity of ochratoxin A alone and that resulting from the combination of ochratoxin A and aflatoxin.

Similar results were found by Kubena et al. (199Oa) with regard to the use of HSCAS for the adsorption of T-2 toxin. Addition of 0.5% HSCAS to the diets of broilers contaminated with the toxin trichothecene T-2 (8 mg kgg ’ feed) and with or without total aflatoxin (3.5 mg kg-’ feed) during 3 weeks demonstrated that HSCAS signifi- cantly diminished some of the toxic effects of aflatoxin when present alone or in combination with T-2, but did not affect the toxicity of T-2 toxin alone at the level used in the study.

Similarly, Kubena et al. (1993a) demonstrated that HSCAS (0.5%) resulted in almost total protection against the effects caused in broilers by aflatoxin alone (3.5 mg kg-’ feed), limited protection against the combination of the same level of aflatoxin with 5.0 mg diacetoxyscirpenol (DAS) per kilogram feed, but no protection against the DAS alone, in a 3 week study. With another trichothecene mycotoxin, the deoxynivalenol (DON), Patterson and Young (1993) found that 0.5 or 1 .O% addition of HSCAS to pig diets contaminated with DON (from 3.5 to 11 mg DON kg-’ feed) resulted in no improvement in pig performance, that is, HSCAS did not influence the average daily gain of the pigs.

Therefore, it can be concluded that although HSCAS has been demonstrated to be very effective with regard to aflatoxin intoxication, its efficacy against zearalenone and ochratoxin A is limited and in the case of the trichothecenes tested, is practically zero.

204 A. J. Ramos, E. Hemcindez / Animal Feed Science Technology 65 (1997) 197-206

3. Conclusion

The use of adsorbent materials against mycotoxicosis is a new field that must be completely explored. First results are very promising with regard to aflatoxins, not only in the case of the most extensively studied sorbent, HSCAS, but also with other natural aluminosilicates, such as montmorillonite or bentonite (Ramos, 1994). The mechanism by which the HSCAS exerts its protective effects on animal performance is not completely known at present but the main hypothesis implicates the formation of a stable complex between the sorbent and the toxins. This complex would not be able to cross the luminal barrier of the intestine, the site of maximum absorption of mycotoxins, and therefore the bioavailability of these toxins could be reduced. One of the main advantages of this method of control of mycotoxicosis is that it could be used in a preventive manner.

Use of HSCAS or another sorbent compound as a feed additive for mycotoxin control requires study of the possible long-term effects of these materials on the utilization of essential nutrients, such as vitamins and minerals. If sufficient quantities of essential nutrients are rendered unavailable to the animals, it could have a non desired effect, such as a reduction in animal performance. If this is true, it may be expedient to include supplemental vitamins and minerals in animal diets when they are supplemented with adsorbent materials.

Although HSCAS is effective in reducing the toxicity of aflatoxin, enthusiasm for the efficacy of this sorbent material must be tempered by the fact that it may not be effective against other mycotoxins that can cause serious problems in animal production. The management of mycotoxin problems associated with animal production using adsorbent materials will only be an effective strategy if these materials have the ability to adsorb a large number of chemically distinct mycotoxins. However, there is currently great interest in overcoming this problem and determining the specificity and mecha- nism of action of HSCAS and other sorbents for aflatoxin and other mycotoxins, and future investigations must be directed to this goal.

Acknowledgements

This work was supported by a grant from the CICYT (AL1 94-0417-CO3). The authors thanks Dr. V. Sanchis, Department of Food Technology, University of Lleida (Spain), for useful discussions and critical review of the manuscript.

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