Philipp J Vet Anim Sci 2009, 35 (1): 49-58 49

EFFECT OF BENTONITE-MONTMORILLONITE ON THE PERFORMANCE AND

HUMORAL IMMUNE RESPONSE OF BROILERS FED AFLATOXIN-
CONTAMINATED DIETS

Sonia P. Acda, Marcela R. Batungbacal and Josephine R. Centeno1

ABSTRACT

A total of 480 day-old chicks (Cobbs) were randomly assigned to three

treatments as follows: 1) aflatoxin-contaminated diets (control); 2) aflatoxin-
contaminated diets + 0.2% bentonite-montmorillonite (BM); and 3) aflatoxin-
contaminated diets + 0.4% BM. The starter and finisher diets contained 3.45
and 21.68 ppb of aflatoxin B1. Results showed that at 28 days of age, average
body weight gain was not significantly different among treatments. Broilers
fed diets with BM were more efficient in feed utilization at 28 days than those
fed diets without BM. Birds in the control group were less immuno-
responsive to Newcastle Disease vaccine than those given aflatoxin-
contaminated diets with BM. BM was able to neutralize the immuno-
suppressive effect and hepatotoxic activity of aflatoxin; hence, birds fed
aflatoxin-contaminated diets with 0.4% BM had normal liver and responded to
ND vaccine with consistently high HI antibody titres.

Key words: aflatoxin, bentonite, broiler, montmorillonite, immuno-response

INTRODUCTION

Feedstuff contaminated with aflatoxin poses a great threat to both farm

animals and human. Animals consuming aflatoxin-contaminated feeds can develop
serious health problems and may pass the contamination to human food. Aflatoxin
exerts its harmful effects either by disrupting certain metabolic processes or by
interfering with proper antibody production (Tilden et al., 1999) thereby, not only
adversely influencing the performance, but also causing suppression of the bird’s
immune system (Huff, 1992).
Since mycotoxins remain long even after the fungi have been irradicated
(Mabbett, 1998), it is nearly impossible to totally eliminate aflatoxin from the feeds.
With the adverse effects that aflatoxin could cause to animal health and
performance, it became a concern of both feed manufacturers and livestock and
poultry raisers to look for ways and means to control and prevent the harmful effects
of aflatoxin in animals. Rose et al. (1983) stated that organic acids can minimize
mycotoxins to almost safe level as long as grains are adequately saturated with the

1
Animal and Dairy Sciences Cluster, College of Agriculture, University of the
Philippines Los Baños, College, Laguna 4031, Philippines (email:
nfc@laguna.net).
50 Acda, Batungbacal and Centeno

chemicals. Adsorbents or toxin binders, on the other hand, have the capacity to
tightly bind and immobilize toxic substances during digestion and/or absorption of
nutrients in order to prevent pathological effects to the animal. However, these
materials capable of sequestering multiple mycotoxins are also able to trap any one
or more nutrients incorporated in feeds (Dale, 1998).
Clay encompasses a diverse family of minerals and have indicated benefits
against mycotoxins. Bentonite-montmorillonite having partially modified surface is
designed to effectively bind polar mycotoxins, either by way of a chemical bond or
physical interaction. Hence, this study aimed to investigate the effects of bentonite-
montmorillonite on the performance and humoral immune response of birds fed
aflatoxin-contaminated diets.

MATERIALS AND METHODS

Broiler chicks
A total of 480 broiler chicks (Cobbs) were used in this study. All chicks were
vaccinated against Newcastle disease (ND) and infectious bursal disease (IBD)
following the recommended vaccination program, and reared according to standard
broiler production procedures. The chicks were randomly assigned to three
treatments. Each treatment was replicated 16 times with 10 chicks per replicate.
The treatments were as follows:
1. Aflatoxin-contaminated diets (control)
2. Aflatoxin-contaminated diets + 0.2% bentonite montmorillonite (BM)
3. Aflatoxin-contaminated diets + 0.4% BM

Experimental diets
The starter and finisher diets were formulated to contain 20.7% and 18.5%
crude protein, respectively. Both diets had a metabolizable energy of 3000 kcal/kg,
and supplemented with required vitamins, minerals and amino acids (Table 1).
Mouldy corn was used in the formulation of the diets. Enzyme-linked
immunosorbent assay (ELISA) was used to detect and measure the amount of
aflatoxins in the finished feeds. Proximate analyses and gross energy of the
formulated diets were done following standard methods of analysis described by the
Association of Official Analytical Chemists (AOAC, 1993).

Mycotoxins-adsorbent agent
The mycotoxins-adsorbent agent used in this study is a mineral powder
made from bentonite-montmorillonite with a partially modified mineral surface
designed to effectively bind polar mycotoxins either by way of a chemical bond or
physical interaction. The mycotoxin adsorbent was added-on to the diets according
to the manufacturer’s recommended rate of inclusion.

Production parameters
The average feed consumption, body weight gain, feed efficiency and
livability were determined at the end of each feeding period and for the entire
 Effect of bentonite-montmorillonite on humoral response of broilers 51

Table 1. Ingredient composition and calculated nutrient content of broiler starter

and finisher diets.

Feed ingredients Starter Finisher

Corn, yellow 40.06 48.22
Soybean meal, US HP 36.22 29.08
Rice bran D1 10.00 5.00
Crude coco oil 5.13 4.44
Copra meal 5.00 10.00
Limestone 1.48 1.26
Monodicalcium phosphate 1.36 1.19
Salt 0.25 0.35
Vitamin premix 0.20 0.20
Mineral premix 0.20 0.20
Choline chloride 0.05 0.05
DL-methionine 0.05 0.01
TOTAL 100.00 100.00
Calculated analysis (as fed):
Crude protein, % 20.70 18.50
Crude fiber, % 3.70 3.87
Crude fat, % 8.51 7.84
Calcium, % 0.90 0.78
Total phosphorus, % 0.82 0.71
Available phosphorus, % 0.45 0.39
Metabolizable energy, kcal/kg 3000 3000
Lysine, % 1.27 1.07
Methionine + cystine, % 0.79 0.67
Tryptophan, % 0.28 0.24
Threonine, % 0.86 0.76

feeding trial.

Serum antibody assay

To determine the antibody response to ND vaccine, ten birds from each
52 Acda, Batungbacal and Centeno

treatment were randomly taken and bled for sera immediately before and after
vaccination at weekly intervals. The antibody titres to ND vaccine were measured
by the hemagglutination-inhibition (HI) test (Carbrey et al., 1974). The HI antibody
titres were expressed as the reciprocals of the end-point of dilutions in logarithms to
the base 2.

Histopathological analysis
At the end of the feeding trial, the birds were slaughtered. The liver of each
bird was weighed and examined for gross lesions. Liver samples were placed
immediately in 5% buffered formalin solution and processed according to standard
procedures. Histological observations were made from Hematoxylin-eosin (H & E)
stained sections.

Statistical analysis
All gathered data were analyzed using the General Linear Model Procedures
of SAS (1985) in a completely randomized design. Treatment means were
compared using Duncan’s Multiple Range Test (DMRT).

RESULTS AND DISCUSSION

Nutrient composition of broiler diets

Mouldy corn was used in the formulation of the broiler diets to replicate what
is the reality in commercial operation. Studies have shown that fungal growth on
grains results in the reduction of nutritional qualities. Chen (2003) reported that in
mouldy corn, the energy content could be reduced by as much as 5%, protein by
7% and fat by as much as 63%. Alteration in the nutrient content of feedstuff due to
fungal development, however, depends on the species of fungus growing on it,
temperature, moisture and extent of storage (Vieira, 2003). The nutrient
composition and calculated analysis of the broiler starter and finisher diets are
presented in Table 1. Chemical analyses showed that the nutrient composition of

Table 2. Nutrient composition and aflatoxins content of broiler starter and finisher
diets.

Starter Finisher
Moisture, % 10.81 10.68
Ash, % 6.88 5.73
Crude protein, % 21.34 19.48
Crude fat, % 5.76 5.03
Calcium, % 1.05 1.04
Total phosphorus, % 0.76 0.77
Aflatoxins, ppb 3.45 21.68
 Effect of bentonite-montmorillonite on humoral response of broilers 53

the diets used in the present study were slightly higher than the calculated analysis
(Table 2) despite inclusion of mouldy corn. The apparent coefficient of
metabolizability of crude protein and energy in the diets was slightly lower than the
standard value; nevertheless, the diets contained sufficient amount of nutrients to
support normal growth of broilers.
The starter and finisher diets were found to contain 3.45 and 21.68 ppb of
aflatoxin B1, respectively. These values were within the reported average levels of
aflatoxin in foods and feedstuffs worldwide from 1989 to 1991, which were 0-50 ppb
for shelled corn and 0-28.6 ppb for milled corn (Wood, 1992). Recent surveys on
the incidence of mycotoxin in corn and its by-products, seeds and other grain by-
products and complete feeds, showed that in Asia the average level of aflatoxin in
these products was 19 ppb during 2006 and 24.8 ppb in 2007 (Chen and Rawlings,
2008). Wood (1992) commented that the worldwide levels of aflatoxin in feeds are
much lower than the levels which have been used in scientific studies.

Growth performance
The growth performance of broilers in the different treatments is presented in
Table 3. The average initial body weight of the chicks assigned to the different

Table 3. Summary of performance of broilers fed aflatoxin-contaminated diets

without or with BM.

Parameter Control 0.2% BM 0.4% BM CV (%)

Average body weight, g
Initial 118a 119a 118a 1.12
a a a
28 d 939 956 964 4.72
a b ab
42 d 1907 1806 1870 3.72
Average body weight gain,
g
28 d 820a 836a 846a 13.98
a b a
42 d 1788 1688 1752 3.83
Feed consumption, g
28 d 1327a 1312a 1317a 4.02
a a a
42 d 3270 3154 3158 5.49
Feed efficiency, g/g
28 d 1.62b 1.57a 1.56a 2.98
a a a
42 d 1.83 1.87 1.80 4.33

Treatment means within a row with the same superscript are not different (P>0.05).
54 Acda, Batungbacal and Centeno

treatments ranged from 118 to 119 g.

At 28 days of age, birds fed starter diet with BM had higher body weight gain
than those in the control group. The difference in body weight gain, however, was
not significant. At 42 days of age, the average body weight gain of broilers fed the
control diets and those fed diets with 0.4% BM was significantly higher (P<0.05)
than that of birds fed diets with 0.2% BM. It is known that young birds are much
more susceptible to the harmful effects of mycotoxins relative to older birds.
Hamilton (1987) claimed no safe level of aflatoxins, and level as low as 10 ppb
could result to economic losses to the broiler industry. Glahn (1993) reported that
aflatoxin at a dose of 2 mg/kg/day given for 10 consecutive days to 3-week old
broilers produced a moderate aflatoxicosis.
Although not statistically significant, broilers fed diets with BM consistently
had lower feed intake than the control. This indicates that the low level of aflatoxins
in the diets was not enough to negatively influence feed consumption of the birds in
the control group. Reddy et al. (1984) reported that 0.75 ppm aflatoxin in the diet
depressed feed consumption of broilers. On the other hand, the relatively low feed
intake of birds in treatments 2 and 3 could have been due to an increase in bulk
density of the feeds brought about by the inclusion of BM in the diets. Birds tend to
eat less with high dense feeds.
Despite the negative effect of BM on feed consumption, broilers fed diets
with the mycotoxin adsorbent were more feed efficient (P<0.05) than the control at
28 days but not at 42 days of age. This could be attributed to their low feed intake
but higher body weight gain relative to those fed diet without BM during the starter
period. The results also imply that BM enhanced the efficiency of feed utilization of
broilers only up to 28 days of age or during the starter period.

Antibody response to ND vaccine

The mean serum HI antibody titres (log2) to ND vaccine of broilers fed
aflatoxin contaminated diets with or without BM are presented in Table 4. The

Table 4. Average HI antibody titre (log2) to ND vaccine of broilers fed aflatoxin-

contaminated diets without or with BM.

Days after vaccination Control 0.2% BM 0.4% BM CV (%)

Primary immune response
7d 3.33a 2.67a 3.33a 15.70
a a a
14 d 2.33 3.33 3.70 38.92
Primary immune response
7d 4.00b 5.20a 5.20a 6.13
b a a
14 d 2.80 4.20 4.70 27.95
b a a
21 d 2.30 7.30 6.70 25.48

Treatment means within a row with the same superscript are not different (P>0.05).
 Effect of bentonite-montmorillonite on humoral response of broilers 55

maternal antibody titre for ND in broilers at day-old was 8.0 (log2) for all treatments.
There were no significant differences among treatments in the mean antibody titres
at 7 and 14 days after the initial ND vaccination.
Although the continuous ingestion of low to moderate levels of aflatoxin did
not have pronounced adverse effects on growth performance, the
immunosuppressive effect of aflatoxins was evident at 7, 14 and 21 days following
revaccination against ND.
The broilers fed the aflatoxin-contaminated diets without BM, consistently
had lower (P<0.05) secondary antibody response to ND vaccine than those given
diets with BM. At 21 days after revaccination against ND, the mean HI antibody titre
of the control birds dropped to 2.30 (log2), while those of the birds in treatments 2
and 3 rose to 7.30 and 6.70, respectively. According to Alexander (1988), HI
antibody titres of 4.0 to 6.0 (log2) would confer complete protection against
challenge with highly virulent ND virus, whereas titres of 2.0 or below means no
protection against challenge.
Mycotoxin-induced immunosuppression also could result in increased
susceptibility to other diseases. In the present study, broilers fed the control diet
without BM incurred a 3.0% mortality, whereas, those fed diets with BM had 98-
100% livability. Birds that died showed gross lesions suggestive of coli septicemia.
Results further indicate that the continuous intake of even small amount of
mycotoxins could be considered unsafe. The adverse effect of aflatoxin on the
immune system or general health may be alleviated by the addition of an adsorbent
such as BM.

Histopathological changes
The hepatotoxic effects of aflatoxin B1 have received a great deal of
scientific attention and shown to be of substantial economic threat to both meat and
egg production (Glahn, 1993). The liver is known to be the primary target for the
toxic action of aflatoxin B1. As shown in Figures 1 and 2, microscopic lesions such
as dilated sinusoids, foci of leukocyte infiltration and necrotic hepatocytes were
observed in histological sections of the liver samples of broilers fed the control diets
without BM and of broilers fed diets with 0.20% BM, whereas, those fed diets with
0.40% BM had normal liver (Figure 3).
Results of the present study showed that low doses of aflatoxins in broiler
diets which do not adversely affect performance may go unnoticed and yet are likely
to represent a continuous threat to animal and human health if not approximately
addressed. Bentonite-montmorillonite at 0.4% inclusion rate was able to neutralize
the immunosuppressive effect and hepatotoxic activity of aflatoxin in broilers fed
diets containing low (3.45 ppb) to moderate (21.68 ppb) doses of this mycotoxin.

CONCLUSION

Although there were no significant differences among treatments in the

mean antibody titres at 7 and 14 days after the initial ND vaccination, the broilers
fed the aflatoxin-contaminated diets with 0.20 and 0.40% BM showed 7.30 and 6.7
HI antibody titres, respectively against 2.30 of that of the control. Furthermore,
56 Acda, Batungbacal and Centeno

NH

DS

NH

LI

Figure 1. Section of liver from chicken (broiler) fed the control diets (T1), showing
necrotic hepatocytes (NH) leukocytes infiltration (LI), dilated sinusoids (DS).
(Hematoxylin and eosin, 100×).

microscopic lesions were observed in histological sections of the liver samples of

broilers fed the control diets and those fed diets with 0.20% BM, whereas those fed
diets with 0.40% had normal liver.
Based on the results, continuous intake of low to moderate dosage of
aflatoxins had no adverse effect on the performance of birds but may develop
serious health problems. The immunosuppressive effect and hepatotoxic activity of
aflatoxin in broilers fed aflatoxin-contaminated diets could be neutralized by addition
of 0.40% BM.

ACKNOWLEDGEMENT

The authors are very grateful to Bayer Philippines, Inc. for the financial
support extended to the project.

REFERENCES

Alexander DJ. 1988. Newcastle disease diagnosis. In: Newcastle Disease.

Alexander DJ (ed). Massachusetts, USA: Kluwer Academic Publishers.
Association of Official Analytical Chemists. 1993. Official Methods of Analysis. 18th
ed. Washington DC: AOAC.
 Effect of bentonite-montmorillonite on humoral response of broilers 57

LI
CV

NH

DS

Figure 2. Section of liver from chicken (broiler) fed diets with 0.2% BM (T2),
showing necrotic hepatocytes (NH), leucocyte infiltration (LI), dilated sinusoids
(DS), central vein (CV). (Hematoxylin and eosin, 100×).

Carbrey EA, Beard CW, Cooper R, Hansen RP and Pomeroy BS. 1974.
Hemagglutination and hemagglutination-inhibition tests with Newcastle virus-
microtiter technique. 17th Ann Proceed Am Assoc Vet Lab Diagnost.
Chen YJ. 2003. Mould and mycotoxins: Control from grain to feedings. Feed Int
47: 22-24.
Chen H and Rawlings R. 2008. How real is the threat of mycotoxins for feed and
poultry producers in Asia? Asian Poultry May 2008, pp. 36-38.
Dale N. 1998. Mycotoxin binders now it is time for real science. Feed Int 42: 22-23.
Glahn R. 1993. Mycotoxins and the avian kidney: Assessment of physiological
function. World Poultry Sci J 49 (3): 242-250.
Hamilton PB. 1987. Why the animal industry worry about mycotoxins. Raiser
Chemicals. pp. 302-331.
Huff WE. 1992. Efficacy of hydrated sodium calcium aluminum silicate to reduce
the individual and combined toxicity of aflatoxin and ochratoxin A. Poultry
Sci 71: 64-69.
Mabbet TT. 1998. Mycotoxin Hazards: More accurate quantification in the field.
Feed Int 42: 42-43.
Reddy DNP, Rao V, Reddy VR and Yadgiri B. 1984. Effect of selected levels of
dietary aflatoxins on the performance of broiler chicken. Indian J Anim Sci
54: 68-73.
Rose GW, Jasper AW and Agrimerica INC. 1983. Moulds in feed and flocks. Feed
58 Acda, Batungbacal and Centeno

CV S H

Figure 3. Section of liver from chicken (broiler) fed diets with 0.40% BM, showing
normal hepatocytes (H) and sinusoids (S), 100x.

Int 37: 24-25.

SAS. 1989. Statistical Analysis Systems User’s Guide. North Carolina: Cary.
Tilden WP, Cullison AE and Cowrey RS. 1999. Feeds and Feeding. New Jersey,
Prentice Hall, Inc.
Vieira SL. 2003. Nutritional implications of mould development in feedstuffs and
alternatives to reduce the mycotoxin problem in poultry feeds. World Poult
Sci J 59 (1): 111-122.
Wood GE. 1992. Mycotoxins in foods and feeds in the United States. J Anim Sci
70: 3941-3949.